agenda, abstracts, & participants...

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Seventh SympoSium on harmful algae in the u.S.

SympoSium ChairS

Leanne Flewelling, Florida Fish and Wildlife Conservation CommissionBarbara Kirkpatrick, Mote Marine Laboratory

loCal organizing Committee

Alina Corcoran, Florida Fish and Wildlife Conservation CommissionKate Kohler, Mote Marine LaboratoryVincent Lovko, Mote Marine LaboratoryBill Richardson, Florida Fish and Wildlife Conservation CommissionKaren Steidinger, Florida Fish and Wildlife Conservation Commission

Steering Committee

Lorraine Backer, CDC National Center for Environmental HealthGreg Doucette, NOAA National Ocean ServiceDeana Erdner, University of Texas at Austin Sonia Joseph Joshi, NOAA Center of Excellence for Great Lakes and Human HealthJudy Kleindinst, Woods Hole Oceanographic InstitutionRaphael Kudela, University of California, Santa CruzKathi Lefebvre, NOAA Northwest Fisheries Science CenterDennis McGillicuddy, Woods Hole Oceanographic InstitutionHans Paerl, University of North Carolina at Chapel Hill Tammi Richardson, University of South Carolina Marc Suddleson, NOAA National Ocean Service

SeSSion ChairS

HABS in a Changing World: Raphael KudelaBloom Dynamics and Ecology: Quay Dortch and Greg DoucetteBloom Prediction, Forecasting, and Modeling: Dennis McGillicuddyMonitoring and Management: Rob Magnien and Vincent LovkoHAB Genomics and Other ‘Omics: Diane GreenfieldToxicity and Pathogenicity: Kathi Lefebvre Fisheries and Foodwebs: Richard PierceHuman and Animal Health: Jan LandsbergPrevention, Control, and Mitigation: Marc SuddlesonHuman Dimensions of HABs: Sonia Joseph JoshiSpeed Talk Sessions: Leanne Flewelling, Barbara Kirkpatrick, and Alina Corcoran

Logo Design: Eli Minaya, Ringling College of Art and DesignProgram Design: Mary Ellen Seyle, designWorks advertising associates

Seventh Symposium on Harmful Algae in the U.S.

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SympoSium SponSorS:

Beagle Bioproducts, Inc.Chiles Restaurant GroupFlorida Fish and Wildlife Conservation CommissionFluid Imaging Technologies, Inc.Jelks Family FoundationMcLane Research Laboratories, Inc.Mote Marine LaboratoryMote Scientific FoundationSaigene Biotech, Inc.STARTUS Food and Drug AdministrationVisit Sarasota County

Student Support:

Fluid Imaging Technologies, Inc. National HAB OfficeThe Phycological Society of America US Food and Drug Administration

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Seventh SympoSium on harmful algae in the u.S.

October 27 – 31, 2013Sarasota, Florida

Agenda, Abstracts, & Participants

Table of Contents

Organizers _________________________________________________ 1

Sponsors __________________________________________________ 2

Agenda Overview ___________________________________________ 4

Symposium Agenda _________________________________________ 7

Poster Schedule ___________________________________________ 17

Keynote Address ___________________________________________ 27

Abstracts of Oral Presentations _______________________________ 29

Abstracts of Speed Talk Presentations __________________________ 85

Abstracts of Poster Presentations _____________________________ 123

Author Index _____________________________________________ 197

Symposium Participants ____________________________________ 207

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Page 4 Seventh Symposium on Harmful Algae in the U.S.

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Wmeal information

Breakfast: 7:00 am – 8:30 amBreakfast will be served each day (Monday through Thursday) in the Boathouse (behind the hotel).

lunch: 12:30 pm – 2:30 pmLunch coupons provided to all registered attendees (one per day, Monday through Thursday) can be used to purchase food (up to $10 per coupon) in the hotel restaurant or at sandwich carts located outside. Seating will be available in the Boathouse and on the Conference Center patio.

dinner is on your own (Monday through Wednesday).

In addition to Sunday afternoon registration hours, the registration desk will be staffed each day from approximately 7:30 am to 2:00 pm for information, T-shirt and poster sales, and other needs.

All talks and poster sessions will be in the Sarasota Ballroom.

Sunday, oCtoBer 27

12:00 pm - 5:00 pm Registration - Convention Center Registration Desk1:00 pm - 5:00 pm Young Investigators’ Symposium - Florida Room4:00 pm - 6:00 pm NHC Meeting - Boardroom 6:00 pm - 9:00 pm Welcome Reception - Florida Room and Poolside

evening aCtivitieS

Tuesday October 29 8:00 pm – 9:30 pm; Sarasota Ballroom

Roundtable: Methods for Evaluating HAB Species and Toxins: Progress, Comparative Studies, and Meeting End-User Needs

Organizers: Dianne I. Greenfield (Univ. of South Carolina) and Kathryn J. Coyne (Univ. of Delaware)

The goal of this forum is to discuss the advancements and challenges of developing various methods and technologies for evaluating HAB assemblages and toxins, including cross-comparison studies. The objectives of this roundtable discussion are to 1) identify the most important criteria end-users consider when selecting a particular HAB monitoring or research approach (e.g., cost, accuracy, sample through-put, etc.), 2) highlight progress on a range of methods for HAB species and toxin detection and quantification, 3) discuss outcomes and challenges faced while performing cross-comparative evaluations, and 4) evaluate the capacity for integrating different technologies within HAB monitoring and research networks.

Thursday, October 31 6:00 pm – 8:30 pm; Sarasota BallroomSymposium Banquet and Student Award Announcements

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Mon October 28

Tue October 29

Wed October 30

Thu October 31

Breakfast 7:30-8:30

8:30-9:45

Opening Remarks Oral Session 4

Prevention, Control, and Mitigation

Oral Session 7 HAB Genomics and Other

'Omics

Oral Session 10 Bloom Prediction, Forecasting, and

Modeling Plenary

Break 9:45-10:15

10:15-12:30

Oral Session 1 HABs in a Changing

World Poster Session 2

Oral Session 8 Human and Animal

Health Speed Talks 3

Break 11:15-11:30

Oral Session 5 Monitoring and

Management (1)

Break 11:15-11:30

Oral Session 2 Human Dimensions

of HABs Poster Session 4 Poster Session 5

Lunch 12:30-2:00

2:00-3:15 Oral Session 3

Bloom Dynamics and Ecology (1)

Oral Session 6 HAB Toxicity and

Pathogenicity

Oral Session 9 Monitoring and

Management (2) Oral Session 11 Bloom Dynamics and

Ecology (2)

Town Hall Meeting

(2:45-4:45)

Break 3:15-3:45 Break 3:15-3:45

3:45-4:45 Speed Talks 1 Speed Talks 2

Oral Session 12 Fisheries and

Foodwebs

Closing Remarks

Evening Activities Poster Session 1

and social mixer 5:00-6:30 pm

Poster Session 3 and social mixer

5:00-6:30 pm

6:00-8:30pm Banquet

(Ballroom)

8:00-9:30 pm Methods Round Table (Ballroom)

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MONDAY, OCTOBER 28, 2013

8:30 Opening Remarks

9:00 Plenary, Karen A. Steidinger: Dinoflagellate HAB science: Then and now

9:45-10:15 Break

oralS SeSSion 1: haBS in a Changing World Chair, raphael Kudela

time presenter title

10:15 Christopher The role of coastal ocean acidification in promoting and exacerbating the Gobler effects of harmful algal blooms

10:30 Hans Paerl Managing harmful cyanobacterial blooms along the freshwater-marine continuum in a world experiencing human and climatically-mediated change

10:45 Patricia Tester Gambierdiscus biogeography and the consequences of climate change

11:00 Ann Jochens Harmful algal bloom integrated observing system for the Gulf of Mexico

11:15-11:30 Break

oralS SeSSion 2: human dimenSionS of haBS Chair, Sonia Joseph Joshi

11:30 H. Kenneth Freshwater management policy update: Implementing the 3rd pillar of Hudnell the CWA, waterbody management, to form a systems approach

11:45 Porter CHANS: Florida red tides and coastal populations as a coupled Hoagland nature-human system

12:00 Jamie Studts Perceptions of beach activities during Florida red tide blooms: A conjoint analysis

12:15 Dianne Shipley Coordinating Florida red tide communication: A case study

12:30-2:00 Lunch

oralS SeSSion 3: Bloom dynamiCS and eCology - part 1 Chair, Quay dortch

2:00 Cynthia Heil Nutrients supporting Karenia brevis blooms: Results of the ECOHAB: Karenia program

2:15 Pat Glibert A new suite of nutrient kinetic relationships for picoplankton blooms: Non-traditional relationships are the new norm

2:30 Michael New insights into the behavior of Alexandrium fundyense life cycle Brosnahan stages through imaging flow cytometry

2:45 Florian Koch Expansion of brown tide blooms caused by Aureombra lagunensis to the east coast of the United States

3:00 Reagan A day in the life of Karenia brevis: Diel variation in cell volume and Errera ladder-frame polyether production

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MONDAY, OCTOBER 28, 2013 (CONTINUED)

3:15-3:45 Break

3:45-4:45 Speed talKS SeSSion 1 Chair, leanne flewelling

presenter title poster numberHABS IN A CHANGING WORLD Avery Tatters High CO2 promotes the production of Paralytic Shellfish C-3 Poisoning toxins by Alexandrium catenella from Southern California waters BLOOM DYNAMICS AND ECOLOGY Mindy Richlen Population structure and genetic dynamics of toxic Alexandrium B-11 fundyense (Dinophyceae) blooms in coastal embayments

Sugandha Shankar Role of ammonium in Alexandrium fundyense blooms in the Gulf B-16 of Maine and Georges Bank

Jennifer Vreeland Estuarine influence on the phytoplankton community structure B-25 during a Karenia brevis bloom on the Florida Gulf coast

Bill Richardson Use of dissolved inorganic and organic phosphorus by the toxic B-6 dinoflagellates Karenia brevis and Karenia mikimotoi (Dinophyceae)

Matthew Garrett Stratification influences on the migratory behavior of the harmful B-3 alga Karenia brevis

Mengmeng Tong The food quality and dissolved inorganic nutrient effect on the B-23 growth and toxicity of Dinophysis acuminata from North America

Marci Savage Nutrient and bloom dynamics before dredging of Lake B-13 Neatahwanta, a shallow, freshwater lake in upstate New York

Christopher Main The Vibrio-HAB connection: Investigating the influence of iron B-5 on formation of Vibrio biofilms and ROS production by Heterosigma akashiwo TOxICITY AND PATHOGENICITY Dalton Steele Purification and characterization of indole alkaloids from a central t-16 Florida isolate of the freshwater cyanobacterium, Fischerella sp., as inhibitors of vertebrate development in the zebrafish (Danio rerio) embryo model

Ryan Cassell Brevetoxin photoaffinity probes for the identification of the t-1 native brevetoxin receptor

Jan Landsberg The application of immuno-histochemistry as a tool for t-10 investigating suspected, historic, or unrecognized HAB-related animal mortality and disease events

4:45-5:00 Break

5:00-6:30 poster Session 1: HABs in a Changing World; Bloom Dynamics and Ecology; Toxicity and Pathogenicity

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TUESDAY, OCTOBER 29, 2013ORALS SESSION 4: PREVENTION, CONTROL, AND MITIGATION Chair, Marc Suddleson

Time Presenter Title

8:30 KevinSellner Optionsinmitigatingcyanobacteriablooms

8:45 Kaytee Inhibitionofthedinoflagellatecellcycleafterinoculationwiththe Pokrzywinski algicidalcompoundIRI-160AAexcretedbyShewanellasp.IRI-160

9:00 MarkWarner Examiningtheresponseofharmfuldinoflagellatestothebacterial algicideIRI-160AA:Differencesingrowth,physiologyandmodeofaction acrosslaboratoryandfieldcollectedsamples

9:15 Meredith Untanglingtheeffectsofanthropogenicversusnaturalnutrientsources Howard andimplicationsforharmfulalgalbloomsintheSouthernCalifornia Bight

9:30 Richard HighCaloosahatcheeRiverandestuarynutrientloadingsandone Bartleson harmfulalgalbloomafteranother

9:45-10:15 Break10:15-11:15 Poster Session 2:BloomDynamicsandEcology;HumanDimensionsof

HABs;MonitoringandManagement

ORALS SESSION 5: MONITORING AND MANAGEMENT - PART 1 Chair, Rob Magnien

11:15 EricaSeubert Implementationofsolidphaseadsorptiontoxintracking(SPATT)forthe monitoringharmfulalgalbloomsinsouthernCalifornia

11:30 Gregory DetectingparalyticshellfishtoxinsusingtheEnvironmentalSample Doucette Processor(ESP):Assaydevelopment&fielddeployment

11:45 Timothy AnewframeworkforpublichealthmonitoringofCHABimpacted Otten drinkingwaterreservoirs:Incorporationofcyanobacterialphysiology andgenotypesuccessionrates

12:00 NiclasEngene Taxonomicdescriptionsofnovelcyanobacterialbiodiversityfor monitoring,surveying,andcontrollingcyanobacterialharmfulalgal blooms(cyanohabs)

12:15 Kathryn Effectsofgrowthphase,dielcycleandmacronutrientlimitationonthe Coyne quantificationofHeterosigmaakashiwousingqPCRandSHA

12:30-2:00 Lunch

ORALS SESSION 6: TOXICITY AND PATHOGENICITY Chair, Kathi Lefebvre

2:00 JonathanDeedsGlobalperspectiveontheimpactsofthemarinetoxinpalytoxin2:15 WayneLitaker Whenhungry,Kareniabrevisgoestoxic

2:30 Alison Linkingfishtohumanillness:confirmationofciguaterafishpoisoning Robertson casesfromSt.ThomasU.S.VirginIslands2010-2012

2:45 JohnBerry IndolealkaloidsfromFisherellasp.isolatedfromFloridafreshwater sourcesaretoxictoaquaticvertebratesandinvertebrates

3:00 AshaJaja- Inhibitionofvertebratedevelopmentinzebrafishembryos(Danio rerio) Chimedza exposedtopolymethoxy-1-alkenesisolatedfromfreshwater cyanobacteria

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TUESDAY, OCTOBER 29 (CONTINUED)

3:15-3:45 Break

3:45-4:45 Speed talKS SeSSion 2 Chair, Barbara Kirkpatrick

presenter title poster numberBLOOM PREDICTION, FORECASTING, AND MODELING Kristen Thyng Physical mechanism for Karenia brevis bloom initiation in Texas p-8

Mary Christman A satellite-derived predictive model of red tide severity for the p-2 West Florida Shelf

Allison Allen NOAA’s Ecological Forecasting Roadmap p-1

HUMAN DIMENSIONS OF HABS Karen Kavanaugh From social media to national weather service products: hd-10 exploring new outreach tools for NOAA’s Harmful Algal Bloom Operational Forecast System

Gary Hitchcock CHANS: Environmental factors influencing Karenia brevis blooms hd-4 on the Florida west coast: Interpretation in support of health and socio-economic impacts

Vincent Lovko CHANS: Development of a hazard measurement system for hd-5 Florida red tide

Katrin Rudge CHANS: The benefits of participating in broader impacts: A high hd-7 school teacher’s perspective

Andrew Reich CHANS: Incidence of Neurotoxic Shellfish Poisonings during hd-6 Florida red tide (Karenia brevis) blooms: Is shellfish management effective at mitigating outbreaks? Margaret Byrne Perceptions of seafood consumption during Florida red tide hd-9 blooms: A conjoint analysis FISHERIES AND FOODWEBS Sheila O’Dea Florida’s first shellfish harvest closure due to domoic acid f-2

Christopher Loeffler Survey of Caribbean ciguatoxins in fish commonly consumed f-1 in St. Thomas, U.S. Virgin Islands

Justine Schmidt Variations in the microcystin content of different fish species f-4 collected from a eutrophic lake

4:45-5:00 Break

5:00-6:30 poster Session 3: Bloom Dynamics and Ecology; Bloom Prediction, Forecasting, and Modeling; Human Dimensions of HABs; Fisheries and Foodwebs

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WEDNESDAY, OCTOBER 30, 2013oralS SeSSion 7: haB genomiCS and other ‘omiCS Chair, diane greenfield


8:30 Allen Place Some assembly required: Multidomain PKS/NRPS genes in dinoflagellates

8:45 Julie Kubanek Metabolomic analysis of harmful algal bloom allelopathy: Deciphering sublethal impacts on competing phytoplankton

9:00 Kevin Meyer What exactly are you growing? An assessment of the bacterial community in commonly used cultures of Karenia brevis and other toxic and non-toxic dinoflagellates

9:15 Frances Global analysis of mRNA half-lives and de novo transcription in the Van Dolah Florida red tide dinoflagellate, Karenia brevis

9:30 Morgan A systems biology approach to understanding Microcystis blooms Steffen

9:45-10:15 Break

oralS SeSSion 8: human and animal health Chair, Jan landSBerg

10:15 Heather Hematologic and biochemical parameters in sea birds with Barron brevetoxicosis in southwest Florida

10:30 Kathi Lefebvre Common effects of chronic domoic acid exposure in zebrafish, sea lions, mice and humans

10:45 John Ramsdell Ciguatoxin and Hawaiian monk seals: monitoring blood toxin levels in northwestern Hawaiian Islands seals and evaluation of foraging under stress using a mouse model

11:00 Lynn Grattan Ciguatera fish poisoning in St. Thomas, U.S.V.I.: Exposure, symptoms, and recovery

11:15-11:30 Break

11:30-12:30 poster Session 4: Bloom Dynamics and Ecology; HAB Genomics and Other ‘Omics; Human and Animal Health; Toxicity and Pathogenicity

12:30-2:00 Lunch

oralS SeSSion 9: monitoring and management - part 2 Chair, vinCent lovKo

2:00 Gregory Boyer MERHAB-LGL: Monitoring and event response in the Lower Great Lakes– 10 years hence

2:15 Corinne Gibble Widespread detection of the freshwater toxin microcystin at the land-sea interface within Monterey Bay, CA

2:30 Neil The Jamestown S’klallam tribe responds to an emerging threat to human Harrington health: Diarrhetic Shellfish Poisoning in Washington state

2:45-4:45 haB Community town hall meeting

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THURSDAY, OCTOBER 31, 2013oralS SeSSion 10: Bloom prediCtion, foreCaSting, and modeling Chair, dennis mcgillicuddy


8:30 Barbara Hickey Pacific Northwest Toxins Project

8:45 Clarissa Forecasting the terrestrial influence on domoic acid production: Anderson a mechanistic approach

9:00 Richard Stumpf Climatological analysis of blooms in Lake Erie and seasonal forecast

9:15 David Millie Great Lakes ‘Big Data’ and HAB-informatics: using artificial intelligence to deconvolve Microcystis-environmental relationships

9:30 Darren Predicted origins of Karenia brevis bloom formation along the coast of Henrichs Texas using an individual-based model

9:45-10:15 Break

10:15 – 11:15 Speed talKS SeSSion 3 Chair, alina Corcoran

presenter title poster numberHAB GENOMICS AND OTHER ‘OMICS Kathleen Pitz Saxitoxin gene structure and representation in non-toxic and o-10 toxic Alexandrium tamarense species

Lisa Campbell Cellular response to osmotic stress in Karenia brevis o-4

David Jayroe Stress response in Karenia brevis: Changes in ribosomal RNA o-7

G. Jason Smith Transcriptome analysis of the diatom Pseudo-nitzschia o-12 australis reveals pathways associated with domoic acid biosynthesis

Holly Bowers Assessing pre-bloom, sub-surface populations of Pseudo- o-2 nitzschia in the San Pedro shelf region of southern California MONITORING AND MANAGEMENT Marco Hatch Real-time detection of harmful algae at a tribal marine m-9 aquaculture site

Brian Gregson Real-time water quality monitoring for HAB events using m-2 autonomous portable water laboratories and data visualization “cloud” analysis

Michelle Tomlinson Quantifying cyanobacteria and high biomass blooms from m-8 satellite

PREVENTION, CONTROL, AND MITIGATION Katherine Perri An investigation into the effects of shoreline use on Cm-6 cyanobacterial abundance in Sodus Bay, Lake Ontario Wenjun Jiang Photocatalytic degradation of microcystin-LR by Rose Bengal Cm-3

Helena Pound Isolation of bacterial capable of degrading microcystin-LR Cm-7

Kevin Owen Proposed harmful algal bloom control technologies: Floating Cm-5 desalination and water pumping plants

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THURSDAY, OCTOBER 31, 2013 (CONTINUED) 11:15-11:30 Break

11:30-12:30 poster Session 5: HAB Genomics and Other ‘Omics; Monitoring and Management; Prevention, Control, and Mitigation

12:30-2:00 Lunch oralS SeSSion 11: Bloom dynamiCS and eCology - part 2 Chair, gregory doucette

2:00 Stephanie Alexandrium bloom ecology in Puget Sound: Cyst dynamics, Moore growth, transport, and climate pathways

2:15 Dennis Georges Bank: A leaky incubator of Alexandrium fundyense blooms McGillicuddy

2:30 John Ryan Planktonic layer phenomena in the ecology of toxigenic Pseudo- nitzschia: examples from coastal California

2:45 Katherine Spatial and temporal variability of toxic Pseudo-nitzschia spp. in the Hubbard Gulf of Maine during summer 2012

3:00 Susanne Linking individual cell behaviors with the population distribution of the Menden- raphidophyte Heterosigma akashiwo Deuer

3:15-3:45 Break

oralS SeSSion 12: fiSherieS and foodWeBS Chair, richard pierce

3:45 Justin Liefer Ecological and environmental factors affecting Caribbean ciguatoxins and their trophic transfer to reef dwelling fishes

4:00 Jennifer Using genetic markers to determine the effect of seeding on the Phillips distribution of a saxitoxin-resistant mutation in Mya arenaria

4:15 Emily Smith Evaluation of potential oyster contamination from cyanobacterial toxins

4:30-4:45 Closing remarks

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POSTER SESSION 1 MONDAY, OCTOBER 28, 2013 5:00 – 6:30 PM

presenter title poster number (*speed talk)

HABS IN A CHANGING WORLD Lora Fleming Linking human health and well being with weather, climate and the C-1 environment with harmful algal blooms (HABs)

Harold Flores Development and evaluation of a direct LC-MS/MS method for C-2Quintana determination of DMSP in biological samples

Avery Tatters High CO2 promotes the production of Paralytic Shellfish Poisoning C-3* toxins by Alexandrium catenella from Southern California waters BLOOM DYNAMICS AND ECOLOGY Matthew Garrett Stratification influences on the migratory behavior of the harmful B-3* alga Karenia brevis

Christopher Main The Vibrio-HAB connection: Investigating the influence of iron on B-5* formation of Vibrio biofilms and ROS production by Heterosigma akashiwo

Bill Richardson Use of dissolved inorganic and organic phosphorus by the toxic B-6* dinoflagellates Karenia brevis and Karenia mikimotoi (Dinophyceae)

Bill Richardson Use of dissolved inorganic and organic nitrogen by the toxic B-7 dinoflagellates Karenia brevis and Karenia mikimotoi (Dinophyceae)

Mindy Richlen Population structure and genetic dynamics of toxic Alexandrium B-11* fundyense (Dinophyceae) blooms in coastal embayments

Marci Savage Nutrient and bloom dynamics before dredging of Lake, B-13* Neatahwanta a shallow, freshwater lake in upstate New York

Sugandha Shankar Role of ammonium in Alexandrium fundyense blooms in the Gulf B-16* of Maine and Georges Bank

Charles Tilney Comparing the diel vertical migrations of sympatric Karlodinium B-21 veneficum (Dinophyceae) and Chattonella subsalsa (Raphidophyceae) in laboratory columns

Mengmeng Tong The food quality and dissolved inorganic nutrient effect on the B-23* growth and toxicity of Dinophysis acuminata from North America Jennifer Vreeland Estuarine influence on the phytoplankton community structure B-25* during a Karenia brevis bloom on the Florida Gulf coast TOxICITY AND PATHOGENICITY Ryan Cassell Brevetoxin photoaffinity probes for the identification of the native t-1* brevetoxin receptorWei Chen Localization of brevetoxins and photoaffinity labeling of target t-2 proteins

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POSTER SESSION 1 (C0NTINUED) MONDAY, OCTOBER 28, 2013 5:00 – 6:30 PMpresenter title poster number (*speed talk)Courtney Cocilova Brevetoxin metabolism and physiology – a freshwater model t-3 of morbidity in endangered sea turtles

Allan Goodman Structure activity relationship of brevenal derivatives t-5

I-Shuo Huang Geitlerinema sp. produces a novel ichthyotoxic cyanobacterial t-7 toxin

Asha Jaja- Bioassay-guided purification of a bioactive carotenoid from t-8 Chimedza the freshwater cyanobacterium, Cylindrospermopsis raciborskii

Jan Landsberg The application of immuno-histochemistry as a tool for investigating t-10* suspected, historic, or unrecognized HAB-related animal mortality and disease events

Christina Lydon Identification of cyanobacterial toxins involved in the apparent t-11 intoxication of dolphins in the Florida Keys

Sean O’Mara Are fatty acid amides responsible for fish mortality during t-12 Prymnesium parvum blooms?

Carlton Quantification of domoic acid and okadaic acid in shellfish and t-13 Rauschenberg algae using an LC/MS/MS (triple quadrupole) approach

Dalton Steele Purification and characterization of indole alkaloids from a central t-16* Florida isolate of the freshwater cyanobacterium, Fischerella sp., as inhibitors of vertebrate development in the zebrafish (Danio rerio) embryo model

Bingxue Zheng Development of capillary electrophoresis as part of a micro-total t-17 analytical system for microcystins

POSTER SESSION 2TUESDAY, OCTOBER 29, 2013 10:15 – 11:15 AM presenter title poster number (*speed talk)BLOOM DYNAMICS AND ECOLOGY Deana Erdner Natural mortality during the decline of Alexandrium blooms B-10

K. David Hambright An experimental assessment of the importance of propagule B-12 pressure and community resistance on the invasion success of the toxigenic haptophyte Prymnesium parvum

Sarah Holmes Exploring the possibility of cheating between strains of harmful B-14 algal bloom-forming Microcystis aeruginosa POSTER SESSION 2 (CONTINUED)

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POSTER SESSION 2 (C0NTINUED) TUESDAY, OCTOBER 29, 2013 10:15 – 11:15 AM presenter title poster number (*speed talk)Jayme Smith The influence of nutrient and light perturbations on natural B-19 phytoplankton assemblages containing the toxin producing diatom Pseudo-nitzschia

William Unprecedented Microcystis bloom in northern California’s B-22 Stringfellow Bay-Delta Estuary and impact on zooplankton abundance

Kathryn Van Environmental factors affecting the release of dopamine and B-26 Alstyne oxidants by the bloom-forming green seaweed Ulvaria obscura HUMAN DIMENSIONS OF HABS Nick Boase Thinking alike? Comparing expert and public perceptions of the hd-1 benefits and risks of consuming shellfish

Lora Fleming A modified conceptual model integrating health and environmental hd-2 impact assessment: Harmful Algal Blooms

Lora Fleming CHANS: Public perception of fertilizer ordinances and Florida hd-3 red tides

Porter Hoagland Linking Florida red tides to human health effects: Data issues and hd-11 preliminary signals

Kate Kohler Where and how do individuals receive and prefer to receive hd-12 information on Florida red tide?

Zongchao Li CHANS: Framing the red tide story: Daily newspaper coverage in hd-8 southwest Florida MONITORING AND MANAGEMENT Ann Abraham LC-MS methodology for biomarkers of brevetoxins in the eastern m-1 oyster (Crassostrea virginica) and hard clam (Mercenaria sp.) exposed to Karenia brevis blooms

Alan Hails Successful ocean observatories at work on a harmful algal bloom m-3 on the West Florida Shelf

Sherwood Hall Facilitating implementation of the receptor binding assay for PSP m-4

Sandra Shumway Biofouling tunicates on aquaculture gear as potential vectors of m-5 harmful algal introductions

Samantha Weber Monitoring nutrients in Sodus Bay New York m-6

Ben Whitenack Assessing the strengths and weaknesses of each Florida HAB m-10 reporting system

Lawrence Younan Determination of phytoplankton groups using Turner Designs’ m-11 PhytoFind

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POSTER SESSION 3 TUESDAY, OCTOBER 29, 2013 5:00 – 6:30 PM

presenter title poster number (*speed talk)BLOOM DYNAMICS AND ECOLOGY Natalie Dou A persistent bloom of Anadyomene J.V. Lamouroux B-1 (Anadyomenaceae, Chlorophyta) in Biscayne Bay, Florida

Amanda Ellsworth Monitoring strategies for the ciguatera-causing benthic B-2 dinoflagellate, Gambierdiscus

Gabriela Hannach Seasonal patterns in nutrients and phytoplankton in Quartermaster B-4 Harbor, an enclosed Puget Sound embayment with recurrent harmful algal blooms

Yoonja Kang Effects of nutrients and grazing mortality on the abundance of B-15 Aureoumbra lagunensis during a Florida brown tide bloom in 2012

Raphael Kudela Stimulation of domoic acid production from transient changes B-17 in nutrients

Alex Leynse Gambierdiscus nutrient uptake kinetics B-20

Laura Markley Potential HAB species found in the Caloosahatchee Estuary, Florida B-24

Regina Radan Growth and toxin production of the mixotrophic dinoflagellate B-27 Alexandrium catenella BLOOM PREDICTION, FORECASTING, AND MODELING Allison Allen NOAA’s Ecological Forecasting Roadmap p-1*

Mary Christman A satellite-derived predictive model of red tide severity for the p-2* West Florida Shelf

Edward Davis Assessment of the eastern Gulf of Mexico Harmful Algal Bloom p-3 Operational Forecast System: A comparative analysis of forecast skill and utilization, 2004-2012

Katherine Derner Assessment of the western Gulf of Mexico Harmful Algal Bloom p-4 Operational Forecast System: Analysis of forecasts and utilization over the first two operational years, 2010-2012.

Jason Lenes Silicon control of diatom competitors against the red tide p-5 dinoflagellate, Karenia brevis, within the eastern Gulf of Mexico

Susan Lubetkin Habitat modeling of Pseudo-nitzschia distribution and toxicity in p-6 the coastal waters of the northwest Pacific using non-parametric multiplicative regression

Grace Maze Time series analysis for periods with high concentration versus p-7 periods with low concentration of Karenia brevis

Kristen Thyng Physical mechanism for Karenia brevis bloom initiation in Texas p-8*

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POSTER SESSION 3 (CONTINUED) TUESDAY, OCTOBER 29, 2013 5:00 – 6:30 PM

presenter title poster number (*speed talk)FISHERIES AND FOODWEBS Christopher Survey of Caribbean ciguatoxins in fish commonly consumed in f-1*Loeffler St. Thomas, U.S. Virgin Islands

Sheila O’Dea Florida’s first shellfish harvest closure due to domoic acid f-2*

Richard Pierce Bioaccumulation of brevetoxins and major metabolites in filter- f-3 feeding and carnivorous mollusks exposed to natural Karenia brevis harmful algal blooms

Justine Schmidt Variations in the microcystin content of different fish species f-4* collected from a eutrophic lake HUMAN DIMENSIONS OF HABS Margaret Byrne Perceptions of seafood consumption during Florida red tide blooms: hd-9* A conjoint analysis

Gary Hitchcock CHANS: Environmental factors influencing Karenia brevis blooms on hd-4* the Florida west coast: Interpretation in support of health and socio-economic impacts

Karen Kavanaugh From social media to national weather service products: Exploring hd-10* new outreach tools for NOAA’s Harmful Algal Bloom Operational Forecast System

Vincent Lovko CHANS: Development of a hazard measurement system for Florida hd-5* red tide

Andrew Reich CHANS: Incidence of Neurotoxic Shellfish Poisonings during Florida hd-6* red tide (Karenia brevis) blooms: Is shellfish management effective at mitigating outbreaks?

Katrin Rudge CHANS: The benefits of participating in broader impacts: A high hd-7* school teacher’s perspective

POSTER SESSION 4 WEDNESDAY, OCTOBER 30, 2013 11:30 AM – 12:30 PM

presenter title poster number (*speed talk)BLOOM DYNAMICS AND ECOLOGY L. Kellie Dixon Karenia sp. in SW Florida – trajectories of nitrogen, silica, other B-8 water quality, and taxonomic components during three extended blooms

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POSTER SESSION 4 (CONTINUED) WEDNESDAY, OCTOBER 30, 2013 11:30 AM – 12:30 PM

presenter title poster number (*speed talk)Alexis Fischer The effect of temperature conditioning on Alexandrium fundyense B-18 cyst germination dynamics in a shallow estuarine system

Ari Nissanka Responses of a natural Karenia bloom and other taxa of the West B-9 Florida Shelf to single nutrient, multiple nutrient, or estuarine water additions

HAB GENOMICS AND OTHER ‘OMICS Scott Anglin Characterizing sense and antisense RNA populations in Karenia o-1 brevis at different times of the diel cycle

Dianne Greenfield Elucidating the genetic diversity of Microcystis aeruginosa within o-3 a toxic bloom in South Carolina coastal ponds using 454-pyrosequencing technology

Grant Jones Involvement of multiple eIF4ES in mRNA recruitment in o-5 dinoflagellates

Darcie Ryan Karenia brevis reference transcripome assembly and transmembrane o-9 protein sequence discovery

Pengfei Sun Epoxide hydrolases in Karenia brevis: Key enzymes in brevetoxin o-11 biosynthesis

April Woods Role of ROS signaling in the domoic acid biosynthetic pathway o-13

HUMAN AND ANIMAL HEALTH Preston Kendrick Effects of chronic low-dose domoic acid exposure on mitochondrial h-1 function in mice

Anne Rolton The effects of field exposure of Crassostrea virginica to Karenia brevis: h-2 Histopathology and brevetoxin accumulation in gametes

Cathy Walsh Immune function in rescued manatees exposed to brevetoxins h-3

TOxICITY AND PATHOGENICITY Andrea Bourdelais Ladder frame polyethers as potential drug transporters t-18

Elizabeth Elliott Uptake mechanism of fluorescent conjugates of Karenia brevis t-19 ladder frame polyether compounds

Nick Fowler A chemical analysis of Karenia papilionacea T-4

Meghan Grandal Determining functional effects of brevetoxin and brevetoxin t-6 antagonists

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POSTER SESSION 4 (CONTINUED) WEDNESDAY, OCTOBER 30, 2013 11:30 AM – 12:30 PM

presenter title poster number (*speed talk)Tanya Hogue Development of an LCMS assay for determination of amino acid t-9 concentration in Karenia brevis

Jen McCall Development of competitive fluorescence-based binding assays for t-14 brevetoxins and brevenal

Nina Neill Localization of brevisin-daunorubicin conjugate in SJCRH30 cells t-20

Susan Niven Isolation and characterization of a brevenal epimer from cultured t-15 Karenia brevis

POSTER SESSION 5 THURSDAY, OCTOBER 31, 2013 11:30 AM – 12:30 PM

presenter title poster number (*speed talk)HAB GENOMICS AND OTHER ‘OMICS Holly Bowers Assessing pre-bloom, sub-surface populations of Pseudo-nitzschia o-2* in the San Pedro shelf region of southern California

Lisa Campbell Cellular response to osmotic stress in Karenia brevis o-4*

Kelly Fridey Translational response to heat stress in the Florida red tide o-6 dinoflagellate, Karenia brevis

David Jayroe Stress response in Karenia brevis: Changes in ribosomal RNA o-7*

Megan Meek Development of PCNA as a molecular biomarker for growth status o-8 of Karenia species in Gulf of Mexico red tides

Kathleen Pitz Saxitoxin gene structure and representation in non-toxic and toxic o-10* Alexandrium tamarense species

G. Jason Smith Transcriptome analysis of the diatom Pseudo-nitzschia australis o-12* reveals pathways associated with domoic acid biosynthesis

Mark Van Asten The development and validation of a multiplex qPCR assay for the o-14 detection of toxin producing cyanobacteria

MONITORING AND MANAGEMENT Danielle Dupuy A synoptic view of Florida inland algal blooms m-7

Brian Gregson Real-time water quality monitoring for HAB events using m-2* autonomous portable water laboratories and data visualization “cloud” analysis

Marco Hatch Real-time detection of harmful algae at a tribal marine aquaculture m-9* site

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POSTER SESSION 5 THURSDAY, OCTOBER 31, 2013 11:30 AM – 12:30 PM

presenter title poster number (*speed talk)Michelle Tomlinson Quantifying cyanobacteria and high biomass blooms from satellite m-8*

PREVENTION, CONTROL, AND MITIGATION Anamari Boyes Innovative drinking water treatment for taste and odor removal Cm-1 with naturally occurring bacteria found in surface water

Amanda Burson Mitigation of a toxic Alexandrium bloom using hydrogen peroxide Cm-2

Wenjun Jiang Photocatalytic degradation of microcystin-LR by Rose Bengal Cm-3*

Kevin O’Shea Photooxidation of domoic acid Cm-4

Kevin Owen Proposed harmful algal bloom control technologies: Floating Cm-5* desalination and water pumping plants

Katherine Perri An investigation into the effects of shoreline use on cyanobacterial Cm-6* abundance in Sodus Bay, Lake Ontario

Helena Pound Isolation of bacterial capable of degrading microcystin-LR Cm-7*

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Karen a. Steidinger

Florida Fish and Wildlife Conservation Commission100 Eighth Avenue SE, St. Petersburg, FL 33701, USA

Early dinoflagellate HAB research was geared toward autecology of the causative organism and was conducted on a local geographic scale. Breakthroughs in phytoplankton culturing methods allowed scientists to bring the organisms and their environments into the laboratory for experimentation and characterization. Neurotoxins and other bioactive compounds were suspected and tested for but often not chemically characterized until years later. Global advances in HAB methodology, instrumentation, technology, and analyses have advanced the science to the point that a species is not just a Latin binomial but a string of multi-character traits of morphology, ultrastructure, biochemical constituents (including pigments, toxins, sterols, etc.), gene sequences, life cycle stages, ecophysiology, geographical distribution, optical signature, and other defining points in space-time. Today, scientists still study toxins and their analogs, toxin gene sequences, stressors that affect toxin production, subtle population differences, metabolic alteration of structure and potency, and the role of bacteria in toxin synthesis. In the 1970s, scientists recognized the importance of life cycle stages and the possibility of seed beds in initiating recurring blooms. Today scientific advancement has resulted in a model that predicts Gulf of Maine Alexandrium fundyense blooms from population dynamics, seed beds, and physical forcing. Yet, benthic resting stages are not the only players in recurring blooms; other life cycle stages such as pellicle cysts and pelagic populations may represent a continuing source of inoculum. Many HAB models involve physical forcing at some stage of bloom development. Indeed, the physics of the coastal oceans, whether it relates to upwelling, tidal fronts, thin layers, or circulation patterns is often critical to predicting HABs. Coupled biophysical models are constantly being pursued to better understand the system. Ocean observing systems including satellite sensors and new tools such as chemical and optical detection devices aboard fixed and mobile platforms are the future for monitoring and forecasting. However, refinement of these and new applications requires basic research on the HAB organisms, their biological consortia, the environment, and feedback loops.

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S S FORECASTING THE TERRESTRIAL INFLUENCE ON DOMOIC ACID PRODUCTION: A MECHANISTIC APPROACH

Clarissa R. Anderson1, Christopher A. Edwards1, Nicole L. Goebel1, and Raphael M. Kudela1

1University of California Santa Cruz, 1156 High St., Santa Cruz, CA 95073, USA

Physical-biological models are an increasingly important tool for expanding our heuristic view of phytoplankton community structure. Ideally, biological models will have a mechanistic component based on first principles of planktonic life cycles and physiology. Several studies from the Monterey Bay and southern California suggest that the dominant HAB-former, the toxigenic diatom genus, Pseudo-nitzschia, is alternately associated with 1) upwelling pulses 2) periods of river runoff and 3) resuspension of seeding populations into the euphotic zone during upwelling and storms. Laboratory manipulations of toxigenic Pseudo-nitzschia species in culture have shown that production of its deadly neurotoxin, domoic acid (DA), is in turn often a function of silicic acid or phosphate limitation at different phases of Pseudo-nitzschia growth (Bates et al. 1991; Fehling et al. 2004; Pan et al. 1996). Increased nutrient inputs from river runoff are expected to reduce water column Si:N ratios on time scales relevant to phytoplankton growth (Kudela 2008). At the same time, these changes in nutrient stoichiometry are likely coupled to increased loading of urea and other regenerated forms of N, which further enhance toxin production by at least some species of Pseudo-nitzschia (Howard et al. 2007; Kudela et al. 2008). We hypothesize that heavy river discharge to the Monterey Bay following the first large storms of the rainy season - “first flush” events - increases the toxicity of Pseudo-nitzschia blooms in response to an adjustment in nutrient stoichiometry. The altered stoichiometry in favor of Si-limitation and regenerative forms of N increases particulate DA concentrations due to a physiological stress response associated with DA production. To examine this, we developed a mechanistic model of DA production, defined as the product of the phytoplankton growth rate, DA-producing phytoplankton biomass, and a DA production factor where the growth rate follows the Michaelis-Menten form for individual nutrient limitation. Various forms of nutrient limitation (Si-lim, N-lim) are tested as adjustments to the DA growth factor. Two tunable parameters determine the maximum rate of production and its rate of decline with increasing growth. Our first-year effort has focused on optimizing model parameters with bootstrapping methods to fit laboratory data from Si-limited chemostats. We have also tested these fits against batch culture data from the literature (Fehling et al. 2004) in order to compare our one-dimensional model with a recently published mechanistic DA model (Terseleer et al. 2013). In this presentation, we show model comparison results and discuss the implications of a carbon-based approach (Terseleer et al. 2013) versus our simpler deterministic model for examining DA production as a function of nutrient ratios and nitrogen source. An advantage to the latter approach is its suitability for incorporation into a wide variety of NPZ-Regional Ocean Model Systems without requiring major modification to the NPZ construct and thereby facilitating model evaluation against observations of “first flush” events in the Monterey Bay.

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HEMATOLOGIC AND BIOCHEMICAL PARAMETERS IN SEA BIRDS WITH BREVETOxICOSIS IN SOUTHWEST FLORIDA

Heather W. Barron, DVM, Dipl. ABVP-avian1, Richard D. Bartleson, PhD2, Katherine B. McInnis, DVM1, Helen L. Ingraham, DVM1 and Carolyn Cray, PhD3

1Clinic for the Rehabilitation of Wildlife (CROW), 3883 Sanibel-Captiva Rd., Sanibel, FL 33957, USA2Sanibel-Captiva Conservation Foundation Marine Lab, Sanibel, FL, 33957 USA3University of Miami Miller School of Medicine, Division of Comparative Pathology, P.O. Box 016960 R-46, Miami, FL 33101 USA.

Morbidity and mortality in sea birds from red tide events appears to be on the rise and thus of immediate concern in conservation medicine. Brevetoxins are algal toxins produced by the dinoflagellate, Karenia brevis (the Florida red tide organism) and other algal species. Animals may be exposed by ingestion or inhalation of the toxin. More than 2,000 birds representing over a 150 different species present annually to the wildlife hospital at CROW. In 2012, 439 birds representing 22 different species presented to CROW with suspected brevetoxicosis, 44 of which were confirmed via ELISA assay. These toxins cause neuronal and muscle cell depolarization, leading to neurologic signs in affected animals including ataxia, loss of palpebral reflex, nystagmus, and tremors. Other clinical signs documented here at CROW that are not routinely reported in the avian literature include bradycardia, melena, hematochezia, paralysis of the nictating membrane, decreased vent tone, coughing, and dyspnea. Clinical signs appear to vary to some degree across species lines. Brevetoxins are metabolized by the liver and excreted by the kidneys, potentially leading to disease in these organs. However, there has been little documentation of the clinicopathologic changes seen in sea birds with brevetoxicosis, which has made effective treatment problematic. Other rehabilitation centers report treatment success rates of 33-46%; the release rate at CROW in 2012 was 55%. This study determined the seroprevalence of brevetoxins in a wide variety of piscivorous and omnivorous birds presented to CROW. Additionally, the clinicopathologic extent of disease in double crested cormorants was determined by documenting changes in hematologic and plasma biochemical profiles, thus enabling more effective treatment. The study also determined the range of clinical signs in a variety of different species of birds positive for brevetoxins to enhance clinical recognition of the disease among biologists, veterinarians, and wildlife centers.

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HIGH CALOOSAHATCHEE RIVER AND ESTUARY NUTRIENT LOADINGS AND ONE HARMFUL ALGAL BLOOM AFTER ANOTHER

Richard D. Bartleson1, Eric C. Milbrandt1, Mark A. Thompson1

1Sanibel-Captiva Conservation Foundation Marine Lab, 900A Tarpon Bay Rd. Sanibel, FL, 33957, USA

Surface runoff has replaced much of the groundwater inflows to the Caloosahatchee Estuary. This runoff in the Caloosahatchee watershed is high in nutrients and colored dissolved organic matter. Freshwater flow from part of the watershed and Lake Okeechobee are regulated by three water control structures. When the water control structure S79 is closed, blooms of cyanobacteria such as Microcystis and Anabaena occur in the stagnant, fresh water section of the river to the east. These blooms and their toxins are sometimes passed downstream when flows through S79 are resumed. When S79 is open, dissolved nutrients from the watershed and the Lake can be transmitted far downstream and into the Gulf due to low light penetration through the dark water. Low densities of submersed plants in the Estuary facilitate the transport of dissolved nutrients. Often, freshwater discharge rates through S79 are high enough to send plumes of high nutrient water great distances into the Gulf. Sometimes when these high discharges are occurring, the offshore phytoplankton populations include Karenia spp. and/or Trichodesmium erythraeum. The high nutrient loading rate into the Gulf during these events can support growth of dense populations of Karenia brevis well away from shore. The nitrogen fixing T. erythraeum, which can provide nutrients to Karenia and may also produce toxins, can also benefit from the high discharge plumes. Blooms of a variety of other phytoplankton species, some of which are potential toxin producers regularly occur at the interface of the Caloosahatchee Estuary and the Gulf of Mexico. In 2011, blooms of two potential brevetoxin producing species, Fibrocapsa japonica and Chatonella subsalsa, discolored the water along Sanibel beaches. Blooms of other potentially toxin producing phytoplankton including Pseudo-nitzschia spp. frequently occur. Thousands of fish and birds and over 200 manatees died in the estuary during the red tide of 2012-2013. A filamentous, nitrogen fixing cyanobacteria, Lyngbya majuscula also occasionally overgrows seagrasses in a large area of the estuary after Karenia blooms. And other macroalgae also reach high biomass levels after red tides kill algal grazers. The presence of problematic nitrogen fixing cyanobacterial species throughout the Caloosahatchee River and Estuary and into the Gulf of Mexico shows the need to substantially reduce phosphorus loading rates which average over 240 metric tons yr-1 and range to 500 metric tons yr-1.

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INDOLE ALKALOIDS FROM FISHERELLA SP. ISOLATED FROM FLORIDA FRESHWATER SOURCES ARE TOxIC TO AQUATIC VERTEBRATES AND INVERTEBRATES

Katherine Walton1, Patrick D. L. Gibbs2, Michael C. Schmale2, Miroslav Gantar3 and John P. Berry1

1Department of Chemistry and Biochemistry, Marine Science Program, Florida International University, 3000 NE 151st Street, North Miami, FL 331812Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 331463Department of Biological Sciences, Florida International University, 11900 SW 8th Ave, Miami, FL 33199

Fischerella and related genera in the Stigonemataceae, a family of branched filamentous cyanobacteria, are widely distributed in freshwater systems in the U.S. and globally. We used the zebrafish (Danio rerio) embryo as a model of vertebrate development - specifically as a relevant freshwater species of teleost fish – to identify, purify and subsequently characterize toxic metabolites from cultures of Fischerella isolated from inland Florida waters. Using this approach, we identified a series of indole alkaloids, including previously described hapalindoles, as well as a novel nitrile-containing congener, as extracellular toxic components in culture medium. Toxicity in the zebrafish embryo was highly variable with structural variant – as evidenced by a range of “toxitypes” - and consequently pointed to apparent structure-activity relationships with respect to developmental toxicity. In addition to the zebrafish embryo, purified metabolites were evaluated for mosquito larvicidal activity in the Aedes aegypti model, as a relevant representative of freshwater invertebrate, and, likewise, found to be toxic. Although indole alkaloids from the Stigonemataceae have been previously identified as putative allelopathic (i.e. anti-algal, antimicrobial) metabolites from this family, this is the first report to suggest toxicity to relevant vertebrate and invertebrate species, and support this family of compounds as cyanoHAB toxins with possible implications for both human and ecosystem health.

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MERHAB-LGL: MONITORING AND EVENT RESPONSE IN THE LOWER GREAT LAKES – 10 YEARS HENCE.

Gregory L. Boyer1, Steven W. Wilhelm2, Joseph Makarewicz3, Mary Watzin4, Joseph Atkinson5, Richard Becker6, Mohammed Sultan7, Charles O’Neill8, Timothy Mihuc9 and Sue B. Watson10

1State University of New York -CESF, Syracuse NY 13210 USA 2University of Tennessee, Knoxville, TN 37996, USA 3The College at Brockport, Brockport NY 14420, USA4North Carolina State University, Raleigh, NC 27695, USA5University of Buffalo, Buffalo, NY 14260, USA6University of Toledo, Toledo OH 43606, USA7Western Michigan University, Kalamazoo, MI, USA8New York Sea Grant, Ithaca NY 14853, USA9State University of New York at Plattsburgh, Plattsburgh NY 12901, USA10Environment Canada, Burlington, ON L7R 4A6 Canada

In 2002-2008, NOAA funded the MERHAB-Lower Great Lakes effort to develop a tier-based monitoring system for cyanobacteria toxins in Lakes Erie, Ontario and Champlain. This effort combined individual working groups on cyanobacteria monitoring, toxin chemistry, new detection methods including molecular biology, hydrodynamic modeling, remote sensing and outreach. MERHAB-LGL participated in 65 research cruises between 2002 and 2008, plus hundreds of weekly, biweekly and spot sampling trips including the first whole lake synoptic and temporal surveys of Lake Ontario, Lake Erie and Lake Champlain. More than 60 publications appeared in scientific journals to date; the project generated 11 M.S. and 5 Ph.D. dissertations, and MERHAB investigators gave more than 200 presentations at scientific meetings or workshops. The MERHAB-LGL effort did not die once funding ended in 2008. The group remains active in all three lakes and the monitoring efforts developed here have transitioned to State monitoring efforts in both Vermont and New York State. Since 2008- MERHAB-LGL investigators have participated 20 additional research cruises looking at cyanobacteria and their toxins in the lower Great Lakes and Lake Champlain, and continue to develop new methods for monitoring and detecting cyanobacteria toxins. This includes the application of ferrybox systems specifically for the detection of cyanobacterial blooms, as well as the deployment of new buoy-based monitoring systems for cyanobacteria. New methods have been developed for the measurement of cyanobacteria toxins in fish that bring into question some of the prior reports on movement of this toxin through the food web. Molecular techniques also continue to expand with application of metagenomics, proteomics and metabolomic techniques to both field and laboratory cultures. The highlights of these advances in monitoring for harmful cyanobacterial algal blooms will be reviewed and new methods currently under development will be discussed.

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NEW INSIGHTS INTO THE BEHAVIOR OF ALExAndRIum FundyEnSE LIFE CYCLE STAGES THROUGH IMAGING FLOW CYTOMETRY

Michael L. Brosnahan1, David K. Ralston1, Heidi M. Sosik1, Robert J. Olson1, and Donald M. Anderson1

1Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.

For two consecutive years, we have deployed an in situ imaging flow cytometer, the Imaging FlowCytobot (IFCB) in Salt Pond, a kettle pond within the Nauset Marsh system (Cape Cod, MA, USA) to study an annually occurring bloom of A. fundyense. Salt Pond is an ideal study site because its A. fundyense blooms are retained by an interaction between the cells’ swimming behavior and the pond’s bathymetry that minimizes advective losses to the A. fundyense population under most conditions. As a consequence, bloom development and termination can be assessed directly from changes in cell abundance. Deployments within the pond have been made possible through the development of a cable-free mooring platform that provides power and communication links to the IFCB. During the first IFCB deployment in 2012, a concerted transition by A. fundyense to the sexual phase of its life cycle was recorded. This transition coincided with increasing stratification in the water column and a rapid increase in the abundance of an Amoebophrya sp. In 2013, the mooring platform was updated with an automated winch that was used to record profiles of conductivity, temperature, depth, chlorophyll fluorescence and photosynthetically active radiation at 15 – 20 minute intervals over the full course of the bloom. The IFCB itself was also modified so that it could sample from two different depths throughout the deployment. Data from 2013 will be used to describe the timing and water column location of several different life cycle stages including vegetative cells, singlet gametes, fusing gametes and planozygotes.

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EFFECTS OF GROWTH PHASE, DIEL CYCLE AND MACRONUTRIENT LIMITATION ON THE QUANTIFICATION OF HETEROSIGmA AKASHIWO USING QPCR AND SHA

Kathryn J. Coyne1*, Christopher R. Main1, Cameron Doll2, Colleen Bianco1 and Dianne I. Greenfield2

1University of Delaware College of Earth, Ocean, and Environment, 700 Pilottown Rd., Lewes, DE 19958; 2 Belle W. Baruch Institute for Marine & Coastal Sciences, 331 Fort Johnson Road, University of South Carolina, Charleston, SC 29412

Quantitative real-time PCR (qPCR) and sandwich hybridization assay (SHA) are two molecular approaches that have been developed for detection and quantification of HAB species. These methods differ in that SHA detects ribosomal RNA (rRNA) from an unpurified and unamplified sample through oligonucleotide hybridization followed by colorimetric detection, whereas qPCR requires isolation of DNA followed by amplification using species-specific primers and probes and fluorometric detection of PCR products. Our overall goals for this project, funded by NOAA MERHAB, are to rigorously compare these methods for enumeration of harmful algal species, using Heterosigma akashiwo as a model alga, and to provide recommendations for the application of these methods in HAB research and management activities. Here, we examined the effects of physiological status on quantification of H. akashiwo by qPCR and SHA. Samples were collected for analysis at several points during the growth phase, over a diel cycle, and from cultures subjected to either nitrogen or phosphorus limitation. Since the same number of cells was collected for each time point or treatment, the results are indicative of the relative content of DNA (for qPCR) or rRNA (for SHA) per cell. During the growth phase experiment, there was a significant increase in both qPCR and SHA signal for cells collected during log phase growth compared to stationary phase, as would be expected for cells with higher growth rates. Over a diel cycle, however, the results for qPCR analysis indicated an increase in DNA content during the light phase, while rRNA content, as measured by SHA, decreased during the light phase. The decrease in rRNA was unexpected, but may be a consequence of managing resources within the cell. Heterosigma akashiwo has a very large nuclear genome, suggesting that cellular resources such as phosphate may be diverted to dNTP synthesis during the light phase for DNA replication. Under macronutrient limitation, only N-stressed cultures exhibited significantly lower nucleic acid content compared to control cultures, resulting in a lower signal for both qPCR and SHA compared to control cultures. Biochemical and photochemical analyses also confirmed higher levels of stress in the N-stressed cultures compared to P-stressed cultures, which were not significantly different from controls. Overall, results of this investigation suggest that growth stage, nutrient stress and diel changes in the cell cycle can have a significant impact on molecular methods for quantitative assessment of HAB species, and that resource managers should take these factors into consideration when evaluating results from SHA and qPCR analysis.

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GLOBAL PERSPECTIVE ON THE IMPACTS OF THE MARINE TOxIN PALYTOxIN

Jonathan R Deeds1

1US FDA Center for Food Safety and Applied Nutrition, 5100 Paint Branch Parkway, College Park, MD 20740, USA

The ancient Hawaiian legend of the “Limu Make o Hana” (deadly seaweed of Hana) recounts the tale of a mythical creature with a shark’s mouth on its back that is burned and its ashes cast into a tidal pool after it terrorizes a village in Muolea, in the district of Hana, on the island of Maui. As the legend goes, after this episode the Limu in the pool became toxic and the pool itself became kapu (taboo) to the Hawaiians who believed that an ill fate would befall anyone who disturbed the sacred pool. In efforts to determine the cause of ciguatera fish poisoning, researchers at the Hawaii Institute of Marine Biology set out to determine the location of this fabled pool where warriors were once said to smear the limu on spear points to make their wounds fatal. On December 31, 1961 the pool was finally visited and found to contain a previously undescribed species of coelenterate zoanthid (colonial anemone), later named Palythoa toxica, from which palytoxin (PLTX) would eventually be isolated. Coincidentally, a fire destroyed the main building of the Hawaii Marine Laboratory on Oahu that very afternoon.

From its original discovery, PLTX was realized to be one of the most potent non-protein toxins ever described. While its toxicity is greatly reduced through oral consumption, fatal poisonings from contaminated seafood have been documented. More recently, it was discovered that Ostreopsis spp. dinoflagellates also produce PLTX and PLTX-like compounds. Furthermore, the frequency and intensity of Ostreopsis blooms appears to be increasing in temperate coastal waters around the world. Blooms of Ostreopsis have now been linked to inhalational PLTX exposure, so called “Algal Syndrome”, in portions of the Mediterranean and Adriatic seas. Lastly, for many years it was believed that the most highly toxic species of coelenterate zoanthids, such as P. toxica, were limited in distribution to remote locations such as the tide pools on Maui. It is now known that a highly toxic variety of zoanthid is commonly available in the home aquarium trade and has been responsible for serious illnesses due to dermal and inhalational PLTX exposure in maintainers of marine aquaria world-wide. In addition, less toxic, but highly abundant, PLTX-containing zoanthid varieties are also known to occur throughout the Pacific and Caribbean regions and have been linked to food-web accumulation of PLTX and PLTX-like compounds.

PLTX is rare among marine toxins in that it poses risks through multiple routes of exposure (oral, inhalational, and dermal), and multiple vectors (planktivorous and carnivorous fish, crustaceans, shellfish, and waters containing Ostreopsis spp. or toxic zoanthids). All of this makes the list of potential biological effects after exposure to PLTXs extensive. The occurrence, chemistry, pharmacology, toxicity, and methods of analysis for PLTXs have all been recently reviewed. Despite this global attention, much remains to be learned about the biological mechanisms involved in palytoxicosis, a serious, potentially life threatening, and sometimes lethal syndrome from exposure to these compounds. This information is critical for the proper assessment of risks and the determination of potential management strategies to reduce human exposure to this unique group of marine toxins.

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DETECTING PARALYTIC SHELLFISH TOxINS USING THE ENVIRONMENTAL SAMPLE PROCESSOR (ESP): ASSAY DEVELOPMENT & FIELD DEPLOYMENT

Jinkeng Asong1, Christina M. Mikulski1, Juliette L. Smith2, Bruce A. Keafer2, Roman Marin III3 Katrina Campbell4, Christopher T. Elliott4, Christopher A. Scholin3, Donald M. Anderson2, and Gregory J. Doucette1

1NOAA/National Ocean Service, Marine Biotoxins Prog., Charleston, SC 29412, USA2Woods Hole Oceanographic Institution, Biology Dept., Woods Hole, MA 02543 USA3Monterey Bay Aquarium Research Institute, Moss Landing, CA 95039, USA4Queen’s University Belfast, Inst. for Global Food Security, Belfast BT9 5BN UK

The emergence of ocean observing systems (OOS) capable of transmitting real or near-real time information from remote locations has the potential to transform the monitoring and management of harmful algal blooms (HABs) and their adverse public health and socioeconomic impacts (Anderson et al. 2012). Achieving such a benefit from OOS will require the development of autonomous, in-water sensors able to detect individual HAB species and the toxins they produce. The Environmental Sample Processor (ESP) is a robotic, subsurface platform that can monitor concentrations of both algal cells and toxins, and provide these data to ship- or shore-based facilities in near-real time. Here we report on the development of a sensor for paralytic shellfish toxins (PST) to complement the existing ability of the ESP to detect toxigenic Alexandrium spp.The PST sensor employs a membrane-based, protein array analogous to that developed previously for detection of domoic acid on the ESP and, similarly, adopts a competitive ELISA format. The PST sensor is unique in that two toxin-protein conjugates (one based on saxitoxin (STX), the other on neosaxitoxin (NEO)) are spotted on the array and a cocktail of two antibodies is used. One of the antibodies was generated against gonyautoxin (GTX) 2/3 and the other against NEO. Our aim was to conduct two assays simultaneously on a single array, one sensitive to the N-1 H toxin group (e.g., STX, GTX2/3, GTX5, C1/2) and the other sensitive to the N-1 OH group (e.g., NEO, GTX1/4, C3/4), thereby enhancing our ability to accurately quantify diverse PST profiles associated with various Alexandrium spp. Currently (modifications are ongoing), both assays show an IC50 value (analyte concentration yielding half-maximal response) in the low to mid nanomolar range and can accurately quantify certain complex toxin profiles generated using both mixed reference standards and Alexandrium culture extracts. We will report on the performance of the PST sensor in its latest configuration and also provide preliminary data on the first field deployment of an ESP with saxitoxin detection capability during spring/summer 2013 in the Gulf of Maine.

ReferenceD.M. Anderson, G.J. Doucette, G. Kirkpatrick, C.A. Scholin , J. Paul, V. L. Trainer, L. Campbell, R.M. Kudela, R.P. Stumpf, J.R. Morrison. 2012. Harmful algal bloom (HAB) sensors in ocean observing systems. IOOS Summit 2012 (http://www.iooc.us/summit/). Community White Paper.

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TAxONOMIC DESCRIPTIONS OF NOVEL CYANOBACTERIAL BIODIVERSITY FOR MONITORING, SURVEYING, AND CONTROLLING CYANOBACTERIAL HARMFUL ALGAL BLOOMS (CYANOHABS)

Niclas Engene1, Sarath P. Gunasekera1 and Valerie J. Paul1

1Smithsonian Marine Station at Fort Pierce, 701 Seaway Dr., Fort Pierce, FL 34949,USA

Cyanobacteria of the genus Lyngbya seasonally form extensive blooms in Southern Florida. The prolific production of bioactive secondary metabolites of many of these bloom-forming Lyngbya can be hazardous for humans as well as the natural environment. In our efforts to provide taxonomic clarity, we show phylogenetically that several of the most prevalent Lyngbya specimens in Southern Florida, in fact, represent novel cyanobacterial genera. Here we characterize and compare the ecology, morphology, evolutionary history, and secondary metabolism of these new taxonomic groups. These taxonomic descriptions provide classification systems that are able to identify the cyanobacteria and predict their production of toxins and other bioactive secondary metabolites. These classification systems are important for monitoring, predicting, or possibly controlling potentially harmful cyanobacterial blooms in Southern Florida.

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A DAY IN THE LIFE OF KAREnIA bREvIS: DIEL VARIATION IN CELL VOLUME AND LADDER-FRAME POLYETHER PRODUCTION

Reagan M. Errera1, Susan Niven2, Andrea Bourdelais2, Lisa Campbell1

1Texas A&M University, 3146 TAMU, College Station, TX, 77843, USA 2University of North Carolina-Wilmington, 5600 Marvin K. Moss Lane, Wilmington, NC 28409

The major harmful alga in the Gulf of Mexico, Karenia brevis, produces a suite of ladder-frame polyethers (LFP), which include toxic (brevetoxins) and nontoxic (brevenal and brevisin) compounds. Light intensity and day length have been shown to be important environmental factors influencing K. brevis growth rate; however, there has been no examination of the effect of diel cycles and exponential growth period on the production of LFPs and cell volume. It has been suggested that variation in LFP production among clones of K. brevis can be clarified through normalizing LFP concentration to cell volume. Due to the plasticity of K. brevis theca, daily and growth curve volume changes need to be explored to verify this hypothesis. Differences in cell size, total parent brevetoxins and brevenal production were evaluated among four K. brevis clones over a diel cycle in the middle of exponential growth and over exponential growth. Cell volume for all four clones varied over the diel cycle. Under a 12:12 (light:dark) photoperiod, all four clones significantly increased cell volume over the light period and decreased cell volume over the dark period, while no significant change in LFP production was detected. During exponential growth, three of the four clones decreased cell volume over the 9-day period, while the fourth clone increased cell volume, again LFP concentrations (pg cell-1) remained constant. Furthermore, there was no correlation between cell volume and LFP production among any of the clones. This suggests LFP production is not related to cell volume and should not be expressed as a concentration of cell volume. Examination of field populations of K. brevis over several months also indicated a change in cell size over the course of the bloom, i.e. from initial state to bloom termination.Differences in cell size, total parent brevetoxins and brevenal production were also evaluated under four different photoperiods (6:18, 12:12, 18:6 and 24:0; light:dark) under an irradiance level of 70 μEin m-2s-1. In both the 12:12 and 24:0 photoperiods, LFP concentrations (pg cell-1) remained constant in all four clones during exponential growth. As photoperiod increased, LFP production increased in all four clones, with the highest concentrations of LFP (pg cell-1) observed under a 24:0 photoperiod. Differences in cell volume during different photoperiods will also be discussed. To determine if increases in light intensity resulted in an increase in LFP production growth rates, total parent brevetoxin and brevenal content of K. brevis were also evaluated at an irradiance level of 140 μEin m-2s-1 for all four clones. Growth rates significantly increased for the four clones at the higher irradiance, however, LFP concentrations (pg cell-1) decreased. The increase of LFP production with increased photoperiod but not with light intensity suggests a connection between diel cycle and LFP production, specifically LFP production maybe dependent on the light period.

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WIDESPREAD DETECTION OF THE FRESHWATER TOxIN MICROCYSTIN AT THE LAND-SEA INTERFACE WITHIN MONTEREY BAY, CA

Corinne M. Gibble1 and Raphael M. Kudela1

1University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA 95064

Harmful algae have a worldwide distribution and can form extensive blooms with toxin production in freshwater, estuarine, and marine habitats. Microcystis aeruginosa blooms and associated toxin microcystin (MCY) are a regular occurrence in freshwater systems throughout California, but until recently have not been detected in marine environments. To investigate potential land-sea transfer of this toxin, 28 sites in and around Monterey Bay were surveyed for evidence of MCY toxin (2010-2011). In years two and three (2011-2013) 4 major watersheds in the Monterey Bay area were surveyed for MCY abundance, nutrients, temperature, and alkalinity to identify potential factors that might be influencing the abundance of MCY at the land-sea interface. In year one, MCY was detected in 11 of 28 sites. Data from years two and three analyzed by multiple regression indicated that coastal nutrient loading (nitrate, phosphate silicate, ammonium, urea) temperature, and time of year were statistically significant predictors of the amount of MCY toxin in the environment. At some sites we found evidence of unexpected seasonality. As anticipated, we predominantly saw large increases of MCY in the autumn; however at some locations we also recorded very high levels of toxin during spring. Because this toxin has the ability to biomagnify and persist within food webs, elevated levels within the watershed may increase potential for illness and death of wildlife and humans in both terrestrial and marine waters. The widespread occurrence of MCY at low to moderate levels throughout the year and throughout the watershed demonstrates the potential difficulty of mitigating these impacts.

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A NEW SUITE OF NUTRIENT KINETIC RELATIONSHIPS FOR PICOPLANKTON BLOOMS: NON-TRADITIONAL RELATIONSHIPS ARE THE NEW NORM

Pat Glibert1

University of Maryland Center for Environmental Science, Horn Point Laboratory, PO Box 775, Cambridge MD 21613, USA

Many coastal and estuarine systems are impacted by EDABs (Ecosystem Disruptive Algal Blooms)- blooms that are not necessarily toxic but which have negative impacts on ecosystem function. Many EDAB species are of the picoplankton size range; for example, Synechococcus blooms in Florida Bay and brown tide blooms in coastal lagoons.Knowledge of nutritional sources supporting such blooms has greatly advanced in the past few years. It has been shown, for example, that many picoplankton EDAB species prefer organic forms of nitrogen (DON) and/or chemically reduced N forms (NH4 or urea) over oxidized forms (NO3) of nitrogen. This pattern ahs been substantiated by measurements of uptake rates and also by the finding that many picoplankton have high proportions of NH4 transporters but few, if any, NO3 transporters. This is in contrast to diatoms that have many copies of NO3 transporters. Moreover, for some cyanobacteria the high affinity transporter for NH4 is not regulated at all, being constitutively expressed.

Here we show the nitrogen uptake kinetic relationships for picocyanobacterially-dominated blooms from Florida Bay. These blooms have been a common feature in this system for many years, although from year to year have varied in intensity (but not in dominant species). The kinetic relationships for NO3, NH4 and urea did not follow classic saturation responses in most cases. The typical response for NO3 was a linear relationship for uptake as a function of increasing concentration. This pattern is consistent with a lack of NO3 regulation at the level of transport across the cell membrane, and is also consistent with diffusion od substrate into the cell. For NH4 and urea there were two common responses. The first was inhibition of uptake as a function of increasing concentration. This pattern is suggestive of down-regulation of nutrient transport so as to maintain a constant flux of nutrient into the cells. The second common pattern was biphasic, with both suppression of uptake at intermediate concentration levels, followed by a secondary increase at much higher concentrations. This pattern suggests that the cells regulated uptake up to a point, but that a diffusional effect occurred at the highest concentrations measured.

These findings underscore that these species are able to thrive under conditions of elevated NH4, urea and organic N. The non-traditional kinetic relationships have important implications for parameterizing these rates in developing ecosystem models; standard Michaelis Menten kinetics would not be appropriate to apply in these conditions. Developing ecosystem models for Florida Bay are beginning to incorporate these non-traditional kinetic relationships.

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THE ROLE OF COASTAL OCEAN ACIDIFICATION IN PROMOTING AND ExACERBATING THE EFFECTS OF HARMFUL ALGAL BLOOMS

Christopher J. Gobler1, Theresa K. Hattenrath-Lehmann1, and Ryan B. Wallace1

1School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794-5000, USA

During the past decade, oceanographers have become acutely aware of the decline in ocean pH associated with the loading of carbon dioxide (CO₂) predominantly from the combustion of fossil fuels. Concurrently, it has become increasingly recognized that coastal habitats within which harmful algal blooms occur are already experiencing acidification, often of a magnitude far exceeding the ‘worst case scenario’ conditions predicted for the open ocean later this century (>1,000 ppm CO2). Excessive nutrient loading to coastal waters often promotes algal blooms, including HABs, and the demise of these algal blooms typically yields high rates of microbial respiration that lowers dissolved oxygen concentrations. A second, often overlooked consequence of microbial respiration is the production of CO2 and a reduction in seawater pH. Since many HABs often occur following the decline of other algal blooms they are more likely to develop in acidified environments that have elevated levels of pCO2. This presentation will highlight the interactions and feedbacks between nutrient loading, HABs, and acidification and how they affect marine organisms and ecosystems. Novel laboratory and ecosystem observations regarding the ability of acidification to significantly increase (p<0.05) the growth and toxicity of North Atlantic isolates and blooms of Alexandrium fundyense will be presented. Data regarding the ability of ecosystem disruptive algal blooms including brown tides caused by Aureococcus anophagefferens and Aureoumbra lagunensis, to manipulate the pH, oxygen, and pCO2 levels within estuaries will be presented. Finally, data demonstrating that the development of HABs within high CO2 ecosystems cause harm to marine animals beyond the effects posed by the elevated CO2 or the harmful algae alone will also be presented.

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CIGUATERA FISH POISONING IN ST. THOMAS, USVI: ExPOSURE, SYMPTOMS, AND RECOVERY

Lynn M. Grattan,1 Sparkle M. Roberts,1 Alison Robertson,2 Elizabeth Radke,3 Margaret Abbott,3 and J. Glenn Morris3

1 University of Maryland School of Medicine, 110 S. Paca Street Street, Baltimore, MD 212012 Gulf Coast Seafood Laboratory, U.S. Food and Drug Administration, 1 Iberville Drive, Dauphin Island, AL 365283 University of Florida, P.O. Box 100009, 2055 Mowry Road, Gainesville, FL 32610

Ciguatera fish poisoning (CFP), a harmful algal bloom syndrome, is the most common, non-bacterial foodborne illness associated with fish consumption in the United States and globally. While the syndrome has been recognized for more than six centuries, more than 50 different symptoms have been associated with the symptom complex with varying levels of consistency. Moreover, patterns of recovery may range from 24 hours to several years. In contemporary thought, syndrome variations are potentially explained by geographic toxin diversity, therefore region-specific studies are indicated.Toward this end, we examined a series of patients who presented to the emergency department at the Roy Schneider Hospital and were diagnosed with CFP.

Exposure was assessed by type and quantity of fish consumed as well as analysis of meal remnant samples. Sixty-two study participants were examined within five days of emergency room discharge, with measures to capture potential symptoms or symptom complaints. This included symptom checklists and standard measures of cognition, mood, and sensory perception. Follow-up evaluations were completed three months and one year post exposure. Preliminary findings indicate no seasonal variation of CFP cases. At least twelve different types of finfish were associated with illness, with the most frequently reported fish consumed being Red Hind (16%), Barracuda (13%), and Red Snapper (13%). Most of the fish associated with illness were purchased from or gifted by local fishers. Where meal remnants were available and analyzed (n=20), 80% were found to be ciguatoxic. The most frequently reported clinical symptoms were diarrhea; upper and lower extremity weakness and tingling; and abdominal pain and nausea. Sensory and neuropsychological screening indicated no abnormalities. With respect to chronicity, 56% of patients presenting to the emergency department with CFP had dermal and/or neurologic symptoms three months later and 13% continued to have unusual and vague neurologic complaints one year post exposure. Finally, 56% of patients had at least one previous episode of CFP. Data collection and anlysis are ongoing.

Meanwhile, it is concluded tht CFP remains a problem in St. Thomas, USVI, with Red Hind, Barracuda, and Red Snapper being the most risky fish. Neurologic and dermal symptoms are the most persistent symptoms and full recovery may be expected within the first year for most people. Ongoing outreach and education are essential to reducing the risk of exposure and illness.

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THE JAMESTOWN S’KLALLAM TRIBE RESPONDS TO AN EMERGING THREAT TO HUMAN HEALTH: DIARRHETIC SHELLFISH POISONING IN WASHINGTON STATE

Neil Harrington1, Vera L. Trainer2, Leslie Moore2, Brian D. Bill2, Jerry Borchert3 and Bich-Thuy L. Eberhart2

1 Jamestown S’Klallam Tribe, 1033 Old Blyn Highway, Sequim, WA 98382 2Marine Biotoxins Program, Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Blvd. E, Seattle, WA 981123Office of Shellfish and Water Protection, Washington State Department of Health, 111 Israel Rd SE, Tumwater, WA 98504

Three people contracted diarrhetic shellfish poisoning (DSP) after eating mussels collected at Sequim Bay State Park on the Olympic Peninsula of Washington State in June 2011. These were the first confirmed cases of DSP in the United States. The emergence of this new threat to public health had an immediate impact on the Jamestown S’Klallam Tribe whose subsistence and commercial shellfish beds were located nearby. In 2012 a collaborative research project on DSP in Sequim Bay and Washington State by NOAA, the Tribe and the Washington State Department of Health (WDOH) looked at the presence of Dinophysis species and concentrations of diarrhetic shellfish toxins (DSTs) in shellfish and phytoplankton. In an effort to provide advanced warning of shellfish toxicity using rapid screening methods for toxins, analysis of extracts using both antibody- and activity-based screening assays were compared to the standard regulatory method, liquid chromatography-mass spectrometry. Weekly sampling illustrated that D. acuminata was the primary species present during toxic events and increases in cell density usually predicted increases in shellfish toxicity. In addition, it was found that dinophysistoxin-1 (DTX-1) was the primary responsible toxin isomer in WA and mussels generally concentrated the toxin more than other species of shellfish. Greater than average snowpack and freshwater runoff in both 2011 and 2012 in the northwest may have contributed to conditions favorable for dinoflagellate blooms. These findings are being used to better utilize monitoring data from the SoundToxins and Olympic Region Harmful Algal Blooms (ORHAB) phytoplankton monitoring groups to prioritize shellfish collection and analyses for DSTs to protect public health.

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NUTRIENTS SUPPORTING KAREnIA bREvIS BLOOMS: RESULTS OF THE ECOHAB: KAREnIA PROGRAM

Cynthia Heil1, Deborah Bronk2, L. Kellie Dixon3, Gary Hitchcock4, Gary Kirkpatrick3, Margaret Mulholland5, Judith O’Neil6, John Walsh7, Robert Weisberg7, Matthew Garrett8, Jason Lenes7, Rachel Sipler2, Bill Richardson8, Lynn Killberg-Thorsen2, Kevin Meyer6, Leo Procise5 and Breanne Walsh6

1Bigelow Laboratory for Ocean Sciences, East Boothbay, ME 04544, USA 2Department of Physical Sciences, The College of William & Mary, Virginia Institute of Marine Science, Gloucester Point, VA, 23062, USA3Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236, USA4RSMAS/MBF, University of Miami, Miami, FL 33149, USA5Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University, Norfolk, VA 23529, USA6Horn Point Laboratory, Univ. of Maryland Center for Environmental Science, Cambridge, MD 21613, USA7College of Marine Science, University of South Florida, St. Petersburg, FL 33701, USA8Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, St. Petersburg, FL 33701, USA

Blooms of the toxic dinoflagellate Karenia brevis predate coastal eutrophication but continue to annually plague the eastern Gulf of Mexico with fish kills, marine mammal mortalities, human respiratory irritation and significant economic losses. The ECOHAB: Karenia Program was a collaborative, multi-faceted approach to identify, assess and model the nutrient sources fueling these blooms. Three blooms were sampled, one at initiation phase (2008), one in a high biomass, nearshore maintenance phase (2007) and a third in a lower biomass, offshore maintenance phase (2009) as well as a ‘no bloom’ year (2010). Nutrient sources supporting K. brevis blooms are multiple, diverse and complex: the largest nitrogen source was Trichodesmium blooms (N2 fixation and release in com-bination with decay and recycling of biomass), followed by nutrient release from zooplankton grazing, decay of red tide-related dead fish, benthic nutrient flux, photochemical nutrient production, nitrification, estuarine inputs and pelagic N2 fixation. Reduced nitrogen forms dominant the sources, many of which were individually sufficient to support observed bloom biomass. Project results suggest that toxins play a significant role in altering nutrient sources and dynamics of larger, high biomass, fish killing blooms compared with smaller, low biomass blooms by reducing some nutrient sources (e.g. zooplankton grazing) while enhancing others (e.g. decay of dead fish). K. brevis cells are physiologically flexible with regards to their nutrient acquisition strategies, which allow them to respond rapidly to changing environmental conditions. Cells can take up and grow on a variety of inorganic and organic N and P sources, grow rapidly (up to1 day-1) under organic enrichment conditions, and possess unique carbon acquisition strategies. Results suggest that management options for K. brevis blooms, while limited for nutrient reduction strategies, should include reduction of controllable nutrient sources through best management practices, continued support and improvement of short-term now-casting and forecasting abilities and products, and targeted educational outreach efforts.

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PREDICTED ORIGINS OF KAREnIA bREvIS BLOOM FORMATION ALONG THE COAST OF TExAS USING AN INDIVIDUAL-BASED MODEL

D. W. Henrichs1, R. D. Hetland1, L. Campbell1,2

1Department of Oceanography, Texas A&M University, College Station, TX 778432Department of Biology, Texas A&M University, College Station, TX 77843

Bloom formation in Karenia brevis is not the result of rapid cell division but rather, a combination of physical and physiological processes. Bloom formation in the eastern Gulf of Mexico is well-studied but the origin of cells contributing to bloom formation remains unknown. The western Gulf of Mexico experiences more infrequent blooms of K. brevis and as a result, the origin of cells contributing to bloom formation has received little attention. A spatially-explicit, individual-based model (IBM) of K. brevis has been developed to identify potential source locations of cells that may become physically concentrated along the Texas coast. The model merges the behavioral model of Liu et al. (2001) with an individual-based population-genetic model incorporating cell division and sensitivity to environmental temperature and salinity. Current velocities, water temperature, and water salinity are obtained from the Gulf of Mexico HYCOM model. To identify potential source(s) of Karenia blooms, the IBM was run in reverse and cells were inserted at locations along the coast of Texas by date according to results obtained from the Imaging FlowCytobot in Port Aransas and count data from Texas Parks and Wildlife Department. Comparisons between bloom years and non-bloom years suggest blooms of K. brevis in Texas originate in the southern Gulf of Mexico, near the Bay of Campeche, an observation supported by recent reports of blooms off the western Yucatan. Pre-bloom identification of cells offshore of Texas, carried north by currents, would help to validate the model results. Running the model in forward time and starting cells in the southern Gulf of Mexico, using forecast currents, coupled with a downwelling index (based on the mean monthly along-shore component of the wind at the coast of Texas) will be used to test the forecasting ability of the individual-based model.

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PACIFIC NORTHWEST TOxINS PROJECT

Barbara Hickey1, Neil Banas2, Michael Foreman3, Raphael Kudela4, Evelyn Lessard1, Parker MacCready1, Diane Masson3, Richard Thomson3, Thomas Connolly5, Nancy Kachel1, Kristen Davis6, Isaac Fine3, Susan Geier1, Sarah Giddings1, Susan Lubetkin1, Ryan McCabe1, and Samantha Siedlecki2

1School of Oceanography, University of Washington, Seattle, WA 98195, USA2 Joint Institute for the Study of the Atmosphere and Ocean, University of Washington, Seattle, WA 98195, USA3 Fisheries and Oceans Canada, Institute of Ocean Sciences, Sidney, BC V8L4B2, Canada4Department of Ocean Sciences, University of California, Santa Cruz, CA 95064, USA5Woods Hole Oceanographic Institute, Woods Hole, MA 02543, USA6Civil and Environmental Engineering and Earth System Science, University of California, Irvine, CA 92697, USA

The goal of PNWTOX is to determine transport routes for toxigenic Pseudo-nitzschia in the Pacific Northwest. The role of the freshwater plume from the Columbia River in bloom initiation, nutrient provision, and transport is a central focus of the project. The study includes data analysis and model development based on an extensive suite of biological/chemical/physical observations acquired on ten 21 d cruises over a 4-year period, from satellites, and from multiple deployments of moored arrays and surface drifters throughout the region. The massive data set, which includes biological growth and grazing rates, nutrients, and oxygen data, has guided the development of a regional ROMS transport and ecosystem model. The model has been thoroughly validated and includes multiple processes, such as Puget Sound and Strait of Georgia/Fraser River estuarine exchange and remotely forced waves, which our studies have shown to be critical for adequate representation of upwelling from the slope to the shelf, and for accurate provision of nutrients to the region. The model captures observed patterns and magnitudes of chlorophyll, nitrate, bottom oxygen, temperature, Columbia plume location and particle transport routes on both seasonal and event (several day) scales with good accuracy. HAB transport routes examined in detail for 2004-2007 reproduced the observational results showing that toxic cells are delivered to the WA coast not from a single source, as suggested in prior studies, but from two sources, one north and one south of the WA beaches where razor clam harvest is impacted by HABs. The existence of sources both north and south of the affected beaches is dependent on the fact that regional currents in the Pacific Northwest reverse seasonally, in contrast to the US East coast currents. The northern source, the Juan de Fuca Eddy region, is important in late summer and fall, when currents are southward; whereas the southern source, Heceta Bank, OR, is the source in late winter/spring, when currents are seasonally northward. The model results also confirm the hypothesis that the Columbia plume at times acts as a barrier to onshore transport, while at other times it enhances alongshelf transport. As a result of the PNWTOX research, the Pacific Northwest regional model, which can easily be applied to other HABs with known source regions, is now also poised to be used in HAB and hypoxia regional forecasting efforts.

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CHANS: FLORIDA RED TIDES AND COASTAL POPULATIONS AS A COUPLED NATURE-HUMAN SYSTEM

Porter Hoagland1, Barbara Kirkpatrick2,3, Gary Kirkpatrick2, Gary Hitchcock4, Kate Kohler2, Vince Lovko2, Steve Ullmann5, Andy Reich6, and Lora Fleming3,4,7

1Marine Policy Center, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA2Mote Marine Laboratory, Sarasota, FL 34236, USA3Department of Epidemiology and Public Health, Miller School of Medicine, University of Miami, Miami, FL 33177 USA4Oceans and Human Health Center, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, Miami, FL5Programs and Center in Health Sector Management and Policy, University of Miami, Miami, FL 33177 USA6Aquatic Toxins Program, Environmental Health, Florida Department of Health, Tallahassee, FL7European Centre for Environment and Human Health, Peninsula College of Medicine and Dentistry, Truro, Cornwall, UK

Coupled nature-human (CNH) systems are now the focus of a growing number of inter-disciplinary research programs worldwide. As implied by the term “coupled,” these systems involve interactions between humans and nature, often affecting the dynamic characteristics of each component. Both natural and social scientists are engaged in developing a deeper understanding of these dynamics, focusing on the linkages and feedbacks affecting the trajectories of coupled system behavior. Several researchers have begun to identify the generic aspects of nature-human couplings. Many of these aspects have been adapted from the field of ecology, where the dynamic characteristics of ecological systems have been studied for decades. These aspects include system heterogeneity, time lags, reciprocal feedbacks, thresholds, surprises, legacies, and resilience. The presence of such aspects has implications for the stability and persistence of particular ecosystem states, leading potentially to further implications for human heath and welfare. This talk reviews a specific type of natural hazard-human coupling that relates to coastal blooms of toxic marine algae, drawing examples primarily from human interactions with blooms of the toxic dinoflagellate Karenia brevis from the eastern Gulf of Mexico. This talk introduces a set of HAB Symposium “speed” presentations relating to different aspects of an ongoing multi-institutional and inter-disciplinary research project that examines Florida red tides as a type of CNH system. We present examples of the generic aspects of CNH systems in the context of Florida red tides, and we discuss also some of the challenges involved in compiling relevant data to support our analytical efforts.

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UNTANGLING THE EFFECTS OF ANTHROPOGENIC VERSUS NATURAL NUTRIENT SOURCES AND IMPLICATIONS FOR HARMFUL ALGAL BLOOMS IN THE SOUTHERN CALIFORNIA BIGHT

Meredith D.A. Howard1, Karen McLaughlin1, Martha Sutula1, David A. Caron2, Yi Chao3,4, Hartmut Frenzel4, Alyssa Gellene2, Kendra Hayashi5, Burton Jones6 , Raphael M. Kudela5, Michael J. Mengel7, Nikolay Nezlin1, George Robertson7, Ashmita Senqupta1 and Erica Seubert2

1Southern California Coastal Water Research Project, 3535 Harbor Blvd., Costa Mesa, CA 92626 USA2University of Southern California, 3616 Trousdale Parkway, Los Angeles, California 90089-0371, USA 3University of California, Los Angeles, 405 Hilgard Ave, Los Angeles, CA 4Remote Sensing Solutions, Inc., 2824 East Foothill Blvd., Pasadena, California 911075University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA 950646King Abdullah University of Science and Technology, Thuwal 23955-6900, Kingdom of Saudi Arabia7Orange County Sanitation District, 10844 Ellis Avenue, Fountain Valley, CA 92708Fountain Valley, CA 92728

Eutrophication of coastal waters is a global environmental issue, with demonstrated links between anthropogenic nutrient inputs and increased frequency and occurrence of harmful algal blooms. However, in upwelling-dominated ecosystems, such as southern California, untangling the relative influence of natural versus anthropogenic nutrient sources on coastal waters has proved to be more complex. In these ecosystems, there has been a perception that anthropogenic nutrient inputs are small relative to upwelling, and thus, have little effect on nearshore productivity. However, recent studies in the Southern California Bight (SCB) have provided evidence to the contrary. A one year study in 2010 determined that anthropogenic nitrogen loads from wastewater effluent discharged to ocean outfalls were equivalent to modeled upwelling nitrogen loads in five of six sub-regions located proximal to the coastline. Furthermore, stable isotope analysis of nitrate, ammonia, and particulate organic matter showed that nitrogen from wastewater effluent comprised up to half of the total nitrogen in phytoplankton and zooplankton located proximal to an ocean outfall in the fall 2012. Similar isotopic analysis is underway for samples collected during a toxic Pseudo-nitzschia bloom in the same location in spring 2013 and will be presented. These findings suggest that anthropogenic nutrients, mainly wastewater effluent, can provide a significant source of nitrogen for algal bloom development in southern California coastal waters.

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SPATIAL AND TEMPORAL VARIABILITY OF TOxIC PSEudO-nITZSCHIA SPP. IN THE GULF OF MAINE DURING SUMMER 2012

Katherine A. Hubbard1,2, Alison Sirois3, Jane Disney4, Leanne Flewelling2, Sheila O’Dea2, Steve Morton5, Alison Robertson6, Harold A. Flores Quintana6, Judith L. Kleindinst1, Dennis J. McGillicuddy1, Donald M. Anderson1

1 Woods Hole Oceanographic Institution, Woods Hole, MA, 025432 Fish and Wildlife Research Institute, 100 8th Ave SE, St. Petersburg, FL 337013 Maine Department of Marine Resources, PO Box 8, West Boothbay, ME, 045754 Mount Desert Island Biological Laboratory, PO Box 35, Salisbury Cove, ME 046725 NOAA/ NOS, Hollings Marine Laboratory, 331 Fort Johnson Rd, Charleston, SC 294126 Gulf Coast Seafood Laboratory, U.S. FDA, 1 Iberville Drive, Dauphin Island, AL 36528

Although repeated shellfish harvest closures for the Amnesic Shellfish Poisoning (ASP) toxin domoic acid (DA) have occurred in eastern Canadian provinces since 1987, the first ASP closure in New England waters occurred in late July 2012. Prior to the closure, increases in Pseudo-nitzschia abundance along the coast of eastern ME from Eastport to Schoodic Peninsula were detected by the Maine Volunteer Phytoplankton Monitoring Program (Maine Department of Marine Resources). By July 31st, phytoplankton and shellfish samples from several small embayments in the region tested positive for DA (with Jellet ASP rapid testing assays), and a closure was issued (area # 64A). Federal partners at NOAA and the FDA respectively conducted SEM characterization of Pseudo-nitzschia species and confirmatory toxin testing of shellfish. Emergency funding provided by NOAA’s CSCOR Harmful Algae Event Response program permitted additional sampling of nearshore and offshore phytoplankton communities, particulate DA, and physicochemical parameters at 25 stations during the closure. The highest cellular abundance (1.3 x 106 cells l-1) occurred in Corea Harbor, and was attributed to a novel species that is morphologically similar to P. calliantha and genetically similar to P. pseudodelicatissima/P. cuspidata. Pseudo-nitzschia abundance decreased with distance from shore, with 104-105 cells l-1 just outside of embayments and 102 -103 cells l-1 2-3 nautical miles offshore. Pseudo-nitzschia became more abundant west of the Schoodic region during September and October, and some but not all samples with high Pseudo-nitzschia abundance tested positive for DA. To assess the effects of species composition on variability in DA toxicity, a modified automated ribosomal intergenic spacer analysis (ARISA) was used to identify and quantify Pseudo-nitzschia species in DNA samples collected during the event, specifically targeting regional species (n=8) that are morphologically similar to P. pseudodelicatissima. Although DA concentrations in shellfish during the bloom peak were quickly determined to be below the regulatory closure limit of 20 ppm and the closure was lifted, the multi-agency response provided timely, critical information to address the regional threat of ASP. This has led to enhanced monitoring efforts in ME, including a pilot study to conduct volunteer-based DNA sampling. As a more extensive regional program for monitoring DA and Pseudo-nitzschia spp. is adopted, it is important for managers, monitors, and researchers to continue to learn more about the toxicity, frequency and magnitude of Pseudo-

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FRESHWATER MANAGEMENT POLICY UPDATE: IMPLEMENTING THE 3RD PILLAR OF THE CWA, WATERBODY MANAGEMENT, TO FORM ASYSTEMS APPROACH

H Kenneth Hudnell1 and Wayne Carmichael2

1Medora Corporation & UNC-CH, 105 Serrano Way, Chapel Hill, NC 27517, USA2Department of Biological Sciences, Wright State University, Dayton, OH 45435, USA

A systems approach to freshwater management is needed to protect and restore freshwater in the near term at a cost much less than that currently spent on watershed management alone. Current US policy fails to protect unimpaired waters or restore impaired waters. Whereas the EPA estimated in 1972 that 10 to 20 percent of lakes and reservoirs were eutrophic, the agency now estimates that about 50 percent are eutrophic or hypereutrophic. A recent EPA assessment of 2008-2009 data for rivers and streams found that 66 percent contained excessive levels of phosphorus, a primary cause of eutrophication, up from 47 percent in 2004. The CWA (Clean Water Act) established three pillars of freshwater protection and restoration, the watershed management point- and nonpoint-source pollutant-input control programs, and the waterbody management (Clean Lakes) program, but the latter program was not implemented to a significant extent. Waterbody management provides the supportive therapy needed to reduce stress on impaired biochemical processes and enable recovery. Only full implementation of the CWA provides a systems approach by addressing not only land-based pollutant inputs, but also atmospheric depositions, existing internal loads, stressed aquatic biochemical processes, and pollutant-induced impairments. A policy of watershed management only requires decades to restore designated uses, if ever, and is excessively expensive. For example, the watershed management plan for Falls Lake, NC, is estimated to cost about $2 billion by 2035. A systems approach will restore impaired waterbodies’ designated uses in the near term by directly targeting the impairments, such as suppressing harmful algal blooms that often produce highly potent toxins and cause chlorophyll-a, pH, and turbidity impairments. Treatments also can remove nutrients, inactivate pathogens, degrade toxic substances, and restore the ecological community balance that historically maintained good water quality. A systems approach also will lower the cost of restoration and protection. The complementary combination of within waterbody treatments and pollutant input reductions that stabilize point source nutrient-input limits (account for only 5-10% of input) and use only the most effective and cost efficient nonpoint source best management practices would reduce the overall cost. An estimation of waterbody management cost in Falls Lake is $25 million by 2035. A national multipronged initiative is underway to align policy with the CWA and establish a systems approach. The initiative includes: 1) improved Federal legislation; 2) ongoing discussion with Federal agencies; 3) improved state policy and legislation and; (4) two new nonprofit organizations; the 501(c)(3) Impaired Waters Restoration Alliance for education and research, and the 501(c)(4) Waterbody Management Association for advocacy. Significant advances and challenges will be reported. Audience input will help direct this initiative’s course of action critical for health, aquatic ecosystems, economies and our nation’s security and wellbeing.

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INHIBITION OF VERTEBRATE DEVELOPMENT IN ZEBRAFISH EMBRYOS (dAnIO RERIO) ExPOSED TO POLYMETHOxY-1-ALKENES ISOLATED FROM FRESHWATER CYANOBACTERIA

Asha Jaja-Chimedza1, Patrick D. L. Gibbs2, Miroslav Gantar3, and John P. Berry1

1Department of Chemistry and Biochemistry, Florida International University, 3000 NE 151st Street, North Miami, FL 33181 USA2Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, 4600 Rickenbacker Causeway, 33149, Florida, USA3Department of Biological Sciences, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA

Aphanizomenon and Cylindrospermopsis are two toxigenic genera of freshwater cyanobacteria known to produce a number of toxic metabolites, particularly including the water-soluble toxins cylindrospermopsin, saxitoxin, and anatoxin-a. Lipophilic extracts of strains of each of these genera were shown to inhibit the development of zebrafish (Danio rerio) embryos. Bioassay-guided fractionation using the zebrafish embryo, as a model of vertebrate development, was subsequently utilized to purify the toxic chemical components. Chemical characterization of the purified compounds, including mass spectrometry and NMR (1H, 13C, DEPT, COSY, HMQC, HMBC), revealed the presence of a homologous series of six polymethoxy-1-alkenes (PMAs). Toxicological data suggests there is an apparent correlation between chain length and/or methoxylation of the PMAs and toxicity in the embryos. In addition, exposure of the embryos to combinations of the PMAs, suggests a possible synergistic effect associated with the toxicity of these compounds. A culture collection of different strains of cyanobacteria was screened by LC-MS for the presence of PMAs, where they were identified in a number of isolates of cyanobacteria, including a Microcystis strain isolated from the Great Lakes, suggesting that these bioactive compounds may be widespread. Due to the frequent occurrence of these cyanobacterial genera in freshwater sources, particularly in association with cyanobacterial harmful algal blooms, they may pose a previously unrecognized environmental and health risk, including possible bioaccumulation of these lipophilic toxins in freshwater food-webs.

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HARMFUL ALGAL BLOOM INTEGRATED OBSERVING SYSTEM FOR THE GULF OF MExICO

Ann E. Jochens1, Barbara A. Kirkpatrick2, Steven H. Wolfe3

1Texas A&M University, 3146 TAMU, College Station, TX 77843, USA2Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236 USA3Florida Institute of Oceanography, 830 1st Street S., St. Petersburg, FL 33701, USA

Building an integrated observing system for harmful algal blooms in the Gulf of Mexico is a high priority for the partners that make up the Gulf of Mexico Alliance (GOMA), led by the five U.S. Gulf States, and the Gulf of Mexico Coastal Ocean Observing System Regional Association (GCOOS-RA), which is a regional component of the U.S. Integrated Ocean Observing System that is the U.S. part of the International Global Ocean Observing System. These two organizations encompass myriad local, state, and federal agencies, private industry, and academic and research institutions. They have joined together to plan, design, and implement an integrated, comprehensive, sustained Harmful Algal Bloom Integrated Observing System (HABIOS) for the Gulf of Mexico. Under the auspices of the GCOOS-RA and GOMA, a series of three workshops have been held specifically to develop the HABIOS, and efforts are underway to identify assets and funding sources to collaboratively build HABIOS as part of the broader Gulf of Mexico Coastal Ocean Observing System (GCOOS-RA) and Gulf Monitoring Network (GOMA). The workshops developed a strategy for HABIOS and identified user groups and needs for HABs data and information. Then, gaps in existing HAB data and products were identified, and the steps for implementation were explored. The primary beneficiaries of a fully-functioning HABIOS are shell and fin fisherman (both commercial and recreational), recreational boaters, and beachgoers, as well as the managers responsible for protecting public and ecosystem health.HABIOS is designed to address four focus areas: (a) prediction of bloom initiation, (b) detection of bloom existence, (c) tracking or monitoring of the bloom, and (d) forecasting of bloom movement and effects. The fully-developed HABIOS will support observing of major harmful algal species, with emphasis initially on prominent species, such as Karenia brevis and Dinophysis spp., that currently are causing shellfish bed closures, marine life kills, or human illness. It will target not only species that have surface chlorophyll signals observable by satellite, such as K. brevis, but also species that do not, such as Pseudo-nitzschia spp. Observing components will include satellite data, moorings, and gliders or other autonomous profiling instrumentation. The system builds on existing efforts by state agencies, academics, and federal agencies such as NOAA. In addition to supporting the broader goals of the GCOOS and GMN, the full HABIOS will supplement the existing HAB monitoring system by using fixed stations and routine glider transects to continuously detect specific HAB species and collect data on a suite of parameters (e.g., nutrients, currents, temperature, salinity, fluorescence, and optical phytoplankton community structure) that would indicate favorable conditions for HAB events.

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ExPANSION OF BROWN TIDE BLOOMS CAUSED BY AuREOumbRA LAGunEnSIS TO THE EAST COAST OF THE UNITED STATES

Florian Koch1, Christopher J. Gobler1, Yoonja Kang1, Dianna L. Berry1, Ying Zhong Tang1, Margaret Lasi2, Linda Walters4, Lauren Hall3, and Jan D. Miller3

1Stony Brook University, School of Marine and Atmospheric Sciences, Southampton, NY, 11968

2St. Johns River Water Management District, Palatka, FL 321773St. Johns River Water Management District, Palm Bay, FL 329094University of Central Florida, Department of Biology, Orlando, Florida 32816

Brown tides caused by the pelagophyte Aureoumbra lagunensis DeYoe et Stockwell have formed ecosystem disruptive algal blooms in shallow lagoons of Texas (TX), USA, for more than two decades but have never been reported elsewhere. During the summer of 2012, a dense brown tide occurred in the Mosquito Lagoon and northern Indian River Lagoon along the east coast of Florida (FL), USA. While chlorophyll a levels in this system have averaged 5 µg L-1 during the past two decades, concentrations during this brown tide reached ~ 200 µg L-1. Concurrently, levels of nitrate were significantly lower than average and levels of dissolved organic nitrogen were significantly higher than average (p<0.001 for both). Sequences of the 18S rRNA gene of the bloom community and of single cell clonal isolates were identical to those of Aureoumbra lagunensis DeYoe et Stockwell from TX. The A. lagunensis brown tide in FL bloomed to densities exceeding 106 cells mL-1 (quantified with a species-specific immuno-label) from July through September, began to dissipate in October, but maintained densities exceeding 105 cells mL-1 in some regions through December of 2012. The decline of the bloom was associated with near-hypoxic conditions and more than 30 fish kills reported within the Mosquito Lagoon in September 2012, a number exceeding all prior reports in this system dating to 1996. Wild Northern quahog populations (a.k.a. hard clam, Mercenaria mercenaria) suffered mass die offs during the brown tide and eastern oysters (Crassostrea virginica) that settled during 2012 were significantly smaller than prior years. Clearance rates of hard clams and eastern oyster were significantly reduced in the presence of Mosquito Lagoon bloom water and A. lagunensis monocultures isolated from the Mosquito Lagoon at densities of ~106 cells L-1. The expansion of harmful brown tides caused by A. lagunensis to these estuaries represents a new threat to the US southeast coast. Dynamics of Aureoumbra lagunensis in these lagoons during the 2013 and details of a novel method for quantifying this algae will also be presented.

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METABOLOMIC ANALYSIS OF HARMFUL ALGAL BLOOM ALLELOPATHY: DECIPHERING SUBLETHAL IMPACTS ON COMPETING PHYTOPLANKTON

Kelsey Poulson-Ellestad1, Christina Jones2, Jessie Roy1, Facundo M. Fernandez2, Brook Nunn3, Jon Byrne4, Mark Viant4, Julia Kubanek1,2

1Georgia Institute of Technology, School of Biology and Aquatic Chemical Ecology Center, 310 Ferst Drive, Atlanta, GA 30332, USA2Georgia Institute of Technology, School of Chemistry & Biochemistry and Aquatic Chemical Ecology Center, 310 Ferst Drive, Atlanta, GA 30332, USA3University of Washington, Department of Genome Sciences, Box 355065, WA 98195, USA4University of Birmingham, School of Biosciences and NERC Metabolomics Facility, Edgbaston, Birmingham B152TT, UK

How individual species come to be dominant members of marine planktonic communities is not deeply understood; however, it is thought that chemistry plays a substantial role. For example, some red tide-forming dinoflagellates produce toxic compounds that are hypothesized to enhance dinoflagellate fitness by acting as grazer deterrents, allelopathic agents, or antimicrobial defenses. In field and lab experiments we have previously shown that the red tide dinoflagellate Karenia brevis is allelopathic, inhibiting the growth of several co-occurring phytoplankton species, but that K. brevis compounds other than well-known brevetoxins are responsible for suppressing most of these species. For phytoplankton competitors, death is a rare outcome of K. brevis allelopathy: more subtle responses predominate, such as reduced photosynthetic output and increased cell permeability. We characterized changes in cellular physiology with NMR and MS-based metabolomics which indicated disruption of primary metabolic pathways in diatoms exposed to K. brevis. Preliminary proteomic analyses further supported the finding that metabolic networks are impacted by red tide allelopathy. A diatom species known to co-occur with K. brevis responded with less dramatic metabolic alteration than a model diatom whose range does not overlap with K. brevis, suggesting adaptation among phytoplankton that frequently experience K. brevis blooms. Overall, systems biology approaches including metabolomics and proteomics offer considerable opportunities for expanding our understanding of plankton ecology, driving novel hypotheses about the molecular mechanisms of chemically mediated interactions.

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COMMON EFFECTS OF CHRONIC DOMOIC ACID ExPOSURE IN ZEBRAFISH, SEA LIONS, MICE AND HUMANS

Kathi Lefebvre1, Lynn Grattan2, Elizabeth Frame1, Preston Kendrick1, Sparkle Roberts2, Emma Hiolski3, Don Smith3, Glen Morris4, and Dave Marcinek5

1NOAA Fisheries, NWFSC, Seattle, WA 98112, USA2University of Maryland School of Medicine, Baltimore, MD 21201, USA3University of California, Santa Cruz, CA 95064, USA4University of Florida, P.O. Box 100009, Gainesville, FL 32610, USA5University of Washington, Seattle, WA 98109, USA

It is well known that acute exposure to high levels of the algal toxin domoic acid (DA) is responsible for a neurotoxic illness known as amnesic shellfish poisoning (ASP) characterized by vomiting, diarrhea, seizures, memory loss, coma and death. Human seafood consumers are protected from high level exposure via the monitoring of seafood and subsequent regulation of harvests based on toxin loads (≥ 20 ppm = harvest closure). However, there are no protections in place for low-level repetitive exposure (< 20 ppm = harvest open) because there is a critical knowledge gap regarding chronic exposure effects. Our research team has investigated the impacts of repetitive subclinical DA exposure in two laboratory human disease model species (zebrafish and mice), one naturally exposed sentinel for human health species (California sea lions), and in “at risk” high seafood consuming humans. Chronic subclinical exposure (below levels that show overt excitotoxic symptoms) has been shown to significantly alter gene expression in the CNS, elicit an immune response, and increase toxin sensitivity in zebrafish. Additionally, the presence of a DA specific antibody was indicated in zebrafish and validated in naturally exposed sea lions, thereby revealing a potential diagnostic biomarker for chronic exposure. Mouse and human studies are currently underway in an effort to verify the utility of the antibody biomarker and relate it to subclinical neurotoxic effects. A synthesis of results from all models will be presented.

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ECOLOGICAL AND ENVIRONMENTAL FACTORS AFFECTING CARIBBEAN CIGUATOxINS AND THEIR TROPHIC TRANSFER TO REEF DWELLING

J.D. Liefer1, A. Robertson1, A.C. Garcia1, T. Smith2, H.A. Flores Quintana1, M. Richlen3, andD.M. Anderson1

1FDA Gulf Coast Seafood Laboratory, 1 Iberville Dr., Dauphin Island, AL 36528, USA2University of the Virgin Islands, #2 John Brewers Bay, St. Thomas, U.S. Virgin Islands 00802, USA3Woods Hole Oceanographic Institution, 266 Woods Hole Rd., Woods Hole, MA 02543, USA

Ciguatoxins (CTXs) are cyclic polyether toxins that originate from benthic dinoflagellates of the genus Gambierdiscus, which often grow in association with common reef macroalgae such as Dictyota. These toxins undergo biotransformation and biomagnification in tropical reef food webs. Consumption of CTX-contaminated fish results in ciguatera fish poisoning (CFP). In the Caribbean, this poisoning syndrome is characterized by a variety of gastrointestinal and neurological symptoms. The US Virgin Islands is an area that has sustained high prevalence of CFP that has impacted human health and important fisheries. To address these impacts, we are examining how microalgal community composition, reef physical conditions, and water quality affect the production, biotransformation, and trophic transfer of CTXs. The long-term goal of these efforts is modeling CTX production dynamics and predicting future outbreaks of CFP in the greater Caribbean region. Four sites near St. Thomas, US Virgin Islands were sampled from Jun 2010 to Dec 2012 to assess the production and trophic transfer of Caribbean CTXs (C-CTX). Epiphytic dinoflagellates were quantified from Dictyota samples collected monthly to determine species composition and composite C-CTX concentration (which includes the precursors gambiertoxins) using N2a cytotoxicity assays and LC-MS. In addition, a variety of fishes representing multiple trophic levels were collected quarterly from each site and analyzed for C-CTX. Gambierdicsus and C-CTX activity in dinoflagellate samples were detected year-round at all four sites. The Gambierdiscus C-CTX cell quotas were highest during colder months, a pattern not previously shown in this region. The Gambierdiscus cell quota of C-CTX was consistently higher at Black Point, a near shore site characterized by relatively less mixing and wave action. Herbivorous fish species also had consistently higher C-CTX concentrations, many above the FDA guidance threshold of 0.1 ppb, at Black Point compared to the other three sites. These results indicate the importance of temperature and physical forcing in regulating Gambierdiscus abundance and the subsequent production and transfer of CTX. We also present an extensive comparison of C-CTX cell quotas with dinoflagellate species composition and environmental conditions such as physical hydrography and nutrient concentrations.

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WHEN HUNGRY, KAREnIA bREvIS GOES TOxIC

D. Ransom Hardison1,2, William G. Sunda1, Damian Shea2, Patricia A. Tester1, R. Wayne Litaker1

1Center for Coastal Fisheries and Habitat Research, National Centers for Coastal Ocean Science, National Ocean Service, 101 Pivers Island, Beaufort, NC, 28516, USA.

2Department of Biology, North Carolina State University, Raleigh, North Carolina, USA

Karenia brevis is the dominant toxic red tide species in the Gulf of Mexico. It produces potent neurotoxins (brevetoxins [PbTxs]), which negatively impact human and animal health, local economies, and ecosystem function. Field measurements have shown that cellular brevetoxins vary from 1‒68 pg/cell but the source of this variability is uncertain. The carbon:nutrient balance (CNB) hypothesis, which was originally developed for terrestrial plants, predicts that nutrient-limited growth will be accompanied by a diversion of fixed carbon into increased levels of carbon-based defensive compounds or structures. Consistent with this hypothesis, data from numerous harmful algal bloom species have shown that nutrient limitation causes an increase in cellular toxin content. Historically, the effect of nutrients on K. brevis toxicity has been controversial. This study was undertaken to examine the effect of nitrogen (N) and phosphorus (P) limitation of growth rate on cellular brevetoxins in diverse K. brevis strains from different geographic locations. N and P were selected because both have been reported to limit algal growth in the Gulf of Mexico. N- and P-limited cells had 2- to 3-fold higher PbTx per unit of cell volume or cell carbon. The percent of total cellular carbon associated with brevetoxins (%C-PbTx) was 1-4% in N-replete cultures and 5-9% in the N-limited cells. Similarly, %C-PbTx in P-replete isolates ranged from 0.7 to 2.1% in P-replete cells, but increased to 1.6‒5% under P-limitation. Because PbTxs are potent anti-grazing compounds, this increased investment in PbTxs should enhance cellular survival during periods of nutrient-limited growth. The %C-PbTx for all isolates was also found to be inversely related to specific growth rate, which is consistent with an evolutionary tradeoff between carbon investment in PbTxs and other grazing defenses, and carbon investment in growth and reproduction. In aquatic environments where nutrient supply and grazing pressure often vary on different temporal and spatial scales, this tradeoff would be advantageous as it would result in increased net population growth rates. The variation in PbTx/cell values observed in this study can account for the range of values observed in the field. These results suggest that N- and P-limitation are important factors regulating cellular toxicity and adverse impacts during at least some K. brevis blooms. The relationship between growth and toxicity is being used in models to predict how the toxicity of K. brevis blooms will change as the bloom densities increase and use up available nutrients. These models show that the combination of high biomass and high toxin to biomass ratios that occur when blooms run out of nutrients results in extreme bloom toxicity, and helps explain why Karenia blooms are often very damaging to ecosystems, human health, and coastal economies. The results also point to the need for direct toxin measurements in affected waters to directly assess toxicity and forecast adverse impacts.

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GEORGES BANK: A LEAKY INCUBATOR OF ALExAndRIum FundyEnSE BLOOMS

Dennis J. McGillicuddy, Jr.1

David W. Townsend2

Bruce A. Keafer3

Maura A. Thomas2

Donald M. Anderson3

1Department of Applied Ocean Physics and Engineering, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.2School of Marine Sciences, University of Maine, Orono, ME 04469, USA.3Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA

A series of oceanographic surveys on Georges Bank document variability of populations of the toxic dinoflagellate Alexandrium fundyense on time scales ranging from synoptic to seasonal to interannual. Blooms of A. fundyense on Georges Bank can reach concentrations on the order of 104 cells l-1, and are generally bank-wide in extent. Georges Bank populations of A. fundyense appear to be quasi-independent of those in the adjacent coastal Gulf of Maine, insofar as they occupy a hydrographic niche that is colder and saltier than their coastal counterparts. In contrast to coastal populations that rely on abundant resting cysts for bloom initiation, very few cysts are present in the sediments on Georges Bank. Bloom dynamics must therefore be largely controlled by the balance between growth and mortality processes, which are at present largely unknown for this population. Based on correlations between cell abundance and nutrient distributions, ammonium appears to be an important source of nitrogen for A. fundyense blooms on Georges Bank.

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LINKING INDIVIDUAL CELL BEHAVIORS WITH THE POPULATION DISTRIBUTION OF THE RAPHIDOPHYTE HETEROSIGmA AKASHIWO

Susanne Menden-Deuer and Elizabeth L. HarveyGraduate School of Oceanography, University of Rhode Island, Narragansett, RI

In the laboratory, we investigated the effects of biotic and abiotic stimuli on the individual behaviors and vertical distribution of Heterosigma akashiwo. Using automated observation and video analysis procedures; we simultaneously investigated the three-dimensional movements of free-swimming cells and the resultant macroscopic population distributions of different strains in tanks with a halocline structure. Induced by both the presence of actively feeding ciliate and dinoflagellate predators and predator-derived chemical cues, H. akashiwo cells exhibited effective fleeing behaviors. At the individual cell level, both vertical velocity and swimming direction of the halo-tolerant HAB alga changed significantly, resulting in population aggregation in low salinity waters, inaccessible to the less halo-tolerant predator. The ciliate predator showed no response to algal exudates but the dinoflagellate displayed significant increases in swimming speed and turning rate when exposed to filtrate from H. akashiwo. Prey cue–induced changes in predator swimming behavior resulted in an 11% increase in encounter rate and a 25% reduction in the necessary ambient prey concentration necessary to meet the daily quota of the predator. Our results provide quantitative evidence for the importance of individual behaviors and chemical cues in modulating H. akashiwo’s vertical distribution and potential predator pressure, both factors that are independent of population growth, but critical in predicting abundance, distribution and bloom formation potential of this HAB alga.

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WHAT ExACTLY ARE YOU GROWING? AN ASSESSMENT OF THE BACTERIAL COMMUNITY IN COMMONLY USED CULTURES OF KAREnIA bREvIS AND OTHER TOxIC AND NON-TOxIC DINOFLAGELLATES

Kevin A. Meyer1, Judy M. O’Neil1, and Byron C. Crump2

1University of Maryland Center for Environmental Science: Horn Point Laboratory, 2020 Horns Point Road, Cambridge, MD, 21613, USA2Oregon State University, 1500 SW Jefferson Way, Corvallis, OR, 97331, USA

Algal cultures are commonly used for experimental studies because of the control over the system, and organism of interest, it provides the researcher. Harmful algal bloom forming species are no exception and in some cases, like the Florida Red Tide dinoflagellate Karenia brevis, were some of the earliest cultures established in the United States. In many cases the bacterial community is left intact in phytoplankton cultures (not axenic), which then subjects the bacterial community to the same selective pressures being used to maintain a single algal species. Bacteria-algal relationships are well documented but are rarely considered during experiments that use HAB cultures. Given that some cultures, like the infamous “Wilson clone” were isolated decades ago, it is likely that bacterial communities in such cultures no longer resemble typical Gulf of Mexico bacterial communities. The bacterial community was investigated within seven different isolates of Karenia brevis, an isolate of Karenia mikimotoi, Prorocentrum minimum, P. rhathymum, Karlodinium veneficum, and Gyrodinium dorsum from Florida Fish and Wildlife Research Institute and MOTE Marine Laboratory. Genetic analysis was done using Illumina sequencing and bacterial growth was measured using 3H-Leucine and Thymidine to better understand the community composition, biological function, and possible relationships between commonly used cultures and co-cultured bacteria.

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GREAT LAKES ‘BIG DATA’ AND HAB-INFORMATICS: USING ARTIFICIAL INTELLIGENCE TO DECONVOLVE mICROCySTIS-ENVIRONMENTAL RELATIONSHIPS

David F. Millie1,2, Gary R. Weckman3,1, Gary L. Fahnenstiel2,4, William A. Young II5, Ehsan Ardjmand3, John A. Fahnenstiel1, Robert A. Shuchman2,4, and Michael J. Sayers2

1 Palm Island Enviro-Informatics LLC, Sarasota, FL 34232, USA2 Michigan Technological University, Michigan Tech Research Institute, Ann Arbor, MI 48105, USA3 Ohio University, Russ College of Engineering and Technology, Department of Industrial and Systems Engineering, Athens, OH 45701, USA4 Michigan Technological University, Great Lakes Research Center, Houghton, MI 49931, USA5 Ohio University, College of Business, Management Systems Department, Athens, OH 45701, USA

Nutrient-enriched waters of the Laurentian Great Lakes are plagued by expansive, recurring blooms of the toxic cyanobacterium, Microcystis aeruginosa. The intensity of these events have been linked to phosphorus–laden tributary discharges and concurrent, optimal meteorological conditions. Although such influences appear to work ‘in tandem’ as controls of HABs, a comprehensive analysis of Microcystis ‘forcing factors’ has yet to be completed for the Lakes.

Monitoring of the blooms - via invasive sampling, instrumental-based observatories, and satellite imagery, has resulted in massive, high-dimensional data streams (i.e. ‘Big Data’) that personnel must search through to identify data structures and relationships. Recent technological advances have provided for the evolution of computational-intensive analytics that optimize for the delineation and reproduction of complex patterns within large databases. Such an ‘informatic’ approach previously has been used by the authors to denote (apparent) phosphorus threshold concentrations for Microcystis blooms within Saginaw Bay, Lake Huron from 1990 to 1996 (J. Phycol., 2011; 7: 714–730).Using diverse databases (imagery, watershed hydrodynamics, meteorology, physical/chemical limnology), we present a neural network–based approach demonstrating the selection and quantitation of environmental attributes for modeling Microcystis abundance dynamics throughout Saginaw Bay (2008-2010) and western Lake Erie (2009-2011). Model formulations provide interpretable, multi-dimensional response surfaces for environmental-Microcystis associations, thereby allowing for visualized, qualitative/quantitative representations of HAB-nutrient relationships and interaction (akin to niche modeling). Such a heuristic methodology affords a mathematically comprehensive, yet pragmatic understanding for factors regulating phytoplankton blooms and becomes a data-driven source upon which to identify ‘target’ nutrient concentrations for management efforts attempting to reduce/eliminate HABs throughout the Lakes.

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ALExAndRIum BLOOM ECOLOGY IN PUGET SOUND: CYST DYNAMICS, GROWTH, TRANSPORT, AND CLIMATE PATHWAYS

S. K. Moore1, B. D. Bill1, L. R. Hay1, K. C. Eldred1, C. L. Greengrove2, J. E. Masura2, N. S. Banas3, E. P. Salathé4, N. J. Mantua4,5, J. A. Johnstone6, D. M. Anderson7, V. L. Trainer1, and J. E. Stein1

1NOAA, Northwest Fisheries Science Center, Seattle, WA; [email protected] of Washington–Tacoma, Tacoma, WA3Applied Physics Laboratory, University of Washington, Seattle, WA4University of Washington, Climate Impacts Group, Seattle, WA5NOAA, Southwest Fisheries Science Center, Santa Cruz, CA6University of Washington, Joint Institute for the Study of the Atmosphere and Ocean, Seattle, WA7Woods Hole Oceanographic Institution, Woods Hole, MA

The Puget Sound Alexandrium Harmful Algal Bloom (PS-AHAB: www.tiny.cc/psahab) program seeks to understand environmental controls on the benthic (cyst) and planktonic life stages of the toxic dinoflagellate Alexandrium catenella, and disentangle the effects of climate pathways on the timing and location of blooms. Spatially detailed mapping of winter cyst distributions in 2011, 2012, and 2013 found the highest cyst concentrations in Bellingham Bay in the north and Quartermaster Harbor in central Puget Sound. However, the viability of cysts at these seed bed areas is low – with fewer than 54% of cysts germinating when incubated at favorable temperatures. This may complicate potential relationships between cyst abundances and bloom magnitude the following season. The time of year that cysts can germinate does not appear to be determined by endogenous controls. The rate of cyst germination is strongly determined by temperature. Passive particles released from these seed beds were tracked using a high-resolution hydrodynamic simulation of Puget Sound and adjacent coastal waters (MoSSea: http://faculty.washington.edu/pmacc/MoSSea/). In two weeks, particles released from Bellingham Bay made it out of the Strait of Juan de Fuca to the outer Washington coast whereas particles released from Quartermaster Harbor mostly stayed in the main basin of Puget Sound. No particles entered Hood Canal, suggesting that physical transport mechanisms may prevent toxic cells from contacting shellfish in this basin of Puget Sound. Laboratory experiments showed that maximal growth rates (~0.3-0.5 μ d-1) occur over a broad range of temperatures (~14-24°C) at salinities typical for Puget Sound (20-35 psu). These ranges were used to define favorable habitat for A. catenella using model output from the MoSSea simulation. A 40-year global climate projection was regionally downscaled and coupled to MoSSea to determine temporal and spatial changes to favorable habitat under the A1B greenhouse gas emissions scenario. A comparison between present-day and circa-2050 conditions allows us disentangle the effects of three climate pathways on favorable habitat for A. catenella in Puget Sound: 1) changing ocean inputs (associated with upwelling winds), 2) changing streamflow magnitude and timing, and 3) increased direct insolation.

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A NEW FRAMEWORK FOR PUBLIC HEALTH MONITORING OF CHAB IMPACTED DRINKING WATER RESERVOIRS: INCORPORATION OF CYANOBACTERIAL PHYSIOLOGY AND GENOTYPE SUCCESSION RATES

Timothy G. Otten1,2, Theo W. Dreher1 and Hans W. Paerl2

1Oregon State University, Department of Microbiology, 220 Nash Hall, Corvallis, OR 97331, USA2University of North Carolina at Chapel Hill, Institute of Marine Sciences, 3431 Arendell St, Morehead City, NC 28557, USA

High throughput sequencing and quantitative PCR were used to characterize the spatiotemporal patterns of cyanobacterial harmful algal blooms (CHABs) in two large river ecosystems - the Klamath River and San Francisco Bay Delta. In recent years both of these systems have been plagued by recurring, seasonal blooms of toxin-producing Microcystis spp., as well as diazotrophic genera (e.g., Aphanizomenon and Anabaena) of relatively unknown toxicity. The sequencing and qPCR data enable us to quantify population succession rates based on a multilocus analysis of allele frequencies over time and space. These data, along with detailed physicochemical analyses, can be used to greatly improve our understanding of how different environmental conditions may select for toxigenic ecotypes over nontoxic variants. Here we propose a public health monitoring framework which takes into account laboratory based analyses of algal physiology (e.g., growth kinetics, cell toxin quotas, stress response) and in situ observations of population turnover rates from cyanobacterial blooms under natural settings. The goal of this research is to create and validate a public health sampling protocol for CHAB impacted reservoirs which minimizes unnecessary costs due to oversampling, without sacrificing the public health benefits of a proactive drinking water monitoring program.

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MANAGING HARMFUL CYANOBACTERIAL BLOOMS ALONG THE FRESHWATER-MARINE CONTINUUM IN A WORLD ExPERIENCING HUMAN AND CLIMATICALLY-MEDIATED CHANGE

Hans W. Paerl1, Timothy G. Otten1, Hai Xu2, Boqiang Qin2, Guangwei Zhu2, Nathan S.Hall1

1University of North Carolina at Chapel Hill, Institute of Marine Sciences, Morehead City, NC 28557, USA 2State Key Laboratory of Lake Science and Environment, Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjing 210008, PR China

Coastal watersheds support nearly 75% of the world’s human population and are experiencing unprecedented urban, agricultural and industrial expansion. The freshwater-marine continua draining these watersheds are increasingly experiencing nutrient over-enrichment, eutrophication, and proliferation of harmful cyanobacterial blooms (CyanoHABs); with negative impacts on higher plant and animal habitats as well as animal and human health. In addressing nutrient input reductions needed to stem and reverse this troubling trend, phosphorus (P) has traditionally received priority in upstream freshwater regions, because some CyanoHABs can fix atmospheric nitrogen (N2) to satisfy their nitrogen (N) requirements. Conversely, controlling nitrogen (N) inputs has been the focus of management strategies in estuarine and coastal waters. However, freshwater, brackish and full-salinity components of the continuum are structurally and functionally connected. Furthermore, eutrophying freshwater and marine systems are increasingly plagued with non N2 fixing CyanoHABs that are N and P co-limited or even N limited. In some systems N loads are increasing faster than P loads. Therefore N and P input constraints are likely needed for long-term CyanoHAB control. Climatic changes, specifically warming, increased vertical stratification, salinization, and intensification of storms and droughts, favor CyanoHABs and thus play synergistic roles in promoting CyanoHAB frequency, intensity, geographic distribution and duration. In particular, rising temperatures cause shifts in critical nutrient thresholds at which cyanobacterial blooms can develop. In practical terms, this means that nutrient input reductions aimed at controlling CyanoHABs may need to be more aggressively pursued in a warming world. Additional control steps that should be considered include 1) altering the hydrology to enhance vertical mixing and/or flushing and 2) decreasing nutrient fluxes from organic rich sediments by physically oxygenating or removing the sediments or capping sediments with clay. These efforts however have met with mixed results and can disrupt benthic and planktonic habitats. In most instances, long-term effective eutrophication and CyanoHAB control must consider both N and P loading dynamics within the context of altered thermal and hydrologic regimes associated with climate change.

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USING GENETIC MARKERS TO DETERMINE THE EFFECT OF SEEDING ON THE DISTRIBUTION OF A SAxITOxIN-RESISTANT MUTATION IN myA AREnARIA

Jennifer Phillips1 and Laurie Connell1

1 School of Marine Sciences, University of Maine, Orono, ME 04469

We have identified a sodium (Na+) channel mutation in softshell clams Mya arenaria that causes a 1,000-fold decrease in binding affinity at the saxitoxin-binding site in the sodium channel pore, thereby conferring a resistance to paralytic shellfish toxins (PSTs) produced by the genus Alexandrium. This mutation results in improved fitness (growth, motility and survival advantage of resistant individuals) during toxic blooms, as well as higher toxin accumulation rates. Therefore, PSTs can act as a strong natural selection agent in nature, leading to spread of toxin resistance in M. arenaria populations. My work has shown that selection for this resistant allele is correlated with the prevalence and intensity of Alexandrium spp. blooms, resulting in higher numbers of resistant individuals in areas with regular HAB events, however the effect of juvenile Mya arenaria seeding on the distribution of resistant and sensitive clams is currently unknown. This study examines the effect of the seeding program at the Downeast Institute in Beals, ME on the genetic composition of seeded sites in the northeastern US with respect to the resistant mutation, using newly characterized M. arenaria microsatellite markers.

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SOME ASSEMBLY REQUIRED: MULTIDOMAIN PKS/NRPS GENES IN DINOFLAGELLATES

Tsvetan R. Bachvaroff1, Ernest Williams1, Charles F. Delwiche2, and Allen R. Place1

1Institute of Marine and Environmental Technology, 701 E. Pratt St., Baltimore MD 212022Department of Cell Biology and Molecular Genetics and the Maryland Agricultural Experiment Station, University of Maryland, College Park, MD 20742 Dinoflagellates are important marine primary producers, infamous for producing toxins, but also significant producers of the beneficial omega three polyunsaturated fatty acids. We undertook Illumina RNA sequencing of two species known to produce PKS-derived toxins: Amphidinium carterae, and Karlodinium veneficum, and a third non-toxic species Akashiwo sanguineum. In these species the toxin and fatty acid biosynthetic genes are likely PKS derived and previous results found only single PKS domain genes that was thought to be the norm for dinoflagellates. Surprisingly, numerous multidomain PKS, NRPS, and hybrid PKS / NRPS genes ranging from 8 - 15 kb were found from all three species. Six different multidomain genes were found: one contained only PKS domains, three were hybrid NRPS /PKS genes, and two were NRPS genes. Introns were found in PCR products amplified from genomic DNA templates in A. carterae for all but the NRPS genes. Similarly, all categories but the NRPS had at least one species with a partial characteristic dinoflagellate spliced leader sequence. Overall sequence assembly or coverage for these large transcripts was poor, requiring iterative assembly techniques in several cases. Phylogeny of KS, adenylation, AT, and KR domains from the complete dataset suggested the multidomain genes were acquired as such, rather than derived from single domain genes. However, the majority of sequences with PKS or NRPS domains were encoded as single domain genes and appeared to be quite diverse in these species. It appears that a combination of cis-multidomain proteins interacting with trans-single domain proteins can provide the diversity of polyketides observed in these remarkable biosynthetic organisms.

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INHIBITION OF THE DINOFLAGELLATE CELL CYCLE AFTER INOCULATION WITH THE ALGICIDAL COMPOUND IRI-160AA ExCRETED BY SHEWAnELLA SP. IRI-160

Kaytee L. Pokrzywinski1, Mark E. Warner1 and Kathryn J. Coyne1

1University of Delaware College of Earth, Ocean, and Environment, 700 Pilottown Rd., 19958 Lewes, DE, USA

Previous work in our laboratory investigated the algicidal activity of a bacterium, Shewanella sp. strain IRI-160. We confirmed that algicidal activity was due to a secreted compound (designated IRI-160AA) that targets dinoflagellates while having little to no effect on other phytoplankton species. We also demonstrated that the algicide induces programmed cell death in dinoflagellates, marked by changes in morphology, extracellular H2O2 production, caspase-like enzyme activity, and loss of asymmetry in the cellular membrane. Cell death induced by algicide IRI-160AA may be a consequence of disrupting the cell cycle. The dinoflagellate genome is characterized by massive amounts of genomic data that contain tandem repeats, no or low histone-like proteins, no nucleosome structures, and a high abundance of transition metals and base modifications (hydroxymethyluracil). In addition, dinoflagellates are unusual in that they retain a permanent nuclear envelope during the cell cycle. The chromosomes also remain condensed throughout the cell cycle and extra-nuclear microtubule spindles traverse through cytoplasmic channels during mitosis. These features of dinoflagellates may be a target for the algicide. Here, we used microscopy and flow cytometric analysis to investigate the effects of the algicide on cell cycle progression in three dinoflagellates: Prorocentrum minimum, Karlodinium veneficum and Gyrodinium instriatum. The effect of the algicide on the cell cycle of the coccolithophore Emiliania huxleyi was also included as a control. Our results suggest a change in cell cycle progression for dinoflagellate cultures treated with the algicide along with nuclear degeneration and massive degradation of chromosomal DNA, supporting the hypothesis that the algicide is targeting dinoflagellate-specific nuclear structures and/or checkpoints that play a role in controlling cell cycle progression of these algae.

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CIGUATOxIN AND HAWAIIAN MONK SEALS: MONITORING BLOOD TOxIN LEVELS IN NORTHWESTERN HAWAIIAN ISLANDS SEALS AND EVALUATION OF FORAGING UNDER STRESS USING A MOUSE MODEL

John Ramsdell1, Jessica Tiedeken1, Marie-Yasmine Bottein1, Zhihong Wang1, Aurelie Ledreux1, Jennifer Fuquay1, Liz Kashinsky2, Angie Kaufman2, Michelle Barbieri2 and Charles Littan2

1Marine Biotoxins Program, National Centers for Coastal Ocean Science, NOAA, 219 Fort Johnson Rd., Charleston, SC 29412, USA2 Hawaiian Monk Seal Research Program, Pacific Islands Fisheries Science Center, NOAA, 1601 Kapiolani Blvd., Honolulu, HI 96814, USA

Ciguatoxin, an algal neurotoxin endemic to circumtropical regions, is believed to be present in the food web of the Hawaiian monk seal. A critically endangered species, the Hawaiian monk seal population is declining in its primary habitat of the Northwestern Hawaiian Islands, and the role that ciguatoxins may play in this decline is being investigated through several means. Several potential ciguatoxin congeners were detected in monk seal liver samples by LC-tandem-mass spectrometry. P-CTX-3C was confirmed by matching peak area ratios of 6 MRM transitions of the suspected protonated P-CTX-3C ion (m/z 1023.6 ® 125.1, 155.1, 447.5, 907.6, 987.6, and 1005.6) with those of P-CTX-3C standard. Ciguatoxin activity (ranging from 0.4 to 5.5 pg/ml) was identified by Neuro2A cytotoxicity assay in whole blood of 19% of 55 free-ranging seals collected between 2007 and 2010. Analysis of archived blood spot cards from an expanded set of animals collected between 2003 and 2011 is ongoing to identify age classes at highest risk of exposure. The decline of the Hawaiian monk seal is evident in low survival of juveniles and may result from poor foraging compounded by the stress of top level predators. We therefore developed a murine model to examine the effect of ciguatoxin exposure and foraging under stress. A bright open field with food in the center was used to imitate a foraging environment and restraint-induced stress a proxy for top level predator stress. C57BL/6 mice accustomed to restraint stress over 3 weeks were exposed to ciguatoxin P-CTX-1B (0.26 ug/kg, ip) or saline. Commencing 4 days after ciguatoxin exposure and repeated ten days thereafter, response and behaviors in the open field were recorded, tracked and observed, noting the time until the animal took the food. Animals 15 and 25 days after ciguatoxin exposure that received the restraint stress immediately prior to testing showed significantly longer time to take the food than matched controls for either restraint stress or ciguatoxin exposure. Taken together, these results indicate that ciguatoxin exposure is common in the Hawaiian monk seal and can induce a disease state in mice that negatively impacts foraging in a stressful environment.

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LINKING FISH TO HUMAN ILLNESS: CONFIRMATION OF CIGUATERA FISH POISONING CASES FROM ST.THOMAS, U.S. VIRGIN ISLANDS 2010-2012

Alison Robertson1, Lynn M. Grattan2, Sparkle Roberts2, Justin D. Liefer1, Ana C. Garcia1, Harold A. Flores Quintana1, Jennifer I. Hooe-Rollman1, Margaret Abbott2, Elizabeth G. Radke3, J. Glenn Morris3

1 Gulf Coast Seafood Laboratory, Division of Seafood Science and Technology, U.S. Food & Drug Administration, Dauphin Island, AL 36528, USA2 School of Medicine, University of Maryland, College Park, MD 20740, USA3 Emerging Pathogens Institute, University of Florida, Gainesville, FL 32610, USA

Ciguatera fish poisoning (CFP) occurs throughout the tropics, particularly in island communities such as the U.S. Virgin Islands where fish are a primary source of protein. The impact of CFP in these communities is difficult to assess due to underreporting of illnesses, misdiagnosis, and lack of clinical tests to confirm ciguatoxin exposure. In St. Thomas, CFP annual incidences have been estimated at 120 per 10,000 residents. While some symptoms of CFP are characteristic, many gastrointestinal and neurological disturbances are quite similar to other toxin poisoning syndromes. Confirmation of CFP cases relies on the availability of a meal remnant or leftover fish portion from which ciguatoxins (CTX) can be confirmed using chemical methods. Retrospective toxin data such as this was used to develop the current FDA safety guidance levels of 0.1 µg Caribbean-CTX-1 (C-CTX-1) equivalents/kg fish tissue. This study focused on CFP cases where meal remnant toxicity data could be directly linked to human illness. Persons reporting to the Roy Schneider Hospital, St. Thomas, between April 2010 and April 2012 with a medical diagnosis of acute CFP were asked to participate in this study. Participants completed a detailed questionnaire designed to provide demographic data and information on the implicated fish including: source, species, and meal preparation. In addition, data on the symptoms, severity, and duration of illness experienced by the participants were collected. Of the 57 total participants during this period, 20 were able to provide a meal remnant or leftover portion of fish that was eaten prior to the onset of acute symptoms. Meal remnant samples were subjected to chemical extraction and examined for the presence of ciguatera-related toxins using the sodium channel-specific mouse neuroblastoma cytotoxicity assay and liquid chromatography tandem mass spectrometry (LC-MS/MS). Of the 20 meal remnants tested, 16 were confirmed to contain C-CTXs ranging from 0.3-2.4 µg C-CTX-1 equivalents/kg fish. Multivariate analyses were used to determine factors (e.g., demography, fish species, fish toxicity), which correlated with the overall patterns of symptom diversity, severity, and duration reported. Snapper and barracuda were most frequently implicated in CFP illness, with highest toxicity reported in a barracuda meal remnant at more than 20 times the FDA guidance level (2.4 µg C-CTX-1 equivalents/kg). Other fish implicated in case illnesses included grouper, king mackerel, horse-eye jack, and triggerfish, which have been previously associated with CFP from this region. These data and associated correlations will be discussed in relation to regulatory analyses and management of CFP.

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PLANKTONIC LAYER PHENOMENA IN THE ECOLOGY OF TOxIGENIC PSEudO-nITZSCHIA: ExAMPLES FROM COASTAL CALIFORNIA

J. P. Ryan1, J. B. J. Harvey1, Y. Zhang1, H. A. Bowers1, C. M. Mikulski2, G. J. Doucette2, C. A. Scholin1

1Monterey Bay Aquarium Research Institute, Moss Landing, CA, 95039, USA2NOAA/National Ocean Service, 219 Fort Johnson Rd., Charleston, SC, 29412, USA

Observations along the eastern North Pacific show that the ecology of toxigenic Pseudo-nitzschia spp. is strongly influenced by phenomena of subsurface layers. Such layers may be responsible for some “cryptic” HAB events, in which the presence of a toxic bloom is first signaled by the appearance of poisoned animals rather than by detection of the causative toxic bloom. Interdisciplinary study of layer phenomena is challenging, particularly when layer vertical scales are fine. Layer ecology research is essential not only to understanding bloom dynamics, but also to developing more effective HAB monitoring, prediction, and management strategies. Using examples from Monterey Bay and San Pedro Bay, California, we describe layer ecology of toxigenic Pseudo-nitzschia spp. Results are derived from experiments conducted between 2000 and 2013. Earlier experiments integrated ship-based sampling of phytoplankton with multidisciplinary synoptic observations from autonomous underwater vehicles (AUVs). More recent experiments have more closely coupled environmental and biological observations, using autonomous environmental and molecular analytical time-series at fixed locations, ship-based sampling, and autonomous feature recognition and targeted sampling onboard AUVs, followed by application of molecular methods to samples. The enhanced resolution of both environmental and biological variation enabled by these methods is contributing to advancing this challenging research. These interdisciplinary observations indicate processes influencing the formation and persistence of phytoplankton layer structures, as well as population diversity, transport, nutrient inputs, and toxicity.

Figure 1. Variations in Pseudo-nitzschia spp. abundance and particulate domoic acid (DA) in Monterey Bay, CA, across a fontal zone where phytoplankton layer phenomena were examined. The AUV section represents the upper 30 m over an area ~150 km2.

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OPTIONS IN MITIGATING CYANOBACTERIA BLOOMS

Kevin G. Sellner1, Allen R. Place2, Michael Paolisso3, Yonghui Gao4, Ernest Williams2, Elizabeth VanDolah3.

1Chesapeake Research Consortium, 645 Contees Wharf Road, Edgewater, MD 21037, USA2IMET-UMCES, 701 E. Pratt St., Baltimore, MD 21202, USA3Department of Anthropology, University of Maryland, College park, MD 20742, USA4HPL-UMCES, 2020 Horns Point Rd., Cambridge, MD 21613, USA

Toxic and non-toxic cyanobacteria blooms are recurrent and increasing problems in many fresh and tidal-fresh systems due to excessive nutrient loads and increasing water temperatures. Local use and exposures threaten citizens, domestic animals, and wildlife, leading to high interest in preventing or mitigating blooms. Research has been conducted for the past several years to adapt several techniques for eventual routine application in bloom mitigation, focusing on the Chesapeake region.

Techniques explored in the current study include modifications of a sediment-flocculant technique successfully employed in China (Pan et al. 2006), flushing, barley straw exposure, peroxide additions, and Phoslock® application. Laboratory successes with sediment-flocculant removals of Microcystis aeruginosa (Certner et al. 2011) have been difficult to reproduce using sediment and flocculant concentrations reported from China. The last year of the project will focus on documenting the most practical and living resource protective use of sediment-flocculant additions as a routine mitigation technology. Post-bloom flushing and early spring deployment of barley straw (Hordeum vulgare) appear quite promising in limiting bloom development and toxin (microcystis and anatoxin) concentrations. The addition of peroxide shows an initial rapid decline in phytoplankton and cyanobacteria, but rapid recovery of the bloom assemblage. The addition of P-binding Phoslock® has some success when DIP levels are high but less dramatically when the nutrient pool is low and the effect is short-lived.

A very interesting aspect of this work is the assessment of citizen perceptions of blooms, toxins, causes, and willingness to mitigate. In one location, high awareness of the bloom and its threat across many sectors led to effective communication with and understanding of the mitigation approaches. In another system, however, poor understanding and communication on HABs across sectors led to discord and rejection of local decisions on watershed development strategies. Implications of these communication networks, or their absence, allow recommendations of future approaches to insure public awareness and confidence in possible bloom mitigation strategies.

Certner, R. et al. 2011. Using sediment flocculation to reduce the impacts of Chesapeake Bay Microcystis aeruginosa harmful algal blooms. Thesis, GEMSTONE Program, University of Maryland, College Park, MD. pp. 194.Pan, G. et al. 2006. Removal of harmful cyanobacterial blooms in Taihu Lake usinglocal soils. III. Factors affecting the removal efficiency and an in situ field experiment using chitosan-modified local soils. Environ. Pollut. 141: 206-212.

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IMPLEMENTATION OF SOLID PHASE ADSORPTION TOxIN TRACKING (SPATT) FOR THE MONITORING HARMFUL ALGAL BLOOMS IN SOUTHERN CALIFORNIA

Erica L. Seubert1, Alyssa G. Gellene1, Paige Connell1, Jayme Smith1, George Robertson2, Astrid Schnetzer3 and David A. Caron1

1University of Southern California, 3616 Trousdale Parkway, Los Angeles, CA, 90089, USA2Orange County Sanitation District, 10844 Ellis Ave, Fountain Valley, CA, 92708, USA3North Carolina State University, 2211 Hillsborough St., Raleigh, NC, 27697, USA

The complex spatiotemporal distribution of harmful algal blooms (HABs) has complicated our understanding of their ecology. HAB monitoring programs have existed in many areas, but the discrete sampling may not occur on the appropriate time scale of bloom development or in the precise location. Technologies that can increase our spatial and temporal monitoring of HABs without requiring a significant increase in financial commitment are of great interest. Solid phase adsorption toxin tracking (SPATT) has been implemented in several locations worldwide for the monitoring of dissolved phycotoxins. The SPATT resin integrates dissolved phycotoxins present in the water over the entire deployment period, providing information on toxin presence in an area between discrete samplings. SPATT deployments in the southern California area began in 2012 at two coastal locations, one in the highly urbanized Los Angeles area and the second on the sparsely developed Catalina Island, as well as the deployment of several temporary offshore moorings in the fall of 2012 and spring of 2013. The SPATT deployment in Los Angeles harbor was in conjunction with a HAB monitoring program in which discrete samples were collected on a weekly basis for chlorophyll a concentrations, particulate DA and STX, dissolved inorganic nutrients, and phytoplankton community composition. DA and STX detected in the SPATT from this location occurred prior to and during periods in which the known producers of DA and STX were present in the discrete samples. Information collected from the Catalina deployment revealed relatively low to below detection concentrations of DA and STX throughout the study period. The highest DA concentration measured in the SPATT bag deployments was from a temporary offshore mooring, at 1 meter depth, deployed from September 9 to September 17, 2012. The concentration of 9,200 ng DA/g resin was markedly higher than the concentration measured in the bag deployed at 7 meters depth on the same mooring, 39 ng DA/g resin. The results from the year and a half of coastal SPATT deployments and the two seasons of offshore SPATT deployments will be presented along with discussion on how the information collected has improved HAB understanding in the region.

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COORDINATING FLORIDA RED TIDE COMMUNICATION: A CASE STUDY

Dianne Shipley1, Liz Bumpus 1,Tom Higginbotham1, Hector Mendez1, Jennifer Clemente1, Quintin Clark1, and Chuck Henry1

1Florida Department of Health in Sarasota County, Sarasota, FL

Sarasota County experiences almost annual blooms of the toxic dinoflagellate, Karenia brevis. As part of the Florida Department of Health, our mission is to protect, promote and improve the health of all people in Florida through integrated state, county and community efforts. Therefore, we continually work toward building connections through partnerships with other agencies and organizations to enhance the health and safety of our community. During the fall of 2012, we instituted a weekly conference call to discuss the current bloom with community partners. Our goal was to provide accurate and updated bloom status reports, facilitate consistent messaging across agencies, monitor media articles for accuracy, and assess any outdoor recreational events (i.e. triathlons, swim meets, etc.) where people could potentially be at high-risk for exposure to the bloom. We invited partners from Mote Marine Laboratory, FWC/Fish and Wildlife Research Institute, NOAA HAB Bulletin, USF Center for Red Tide Prediction, Florida Department of Health Aquatic Toxins Program, Florida Department of Aquaculture and Consumer Services, Sarasota County Government Communications, Sarasota County Environmental Utilities, four area chambers of commerce, and Sarasota Convention and Visitor’s Bureau (Visit Sarasota County) to participate on the conference calls. As the bloom expanded, we invited partners from other impacted counties to join as well. This is the first time an effort for consistent health messaging on Florida red tide has been conducted across five counties (Manatee, Sarasota, Collier, Lee and Monroe). We were able to utilize the expertise of many different people which provided us with a significant pool of knowledge that helped in making our message as reliable and well-informed as possible. We believe the calls decreased reactionary media reporting, and improved resident and visitor knowledge about how to stay healthy during a Florida red tide. The calls concluded in March 2013 as the bloom also ended.

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EVALUATION OF POTENTIAL OYSTER CONTAMINATION FROM CYANOBACTERIAL TOxINS

Emily A. Smith, Chase Weidert, Sibel Bargu

Department of Oceanography and Coastal Sciences, School of the Coast and Environment, Louisiana State University, Baton Rouge, LA, 70803

The occurrence of harmful algal blooms in estuaries has increased around the world with the potential for their toxins to enter the food web of these ecosystems. One type of vector through which algal toxins can enter the estuarine food web is filter-feeding organisms such as mussels and oysters. Northern Gulf of Mexico, especially Louisiana (LA) has a very large oyster market. The oysters are harvested from the estuaries that consist of fresh water in the upper areas and have an increasing salinity gradient as they approach the Gulf of Mexico. Oysters are usually found in the 10-20ppt area of the estuary. Freshwater cyanobacteria move into the outer more saline waters when diversions from the Mississippi River are opened in the spring. The question then arises as to whether the toxin producing cyanobacteria can survive in higher saline waters and whether the oysters can uptake their toxins. One of the most commonly seen toxin producing cyanobacteria, Anabaena, was grown at increasing salinities (0-10ppt) for 10 days. Anabaena grew well at 7ppt or lower salinity. It did not grow, but did not decline in biomass at 8ppt and above indicating the ability to maintain biomass even in higher saline waters. Particulate (intracellular) and dissolved (extracellular) toxins were measured at the beginning and end of the experiment. Particulate toxins initially were below the detection limit but increased over time in all salinities except 9 and 10ppt. Dissolved toxins were also below the detection limit initially, but were then detected at the end of the experiment with the highest concentration in 10ppt (0.74 ± 0.49 microcystins µg/L) treatment. Oysters collected from Breton Sound Estuary, LA, were then allowed to feed on toxin producing Anabaena over a three-hour period. The experimental oysters’ initial consumption rate was 3.5x105 ± 2.0x105 cells consumed oyster-1 minute-1 but decreased over time. Analysis of the oyster viscera at the beginning and end of the experiment showed an increase amount of toxins in the oysters. The results indicate the ability of Anabaena to not only grow and produce toxins at higher salinities, but also that oysters can feed on these toxin producing cyanobacteria and contain their toxin. Since toxin producing cyanobacteria can be natural members of the phytoplankton community in estuaries, threat of contamination due to their toxins is imminent and monitoring should be a priority for these ecosystems.

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A SYSTEMS BIOLOGY APPROACH TO UNDERSTANDING mICROCySTIS BLOOMS

Morgan M. Steffen1, T. Chad Effler1, Loren J. Hauser1,2, Zhou Li1, Rachel M. Adams1, Brian D. Dill2,3, Nathan C. VerBerkmoes2,4, Stephen P. Dearth1, Amanda L. May1, Shawn R. Campagna1, B. Shafer Belisle1, Gregory L. Boyer5, Sue B Watson6, Richard A Bourbonniere6, Steven W. Wilhelm1

1University of Tennessee, Knoxville, TN 37996, USA2Oak Ridge National Lab, Oak Ridge, TN 37830 USA3University of Dundee, Dundee DD1 5EH, United Kingdom4New England BioLabs, Ipswich, MA 01938 USA5State University of New York, Syracuse NY 13210 USA6 Environment Canada, Burlington, ON L7R 4A6 Canada

Recurrent blooms of the toxic cyanobacterium Microcystis have plagued Lake Erie for decades. Recent expansion in size and duration of toxic bloom events has galvanized the effort to identify the factors that drive the success of Microcystis and other toxic cyanobacteria in the environment. To obtain a better understanding of the molecular response of Microcystis to different environmental conditions, we have taken a comprehensive systems biology approach. The suite of tools employed include metagenomics, metatranscriptomics, metaproteomics, and metametabolomics, applying each to cultured isolates and field samples collected from Lake Erie. Analyses have focused on the interactions between Microcystis and available nutrients, but include genomic information on how the co-occurring microbial community responds to, and perhaps shapes, environmental conditions. Initial findings resulting from metaproteomics implicated upregulation of the Microcystis urease enzyme in the environment. Recent advances in metatranscriptomics and metametabolomics indicate a complex cellular response when the compound urea is available to Microcystis as a nitrogen source. To obtain these data, triplicate samples from sites in Lake Erie were sequenced on the Illumina® HiSeq platform. Sequences were then analyzed to compare expression differences of Microcystis across multiple environmental gradients, as well as to simulated variable nutrient conditions tested on culture isolates. Differences in composition and expression patterns of associated heterotrophic bacteria were also considered, as these are thought to influence the response of Microcystis to a variable environment. Comprehensive surveys of total small metabolites were run in tandem with metatranscriptomes to generate a secondary measure of physiological response to diverse environmental conditions. Taken together, these observations provide novel and extensive insight into the complex cellular and community interactions that take place during cyanobacterial blooms and may highlight previously unknown molecular mechanisms driving Microcystis bloom events.

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PERCEPTIONS OF BEACH ACTIVITIES DURING FLORIDA RED TIDE BLOOMS: A CONJOINT ANALYSIS

Jamie L Studts1, Margaret M Byrne2, Kate Kohler3, Barbara Kirkpatrick2,3,4

1 Dept of Behavioral Science, Univ. of Kentucky College of Medicine, Lexington, KY 2 Dept of Public Health Sciences, Miller School of Medicine, Univ. of Miami, Miami, FL3 Mote Marine Laboratory, Sarasota, FL4 Rosenstiel School of Marine and Atmospheric Sciences, Univ. of Miami, Miami, FL

Background: Communicating accurate information regarding harmful algal blooms (HABs) presents a significant public health challenge. This is certainly true with Florida red tide, i.e., Karenia brevis, blooms. In recent research supported by the National Institute of Environmental Health Sciences (NIEHS) and others, the investigative team found that Florida red tide caused both acute and possibly chronic health effects, as well as significant socioeconomic impacts to coastal residents and visitors. Pilot work revealed that although approximately 70-80% of residents and tourists reported knowledge of Florida red tide, fewer than 50% had correct information about the potential health risks of Florida red tide for asthmatics.Methods: We conducted semi-structured interviews to establish the key concerns about beach activities during a Florida red tide. Subsequently, we developed a conjoint survey and administered it to 303 local and seasonal residents (i.e., snowbirds) using Sawtooth Software. The sample was also stratified by pulmonary health status (healthy vs. asthmatic). Surveys were conducted from June 2011 through May 2012. The beach activities portion of the conjoint valuation included 25 scenarios, depicting six key attributes of decisions to go to the beach during a bloom: amount of dead fish on the beach, posted beach warnings, odor, parking availability, and wind direction.Results: The relative propensity to go to the beach during a bloom was 3.18 (±1.11) on a Likert scale of 1 (low) to 9 (high). The top utilities measuring the attribute desirability were the presence or absence of odor (81.99), the absence of dead fish (80.92), the presence of many (versus few) dead fish (63.72), and the lack of a beach warning sign (61.10). The relative importance scores for going to the beach during a bloom were highest for the amount of dead fish on the beach and odor. Combining these two attributes explained 64% of the variance in consumer decision making. When examining associations between group status, demographic characteristics, health status, and decision making utilities, a number of interesting patterns emerged. Discussion: Various systems are in place to warn of the impact of Florida red tide on beach conditions. Future studies are needed to examine the utility of these systems and explore novel methods to disseminate accurate risk information to the beach going community. Data from this study suggest the important role of beach clean-up efforts that may be very relevant to the tourism industry. Conclusion: Under-protective and overprotective reactions to a Florida red tide bloom can adversely impact health and economic considerations, respectively. It is important to understand factors that influence individuals’ decisions during a Florida red tide, so that accurate risk information can be communicated and steps can be taken to mitigate barriers to beach-going under appropriate conditions. Educational interventions targeting community stakeholders responsible for beach maintenance and clean up may be particularly important.

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CLIMATOLOGICAL ANALYSIS OF BLOOMS IN LAKE ERIE AND SEASONAL FORECAST

Richard P. Stumpf1, Timothy T. Wynne1, Michelle C. Tomlinson1, R. Peter Richards2, David Baker2.

1NOAA National Centers for Coastal Ocean Science, Silver Spring, MD 20910, USA2Heidelberg University, National Center for Water Quality Research, Tiffin OH 44883, USA

Starting in 2003, Lake Erie’s Western Basin saw a recurrence of annual cyanobacterial blooms. The summers of 2008 to 2011, in particular, had severe blooms. In contrast, 2012 had a respite with a milder bloom. Using data from the MERIS and MODIS sensors, we have characterized the severity and patterns of the blooms through each summer from 2002 to 2012 in order to understand the blooms. The analysis allows estimates of the area and the biomass of the blooms. Combining this data with discharge and nutrient data from the Maumee River (the major tributary to the Western Basin) has allowed development of a model to predict bloom severity. The inter-annual variation in bloom biomass or severity is described by variations in spring (March to June) discharge and phosphorus loads from the Maumee River, with the major bloom years of 2008-2011 having the highest spring flows. 2011 had both the most severe bloom and the most extreme spring river loadings. The seasonal model has an uncertainty of ~15% of the observed bloom intensity in years having strong blooms. The model was was used to forecast the 2012 bloom. The forecast correctly identified a milder bloom than 2011, however, the bloom was stronger than estimated by the model. 2012 appears to have been an unusual year in several regards. The Maumee River had the lowest spring river discharge since 1975 and one of the lowest total phosphorus loads of the past 15 years. The 2012 season followed the most severe bloom in decades (2011 event), occurred following a winter in which the lake did not freeze and, and also followed fall and winter seasons having extremely high river flows and loadings. This combination of events has not been previously observed. Mild blooms have occurred during years with no ice (2002 and 2006), and during the summer following a wet winter (2007). A forecast was planned to be made in July for the 2013 bloom severity. As the winter of 2013 had typical loadings from the Maumee and the lake had ice cover, 2013 will provide insight into stability and robustness of the model for predicting bloom severity.

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GAmbIERdISCuS BIOGEOGRAPHY AND THE CONSEQUENCES OF CLIMATE CHANGE

Patricia A. Tester1, Steven R. Kibler1, William C. Holland1 and Mark W. Vandersea1, Emma L. Hickerson2 and R. Wayne Litaker1

1Center for Coastal Fisheries and Habitat Research, National Centers for Coastal Ocean Science, National Ocean Service, NOAA, 101 Pivers Island, Beaufort, NC, 28516, USA. 2Office of National Marine Sanctuaries, Flower Garden Banks National Marine Sanctuary, NOAA. 4700 Avenue U, Galveston, Texas, 77551, USA

Gambierdiscus is the dinoflagellate genus identified most closely with the production of ciguatoxins and the outbreaks of ciguatera fish poisoning (CFP). While CFP has not had the attention or visibility of other HAB-related illnesses, it claims more victims than all other HAB-caused maladies combined. Following the taxonomic revision of the genus and the development of species-specific qPCR assays there has been a resurgence of interest in Gambierdiscus research. It is now possible to examine Gambierdiscus species diversity in tropical areas and expand our ideas of suitable habitat to include the Mediterranean Sea, Canary Islands and the main islands of Japan. Currently, G. australes, G. pacificus, G. polynesiensis, G. toxicus and G. yasumotoi appear to be endemic to the Pacific while G. excentricus, G. ruetzleri and Gambierdiscus ribotypes 1 and 2 are known from the Caribbean Sea, Atlantic Ocean and Mediterranean Sea. Gambierdiscus belizeanus, G. caribaeus, G. carolinianus and G. carpenteri have been found in both the Atlantic and Pacific Oceans.

In regional scale studies it is not unusual to see a suite of Gambierdiscus species in small areas or with overlapping distributions of toxic and non-toxic species with different temperature preferences. For example, the Flower Garden Banks National Marine Sanctuary (27°56′N, 93°36′W) in the northern Gulf of Mexico has an area of only150 km2 but this habitat supports six of the seven Gambierdiscus species (and ribotypes) found in the Caribbean. While there is species-specific variation, Gambierdiscus growth rates typically increase significantly as temperatures rise from 25 to 31o C. Projections from a variety of climate models indicate the northern Gulf of Mexico will experience an increase of 2.1-2.2 °C to depths of 200 m by 2100. A one degree increase in water temperature is projected to add ~26 days of optimal growth conditions for Gambierdiscus species per year in the northern Gulf of Mexico seaward of the 100 meter isobath. The more substantial projected increase of ~2 °C will add an average of 51-55 days of optimal growth conditions and reduce or eliminate temperatures cold enough to cause Gambierdiscus mortality. It is important to note that even the slight temperature increases projected for the northern Gulf of Mexico within the next few decades portend greater CFP risk.

http://gambierdiscuswiki.wikispaces.com/

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GLOBAL ANALYSIS OF MRNA HALF-LIVES AND dE nOvO TRANSCRIPTION IN THE FLORIDA RED TIDE DINOFLAGELLATE, KAREnIA bREvIS

Frances M. Van Dolah and Jeanine S. Morey

NOAA Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC 29412

The regulation of gene expression underlies all cellular processes resulting in the growth, toxicity, and demise of harmful algal blooms. In most eukaryotes, transcription is a principal point of gene regulation. However, dinoflagellates possess many processes that appear to be under post-transcriptional control, and number of characteristics that suggest that gene expression is regulated predominantly at the translational level. However, the extent to which their genes are regulated post-transcriptionally remains unresolved. To gain insight into the relative roles of differential mRNA stability and de novo transcription in dinoflagellates, we biosynthetically labeled RNA with 4-thiouracil to isolate newly transcribed and pre-existing RNA pools in Karenia brevis. The isolated fractions were then used for analysis of global mRNA stability and de novo transcription by hybridization to a K. brevis microarray. Global K. brevis mRNA half-lives were calculated from the ratio of newly transcribed to pre-existing RNA using the online software HALO (Half-life Organizer). Overall, mRNA half-lives were substantially longer than reported in other organisms studied at the global level, ranging from 42 minutes to greater than 144 h, with a median of 33 hours. Consistent with well-documented trends observed in other organisms, housekeeping processes, including energy metabolism and transport, were significantly enriched among the most highly stable messages. Shorter-lived transcripts included a higher proportion of genes involved in RNA regulation, stress response, and other response/regulatory processes. One such family of proteins involved in post-transcriptional regulation in chloroplasts and mitochondria, the pentatricopeptide repeat (PPR) proteins, had dramatically shorter half-lives when compared to the arrayed transcriptome. We have previously observed that PPR protein transcripts are among the most rapidly increasing transcripts in response to nutrient addition. We therefore queried the newly synthesized RNA pools at 1 and 4 h following nitrate addition to N-depleted cultures to determine if these changes were the result of de novo transcription or increased RNA stability. Transcriptome-wide there was little evidence of changes in the rates of de novo transcription during the first 4 h following N addition, and no evidence for increased PPR protein transcription. This suggests that their rapid increase in abundance following N addition results from increased message stability, not new transcription. These results lend support to the growing consensus of post-transcriptional control of gene expression in dinoflagellates.

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ExAMINING THE RESPONSE OF HARMFUL DINOFLAGELLATES TO THE BACTERIAL ALGICIDE IRI-160AA: DIFFERENCES IN GROWTH, PHYSIOLOGY AND MODE OF ACTION ACROSS LABORATORY AND FIELD COLLECTED SAMPLES

Mark E. Warner, Charles L. Tilney, Kaytee L. Pokrzywinksi, Kathryn J. Coyne

University of Delaware, College of Earth, Ocean, and Environment, 700 Pilottown Rd., Lewes, DE 19958, USA

As harmful algal blooms continue to be a persistent problem in many coastal areas, effective algicidal agents are one tool that may prove useful in controlling some algal species. However, establishing the mode of action and specificity of response for particular phytoplankton as well as efficacy in natural mixed algal communities remains challenging. We have begun to characterize the algicidal activity of several small compounds found in the filtrate of the bacterium Shewanella sp. IRI-160AA that have a high specificity for inhibiting growth of at least 12 dinoflagellates, including Gyrodinium instriatum, Karlodinium veneficum, Prorocentrum minimum, and Alexandrium tamarense. While there is a strong relationship between the presence or degree of thecal armoring and the level of algicidal activity, the temporal nature of inhibition and pathways of cellular damage appear different across these species. Growth arrest as well photoinactivation followed a dose dependent response in G. instriatum and K. veneficum. Proxies for programed cell death, photoinactivation, and loss of membrane integrity appear rapidly and occur simultaneously in G. instriatum, while a dark dependent mode of photoinactivation occurs in K. veneficum prior to the loss of membrane integrity and cell lysis. Further, microscopic examination of K. veneficum following higher algicide dose showed changes in chloroplast morphology, followed by chloroplast expulsion. Short-term algicide applications were tested against freshly collected samples of natural blooms of G. instriatum, K. veneficum and P. minimum. Contrary to the expected results, there was a slight increase in P. minimum cell density following application of the algicide, and growth arrest took longer in samples of G. instriatum and K. veneficum compared to laboratory culture experiments. Further analysis of dose response also indicated a much higher concentration of the algicide was needed as compared to laboratory tests. Molecular analyses of the plankton community showed an increase in raphidophyte and diatom biomass over time that subsequently obscured our ability to accurately detect any algicide dependent photochemical changes to the target dinoflagellates. In addition to changes in phytoplankton number and type, several experiments resulted in an increase in the number of bactivorous ciliates (e.g. Paraphysomonas spp. and Euplotes sp.) that may have resulted from increased bacterial abundance following the release of DOM by dinoflagellates. While the initial tests with natural bloom samples were not as clear as those with isolated laboratory cultures, there was still promising evidence for specific dinoflagellate algicidal activity that will be examined at a larger scale in in situ mesocosm experiments.

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SNOAA’S ECOLOGICAL FORECASTING ROADMAP

Allison L. Allen1, Paul A. Sandifer2, and Richard P. Stumpf1

1 National Oceanic and Atmospheric Administration, 1305 East-West Highway, Silver Spring MD 20910, USA2 National Oceanic and Atmospheric Administration, 331 Fort Johnson Road, Charleston SC 29412, USA

For more than a decade, a number of NOAA offices have been researching ecological processes and developing experimental forecasts for a variety of ecosystem components, including harmful algal blooms, pathogens, jellyfish, brown shrimp, hypoxia, distributions of habitat and key species, sea level change, wave energy, and ocean acidification (oceanservice.noaa.gov/observations/ecoforecast). The most mature of these are harmful algal bloom (HAB) forecasts, which are fully operational in the Gulf of Mexico with the HAB Operational Forecast System (HAB-OFS) .

Historically, however, NOAA has lacked a formal Ecological Forecasting Program, which raises challenges in identifying corporate priorities, securing long-term resource commitments and maximizing the efficiency of existing forecasting abilities. In 2012, NOAA developed the framework for an Ecological Forecasting Roadmap a prioritized, agency-wide approach for coordinating NOAA’s existing capabilities and operational environmental prediction and service delivery infrastructures. Such a broadly-supported NOAA strategy for ecological forecasting will offer management solutions to assure protection, maintenance and restoration of the health and productivity of ocean, coastal and Great Lakes ecosystems, for both natural resources and human communities. This agency-wide effort will also help connect internal-NOAA activities to related efforts in the external academic community and private sector.

Within the Roadmap, there are three initial NOAA priorities, HABs, hypoxia and pathogens. These were chosen based on the following: their economic, environmental and health effects, importance to constituents and to the environment, and apparent increasing prevalence, distribution and intensity in numerous areas of the country. The Roadmap will address the key issues relevant to HAB forecasts, including: sustained resources, improved models to improve forecast accuracy and resolution, additional data collection from both remote and in situ sensors, upgraded computing capacity to process these data and enhanced forecast dissemination using NOAA-wide communications assets.

The outcome of the Roadmap will be a suite of more accurate and reliable forecasts in all regions of the country concerned with harmful algal blooms, hypoxia, and pathogens. As the Roadmap effort progresses, customers should see more dependable, higher quality products on a broader scale than currently available under the current ad hoc development and delivery model.

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S S ASSESSING PRE-BLOOM, SUB-SURFACE POPULATIONS OF PSEudO-nITZSCHIA IN THE SAN PEDRO SHELF REGION OF SOUTHERN CALIFORNIA

Holly A. Bowers1, Roman Marin III1, John P. Ryan1, G. Jason Smith2, Raphael Kudela3, Christina M. Mikulski4, Gregory J. Doucette4 and Christopher A. Scholin1.

1Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, CA 95039, USA2Moss Landing Marine Laboratories, 8272 Moss Landing Road, Moss Landing, CA 95039, USA 3Ocean Sciences Department, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA 95064, USA 4NOAA/National Ocean Service, P.O. Box 12607219 Ft. Johnson Road, Charleston, SC 29412, USA

During the past decade, the San Pedro shelf (SPS) region in southern California has become a hotspot for Pseudo-nitzschia blooms and domoic acid (DA) intoxication of marine mammals and birds. Interestingly, harmful impacts may “appear” suddenly, with no indication of increasing cell abundances or DA from routinely monitored shore-based stations. In part, this discrepancy is due to widespread spatial and temporal distribution of blooms in that region. However, other factors are hypothesized to be involved, including advection of sub-surface populations. The presence of Pseudo-nitzschia populations in sub-surface layers has been described along the greater NE Pacific. Layer ecology for these species in Monterey Bay and the SPS region is an active research area (see Ryan et al. and Smith et al., this conference).

In order to better understand the diversity and spatial distribution of these sub-surface populations on the SPS using molecular methods and microscopy, water samples were collected via shipboard net tows, CTD casts, and autonomously targeted AUV deployments during spring 2013. During the mission, samples were collected using an adaptive sampling strategy that permitted us to explore diversity throughout features of interest (e.g., high chlorophyll patches) across shelf waters and the adjacent deep channel. The experiment fortuitously spanned a pre-bloom period in which numerous Pseudo-nitzschia spp. comprised a fraction (less than twenty percent) of the diatom-dominated phytoplankton assemblage. This non-bloom scenario allowed us to establish clonal cultures from a wide variety of Pseudo-nitzschia species and to interrogate those for DA production. These findings were compared to samples collected in the area a few weeks later during a bloom characterized by high Pseudo-nitzschia cell concentrations and DA.

In situ detection of Pseudo-nitzschia and DA during these types of missions is carried out on moored Environmental Sampling Processors (ESPs). The probe-based array for Pseudo-nitzschia detection on the ESP targets two common DA producers: P. australis and P. multiseries. During several ESP deployments, including the most recent one at the SPS, we have observed a discrepancy in the molecular probes used to detect P. multiseries in conjunction with spikes in DA, indicating the possible presence of ‘cryptic’ toxigenic species. Since shipboard sampling had ended before the spring 2013 ‘event’ in San Pedro Bay, we explored recovery of frustules from spent ESP extraction filters for TEM/SEM to identify ‘cryptic’ Pseudo-nitzschia species.

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SPERCEPTIONS OF SEAFOOD CONSUMPTION DURING FLORIDA RED TIDE BLOOMS: A CONJOINT ANALYSIS

Margaret M Byrne1, Jamie L Studts2, Kate Kohler3, and Barbara Kirkpatrick1,3,4

1 Dept of Public Health Services, Miller School of Medicine, Univ. of Miami, Miami, FL2 Department of Behavioral Science, University of Kentucky College of Medicine, Lexington, KY3 Mote Marine Laboratory, Sarasota, FL4 Rosenstiel School of Marine and Atmospheric Sciences, Univ. of Miami, Miami, FL

Background: Misperceptions about the causes and effects of harmful algal blooms (HABs) abound. These misperceptions are especially of concern when they affect individuals’ actions in such a way that social or economic harms are incurred. Our pilot work found that only about 50% of tourists and residents had correct information about the safety of eating seafood during a Florida red tide, i.e., Karenia brevis.Methods: Semi-structured interviews established the key concerns citizens have regarding eating seafood during a Florida red tide. A survey including a conjoint valuation analysis (CVA) was developed and conducted using Sawtooth Software among 303 local residents and seasonal residents (i.e., snowbirds), stratified by lung health. Participants had to eat seafood to participate in the study. Surveys were conducted from June 2011 through May 2012. The attributes of the 24 seafood CVA scenarios were: government warnings on seafood safety, trust in the retailer, price, local versus chain retailer, and origin of the seafood (Gulf of Mexico or not).

Results: General propensity to eat seafood during a bloom was 4.27 (stdev 1.14) on a Likert scale of 1 (low) to 9 (high). The most important attributes from the CVA in decisions about seafood were a government warning explaining 41% of the variation in decisions, and trust in the merchant, 24%. Price was 3rd in importance, explaining 18% of variance. Logistic regressions with importance scores as the dependent variable were conducted to assess whether demographics were associated with importance of attributes. We found that purchasing locally was significantly less important for asthmatics than those with healthy lungs (p<0.047), and that trust in the retailer was significantly more important for younger respondents (p<0.026). General propensity to eat seafood during a Florida red tide was significantly lower for women (p<0.001).

Discussion: Florida Division of Aquaculture and Florida Fish and Wildlife Research Institute are tasked with monitoring and closing of shellfish beds during Florida red tide blooms. We found that warnings or reassurances from the government were by far the most important factor in individuals’ decisions regarding eating seafood. However, overall propensity to eat seafood was relatively low. Better messaging, possibly targeted for some demographic populations, on the safety of consuming seafood that is commercially harvested is important to avoid deleterious economic effects during a Florida red tide.

Conclusion: It is important to understand what affects individuals’ decisions during a red tide, so that appropriate education and outreach can be developed. Inappropriate reactions to a Florida red tide event can have negative economic consequences.

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S S CELLULAR RESPONSE TO OSMOTIC STRESS IN KAREnIA bREvIS

Lisa Campbell1, Reagan Errera1, Darcie Ryan1, and Andrea Bourdelais2

1Texas A&M University, 3146 TAMU Dept. Oceanography, College Station, TX 778432University of North Carolina, Center for Marine Science, Wilmington, NC 28409 Osmotic stress initiates a rapid response by an organism to minimize cellular damage, followed by a longer-term response to re-equilibrate the cell with its new environment.Previous studies have demonstrated that in response to a rapid decrease in salinity, Karenia brevis, the major harmful dinoflagellate species in the Gulf of Mexico, produced up to 53% more brevetoxin. While the role of brevetoxin in this response is not known, the correlation between brevetoxin production and salinity suggests that osmotic sensing is occurring. Ion transport would be an integral component of the osmotic response to the stress.

An interdisciplinary approach combining physiological experiments, imaging-in-flow cytometry, next generation sequencing, and metabolomic profiling was employed to examine the responses of three clones of K. brevis to osmotic changes. Transcriptome analysis identified two types of transmembrane proteins that facilitate osmoacclimation in all clones: aquaporins and voltage-gated ion channels. Aquaporins are a family of major intrinsic proteins that are sensitive to osmotic pressure and provide a mechanism for rapid transport of water across a membrane to adjust cell volume. A number of voltage-gated ion channels (K+, Na+, Ca2+) were also identified. Because voltage-gated ion channels are sensitive to ion gradients, they would also be involved in osmolytic adjustment. To examine the potential for a specific ion (K+, Cl-, Na1+, Ca2+) to trigger the osmotic response, we systematically diluted one ion while keeping the remaining ions at equivalent molar concentration for salinity of 35. Dilution of K+ triggered a significant increase in the production of brevetoxins, while dilution of Cl- and Ca2+ did not trigger a significant change. These observations suggest that K. brevis may be responding to changes in membrane potential established by the dilution of K+ during osmotic stress.

Transcriptome results also included one predicted monogalactosyldiacylglycerol (MGDG) synthase. MGDG is the most abundant lipid in thylakoid membranes. The observed 2-fold change in comparison with control indicated upregulation of the MGDG synthase 1 hr after hypoosmotic stress. Initial metabolome results for the hypoosmotic stress experiments with K. brevis reported a product with similar retention time and atomic mass as MGDG that was not seen in the control. Since glycolipids such as MGDGs are found in the cell and chloroplast membranes, it is possible that the K. brevis cells can also change the composition of their cell membranes rapidly in response to salinity changes. Additional results from metabolome analyses will be discussed. Focus on changes in the metabolome of stressed treatments vs. controls will complement the transcriptome results by providing validation of signaling/metabolic pathways.

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SBREVETOxIN PHOTOAFFINITY PROBES FOR THE IDENTIFICATION OF THE NATIVE BREVETOxIN RECEPTOR

Ryan Cassell and Kathleen S. Rein

Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199

The dinoflagellate Karenia brevis produces brevetoxin, a potent neurotoxin responsible for substantial amounts of marine mammal and fish mortalities. When the toxin is consumed in humans, it causes Neurotoxic Shellfish Poisoning (NSP). The native function of brevetoxin has remained mysterious since its discovery. It has been reported that an increase in toxin production is triggered from changes in salinity and osmotic stress implicating a role of osmoregulation within the organism. Photoaffinity labeling has become increasingly popular in identifying ligand receptors. By attaching ligands to these photophors, one is able to activate the molecule (generate a carbene by exposure to UV light) after the ligand binds to its receptor to obtain a permanent linkage between the two. Subsequent purification will provide the protein with the ligand directly attached. The synthesis of a brevetoxin photoaffinity probes and their application to identify a native brevetoxin receptor will be described.

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S S A SATELLITE-DERIVED PREDICTIVE MODEL OF RED TIDE SEVERITY FOR THE WEST FLORIDA SHELF

Mary C. Christman1, John Walter2, Jan Landsberg3, Brian Linton2, Karen Steidinger3, Richard Stumpf4 and Jacob Tustison3

1 MCC Statistical Consulting LLC. 2219 NW 23rd Ter. Gainesville, FL 326052 Southeast Fisheries Science Center. 75 Virginia Beach Drive, Miami, FL 331493 Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, 100 Eighth Ave SE, St. Petersburg, FL 337014 NOAA, National Ocean Service, 1305 East-West Highway, Room 9115, Silver Spring, MD, 20910

A vexing problem for HAB research is to determine the relative intensity of blooms from one year to another. Given the event-response nature of most monitoring, cell count data usually does not unbiasedly index bloom intensity nor does it have comprehensive spatial and temporal coverage. In this paper we model the probability of a bloom (cell counts >10E5 cells/liter) as a function of a suite of satellite-derived remote sensing products and other covariates using a generalized additive model (GAM). The model is derived using matched SeaWIFS satellite data products and FWRI harmful algal bloom (HAB) samples for 1998-2010. The GAM approach incorporates non-linear relationships and interactions between model factors. Predictions from the model using daily satellite imagery provides spatial and temporally explicit mapping of the probability of a bloom and associated prediction error over a grid covering the West Florida Shelf. When restricted to offshore waters (>10 meters), model performance was quite strong with 44% of the total deviance explained with an overall misclassification rate of 17.4%. In inshore waters the model performance degraded as satellite observations were increasingly confounded by littoral dynamics as evidenced by higher prediction errors and poorer overall performance of the model (37% explained deviance and 21% overall misclassification rate) when estimated for the entire region. Averaging spatial and temporal predictions over several different spatial domains (inshore, offshore of Tampa Bay, entire region) provided annual indices of red tide intensity. Over the 12 year time period, the 2005 probability of a bloom averaged over all locations was 37% higher than the next highest year (2003) confirming the severity of the 2005 red tide event and quantitatively distinguishing it from other years.

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SSTRATIFICATION INFLUENCES ON THE MIGRATORY BEHAVIOR OF THE HARMFUL ALGA KAREnIA bREvIS

Matthew J. Garrett1, Gary L. Hitchcock2, Kendra L. Daly3, Mark E. Luther3

1Florida Fish & Wildlife Conservation Commission, 100 8th Avenue SE, St. Petersburg, FL 33701, USA 2University of Miami, Rosenstiel School of Marine and Atmospheric Science, 4600 Rickenbacker Cswy, Miami, FL 33149, USA 3University of South Florida, 830 1st St. SE, St. Petersburg, FL 33701, USA

Migratory behavior has been documented in many phytoplankton species, including the harmful dinoflagellate Karenia brevis. Diel vertical migration may offer a competitive advantage to K. brevis population by increasing exposure to favorable nutrient and light regimes. However, the degree to which the strength of stratification in the physical environment influences migratory behavior of K. brevis is not well understood. In this study, we examined the effects of a weak and strong stratification on the migratory behavior of K brevis through the introduction of a thermocline in a controlled experimental water column. Experiments were conducted experiments in a 2m PVC cylinder in a temperature (25°C) and light (110 μE/cm-1sec-1) controlled incubator. Migratory behavior was documented under three conditions: 1) homogenous and unstratified, 2) weak thermally stratified, and 3) strong thermally stratified. Vertical stratification was established by wrapping tubing around the lower half of the column and circulating water through a thermal chiller such that the temperature of the lower column was controlled to create a two temperature and density layered system The column was filled with nutrient replete water, inoculated with a culture of K. brevis, and the experiment was run for 48 hours. To measure the vertical position of K. brevis, we used four continuously recording Turner Cyclops 7 fluorometers affixed at different depths. Stratification altered the migratory behavior of K. brevis. This work will contribute to an understanding the alteration of migratory behavior of naturally occurring blooms in the context of predictive modeling.

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S S REAL-TIME WATER QUALITY MONITORING FOR HAB EVENTS USING AUTONOMOUS PORTABLE WATER LABORATORIES AND DATA VISUALIZATION “CLOUD” ANALYSIS

Brian Gregson1, Katherine Hubbard2, Paul Janecek1 and David Fries1

1Spyglass Technologies, Inc., 101 16th Ave South, Ste 4A, St. Petersburg, FL 33701 USA2Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, 100 8th Avenue SE, St. Petersburg, FL 33701 USA

A number of unique automated water sampling and analytical platforms incorporate biological and chemical sensors to broaden utility for water quality monitoring applications, and are currently in use in harmful algal bloom (HAB) monitoring applications. Presented here is a review of novel monitoring instruments as well as ancillary equipment and data analytical services to support water quality monitoring activities, especially related to HABs.

The Spyglass Environmental Sample Processor (ESP) has been deployed in open ocean and coastal environments since 2001. Analytical methods available for the ESP are designed to target taxa of interest, and include molecular probe arrays, immunoassays (cELISA), and quantitative Polymerase Chain Reaction (qPCR). Reagent manufacturing services are available in accordance with Good Laboratory Practices Guidelines, as well as support equipment for lab-based development and ground-truthing of new and/or improved assays.

The Porifera Automated Field Sampler allows for solid-phase concentration, archival and retrieval of chemical, biological and particulate samples for in-situ fluorescence and/or shored-based interdisciplinary analyses.

Underwater mass spectrometers (UMS), both ion-trap and quadrupole, employ proprietary novel sample inlets for high-fidelity real-time in-situ monitoring of volatile (and limited semi-volatile) chemical targets of interest. Results are shown demonstrating utility of UMS for HAB monitoring applications.

To enable actionable responses based on data provided by such a diverse “fleet” of instrumentation, the Spyglass Water Information Portal provides visualization of data feeds from the described equipment, or as a standalone service for 3rd-party sensors. The interactive and user-friendly cloud-based software service provides researchers, regulatory managers and other stakeholders powerful and rapid decision-making tools.

This flexibility in instrumentation and support services facilitates routine detection of HAB-relevant compounds as well as relevant water quality parameters—in situ and in near real-time—thus providing novel insight into the dynamics and ecology of HAB species.

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SREAL-TIME DETECTION OF HARMFUL ALGAE AT A TRIBAL MARINE AQUACULTURE SITE

Marco B.A. Hatch1, Stephanie K. Moore2, Linda Rhodes2, William Nilsson2, Chris Scholin3, Steven Hallam4, Annie Cox4, Karl Mueller5, Vera Trainer2 and Mark Strom2

1Salish Sea Research Center, Northwest Indian College, 2522 Kwina Road, Bellingham, WA 98226 2Northwest Fisheries Science Center, 2725 Montlake Blvd E, Seattle, WA 98112 3Monterey Bay Aquarium Research Institute, 7700 Sandholdt Rd, Moss Landing, CA 95039 4University of British Columbia, Department of Microbiology and Immunology, Life Sciences Center, 2350 Health Sciences Mall, Vancouver, BC V6T-1Z3 5Lummi Natural Resources, 2616 Kwina Road, Bellingham, WA 98226

Cultured marine shellfish and finfish are vulnerable to naturally occurring harmful algal blooms (HABs) that produce toxins. The economic cost of toxic HABs to coastal tribal communities may be more difficult to evaluate; however, given the importance of seafood to tribal culture and economies, it is likely to be significant. In Puget Sound, concern over increasing HAB frequency and the emergence of new HAB threats calls for improvements in sample collection and analysis.

The Environmental Sample Processor (ESP) is an autonomous sampling and analysis unit that employs DNA-based technology to detect microorganisms in water samples. It is positioned on site, and relays data by telemetry. The ESP uses sensitive and specific molecular assays to produce quantitative results. In 2012, researchers deployed an ESP at Friday Harbor, north of Puget Sound, for the very first time and targeted one of the primary fish-killing HAB species, Heterosigma akashiwo. H. akashiwo was detected in the water column at low levels during the entire 44-day deployment from June through July. Twice during the deployment, H. akahsiwo abundance approached levels known to kill fish. The ESP provided early warning of these events and the information was rapidly disseminated to stakeholders and triggered increased site surveillance at fish farms throughout Puget Sound and British Columbia.

In 2013, two ESP units will be deployed in tandem in Lummi Bay, in northern Puget Sound, for a prolonged real-time assessment of Heterosigma, Alexandrium spp., Pseudo-nitzschia spp. These units will be in position from June through August, sampling daily. Marine waters and shellfish will be sampled weekly for toxin levels. The primary objective is to determine if the ESP can provide early warning of outbreaks of pathogens that impact shellfish and finfish aquaculture at a tribal shellfish and finfish hatchery in an area with extensive subsistence harvesting and a history of the targeted HABs. A secondary objective is to determine if relationships exist between HAB levels in marine waters and toxin levels in shellfish tissues. The ESP enables extensive high frequency sampling that is necessary to determine such relationships and to detect increases in populations of targeted pathogens in marine waters before they are able to contaminate or harm shellfish or finfish. This collaborative effort brings researchers from NOAA, UBC, MBARI, Northwest Indian College, and Lummi Natural Resources together to address issues that may limit exercising of inherent rights. Two American Indian students from Northwest Indian College will also act independent researchers on this project.

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S S CHANS: ENVIRONMENTAL FACTORS INFLUENCING KAREnIA bREvIS BLOOMS ON THE FLORIDA WEST COAST: INTERPRETATION IN SUPPORT OF HEALTH AND SOCIO-ECONOMIC IMPACTS

Gary Hitchcock2, Vincent J. Lovko1, Gary Kirkpatrick1, Barbara Kirkpatrick1, Kate Kohler1, Zoe Shoesmith2, Amy Clement2.

1 Mote Marine Laboratory, 1600 Ken Thompson Drive, Sarasota, FL 2RSMAS, 4600 Rickenbacker Causeway, University of Miami, Miami, FL

As a component of the NSF Coupled Natural and Human Systems program, our focus is to elucidate linkages between harmful algal blooms and associated impacts on human health, social, and economic aspects of the coastal communities between Tampa and Ft Myers, FL. Identifying these linkages is challenging, since it also requires an understanding of the potential human influence on coastal physical, chemical, and biological processes that impact bloom dynamics. One major objective is to determine if relationships exist that are potentially mediated through management actions. A major goal of this objective is to determine if relationships exist between precipitation, watershed discharge and bloom dynamics. An initial study examined twelve watersheds along the Florida west coast from 2002 to 2008. During that interval there were two years each with low, average, and above-average precipitation. In general, the magnitude of annual bloom maximum cell abundance exhibited a weak, but positive, relationship with total coastal precipitation and watershed discharge. However, there was a very limited correspondence between the cell abundance and distribution with precipitation, discharge at monthly time scales. A second goal is to determine if specific environmental conditions are related to indices of respiratory irritation. A change from no detectable to mild respiratory irritation at several west Florida beaches was found to correspond to air temperature, water temperature, and relative humidity. These factors suggest that the environmental factors that regulate aerosolization influence the transfer of brevetoxin from the ocean’s surface to the coastal atmosphere. We also assisted the project goals through the interpretation and sorting of cell counts via the FWC/Florida Wildlife Research Institute to assure the cell counts used in the analysis are pertinent to the project, since the database is a compilation of all sampled cell counts. As an example, cell counts were sorted from the East coast of Florida in the bloom of 2007 to compare the bloom movement from north Jacksonville) to south with the Agency for Health Care Administration (AHCA) inpatient admissions for the same time period.Multidisciplinary projects such as this one challenge the team to discuss and analyze data sets together to assure accurate data analysis and interpretation.

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SSTRESS RESPONSE IN KAREnIA bREvIS: CHANGES IN RIBOSOMAL RNA

David S. Jayroe1 and Timothy I. McLean1

1University of Southern Mississippi, Department of Biological Sciences, 118 College Drive #5018, Hattiesburg, MS, 39406-0001, USA

Karenia brevis is a marine dinoflagellate that causes harmful algal blooms in the Gulf of Mexico. These blooms are responsible for massive fish kills, shellfish bed contaminations, adverse human health effects, and vast economic loss. In order to understand higher order bloom behavior and dynamics it is imperative to understand K. brevis at the cellular level. In growing K. brevis in the laboratory under a variety of conditions, we have noted a distinct shift in the size of both ribosomal RNAs upon culturing cells under “stress” conditions, namely depleted nutrients, cold shock, and decreased salinity. Interestingly the large ribosomal subunit becomes larger in size, and the small ribosomal subunit becomes smaller. This response by K. brevis had not been previously described in the literature. These RNAs, pre- and post-stress, are being fully sequenced to determine how they are different, and what mechanisms may be responsible for producing them: alternative splicing? different transcriptional initiation or termination sites? different loci? These results will help us understand the molecular events surrounding K. brevis survival under certain environmental conditions, which may have implications regarding K. brevis biogeographical distribution and bloom termination.

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S S PHOTOCATALYTIC DEGRADATION OF mICROCySTIn-LR BY ROSE BENGAL

Wenjun Jiang1, Dionysios D. Dionysiou2, Kevin E. O’Shea1

1 Department of Chemistry and Biochemistry, Florida International University, Miami, FL, 33199, USA 2 Environmental Engineering and Science Program, University of Cincinnati, Cincinnati, OH 45221-0012, USA

The bloom-forming cyanobacteria can produce and release cyanobacterial toxin in freshwater bodies, and microcystin (cyclic heptapeptides) is the most common toxin. Microcystins are reported to be potential tumor promoters. While approximately 80 different variants of microcystins have been isolated and identified, microcystin-LR (MC-LR) is one of the most toxic and problematic variants.

Conventional water treatment methods are often not effective or not practical for the removal of MC-LR. In this study, we employed rose bengal (RB), a singlet oxygen (1O2) sensitizer, to study the photo-oxidative destruction of MC-LR. 1O2 is reactive oxygen species, which can react with unsaturated organic compounds via ene-type reaction, [2+2] cycloaddition, and Diels-Alder reaction.

Under photolysis by visible or solar light no measureable degradation of MC-LR was observed in the absence of RB, however the concentration of MC-LR is significantly reduced under such conditions in the presence of RB. The degradation of MC-LR in presence of 500 µM RB follows the pseudo-first order kinetic model nicely. These results indicate RB can be used as a photosensitizer (catalyst) to effectively degrade MC-LR. Rate of degradation increase three fold under oxygen saturated conditions compared to argon-saturated conditions, demonstrating that photo-oxidation is the predominant degradation process. Under argon saturation the degradation is likely due to the energy transfer between the RB* and MC-LR. The results of detailed kinetic studies using furfuryl alcohol and ongoing product studies will be presented. The results can also help understand the photochemical transformation of MC-LR by dissolved organic matter in natural environments.

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SFROM SOCIAL MEDIA TO NATIONAL WEATHER SERVICE PRODUCTS: ExPLORING NEW OUTREACH TOOLS FOR NOAA’S HARMFUL ALGAL BLOOM OPERATIONAL FORECAST SYSTEM

Karen Kavanaugh1, Edward Davis1, Katherine Derner2, and Adria Schneck-Scott1

1NOAA, National Ocean Service, Center for Operational Oceanographic Products and Services (CO-OPS), Silver Spring, MD 20910, USA 2NOAA, National Ocean Service, CO-OPS, 672 Independence Parkway, Chesapeake, VA 23320, USA

Since it was transitioned from research to operations in 2004, NOAA’s Harmful Algal Bloom Operational Forecast System (HAB-OFS) has provided year-round operational forecasts of the potential for Karenia brevis bloom formation, transport, intensification and associated respiratory irritation for the Gulf of Mexico region. Although the HAB-OFS has focused on maintaining high quality forecasts and refining the forecast system through research and development, the continued success of the HAB-OFS is also dependent on reaching its target audiences with useful information. The forecasts are communicated through two main products, the public conditions report and the technical HAB bulletin. These products are both intended as decision support tools: one for the general public and the other for a more technical audience that includes coastal resource managers, public health officials and researchers. After almost ten years in operations, it remains a challenge to sustain communication with current audiences, while attracting new interest.

Beginning in August 2012, the HAB-OFS team launched a coordinated outreach strategy with the goal of increasing awareness of the HAB-OFS program and its products among potential HAB bulletin users and the general public. First, the HAB-OFS Quarterly Newsletter was introduced as a way of providing updates on activities and new product developments, answering frequently asked questions, and giving additional product background. Next, a Facebook page was created as another means to disseminate public conditions reports, network with HAB-OFS partners and inspire interest in HABs from non-scientific individuals, especially those who live in or visit the Gulf of Mexico. Most recently, beginning in February 2013, in cooperation with NOAA’s National Weather Service (NWS), HAB forecasts for “high” levels of respiratory irritation have joined other hazards covered by the NWS Beach Hazards Statement test product, allowing the HAB-OFS to reach an even broader audience with important public health information through a variety of means including the NWS home page and NOAA Weather Radio. Preliminary results of these outreach efforts already show a greater than 15% increase in HAB bulletin subscriptions, approximately 1.5x the number of visits to the HAB-OFS website, and a consistent monthly increase in the number of people interacting on the Facebook page. In order to develop a sustainable and successful outreach strategy, the HAB-OFS has and will continue to rely on collaboration, coordination and feedback from our partners on location in Florida and Texas. Through increased communication with our partners and the public, these outreach efforts have already resulted in important refinements to the original HAB-OFS products and identified goals for future enhancements.

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S S THE APPLICATION OF IMMUNO-HISTOCHEMISTRY AS A TOOL FOR INVESTIGATING SUSPECTED, HISTORIC, OR UNRECOGNIZED HAB-RELATED ANIMAL MORTALITY AND DISEASE EVENTS

Jan H. Landsberg1, Theresa Cody1, and Patrick Wilson1

1Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, 100 Eighth Avenue SE, St. Petersburg, FL 33701, USA

Immuno-histochemistry (IHC) is a useful histological technique that is used extensively in medical and veterinary diagnostics to visualize the presence of specific targeted antigens in tissues. IHC can identify the distribution and organotropism of toxins in animals and can aid in the assessment and interpretation of toxin-associated histopathological effects. IHC is also useful to supplement or support confirmatory analytical methods such as LC-MS or HPLC. For example, IHC can visually demonstrate the presence of toxins, such as microcystins, that are covalently bound in tissues, and which are normally difficult to extract for analysis by rapid or confirmatory assays such as ELISA or LC-MS. In some cases, IHC may have application in the detection of low levels of toxins where traditional analyses are constrained by minimal levels of detection.

We have successfully used IHC to visualize the presence of microcystins and brevetoxins in a range of animal species (fish, birds, and marine mammals) and tissues during and after HAB events. This technique has application for investigating unexplained animal mortality or disease events where HABs were suspected or unknown, but toxins could not be confirmed or results were equivocal in tissues. Further, IHC can also be used for routine animal mortality investigations as a screening tool for differential diagnosis and for toxin rule outs. IHC can be used to retrospectively detect toxins (if stable) in archived histological tissues from historical cases with unsolved or suspected HAB etiologies. IHC is also useful for detecting subtle histopathological changes associated with chronic or low level exposures to phycotoxins and to discern their sublethal effects on animals. While IHC has its advantages, it should not be used exclusively without other confirmatory analyses for toxin presence, and considerable methods development and testing is required to avoid misinterpretation of false positives, especially in the absence of field supported data. Further, there are differences in species’ toxicokinetics, cross-reactivities, and histopathological responses to HAB toxins that can confound the interpretation of the results, demonstrating the need for validation of the IHC with experimental exposures.

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SSURVEY OF CARIBBEAN CIGUATOxINS IN FISH COMMONLY CONSUMED IN ST. THOMAS, US VIRGIN ISLANDS

Christopher R. Loeffler1, David A. Olsen2 and Alison Robertson1

1FDA, Gulf Coast Seafood Laboratory Dauphin Island, AL 36528, USA2St. Thomas Fishermen’s Association, St. Thomas, US Virgin Islands 00802, VI

Ciguatera fish poisoning (CFP) is endemic to the US Virgin Islands (USVI). Illness estimates in St. Thomas range from 600-4000 people annually. In St. Thomas, the primary CFP prevention measure is to utilize local fishermen’s knowledge and experience in avoiding species and fishing grounds previously implicated in CFP. Unfortunately, illnesses are still relatively common and without the benefit of reliable rapid field tests and species-specific toxin prevalence rates, reducing the risk of CFP will be difficult. This study employed four geolocations to evaluate the prevalence of Caribbean ciguatoxins (C-CTXs) in four fish species that are commonly found in local markets of St. Thomas, USVI; Balistes vetula (queen triggerfish), Ocyurus chrysurus (yellowtail snapper), Epinephelus guttatus (red hind), and Haemulon plumierii (white grunt). These species were targeted for collection by fishermen using standard fishing practices, at historic and commonly utilized locations. C-CTXs were extracted from 100 g sub-samples of homogenized fish muscle. Extracts were analyzed by neuro-2a neuroblastoma cell assay, and confirmed using liquid chromatography tandem mass spectrometry. High variability in C-CTXs was observed between and within geolocations, resulting in a sporadic distribution of fish containing detectable levels of toxins. Of the four species surveyed to date, queen trigger and red hind had the highest prevalence of ciguatoxins (~40%). The identification of C-CTXs in fish commonly marketed in St. Thomas highlights a potential exposure risk to local consumers. This information will inform local fisheries resource managers and public health agencies in the development of CFP prevention strategies.

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S S CHANS: DEVELOPMENT OF A HAZARD MEASUREMENT SYSTEM FOR FLORIDA RED TIDE

Vincent J. Lovko1, Derrick S. Hudson1,2, Gary Hitchcock3, Porter Hoagland4, Barbara A. Kirkpatrick1,3,5, Gary Kirkpatrick1, Richard P. Stumpf6

1 Mote Marine Laboratory, Sarasota, FL 2 University of South Florida, Tampa, FL3 Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, Miami, FL 4 Marine Policy Center, Woods Hole Oceanographic Institution, Woods Hole, MA5 Dept of Epidemiology and Public Health, Miller School of Medicine, University of Miami, Miami, FL6 NOAA National Ocean Service, Silver Spring, MD

The impact of natural hazards on human and natural resources can be difficult to measure. The use of hazard scales and indices (such as those used for wind events, tropical storm systems, and earthquakes) provide both a method of classifying the severity of specific hazard events and a predictive capability for determining impact. The nearly seasonal occurrence of blooms of Karenia brevis on the West Florida shelf are typically reported as a location-specific measure of cell abundance. While this measure can provide an indication of the severity and extent of a bloom and of the immediate potential risk of respiratory illness, it is not sufficient to provide a comprehensive assessment of the overall severity of a bloom or the longer-term predictive capability of hazard risk. As a component of a comprehensive, multi-institutional coupled human and natural systems (CHANS) research effort, we describe the initial development of a harmful algal bloom hazard measurement system to assist in assessing and interpreting the impacts of Florida red tide on the communities and natural resources of the central west coast of Florida. As an initial step in this effort, we use measures of the temporal and spatial aspects of bloom manifestation, including aerial coverage of bloom, duration of bloom stages, bloom intensity (based on cell and toxin concentrations) and proximity to coastal resources, to ascribe a bloom severity value to recent and past bloom events. Using impact data derived from multiple sources (e.g. Beach Conditions Reporting System, economic data collected as part of the overall CHANS effort) we examine the correlation of this bloom severity value to effects on human resources. When fully developed, this tool could serve as a public outreach product to respond to media and community queries concerning the comparative severity of current and past bloom events. Further development, application, and assessment of this hazard evaluation system will be discussed.

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THE vIbRIO-HAb CONNECTION: INVESTIGATING THE INFLUENCE OF IRON ON FORMATION OF vIbRIO BIOFILMS AND ROS PRODUCTION BY HETEROSIGmA AKASHIWO

Christopher R. Main1 and Kathryn J. Coyne1

1University of Delaware College of Earth, Ocean and Environment, Lewes, DE 19958

Bacteria within the genus Vibrio are found throughout coastal and marine environments where they often form associations, such as biofilms, with phytoplankton and other eukaryotic organisms. Prior research in our lab demonstrated a close association between Vibrio cholerae and the raphidophyte, Heterosigma akashiwo during blooms of this species, and microscopic examination confirmed the attachment and formation of biofilms by Vibrio cholerae on a local strain of H. akashiwo. Iron is required for the formation of biofilms by Vibrio spp. and enhances the growth and persistence of Vibrio in the natural environment. Bacterially produced siderophores, high affinity iron ligands, also increase planktonic uptake of iron and may influence iron availability in the euphotic zone. Photolysis of the siderophore vibrioferrin, for example, releases iron as Fe(II), and oxidation of the Fe(II) to Fe(III) allows for uptake by both the algae and associated bacteria. Low iron concentrations have also been shown to enhance the production of reactive oxygen species in the harmful raphidophyte, Heterosigma akashiwo, which may contribute to its toxicity toward fish and shellfish. Here, we investigated the role of iron on the formation of biofilms by Vibrio cholerae and the interaction of iron and Vibrio on production of ROS by Heterosigma akashiwo. Cultures of H. akashiwo were acclimated to a range of Fe concentrations and mixed with non-pathogenic Vibrio cholerae. Samples were collected prior to inoculation, and at specific intervals up to 24 hours after inoculation for determination of ROS production. Cellular attachment of V. cholerae was then evaluated at 24 hours after inoculation and the ratio of attached V. cholerae to H. akashiwo was determined by qPCR. Research is ongoing, but preliminary results indicate a negative correlation between biofilm formation of Vibrio on Heterosigma cells and ROS production. The influence of iron and presence of Vibrio on the transcriptome of H. akashiwo is also being investigated and results will be presented. Results of this research will increase our understanding of potential health risks associated with Heterosigma blooms and inform future management practices in this region.

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S S FLORIDA’S FIRST SHELLFISH HARVEST CLOSURE DUE TO DOMOIC ACID

Sheila N. O’Dea1, Leanne J. Flewelling1, Jennifer Wolny1, Julie Brame1, Karen Henschen1, Paula Scott1, Katherine A. Hubbard1, Jeff Wren2, Carrie Jones2 Christopher Knight2, and Christopher Brooks2

1Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 100 Eighth Avenue Southeast, St. Petersburg, FL 33701, US2Florida Department of Agriculture and Consumer Services, Division of Aquaculture1203 Governor’s Square Blvd, Suite 501, Tallahassee, FL 32301, US

Domoic acid (DA), a potent neurotoxin produced by members of the marine diatom genus Pseudo-nitzschia, has the potential to cause amnesic shellfish poisoning. High abundances of Pseudo-nitzschia (> 106 cells/L) occur frequently throughout the year along Florida’s Gulf Coast although concentrations of DA in seawater and biota typically remain low. In late April 2013, high Pseudo-nitzschia cell densities (> 4.9 x 106 cells/L)

in St. Joseph Bay in the Florida Panhandle triggered shellfish collection by the Florida Department of Agriculture and Consumer Services. Oysters (Crassostrea virginica) collected on May 6 contained 76 µg DA/g shellfish (almost 4-fold higher than the federal action limit of 20 µg DA/g) initiating the first closure of a shellfish harvesting area in Florida due to DA.

The Pseudo-nitzschia bloom peaked in early May and was relatively short-lived. The maximum observed Pseudo-nitzschia abundance during the bloom was 14.7 x 106 cells/L, and total DA concentrations reached up to 31.2 µg DA/L in seawater. Electron microscopy and genetic analyses indicated that P. cuspidata was the dominant species. By mid-May, multiple Pseudo-nitzschia species were present and cell densities had decreased to 3.6-8.5 x 105 cells/L. The bloom had subsided by late May, with DA either not detected or present at only trace levels in seawater.

Throughout May and June, DA was monitored in oysters, bay scallops (Argopecten irradians), and pen shells (Atrina rigida). During the bloom, DA concentrations exceeded the action limit in all three shellfish species. Immediately following the bloom decline in late May, DA concentrations in oysters and scallops decreased to below the action limit (0.1- 5 µg DA/g). Concentrations in pen shells, however, remained elevated for several weeks, and slowly decreased to acceptable levels by June 25. In late May, finfish representing at least 11 species and covering a range of trophic levels were collected. Domoic acid was detected at low levels in 93% (51/55) of fish tested (individual or pooled samples), with average concentrations of 0.07 µg DA/g in muscle and 0.2 µg DA/g in viscera. St. Joseph Bay was reopened for shellfish harvest in early July. This brief but significant event marks the first ever closure of a shellfish harvesting area in the Gulf of Mexico due to DA. Given that Pseudo-nitzschia blooms are occurring more frequently in the northern Gulf (Parsons et al., 2002) and DA production by Pseudo-nitzschia can vary with conditions, monitoring of shellfish for DA in this region should be enhanced.

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SPROPOSED HARMFUL ALGAL BLOOM CONTROL TECHNOLOGIES: FLOATING DESALINATION AND WATER PUMPING PLANTS

Kevin C. Owen1, Jenna L. Owen2, and Daniel P. Owen3

1Port Dolphin Energy, LLC, Höegh LNG AS, 400 North Tampa Street, Tampa, FL 336022Tinedale-Oliver & Associates, Inc., 1000 N. Ashley Dr, Tampa, FL 33602 3Department of Biological Sciences, Florida State University, 600 W College Ave, 32306, USA

Over the last several decades, there has been a dramatic increase in the areas affected by harmful algal blooms (HABs); the number and frequencies of the HABs that have occurred; and the economic losses attributed to HABs. The number of toxic species identified as forming HABs and the number of toxins identified in HAB-causing species have also increased.

A number of mechanical, biological, chemical, genetic, and environmental HAB control technologies have been proposed. Given the characteristics of HABs and dinoflagellate life cycles, the primary objectives of HAB control strategies should be to disrupt the environmental conditions that support the persistence and propagation of HABs; stop dinoflagellate cell division and, thereby, HAB population growth; induce the formation of temporary cysts for dinoflagellates that have this life cycle stage; and, most drastically, cause cell mortality in the HAB-causing dinoflagellates.

The authors of this paper suggest using floating desalination plants to treat or control HABs. In a strongly stratified water column, floating water pumping plants can be used for control. HAB-producing dinoflagellates are sensitive to such physical and chemical conditions as changes in temperature and salinity. The discharges from a floating desalination and water pumping plant can rapidly change the water temperature and salinity. These changes could be expected to induce encystment in the dinoflagellate species that form cyst and could cause mortality in those species unable to form temporary cysts. Preventing population growth, inducing encystment, or causing mortality would effectively end a HAB.

Discharges from a desalination plant are temporary in nature and include hypersaline water, freshwater (hyposaline water), and heated water. The water intake, coupled with the hyposaline and hypersaline discharges, would create advection currents and alter the water temperature, salinity and stratification. In a strongly stratified water column exists, pumping water could alter the temperature and salinity and disrupt stratification without the need for additional desalination. These sudden and significant environmental manipulations could stress and disrupt a HAB. With continued mixing after discharge, the water would return to ambient temperature and salinity relatively quickly with minimal effect on the marine environment. The temperature and salinity changes created by a floating desalination or water pumping plant would be achieved without the discharge of chemicals or other foreign materials into the environment. Since dinoflagellates have been shown to react quickly to environmental changes, the temperature and salinity of the discharges could be controlled to target the HAB species and reduce adverse impacts on other marine organisms. Bench-scale and field tests should establish optimal temperature and salinity ranges for HAB treatment and control.

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S S AN INVESTIGATION INTO THE EFFECTS OF SHORELINE USE ON CYANOBACTERIAL ABUNDANCE IN SODUS BAY, LAKE ONTARIO

Katherine A. Perri1 & Gregory L. Boyer1

1State University of New York - College of Environmental Science and Forestry, 1 Forestry Drive, Syracuse, NY 13210, USA

Greater Sodus Bay is a large shallow embayment located on the southern shore of Lake Ontario. Sodus Bay is also home to a large recreational boating fleet, serviced by nine marinas located around the bay. In the last three years, the Bay has experienced repeated outbreaks of toxic cyanobacteria including Microcystis and Anabaena species. To evaluate the impact of shoreline use on these pelagic algal blooms, water samples were collected weekly for nutrient, pigment and toxin analysis from four marina sites, two residential sites and the public beach located in the Village of Sodus Point during the summers of 2011 and 2012. The Bay experienced several cyanobacterial blooms in both summers and differences were observed between the two years. Cyanobacterial abundance peaked in September of both years however smaller isolated blooms were often observed early in the season. Toxin concentrations for microcystins were higher in 2011 than in 2012. Total microcystin concentrations ranged between 0.3 - 17 µg/L and there was considerable variability between the different marina, residential, and beach sites. However, correlations between the toxin concentrations, nutrients, and environmental variables at these separate sites did not support the conclusion that marina operations were a strong source of nutrients to drive these cyanobacterial blooms. Therefore, other factors such as weather conditions and variable anthropogenic activities will need to be considered.

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SSAxITOxIN GENE STRUCTURE AND REPRESENTATION IN NON-TOxIC AND TOxIC ALExAndRIum TAmAREnSE SPECIES

Kathleen Pitz1, Michael Brosnahan1, and Don Anderson1

1Woods Hole Oceanographic Institution, 266 Woods Hole Road, Woods Hole, MA 02543 USA.

Several dinoflagellates within the species complex Alexandrium tamarense produce saxitoxins, a group of neurotoxins that cause paralytic shellfish poisoning, arguably the most widespread HAB poisoning syndrome worldwide. We explored saxitoxin gene diversity over the entire phylogeny of A. tamarense as well as in Alexandrium affine and Alexandrium ostenfeldii. We compared the sequences of assembled contigs that had strong homology to three recently described dinoflagellate genes (sxtA, sxtB, and sxtG) that are likely to be involved in saxitoxin biosynthesis. The contigs were collected from meta-assemblies of fifteen A. tamarense transcriptomes representing all five A. tamarense species: eight toxic Group I clones, one non-toxic Group II clone, three non-toxic Group III clones, two toxic Group IV clones and a non-toxic Group V clone. The contigs were recovered from the meta-assemblies by reciprocal BLAST searches using the recently described dinoflagellate homologs as queries. Strand-specific sequencing was used on eleven of these transcriptomes and reads were mapped back onto contigs in order to determine presence of antisense RNA transcripts. A certain level of uncertainty exists in meta-assembly analysis due to errors including fragmentation and misassembly of expressed genes. We verified presence of saxitoxin genes in genomic DNA and further examined an A. tamarense proteome for presence of saxitoxin proteins. By examining relationships among these groups and their complement of saxitoxin genes we can establish differences between the toxic and non-toxic species, illustrating genetic changes that may be important for toxin biosynthesis and further elaborating on the complexity of the saxitoxin gene cluster in dinoflagellate transcriptomes.

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S S ISOLATION OF BACTERIA CAPABLE OF DEGRADING MICROCYSTIN-LR

Lauren E. Krausfeldt1, Robbie M. Martin1, Helena L. Pound1, Justine R. Schmidt2, Gary R. LeCleir1, Gregory L. Boyer2, Steven W. Wilhelm1

1Department of Microbiology, The University of Tennessee, Knoxville, TN. USA.2Department of Chemistry, State University of New York, Syracuse, NY. USA.

Cyanobacterial harmful algal blooms (cHABs) occur worldwide, causing detrimental effects to aquatic ecosystems and impacting local economies. These cHABs can produce toxins that pose threats to human health and limit potable water supplies. Microcystin-LR (MCLR) is a potent hepatotoxin and protein phosphatase inhibitor produced by Microcystis sp. and other genera of cyanobacteria. Conventional municipal water treatment techniques are ineffective at removing MCLR from water sources. Heterotrophic bacteria co-occurring with harmful algal blooms have been found to degrade and utilize MCLR as a carbon source. Published protocols using ethanol as carrier solvent for MCLR were used to screen for and isolate potential MCLR-degrading bacteria. High performance liquid chromatography (HPLC) analysis revealed that ethanol was not a suitable carrier, as it provided an additional source of carbon. Using deionized, sterilized water as the MCLR solvent, additional screenings were conducted. Several strains of bacteria were successfully isolated from water samples of Lake Tai, China, which has a history of recurring harmful cyanobacterial blooms dominated by Microcystis sp. One strain, identified as Pseudomonas alcaligenes, degraded up to 90% of MCLR toxin over a 10-day period. Ongoing degradation assays will characterize MCLR degradation rates and byproducts with the long-term goal of identifying a bacterial consortium that can be deployed as part of engineered toxin remediation strategies.

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SCHANS: INCIDENCE OF NEUROTOxIC SHELLFISH POISONINGS DURING FLORIDA RED TIDE (KAREnIA bREvIS) BLOOMS: IS SHELLFISH MANAGEMENT EFFECTIVE AT MITIGATING OUTBREAKS?

Jeremy Faris1, Andrew Beet2, Rebecca Lazensky3, Andrew Reich3 and Porter Hoagland2

1University of Florida; College of Veterinary Medicine; College of Public Health and Health Professions, Gainesville, Florida, US2Marine Policy Center, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, US3Bureau of Epidemiology, Florida Department of Health, Tallahassee, Florida, US

Neurotoxic shellfish poisoning (NSP) is a poisoning in humans and other marine animals with a range of acute neurologic, gastrointestinal, respiratory, and cardiac symptoms. NSP can cause significant morbidity; whether it can cause chronic illness in humans is unknown. NSP is caused by the consumption of molluscan shellfish that contain accumulated neurotoxins, called brevetoxins, produced by the marine dinoflagellate, Karenia brevis. This algae causes “Florida red tides” a type of harmful algal bloom (HAB), which commonly occurs off Florida’s southwest coast. Historically, it has been observed that most Florida NSP cases have been associated with the consumption of hard clams (Mercenaria mercenaria) collected from shellfish harvesting areas that were closed during active Florida red tides.

In order to determine whether NSP cases occur when Florida shellfish harvesting areas are closed due to K. brevis-related blooms, a dataset of NSP cases reported from 1997-2009 was compiled using case reports found in the Florida Department of Health’s (FDOH) electronic notifiable disease database, “Merlin.” Onset-of-illness dates were compared to shellfish harvesting area closure periods. The number of NSP illnesses was small (n=21), representing a conservative estimate, as only those cases reported to FDOH and meeting the strict case definition for NSP have been included. The implementation of shellfish bed harvest closures during red tide events as a management tool provides significant public health benefits.

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S S USE OF DISSOLVED INORGANIC AND ORGANIC PHOSPHORUS BY THE TOxIC DINOFLAGELLATES KAREnIA bREvIS AND KAREnIA mIKImOTOI (DINOPHYCEAE)

Bill Richardson

Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission100 8th Avenue SE, Saint Petersburg, Florida 33701

Large harmful algal blooms of Karenia brevis and Karenia mikimotoi are thought to persist, in part, due to their ability to use various forms of dissolved organic and inorganic phosphorus (DOP, DIP). These species’ ability to grow on different forms of phosphorus (P) was evaluated in the laboratory using two K. brevis clones (CCFWC 257, CCMP 2281) and a single K. mikimotoi clone (CCFWC 67) grown axenically and non-axenically on 28 different P compounds. Axenic and non-axenic cultures grew similarly well on all forms of DIP and the DOP phosphomonoester compounds, although final yields on DIP generally exceeded those on DOP. Notably, no single compound provided exceptionally superior growth rate or yield. Axenic and non-axenic cultures differed in their ability to use certain compounds. The axenic clones were unable to grow on the phosphomonoester phytate, any of the phosphodiesters, or phosphonates, whereas the non-axenic clones grew on phytate, most of the phosphodiesters, phosphonates 2-aminoethylphosphonic acid, and phosphonoacetic acid. Furthermore, differences between axenic and non-axenic cultures suggested that bacterial degradation of some DOP compounds was necessary before P use by Karenia, and that bacterial assemblages in non-axenic Karenia cultures differed in their exploitation of DOP compounds. Results suggest that large blooms of K. brevis and K. mikimotoi on the West Florida shelf are not the result of exceptional growth on or the exclusive use of any specific inorganic or organic P compound, but rather an ability of these species to grow well on a broad spectrum of naturally occurring DIP and DOP compounds - either through their direct use (e.g. DIP and phosphate monoesters) or indirectly following bacterial mineralization (e.g. phosphodiesters and phosphonates). The P requirements of K. brevis and K. mikimotoi blooms may in part be met by the mineralization of the more refractory constituents of the DOP pool by the co-occurring bacterial community.

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SPOPULATION STRUCTURE AND GENETIC DYNAMICS OF TOxIC ALExAndRIum FundyEnSE (DINOPHYCEAE) BLOOMS IN COASTAL EMBAYMENTS.

Mindy L. Richlen1, Bibiana G. Crespo1,2, Jillian Adams1, Bruce Keafer1, Deana L. Erdner3 and Donald M. Anderson1

1Woods Hole Oceanographic Institution, 266 Woods Hole Rd., MS 32, Woods Hole, Massachusetts 02543, USA2Departament de Biología Marina i Oceanografía, Institut de Ciències del Mar, CSIC, 08003 Barcelona, Spain3University of Texas Marine Science Institute, Port Aransas, Texas, 78373, USA

Alexandrium blooms associated with Paralytic Shellfish Poisoning in the northeastern USA generally occur in two habitats or systems: open coastal waters, spanning hundreds of kilometers, and coastal embayments, characterized by restricted circulation and limited tidal flushing. Differences in the environmental and hydrodynamic characteristics of these systems provide the opportunity for comparative studies of the mechanisms influencing the genetic structure and diversity of bloom populations. To examine the effects of estuarine hydrodynamics on the population genetics of Alexandrium fundyense blooms, we carried out extensive temporal and spatial sampling in the Nauset Marsh System (NMS) on Cape Cod (MA, USA) in 2009, and again in 2012. Within the NMS, A. fundyense populations are largely restricted to salt ponds or coves at the distal ends of the system by hydrographic processes, thus permitting the investigation of how physical barriers to dispersal influence population substructure of blooms within the embayments. Temporal sampling was carried out during all phases of the bloom to determine how the genetic composition of A. fundyense populations changed over time within a geographically restricted area, when population mixing is limited. These studies revealed significant spatial differentiation among populations within the NMS, indicating that population mixing among the terminal endpoints of the system is restricted. Furthermore, rapid and significant temporal differentiation was observed, with changes in population structure occurring over the course of the bloom. Results of these investigations will be discussed, along with the environmental conditions and biological interactions potentially driving differentiation.

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S S CHANS: THE BENEFITS OF PARTICIPATING IN BROADER IMPACTS: A HIGH SCHOOL TEACHER’S PERSPECTIVE

Katrin Rudge1, Kate Kohler2, Porter Hoagland3, and Barbara Kirkpatrick2,4,5

1Riverview High School, Sarasota, FL 2Mote Marine Laboratory, Sarasota, FL3Marine Policy Center, Woods Hole Oceanographic Institute, Woods Hole, MA4Dept of Epidemiology and Public Health, Miller School of Medicine, University of Miami, Miami, FL5Oceans and Human Health Center, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, Miami, FL

The broader impacts activities of the National Science Foundation Coupled Natural and Human System (CNH) grant: Collaborative Research: Modeling the Dynamics of Harmful Algal Blooms, Human Communities, and Policy Choices along the Florida Gulf Coast include innovative components for engaging with and educating high school students about our highly cross disciplinary research project.

In a cooperative project, Mote Marine scientists and Riverview High School staff, introduced students the NIEHS “AMBIENT” curriculum, with main focus on the waterborne disease segment of the curriculum. Below are examples of how students were engaged and assessed in this teaching unit:

As an introductory lesson students used technology to create posters about waterborne diseases in a jigsaw pairing activity with their classmates. Next, students were introduced to the concept of a dichotomous key with several simplified key examples. A lab activity incorporated use of a dichotomous key along with microscopic examination and identification of waterborne disease specimens. Follow-up lessons included a hand washing, glow germ/black light activity which showed students the importance of proper hand washing. Once assessed on their knowledge of these diseases a final field trip was planned to a nearby sewage treatment facility to examine how water is safely processed and clarified to eliminate chances of spreading waterborne diseases to the general public.

As a second step in this cooperative learning venture, students were introduced to the diverse backgrounds of the project investigators in this grant via Safari Live. The lectures provided students insights into unique career opportunities; varied stages of careers, from early investigator to emeritus status; differences in institutions from Academia, to State agencies and non-governmental organizations; incorporating not only interdisciplinary but also cross disciplinary research examples. Diverse disciplines students were introduced to included: marine policy, marine biology, engineering, public health, environmental health, economics, and business/health administration.

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SNUTRIENT AND BLOOM DYNAMICS BEFORE DREDGING OF LAKE NEATAHWANTA, A SHALLOW, FRESHWATER LAKE IN UPSTATE NEW YORK

Marci L. Savage1 and Gregory L. Boyer2

1State University of New York – Environmental Science and Forestry School, 1 Forestry Drive, NY 132102 State University of New York – Environmental Science and Forestry School, 1 Forestry Drive, NY 13210

Cyanobacterial species present during bloom conditions can be dictated by the amount and type of nutrients present. Management plans for shallow lakes include physical measures such as dredging to remove bottom sediments and reducing the phosphorous pool. Dredging studies have been published in the past few decades with results varying from the desired outcome – reduced nutrient levels, increased diversity, decreased toxin levels, and absence of a bloom, to no significantly discernable differences. Lake Neatahwanta (Fulton, NY) is one lake that has been plagued by cyanobacterial blooms seasonally, and this lake is scheduled for dredging in late summer 2013. By monitoring several parameters including pH, temperature, conductivity, dissolved oxygen, nitrogen, and phosphorous, a baseline can be prepared for the pre-dredge state of the lake. We will incorporate these physical parameters along with measurements of algal biomass and diversity to compare with the post-dredged lake. The change in the species present affected by this event, and subsequent nutrient changes, will allow a unique opportunity to model the effect that dredging will have on the species present and toxin levels. This approach may provide a template that can be applied to other systems for monitor and predict bloom occurrence and toxicity.

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S S VARIATIONS IN THE MICROCYSTIN CONTENT OF DIFFERENT FISH SPECIES COLLECTED FROM A EUTROPHIC LAKE

Justine R. Schmidt 1, Mylynda Shaskus 2, John F. Estenik 2, Carl Oesch 3, Roman Khidekel 3 and Gregory L. Boyer 1

1 Department of Chemistry, State University of New York, College of Environmental Science and Forestry, Syracuse, NY 13210, USA2 Ohio EPA, Division of Surface Water, Lazarus Government Center, P.O. Box 1049, 50 West Town Street, Suite 700, Columbus, OH 43216, USA; 3Ohio EPA, Division of Environmental Services (Laboratory), 8955 East Main Street, Reynoldsburg, OH 43068, USA

Microcystins produced from cyanobacteria can accumulate in fish tissues. Liquid chromatography coupled with tandem quadrupole mass spectrometry (LC-MS/MS) is an attractive alternative to immunoassays for the determination of low concentrations of microcystins in tissues. Fish taken from Grand Lake St. Marys, a eutrophic lake in Ohio, USA, were analyzed for microcystin-LR in their fillets using LC-MS/MS. Of 129 fish tested for microcystins, only black crappie (Pomoxis nigromaculatus) and common carp (Cyprinus carpio) tested positive for microcystin-LR. Less than 10% of Pomoxis and 7% of Cyprinus samples contained measurable levels of microcystin-LR. Statistical analysis yielded a p-value of 0.07 between Pomoxis and the pooled results of the other four fish species. However, this comparison was complicated by the large difference in sample size between species. Further sampling in Grand Lake St. Marys for microcystin-LR would help determine if microcystin-LR exposure occurs through foodweb transfer.

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SROLE OF AMMONIUM IN ALExAndRIum FundyEnSE BLOOMS IN THE GULF OF MAINE & GEORGES BANK

Sugandha Shankar, David. W. Townsend, Maura. A. Thomas

University of Maine, School of Marine Sciences, 5706 Aubert Hall, Orono, ME 04469, USA

We present the results of a study designed to test the hypothesis that ammonium is the more important form of dissolved inorganic nitrogen supporting annual summertime blooms of the PSP-producing dinoflagellate Alexandrium fundyense in the Gulf of Maine – Georges Bank region. It is already known that compared to nitrate and nitrite, ammonium is the more preferred nitrogenous nutrient for phytoplankton because of its chemically reduced state. Previous oceanographic surveys in the study region have shown that deep-water nitrate injections into surface waters of the eastern Gulf of Maine and on the Northern Flank of Georges Bank lead to diatom blooms some distance downstream in the residual flow field, followed by dinoflagellate blooms, including populations of A. fundyense. These A. fundyense populations continue to grow and to maintain high cell concentrations after nitrate is depleted in the surface waters. Rapid remineralization of particulate nitrogen to ammonium and strong coupling with subsequent uptake typically keeps ambient ammonium concentrations low; nonetheless, even very low ammonium concentrations can sustain phytoplankton production. Results of laboratory culture experiments and multivariate statistical analyses of field measurements of distributions of A. fundyense and planktonic heterotrophs are presented, which together support the contention that heterotrophic regeneration of ammonium is sufficient to sustain continued growth during summertime blooms of A. fundyense in the Gulf of Maine and on Georges Bank.

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S S TRANSCRIPTOME ANALYSIS OF THE DIATOM PSEudO-nITZSCHIA AuSTRALIS REVEALS PATHWAYS ASSOCIATED WITH DOMOIC ACID BIOSYNTHESIS

G. Jason Smith1, Thomas J. Savage2, Raphael Kudela3, Kendra Hayashi3 and Holly A. Bowers4

1Moss Landing Marine Laboratories, 8272 Moss Landing Rd, Moss Landing, CA 95039, USA2Department of Chemistry, California State University Sacramento, 6000 J Street, Sacramento, CA 958193Ocean Sciences Department, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA 95064, USA4Monterey Bay Aquarium Research Institute, 7700 Sandholdt Road, Moss Landing, CA 95039, USA

Numerous physiological studies have pointed to a dependence of domoic acid biosynthesis on the growth status of Pseudo-nitzschia spp, such that DA accu-mulation is generally enhanced during growth limitation by a range of stressors. While DA accumulation can be affected through manipulation of growth rate in this diatom genus, such manipulations confound interpretation of DA biosynthetic patterns and pathway reconstruction. Here we report on transcriptomes generated from a Monterey Bay isolate of P. australis (10249_10AB) grown under different N-source supplementations (NO3, NH4, UREa, GLUtamate) at a common dilution rate. Batch subcultures of antibiotic treated 10249_10AB were conditioned for 6 passages (ca. 2 months) with specific N supplements at 40 μM-N and f/2 levels of other constituents. Log phase pre-conditioned cultures were used to establish continuous cultures under constant light at 15oC with the same N supplementation supplied at 0.27/d. Cultures were sampled after 10 days of growth. Total DA accumulation (pg/cell) was highest in the GLU culture (0.253) and lowest under URE supplementation (0.100) with a rank order of GLU>NO3>NH4>URE.

DNA-free RNA was submitted to the National Genome Resource Center for RNA-seq library construction, sequencing and annotation as part of the Marine Microbial Eukaryote Transcriptome Sequencing Project (Project IDs: MMETSP_139_2, 140_2, 141_2, 142_2). An average of 23,317 contigs (2178 bp median length) were machine assembled from each library with 80% having defined protein coding sequences. Of these putative genes an average of 3899 (22%) sequences per library had significant homologies (E<10-10) to known proteins enabling pathway reconstruction. P. australis 10249_10AB expressed transcripts encoding enzymes required for complete mevalonate (hmgr-dependent) and methyl-erithrytol phosphate (dxs-dependent) pathways for isoprene generation. RNA-seq enables use of normalized read frequency (RPKM) as an index of relative transcript abundance. Compared to hmgr, dxs abundance was strongly correlated with DA accumulation (r=0.96) indicating the MEP pathway as the likely source of the isoprene group on DA. Involvement of the ornithine cycle in supply of substrates for DA biosynthesis is again indicated by correlation of argD abundance with DA (r=0.90). Combined with physiological data, this transcriptome database points to the power of RNA-seq analysis for metabolic network reconstruction and analysis of toxin biosynthesis.

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PURIFICATION AND CHARACTERIZATION OF INDOLE ALKALOIDS FROM A CENTRAL FLORIDA ISOLATE OF THE FRESHWATER CYANOBACTERIUM, FISCHERELLA SP., AS INHIBITORS OF VERTEBRATE DEVELOPMENT IN THE ZEBRAFISH (dAnIO RERIO) EMBRYO MODEL

Dalton D. Steele, Asha Jaja-Chimedza and John P. Berry

Department of Chemistry and Biochemistry, Marine Science Program, Florida International University, 3000 N.E. 151st Street, North Miami, FL 33181

Cyanobacteria, also known as the “blue-green algae”, are recognized to produce a vast array of toxic or otherwise bioactive secondary metabolites. As part of on-going research focused on identifying bioactive metabolites from cyanobacteria, particularly as naturally occurring environmental toxicants, extracts of freshwater cyanobacterial isolates from South and Central Florida were previously screened using the zebrafish (Danio rerio) embryo as a model of vertebrate development. Using this approach, extracts from biomass of Fischerella 52-1, a strain of freshwater cyanobacteria isolated from Central Florida, were found to contain so-called “developmental toxins” that inhibit or impair pathways or processes in the developing embryo. The present study focused on using the observed developmental effects, as a means of bioassay-guided fractionation, towards purification of relevant metabolites. Recognizing the production of a diverse repertoire of bioactive indole alkaloids by Fischerella and related genera, we additionally employed Ehrlich’s spray reagent, as a test for the presence of indoles, as well as characteristic UV spectroscopic absorbance associated with the indole chromophore, to specifically target the indole alkaloids as likely contributors to the observed developmental toxicity. Fractionation was performed using silica-gel column chromatograpy, and subsequent preparatory thin-layer chromatography (TLC), followed by purification using reverse-phase high performance liquid chromatography (HPLC). Indole-containing compounds from the bioactive fractions purified in this way are currently being characterized chemically by spectroscopic techniques (i.e. mass spectrometry, NMR, IR), and toxicologically in the zebrafish embryo model. Based on initial characterization data, one of the purified bioactive compounds is suspected to be a hapalindole. Further progress in purification, toxicity evaluations and structure elucidation of relevant metabolites will be reported.

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S S HIGH CO2 PROMOTES THE PRODUCTION OF PARALYTIC SHELLFISH POISONING TOxINS BY ALExANDRIUM CATENELLA FROM SOUTHERN CALIFORNIA WATERS

Avery O. Tatters1, Leanne J. Flewelling2, Feixue Fu1, April A. Granholm2, David A. Hutchins1

1 Department of Biological Sciences, University of Southern California, 3616 Trousdale Parkway, Los Angeles, CA 900892 Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 100 8th Ave. SE, St. Petersburg, FL 33701

Marine dinoflagellates are important constituents of microalgal communities in coastal oceans worldwide. A fraction of them are able to biosynthesize deleterious chemicals that are capable of impacting human and environmental health. As anthropogenic CO2 emissions continue to escalate, drawdown of this gas into seawater is leading to a progressively lower mean pH, referred to as ocean acidification. Concurrently, increases in sea surface temperatures are being recorded and are projected to continue. Despite the recognized importance of toxic dinoflagellates, their response to these climate/global change variables has been relatively unexplored. A clone of paralytic shellfish poisoning toxin producing Alexandrium catenella (A-11c) was conditioned to combinations of temperature, CO2 and phosphate concentrations for a period of eight months. These variables influenced growth and carbon fixation rates and although these variations only elicited minor differences in toxin composition (on a % basis) or ratios, the effect on total cellular toxicity was dramatically altered in all treatments. Cells conditioned to the high CO2, low phosphate at low temperature were the most toxic and compared to the low temperature of 15 °C, the warmer 19 °C alleviated some of this toxicity. Overall increased CO2 generally led to enhanced potency. Paralytic shellfish poisoning toxins are able to vector throughout marine food webs, thus posing a widespread potential risk to humans, marine inhabitants and their environments. To our knowledge, this is the first report of how A. catenella may respond to future marine conditions in Central/Southern California waters. Based on these results further investigation is warranted.

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SPHYSICAL MECHANISM FOR KAREnIA bREvIS BLOOM INITIATION IN TExAS

Kristen M. Thyng1, Robert D. Hetland1, Marcus T. Ogle, Xiaoqian Zhang1, Fei Chen, Lisa Campbell1

1Department of Oceanography, MS 3146, Texas A&M University, College Station, Texas 77843-3146

Karenia brevis blooms are known to occur sporadically in Fall, about once every 2-5 years, along the Texas coast. Circulation in the northwestern Gulf of Mexico is suspected to underlie K. brevis bloom initiation along the Texas coast, and control interannual variability in bloom presence or absence. This circulation, primarily caused by prevailing seasonal winds, is investigated using simulated surface drifters in a numerical model run in the Regional Ocean Modeling System (ROMS). Drifters are moved both forward and backward in time to understand the movement and origination of drifters for years in which harmful algal blooms did and did not occur. Bloom occurrence is correlated with weaker mean downcoast winds in September, as found using station wind data, and with the presence of southern waters near the Texas coastline, as seen through numerical drifter experiments. Further understanding of the basic mechanisms of bloom initiation may lead to better forecasting of these events in the future.

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S S QUANTIFYING CYANOBACTERIA AND HIGH BIOMASS BLOOMS FROM SATELLITE

Michelle C. Tomlinson1, Danielle Dupuy2, Dan Canfield3, Mark Hoyer3, John Hendrickson4, Rolland Fulton4, Robert Burks4, Erich Marzolf4, Travis Briggs1, Richard P. Stumpf1

1NOAA National Centers for Coastal Ocean Science, Silver Spring, MD 20910, USA2CSS-Dynamic, Fairfax, VA 22030, USA 3University of Florida, Gainsville, FL 32653, USA4St. Johns River Water Management District, Palatka, FL 32178, USA

Algal blooms of high biomass and cyanobacteria are on the rise, occuring both nationally and internationally. These blooms can foul beaches, clog water intakes, produce toxins that can contaminate drinking water, and pose a threat to human and domestic animal health. A quantitative tool can aid in the management need to respond to these issues. These blooms can affect many lakes within a state management district, pointing to the need for a synoptic and timely assessment. The 300 m Medium Resolution Imaging Spectometer (MERIS) satellite imagery provided by the European Space Agency from 2002 to 2012 has led to advances in our ability to monitor these systems. Algorithms specific to quantifying high biomass blooms are being developed for use by state managers through a comparison of field radiometry, water quality and cell enumeration measurements, and remotely-sensed satellite data. Initial evaluations were conducted for Florida lakes and the St. Johns River. These algorithms are designed to be detect blooms even with atmospheric interference and suspended sediments. Algorithm tuning is being evaluated against tunings made for other areas, such as Lake Erie. The results show that cyanobacteria blooms, especially of Microcystis, can be identified and their biomass can be estimated (as chlorophyll concentration and other metrics).

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STHE FOOD QUALITY AND DISSOLVED INORGANIC NUTRIENT EFFECT ON THE GROWTH AND TOxICITY OF dInOPHySIS ACumInATA FROM NORTH AMERICA

Mengmeng Tong1,2, Juliette L. Smith2, David M. Kulis2, Donald M. Anderson2

1Department of Ocean Science and Engineering, Zhejiang University, Hangzhou, China, 3100072Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA

Dinophysis acuminata, a producer of diarrhetic shellfish poisoning (DSP) toxins and pectenotoxins, is a mixotrophic species. The ciliate prey and light are required for the physiological activity of Dinophysis. However, how food quality (i.e. particulate nutrient) or dissolved inorganic nutrient affects the metabolism of Dinophysis is still unknown. Here we investigated the growth and DSP toxin production of D. acuminata under two nutritional sources: food quality and ambient dissolved inorganic nutrient on three nutritional levels: nutrient replete (N/P=17.3), phosphate limited (N/P=47.4) and nitrogen limited (N/P=7.0) conditions. Phosphate limitation of prey was accomplished in the study. The growth rate and cell toxin content of okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-2 (PTX2) did not show any difference in all treatments, with the cellular OA, DTX1 and PTX2 yielded from 0.18~0.58, 2.2~8.8 and 6.4~15.3pg/cell, respectively. Toxin was produced at exponential phase. No difference was found in net toxin production rates in the three nutritional treatments. However, the total toxin quotas (ng/mL) were significant higher in P-limited treatment when cell aged (at late plateau phase), with the maximum OA, DTX1 and PTX2 quotas of 9.3±0.5, 91.0±22.7 and 74.0±9.6ng/mL respectively, than that in other two treatments. The higher total toxin content was driven by the higher biomass of D.acuminata in P-limited condition (4293cells/mL VS 3986 and 3357cells/mL in nutrient replete and N-limited), which in return, was driven by the higher biomass of ciliate prey in the mixed culture (1355cells/mL VS 1078 and 1035cells/mL in nutrient replete and N-limited), indicating the indirect effect of ambient phosphate substances on the total DSP toxin concentrations in the culture even no dissolved inorganic nutrient was required by Dinophysis. These results supported us important information on nutrient availability to dynamic of Dinophysis blooms and DSP outbreaks in the field.

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S S ESTUARINE INFLUENCE ON THE PHYTOPLANKTON COMMUNITY STRUCTURE DURING A KAREnIA bREvIS BLOOM ON THE FLORIDA GULF COAST

Jennifer M. Vreeland1, Vincent J. Lovko1, Gary J. Kirkpatrick1, Ari Nissanka1, Val Palubok1, Susan Launay1, Kellie L. Dixon1 1Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL, 34236, USA

In 2012-2013 the southwest coast of Florida experienced a significant bloom of the ‘red tide’ dinoflagellate Karenia brevis causing massive fish kills, record manatee deaths, and human respiratory impacts in coastal areas. Bloom initiation is considered to occur in offshore oligotrophic waters, but less is known about the variety of nutrient sources that support growth and maintenance as the bloom moves towards the coast. The estuaries of the central west Florida coast, prominent sources of nutrients to coastal waters, differ widely in their physical, hydrological and chemical parameters. The scope of this research was to observe how nutrient levels affect phytoplankton communities in relation to the development and strength of Florida ‘red tide’ blooms. Throughout the progression and entirety of the most recent bloom, Mote Marine Laboratory conducted six nearshore -surveys along the 10 m isobath from Tampa Bay to San Carlos Bay from August 2012 to April 2013, as part of a cooperative monitoring and research program in conjunction with the Florida Fish and Wildlife Research Institute (FWRI). Four stations along this transect were located just outside of the inlets of four major estuarine systems (Tampa Bay, Sarasota Bay, Charlotte Harbor, San Carlos Bay). Samples collected at these stations were processed for K. brevis abundance, phytopigments (HPLC), nutrients and physical water structure. The pigment data were analyzed by chemotaxonomic analysis (Chemtax) to provide estimated results of the taxonomic structure of the phytoplankton communities. Primer v6 analyses were used to determine spatial differences and temporal trends in community structure over the course of the bloom and to associate community composition (with a focus on K. brevis) and succession with physical and nutrient concentrations over the duration of the bloom. The statistical relationships found were interpreted in the context of the effect of nutrients on the maintenance of K. brevis blooms.

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SLC-MS METHODOLOGY FOR BIOMARKERS OF BREVETOxINS IN THE EASTERN OYSTER (CRASSOSTREA vIRGInICA) AND HARD CLAM (mERCEnARIA SP.) ExPOSED TO KAREnIA bREvIS BLOOMS

Ann Abraham, Edward L.E. Jester, Kathleen R. El Said, Steven M. Plakas and Robert W. Dickey

FDA, Division of Seafood Science and Technology, Gulf Coast Seafood Laboratory, Dauphin Island, AL 36528

Neurotoxic shellfish poisoning is caused by the consumption of shellfish contaminated with brevetoxins. Traditionally, shellfish toxicity is assessed by mouse bioassay. Efficient in vitro methods are needed for improved monitoring and management programs. We report the selection of biomarkers of brevetoxin and the development of LC-MS methodology for applications in regulatory monitoring of brevetoxins in the Eastern oyster and hard clam. Molluscan shellfish exhibit species-specificity in metabolism of parent algal brevetoxins when exposed to Karenia brevis blooms. We identified biomarkers of brevetoxins representative of composite B-type brevetoxins in Eastern oyster and clam. For oysters, selected biomarkers were the cysteine conjugates BTX-B2 and S-desoxy-BTX-B2. For clams, biomarkers included the cysteine conjugates (BTX-B2 and S-desoxy-BTX-B2), a taurine conjugate BTX-B1, as well as the oxidation product BTX-B5 and its open A-ring derivative. Oysters and clams collected before, during, and after K. brevis blooms were analyzed by LC-MS and a commercial ELISA kit. The composite B-type brevetoxin concentrations as determined by ELISA were highly correlated with the sum of the biomarker concentrations determined by LC-MS (r2 = 0.94 for both oyster and clam). Comparisons with results of mouse bioassay support the use of ELISA and LC-MS as rapid screening and determinative/confirmatory methods, respectively, for monitoring of brevetoxins and assessing toxicity in these species.

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CHARACTERIZING SENSE AND ANTISENSE RNA POPULATIONS IN KAREnIA bREvIS AT DIFFERENT TIMES OF THE DIEL CYCLE

Scott B. Anglin1 and Timothy I. McLean1

1University of Southern Mississippi, Department of Biological Sciences, 118 College Drive #5018, Hattiesburg, MS 39406-0001

Karenia brevis is the responsible agent for generating periodic, if not annual, harmful algal blooms in certain coastal areas of the Gulf of Mexico. In an effort to better understand the biology of this organism, a functional genomics project was initiated. As part of that project, it was determined that a significant number of natural antisense transcripts (NATs), long non-coding RNAs with reverse complementarity to mRNAs, exist within the transcriptome of K. brevis. Based on the role of NATs in other organisms, we hypothesize that K. brevis NATs play a role in regulating gene expression at a post-transcriptional level. To test this prediction, we have extracted total RNA from K. brevis cells at different points in the diel cycle and sequenced all polyA-containing transcripts from each sample using the Illumina platform. Bioinformatic analyses will reconstruct the entire transcriptome of cells at each time point, and then we will cluster, annotate and determine the relative expression level of each transcript corresponding to the respective time of day. Specifically, we will assess the relative ratio of each member of any complementary pairs of mRNA and NAT to determine if the data support or falsify the hypothesis. The results of these experiments will possibly lead to a better understanding of environment-gene interactions for this organism, which in turn, will aid our ability to understand the factors and mechanisms associated with cell growth and bloom formation.

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STHINKING ALIKE? COMPARING ExPERT AND PUBLIC PERCEPTIONS OF THE BENEFITS AND RISKS OF CONSUMING SHELLFISH

Nick Boase1, William Gaze1, Clare Redshaw1,2, Mathew White1, Lora E Fleming1

1European Centre for Environment and Human Health, University of Exeter Medical School, Knowledge Spa, Royal Cornwall Hospital, Truro, Cornwall, TR1 3HD, UK2School of Geography, Earth and Environmental Sciences, University of Plymouth, Drake Circus, Plymouth, Devon, PL4 8AA, UK

The shellfish industry is an important part of coastal economies worldwide, including the UK where shellfish generated 280 million pounds sterling at first point of sale in 2011. Shellfish can also provide a variety of potential health benefits to consumers. However, as with many food types, shellfish can also present health risks, such as impacts on human health from consumption of shellfish contaminated with HAB toxins. UK incidences of shellfish poisoning as a result of HABs are low but consistent, and harmful algae present in UK waters include Alexandrium spp., Dinophysis spp., and Pseudo-nitzschia spp. However, little is known about how the UK public perceives the risks and benefits associated with consuming shellfish, and how this compares with expert knowledge. Understanding this is important to ensure effective, well-targeted communication of the risks and benefits from consuming shellfish, resulting in benefits to both the public and to industry.

Aim: This project aims to develop and compare how the public and experts perceive the risks and benefits (including from HABs) of consuming shellfish, ultimately leading to the development of effective evidence-based communication about shellfish.

Methods: The well-established Mental Models Approach to Risk Communication, which establishes people’s perceptions for a specific subject, is used to guide this project. Semi-structured interviews are underway with experts from across the shellfish supply chain (e.g. producers) and associated organisations (e.g. government) in the UK, and also the public. Following analysis of the interview transcripts, influence diagrams will be constructed to represent the mental models for each of these two groups, and then compared. In addition, language used by the public and experts to describe risks and benefits will be discussed.

Results/Conclusions: Findings from initial pilot studies indicated potential misconceptions amongst the public over some of the risks and benefits from consuming shellfish; for example, in recognising the difference in potential risk exposure between self-harvesters and those sourcing from a commercial supply chain. The findings are expected to inform communications about shellfish between industry and the public to resolve such misconceptions. In addition, this project will inform a subsequent large-scale survey of the UK public to understand the prevalence of these perceptions, and how these relate to shellfish eating behaviour.

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LADDER FRAME POLYETHERS AS POTENTIAL DRUG TRANSPORTERS

Andrea Bourdelais1, Elizabeth Elliott1, Karl Jacocks1, Allan Goodman1, Vinita S. Chauan2, Ian Marriott2 and Daniel G. Baden1

1University of North Carolina at Wilmington, Center for Marine Science Wilmington, NC 28409 USA2University of North Carolina at Charlotte, Department of Biology, Charlotte, NC 28223 USA

Ladder framed polyethers (LFPs) are a class of naturally occurring compounds that are produced by a variety of marine dinoflagellates. Many of the LFPs are highly toxic and most show selective binding to receptor sites on ion channels. Examples of LFPs include; brevetoxins, hemibrevetoxin B, brevenal, brevisin, ciguatoxins, maitotoxins, gambierols and yessotoxins.

Perhaps the most well characterized LFPs originate from the marine dinoflagellate Karenia brevis and include the brevetoxins, which has been shown to modulate voltage sensitive sodium channels (VSSC’s). Brevetoxins activate VSSC’s and exposure to these toxins via consumption of contaminated seafood can result in neurotoxic shellfish poisoning. In an attempt to fully understand the functional activity of the brevetoxins, development of a simple fluorescence based receptor binding assay for brevetoxin in living cells was attempted. Fluorescent conjugates of brevetoxin were prepared using a variety of the fluorophores (BODIPY®, Texas red, 6-TAMRA, D355, C356, A10439, D100, D355, P101, and 4-hydroxybenzhydrizide),. Competitive binding studies using rat brain synaptosomes showed most of the fluorescent brevetoxin conjugates retained good affinity for the receptor. High affinity compounds were then tested for their brightness and fade resistance in SJCRH30 human rhabdomyosarcoma cells. The fluorophore conjugates of BODIPY, 6-TAMRA, and C356 were very bright and easily visualized in living cells. C356, a fluorescein derivative proved to fade quickly, therefore, BODIPY and 6-TAMRA were chosen for further studies. Higher magnification of cells treated with the fluorophore-brevetoxin conjugates showed that the compounds were not localized on the cell surface but had been internalized, invalidating the development of a fluorescence based receptor binding assay. Although their utility for identifying receptor binding sites in whole cells was limited, the rapid uptake of membrane impermeable fluorophores when conjugated to brevetoxin led to the hypothesis that brevetoxins and, possibly, other LFPs may have utility to rapidly transport compounds, with low membrane permeability, across cell membranes.

Brevetoxin’s innate toxicity would likely preclude it from being suitable for drug delivery, so BODIPY-LFP conjugates were also prepared for brevenal and brevisin and studied for a similar ability to act as drug transporters. These smaller, less toxic, LFPs transport membrane impermeable compounds into cells as quickly and as efficiently as brevetoxin. In addition to the previous experiments we have tested the BODIPY and 6-TAMRA-LFP conjugates in 9 different cell lines, determined the EC50 in cytotoxicity assays and determined subcellular localization. Additionally, BODIPY-brevisin was administered to mice and whole body distribution determined. The results from these experiments will be presented.

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SINNOVATIVE DRINKING WATER TREATMENT FOR TASTE AND ODOR REMOVAL WITH NATURALLY OCCURRING BACTERIA FOUND IN SURFACE WATER

A.J. Boyes, K. Gilmore, B. MacLeod, A. Shawvner-Karnitz, and N. Moran

Manatee County Water Treatment Plant 17915 Waterline Rd. Bradenton FL, 34212 USA

Taste and odor (T&O) compounds found in surface waters globally are produced as secondary algal metabolites by certain cyanobacteria. These compounds, 2-methylisobrneol (MIB) and geosmin are tertiary alcohols which impart earthy-musty tastes and odors during cyanobacterial blooms. These odorants are not removed by conventional treatment at drinking water facilities, and can be detected in by consumers at concentrations as low as 5-10 ng L -1. Traditionally, powder activated carbon (PAC) is used as additional treatment to mitigate the T&O effects but it is a costly process and is sometimes unsuccessful at removing the offending odorants. Drinking water industries are facing the need for more innovative and cost effective technologies for water treatment and purification to remove these compounds.

A bench-scale study initiated in 2006 at the Manatee County Water Treatment Plant (Bradenton, Florida) and later a pilot-scale study starting in 2009, investigated direct biological filtration of raw surface water prior to conventional treatment for removal of these T&O compounds. Results indicated that spent granular activated carbon (GAC) or anthracite contactors which were allowed to colonize with bacteria from the Lake Manatee achieved successful removal of the odorants. When applying a spike of 2476 ng L-1 of geosmin to the pilot columns, 99.8% of the odorant was removed in a 12.6 minute empty bed contact time. This value of geosmin spike represents the maximum concentration in the past 10 years of historical occurrences. Direct biological filtration was shown to be an effective treatment option for removal of the odorants MIB and geosmin from warm Florida waters with high TOC content.

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MITIGATION OF A TOxIC ALExAndRIum BLOOM USING HYDROGEN PEROxIDE

Amanda Burson1, Hans C.P. Matthijs1, Renee Talens2, Wilco de Bruijne2, Ron Hoogenboom3, Arjen Gerssen3, Kees Steur4, Yvonne van Scheppingen4, Anne Fortuin4, Petra M. Visser1, Maayke Stomp1, and Jef Huisman1

1Aquatic Microbiology, Institute for Biodiversity and Ecosystem Dynamics, University of Amsterdam, The Netherlands2 Arcadis Netherlands 3RIKILT Wageningen UR, The Netherlands 4Waterschap Scheldestromen, The Netherlands

The dinoflagellate Alexandrium ostenfeldii is a well known harmful algal bloom (HAB) species that can cause paralytic shellfish poisoning (PSP). Although detrimental bloom events of A. ostenfeldii have occurred in Western Europe, the Netherlands have historically not shared this problem. However, in August of 2012 a dense bloom occurred in the brackish Ouwerkerkse Kreek (Zeeland) and its connecting channels. The A. ostenfeldii strain produced both saxitoxins and spirolide toxins which killed a dog via ingestion. The Ouwerkerkse Kreek routinely discharges water via a pump station into the adjacent Oosterschelde estuary, and an immediate call for remediation of the bloom was therefore required to avoid contamination of extensive shellfish grounds. Previously, treatment of infected waters with hydrogen peroxide (H2O2) successfully suppressed cyanobacterial blooms in Dutch lakes. Therefore, we adapted this treatment to fight off the Alexandrium bloom using a three-step approach. First, we investigated the required H2O2 dosage in laboratory experiments with Alexandrium. Second, we tested the method in a small isolated channel adjacent to the Ouwerkerkse Kreek. Finally, we applied the optimized method to the entire Kreek. Vegetative cell numbers were depleted up to 99% within 48 hours, and toxins in the water were reduced below the safety requirements of 15 ng/mL. The added H2O2 decayed to water and oxygen within 72 hours and fish mortalities were minimal. This is the first application of H2O2 to suppress a marine HAB species. This research provides a possible option for management of Alexandrium spp. blooms and other HAB events.

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SLOCALIZATION OF BREVETOxINS AND PHOTOAFFINITY LABELING OF TARGET PROTEINS

Wei Chen, Ryan Cassell and Kathleen Rein


Karenia brevis, the major harmful algal blooms (HABs) dinoflagellate of the Gulf of Mexico, plays a destructive role in the region. Because Karenia brevis produces potent neurotoxins, known as the brevetoxins, it has negative impacts on coastal economies, marine life and public health. Brevetoxins are ladder-shaped polyether (LSP) compounds. The brevetoxins have caused widespread marine animal mortalities and human poisonings. This has been attributed to their affinity for voltage-sensitive sodium ion channels causing channel opening and depolarization of excitable cell membranes. The endogenous role of brevetoxins is unknown. However, it was recently proposed that brevetoxins play an important role in osmoregulation by Karenia brevis. Using fluorescently labeled brevetoxin and brevetoxin photoaffinity probes, we will attempt to localize brevetoxin to a subcellular organelle or a target protein. This may implicate a specific role for the brevetoxins and other PE ladder type molecules.

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BREVETOxIN METABOLISM AND PHYSIOLOGY – A FRESHWATER MODEL OF MORBIDITY IN ENDANGERED SEA TURTLESCourtney Cocilova1, Jennifer Yordy2, Courtney Bennett3, Gregory Bossart4, Leanne Flewelling5, Catherine Walsh6 and Sarah Milton7

1Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431, USA2Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236, USA3 Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236, USA4Georgia Aquarium, 225 Baker Street NW, Atlanta, GA 30313, USA5Florida Fish and Wildlife Conservation Commission, 100 Eighth Ave SE, St. Petersburg, FL 33701, USA6Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236, USA7Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431, USA

The dinoflagellate Karenia brevis is a key organism in harmful algal blooms (HABS, Florida Red tide) that occur off the coast of Florida. K. brevis produces a suite of neurotoxins which are collectively referred to as brevetoxins (PbTx). PbTx-3 is one of the brevetoxin congeners and is known to bind voltage-gated sodium channels which affect cellular permeability, resulting in a cascade of events leading to cell death. Brevetoxin exposure affects marine life by interrupting neurological functions, decreasing immune function, inducing inflammation and causes respiratory effects in humans. HABs are increasing in frequency and distribution and are not only of immediate concern to species that inhabit areas where these blooms occur, but may have long term effects due to biomagnifications and bioaccumulation. In 2005, at least 109 loggerhead sea turtles in Florida were affected by red tides with over 70 impacted during a 2006 bloom. However, brevetoxicosis is difficult to treat in sea turtles as the physiological impacts have not been investigated and magnitude and duration of brevetoxin exposures, as well as specific toxin congeners involved are generally unknown. We are using fresh water turtles (Trachemys scripta) as a model for brevetoxin exposure in marine species. Turtles were exposed to 3.12 mg/kg PbTx-3 by intratracheal instillation (0.05ug/ul) every other day for one week and harvested after different times post-exposure (24h, 48h, 1w, 2w, 1m). Tissues from the heart, kidneys, brain, fat, intestine, liver and lymphoid tissues including the spleen and lungs were frozen in liquid nitrogen for ELISA analysis to investigate uptake, tissue distribution and routes of excretion as well as for histology. Blood samples were collected and used for immune analysis studies. Short-term exposures resulted in few immunological or pathological changes in T. scripta, though the data did suggest that severe interstitial pneumonia with edema may be a specific condition related to PbTx-3 exposure in this species. Blood analysis did not show any changes in hematocrit or plasma (lysozyme, superoxide dismutase, or glutathione-S-transferase) enzyme activity. Some immune function parameters were altered with exposure, including a significant decrease in phagocytosis after 48 h exposure and reduced lymphocyte proliferation after 1 month exposure. Plasma protein electrophoresis profiles showed a decrease in plasma albumin and corresponding A:G after 24 h. Longer exposure times to PbTx-3 will be necessary to accurately determine which organ systems become most severely impacted. Future studies will include in vitro and in vivo work to further determine the role of PbTx-3 as well as devising appropriate treatment plans to implement when such blooms occur.

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SASSESSMENT OF THE EASTERN GULF OF MExICO HARMFUL ALGAL BLOOM OPERATIONAL FORECAST SYSTEM: A COMPARATIVE ANALYSIS OF FORECAST SKILL AND UTILIZATION, 2004-2012

Edward Davis1, Karen Kavanaugh1, Katherine Derner2 and Cristina Urizar3

1 NOAA, National Ocean Service, Center for Operational Oceanographic Products and Services (CO-OPS), Silver Spring, MD 20910, USA2 NOAA, National Ocean Service, CO-OPS, 672 Independence Parkway, Chesapeake, VA 23320, USA3 NOAA, National Ocean Service, CO-OPS, 263 13th Avenue South, St. Petersburg, Florida 33701, USA

To aid early bloom identification and response efforts, in 2004 NOAA transitioned a successful forecast system for harmful algal blooms (HABs) from research to operations along the Gulf coast of Florida. NOAA’s Gulf of Mexico HAB Operational Forecast System (HAB-OFS) issues weekly bulletins that serve as decision support tools for coastal resource managers, federal and state agencies, and academic institutions. In an ongoing effort to improve the HAB-OFS, bulletin utilization and forecast quality are evaluated regularly. Utilization is measured by user feedback and observed or reported mitigating actions from public resource managers in response to bulletin information. Forecast accuracy, reliability and skill (i.e. relative accuracy) are calculated for each of the following forecast components: bloom transport, bloom intensification, and the daily potential for respiratory irritation along the coast.

Blooms of the toxic dinoflagellate Karenia brevis were present alongshore southwest Florida in the Gulf of Mexico four out of five years during the assessment period from May 1, 2008 to April 30, 2012, impacting public health, ecosystems, and regional economies. During this time, over 200 bulletins were issued for the eastern Gulf of Mexico, covering 4 individual bloom events, totaling 526 bloom days. Preliminary analysis shows that forecast accuracy each year was highest for respiratory irritation forecasts, with the highest relative accuracy amongst the “moderate” and “high” level impacts. However, all ‘very low’ respiratory irritation forecasts were unassessable. These results highlight challenges faced by the HAB-OFS team. Confirmation of respiratory irritation relies on a network that does not cover the entire forecast area and therefore cannot adequately confirm all respiratory forecasts. At least 90% of respiratory irritation forecasts could not be assessed due to lack of information. Additionally, the ‘very low’ respiratory irritation category is difficult to assess because it corresponds with a level of irritation that is limited to individuals with chronic respiratory conditions, such as asthma. These results will be compared to the assessment of previous operational years (2004 to 2008) and used as guidance to improve forecasting protocols for the HAB-OFS program in the future.

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ASSESSMENT OF THE WESTERN GULF OF MExICO HARMFUL ALGAL BLOOM OPERATIONAL FORECAST SYSTEM: ANALYSIS OF FORECASTS AND UTILIZATION OVER THE FIRST TWO OPERATIONAL YEARS, 2010-2012

Katherine Derner1, Karen Kavanaugh2, Edward Davis 2, and Cristina Urizar 3

1 NOAA, National Ocean Service, Center for Operational Oceanographic Products and Services (CO-OPS), 672 Independence Parkway, Chesapeake, VA 23320, USA

2 NOAA, National Ocean Service, CO-OPS, Silver Spring, MD 20910, USA3 NOAA, National Ocean Service, CO-OPS, 263 13th Avenue South, St. Petersburg, FL

33701, USA

Blooms of the toxic dinoflagellate, Karenia brevis, occur nearly every year in coastal regions of the Gulf of Mexico causing impacts on public health, ecosystems, and regional economies. Following the successful transition of the eastern Gulf of Mexico forecast system for harmful algal blooms (HABs) from research to operations along the gulf coast of Florida in 2004, NOAA expanded its Gulf of Mexico HAB Operational Forecast System (HAB-OFS) to include the coast of Texas (western Gulf of Mexico) in 2010. NOAA’s Gulf of Mexico HAB-OFS issues weekly bulletins that serve as decision support tools for coastal resource managers, federal and state agencies, and academic institutions. The Texas bulletins include three forecast components: bloom transport direction, transport distance and the daily potential for respiratory irritation along the coast. In order to continually improve the HAB-OFS, forecast skill and bulletin utilization are evaluated regularly. Each of the forecast components is statistically compared to observational data. Utilization is measured by user responses and observed or reported mitigating actions based on bulletin information.

This analysis details the assessment of bulletin utilization and forecast skill for the first two years of operational status along the Texas coastline, encompassing 106 total bulletins issued for the western Gulf of Mexico from October 2010 through April 2012. Of those, 44 bulletins were issued during the 2011 Texas red tide, one of the longest lasting and largest blooms on record in Texas, covering the coastline and inshore bays and waterways from South Padre Island to the Galveston region. Preliminary analysis for the first two operational years shows a relatively high degree of forecast skill for respiratory irritation forecasts, with greater than 70% confirmed correct, and greater than 80% of all moderate and high level forecasts confirmed correct. Heidke skill scores for respiratory irritation forecasts ranged between >.30 to .90, indicating a >30% to 90% improvement in forecast accuracy when compared to chance. These results also highlight the special challenges in the assessment of each forecast component. The majority of respiratory irritation forecasts, which are reliant on daily reports of field observations, could not be assessed due to a lack of information. The high amount of resuspension present along the Texas coastline also presents challenges in the assessment of transport, which is dependent upon the ability to discern and track distinct features through satellite imagery. A comparison in forecast assessment between the eastern Gulf of Mexico (Florida) and the western Gulf of Mexico (Texas) is also explored to highlight possible enhancements in product operations and assessment.

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SKAREnIA SP. IN SW FLORIDA – TRAJECTORIES OF NITROGEN, SILICA, OTHER WATER QUALITY, AND TAxONOMIC COMPONENTS DURING THREE ExTENDED BLOOMS

Kellie Dixon1, Ari Nissanka1, Jason M. Lenes2, Jennifer M. Vreeland1, and Gary J. Kirkpatrick1

1Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236, USA2University of South Florida, 140 7th Avenue South, St. Petersburg, FL 33701, USA

Three harmful algal blooms of Karenia brevis in the coastal waters of SW Florida were investigated for behavior over time of K. brevis and the biomass of other phytoplankton taxonomic groups, with the progressions statistically related to a variety nutrient and physical parameters, and with a particular emphasis on available nitrogen and silica supplies. Allocation of chlorophyll a among the various taxa (K. brevis, diatoms, haptophytes, chlorophytes, cyanophytes, prochlorophytes, cryptophytes, and other dinoflagellates) was performed via HPLC and Chemtax analyses. Available data were limited to coastal samples with K. brevis cell counts greater than 2000 cells L-1 to emphasize bloom growth and maintenance phases rather than non-bloom and bloom initiation phases. The blooms evaluated represented a range of conditions; two more ‘typical’ occurrences beginning in late summer through early winter (2006, 2012-2013), and a less common occurrence initiating during the winter of 2005. Some taxonomic groups (cyanophytes and prochlorophytes) displayed reproducible seasonal groupings, while others varied over time but in less correlation with thermal cues. At times, K. brevis biomass comprised nearly 90% of all phytoplankton, predominantly displacing diatoms, cyanophytes, and chlorophytes. In a monthly progression for each bloom, K. brevis dominance was reduced as DIN:DSi ratios increased from <0.3 to >0.5 μM:μM, although the replacement species was not always diatoms. A multivariate analysis (Primer V6) determined that samples were distinguished predominantly by biomass due to K. brevis, diatoms, and followed to a much lesser extent by cryptophytes and chlorophytes. Community similarity among samples (Bray-Curtis) was significantly associated with selected nutrients and physical parameters, with results varying among blooms. Significant explanatory parameters variously included temperature, inorganic phosphorus, silica, and ammonia concentrations.

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A PERSISTENT BLOOM OF AnAdyOmEnE J.V. LAMOUROUx (ANADYOMENACEAE, CHLOROPHYTA) IN BISCAYNE BAY, FLORIDA.

Ligia Collado-Vides1, Natalie Dou1 Christian Avila2, Stephen Blair2, Frederik Leliaert3; Dení Rodriguez4, Sabrina Schneider1, Pamela Sweeney5, Diego Lirman6

1. Florida International University, Miami, FL 33199, US2. DERM Miami-Dade County, FL 33136, US3.Ghent University, Krijgslaan 281 S8, 9000, Ghent, Belgium4. Univerisdad Nacional Autónoma de México. D.F. 04510, México5. Department of Environmental Protection, Miami, FL 33138, US6. University of Miami, 4600 Rickenbacker Cswy., Miami, FL 33149, US

Green macroalgal blooms are becoming a common problem in coastal waters and estuaries. This study describes the first occurrence of a persistent macroalgal bloom of the genus Anadyomene J.V. Lamouroux (Cladophorales, Anadyomenaceae) in the world and particularly in Biscayne Bay, Florida, USA. The morphological-based identification of species was verified by a molecular analysis that sequenced the variable C1D2 region of the large subunit (LSU) nrDNA. Results indicate that the bloom is composed of two species: Anadyomene stellata, reported previously for Florida, and a diminutive perforate undetermined species, Anadyomene. sp. Morphological traits of Anadyomene sp. are similar to Anadyomene linkiana, however the lack of vein-encircled perforations and molecular data of the original species impede us to give a definitive identification to these species. However it is potentially representing an introduction in the area. General surveys in Biscayne Bay based on a stratified random design, to visually estimate the percent cover of submerged aquatic vegetation, date from 1999, using the same methods recent intensive surveys of the detected bloom were conducted once a year from 2010 to 2012. Results show that the Anadyomene bloom densities have persisted since 2005 through 2012 covering an area of approximately 60 km2 of seagrass habitats. The spatial distribution of the bloom is restricted to the central inshore section of the Bay, an area affected by canals and groundwater discharges. The persistent 75 % cover reported for several sites, has caused significant negative impacts to seagrass beds. This bloom occurring adjacent to metropolitan Miami, adds to the world trend of increasing green macroalgal blooms occurring at enriched coastal waters.

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SA SYNOPTIC VIEW OF FLORIDA INLAND ALGAL BLOOMS

Danielle Dupuy1, Michelle C. Tomlinson2, Rolland Fulton3, John Hendrickson3, Robert Burks3, Erich Marzolf3, Travis Briggs2, Richard P. Stumpf2

1CSS-Dynamic, Fairfax, VA 22030, USA

2NOAA National Centers for Coastal Ocean Science, Silver Spring, MD 20910, USA

3St Johns River Water Management District , Palatka, FL 32178, USA

Toxic cyanobacterial blooms have become an increasing occurrence in Florida’s lakes, rivers, and estuaries. Cyanobacterial blooms pose a threat to human and animal health and degrade water quality, which can have substantial effects on aesthetic, ecological, and recreational values. Expansive monitoring is essential for the successful management of these resources. High resolution remote sensing products, like the 300m ocean color products available from the medium-spectral resolution imaging spectrometer (MERIS) have shown success in capturing Florida’s smaller water bodies. These remotely-sensed products and in situ data acquired from various sources, which include Florida LAKEWATCH and St Johns River Water Management District (SJRWMD), provide a comprehensive view of Florida’s inland algal blooms from 2009 -2011. Seasonal and interannual patterns in bloom activity can be tracked throughout the state, as well as the development of individual bloom events, such as the cyanobacterial bloom in the St Johns River in 2011. This information is a useful supplement to current agency monitoring programs for directing field sampling and management efforts.

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UPTAKE MECHANISM OF FLUORESCENT CONJUGATES OF KAREnIA bREvIS LADDER FRAME POLYETHER COMPOUNDS

Elizabeth A. Elliott, Allan Goodman, Daniel Baden and Andrea Bourdelais

Center for Marine Science, UNCW, 5600 Marvin K. Moss Ln, Wilmington, NC 28409

The marine dinoflagellate Karenia brevisproduces a host of toxins (brevetoxins) and non-toxic natural compounds (brevenal and brevisin) classified as ladder frame polyethers (LFPs).In an attempt to develop a fluorescentreagent for use in the develop-ment of a whole cell receptor binding assay, a series of LFP-fluorophore conjugates were prepared using cell impermeable fluorescent compounds. However, when cells were treated with these conjugates, it was found that the compounds were rapidly transported into the cell. Based on the chemical structure of the fluorophore, the LFP conjugates were internalized to different organelles within the cell. The ability for the LFPs to facilitate the uptake of cell impermeable compounds has led us to dub them as Escortins.

Studies were initiated to determine whether the conjugates were taken up into the cellsvia simple passive diffusion or through active transport mechanisms.Live SJCRH30cells at either 4 °C or 37 °C were treated with LFP-fluorophore conjugate and the uptake of the compound was measured using live cell imaging andfluorescence intensity scoring software.Results showed a significant reduction in uptake at the lower temperature, suggesting that uptake was not through passive diffusion. To further confirm this, studies utilizing 2-deoxy-D-glucose to reduce all active transport mechanisms were performed. In this study, cells werepretreated with the deoxyglucose to deplete the cells of an energy source. The cells werethen treated with the LFP-fluorophore conjugates and the uptake measured as before. Again, comparison of these results with controls grown under normal conditions, showed a decrease in LFP-fluorophore uptake, further supporting an active transport mechanism.

To discern which active transport mechanism the LFP-fluorophores were utilizing, a series of experiments were performed using selective uptakeinhibitors. From this, it was determined that brevetoxin, brevenal and brevisin all enter the cell via dynamin dependent mechanisms. However, using more specific inhibitors, it has been discovered that all three LFPs utilize slightly different pathways within the dynamin mediated mechanism. Results show that brevetoxin and brevisin are facilitated by caveolin dependent mechanisms, but appear to have subtle differences in their specific pathways, whereas brevenal appears to utilize a separate mechanism. These findings suggest that the different LFP conjugates may offer the tools necessary to better understand these different uptake mechanisms. A complete description of methods and results from these studies will be presented.

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SMONITORING STRATEGIES FOR THE CIGUATERA-CAUSING BENTHIC DINOFLAGELLATE, GAmbIERdISCuS

Amanda Ellsworth, Ashley Brandt and Michael L. Parsons

Florida Gulf Coast University, 10501 FGCU Boulevard South, Fort Myers, FL 33965, USA

Ciguatera is the leading form of phycotoxin-related seafood poisoning in humans worldwide. Ciguatera is caused toxins produced by some members of the benthic, epiphytic dinoflagellate genus, Gambierdiscus. Bioaccumulation of these toxins in coral reef food chains ultimately affects human health and food seafood safety.

Gambierdiscus has been shown to exhibit preferences for some host macrophytes (e.g., filamentous rhodophytes) over others (e.g., chlorophytes). While such preferences may reflect differences in chemical attraction/revulsion between different host species, the difference in preference may simply be a function of surface area (i.e., a filamentous thallus will have more surface area than a blade). Differences in host preference, coupled with a research need to compare Gambierdiscus populations between different sites and regions (where different host species are likely to be present), have led to efforts to use neutral (artificial) substrates as a proxy for host macrophytes. In this study, three different artificial substrates (burlap, nylon screen, and PVC tiles) were compared at four sites in the Florida Keys, in the vicinity of Long Key. The substrates were deployed monthly over the course of one year to determine which substrate was best representative of the predominate macrophytes indigenous to those areas. Results demonstrated that Gambierdiscus cell densities from the PVC tiles were better correlated with the Gambierdiscus cell densities on the macrophytes than the other substrates. For this reason, coupled with their reliability, durability, and ease of use, tiles were chosen over the other two artificial substrates for the continued monitoring of Gambierdiscus populations around Long Key. Monthly monitoring of Gambierdiscus populations using macrophytes and artificial substrates showed seasonal variation, possible causes of which will be discussed.

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NATURAL MORTALITY DURING THE DECLINE OF ALExAndRIum BLOOMS

C.J. Choi and D.L. Erdner

The University of Texas at Austin, Marine Science Institute, Port Aransas, TX 78373 USA

While our knowledge of factors controlling the development of phytoplankton blooms is increasing, the mechanisms leading to bloom decline and termination are not well characterized. It has been assumed that grazing and sedimentation can cause a decline of phytoplankton populations, and more recently, lytic viruses and programmed cell death (PCD) have been recognized as sources of mortality. However, the relative significance of the various mechanisms is not well understood. To distinguish factors regulating bloom decline, we studied blooms of Alexandrium spp. in Mill Pond and Salt Pond within the Nauset Marsh System (Cape Cod, MA) where this species forms massive blooms annually. Weekly samples were collected at four depths (1m, 3m, 5m, and 7m) from March to May 2013, to investigate the spatial and temporal extent of bloom initiation, development, and decline. We focused specifically on mortality caused by PCD as a factor in bloom decline. A greater than 10% increase in dead Alexandrium cells was observed during the bloom decline, highlighting the potential for natural mortality to contribute to Alexandrium bloom termination. In addition to quantifying PCD, we investigated the role of reactive oxygen species (ROS), known mediators for PCD. No substantial cell death was observed during the peak of the blooms, and evidence of other potential factors for bloom demise, e.g. induction of sexuality was observed. The contribution of these different mechanisms to bloom termination will be discussed.

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STHE EFFECT OF TEMPERATURE CONDITIONING ON ALExAndRIum FundyEnSE CYST GERMINATION DYNAMICS IN A SHALLOW ESTUARINE SYSTEMAlexis D. Fischer1 and Donald M. Anderson1

1Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA

The flux of cells from germinated cysts is critical to the population dynamics of many harmful algal bloom species. A distinctive feature of the widespread, open coastal blooms of Alexandrium fundyense in the Gulf of Maine region is an endogenous annual clock that restricts cyst germination to a discrete interval in the spring and early summer. Relatively little is known of the mechanism regulating the seasonality of excystment of A. fundyense from shallow water habitats. Through cyst germination experiments, the control of seasonal excystment was examined in the Nauset Marsh System (NMS), a shallow estuary on Cape Cod, Massachusetts (USA) with recurrent blooms tied to cyst seedbeds. No endogenous clock was evident in the germination of cysts produced in the previous spring bloom and stored under cold, dark, anoxic conditions for 18 months. To investigate seasonal temperature regulation, freshly sampled cysts from the NMS were isolated monthly into well plates and incubated at the ambient temperature, with excystment monitored weekly. Regardless of the incubation temperature and month isolated, 68(±6)% of the cysts had germinated after 120 days. Excystment success after 30 days of incubation had an autumn minima and spring maxima. Maximum values were coincident with the water temperature increasing from its annual minimum in January. Minimum values were coincident with the water temperature decreasing from its annual maximum in August. Seasonal excystment patterns were independent of a temperature function. Although the same ambient temperature (2°C) was used to incubate both January and February cyst populations, the January cysts required more than twice as many days of incubation to achieve 10% germination, yet the same length of incubation was required by both populations to attain 50% excystment. This lag experienced by the January cysts can be explained by the additional month of field conditioning the February cysts experienced. Excystment success of A. fundyense in the NMS seems to be modulated by a history of the seasonal temperature patterns. Like the Alexandrium cysts of Cork Harbor, Ireland and the seeds of higher plants, secondary dormancy is likely a strategy used by A. fundyense cysts in the NMS to prevent germination during conditions unfavorable for growth. With secondary dormancy, cyst germination timing is more flexible than is the case with endogenous clock regulation, and thus species with this strategy would be best suited for shallow water systems where interannual variation in environmental conditions are significant. This strategy would also allow the species to be more adaptable to climate-related environmental fluctuations.

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A MODIFIED CONCEPTUAL MODEL INTEGRATING HEALTH AND ENVIRONMENTAL IMPACT ASSESSMENT: HARMFUL ALGAL BLOOMS Lora E. Fleming1, Stefan Reis2, George Morris1, Sheila Beck3, Michael H. Depledge1, Melanie Austen4, Mathew White1, Tim Taylor1

1European Centre for Environment and Human Health, University of Exeter Medical School, Knowledge Spa, Royal Cornwall Hospital, Truro Cornwall, TR1 3HD, UK2Centre for Ecology & Hydrology, Bush Estate, Penicuik, EH26 0QB, UK3NHS Health Scotland, Meridian Court, 5 Cadogan Street, Glasgow G2 6QE UK 4Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, PL1 3DH, UK

Background - Methodologies for risk and impact assessment of human and environmental health effects often incorporate the use of conceptual models for problem framing and for the identification and evaluation of interventions. As the understanding of the intricate relationships between human health and the environment grows - particularly with regard to humans as part of ecosystems - the need for integration across scientific and professional disciplines and policy communities increases.

Aims - Through linking environmental health and ecosystem services, we aim to address inefficiencies introduced by discipline and policy barriers and so foster mutual understanding and improvements to human health and well-being.

Methods/Results - We propose the “Ecosystem-Enriched Driver-Pressure-State-Exposure-Effect (eDPSEEA) model,” to overcome the fragmentation of scientific disciplines and policy areas. It recognises positive and negative connections between the health of both humans and the environment. eDPSEEA incorporates socio-economic and other contextual modifiers of exposure and exposure-effect relationships, and recognises the importance of feedback-loops, trade-offs, and synergies between actions. As an illustration, we discuss how harmful algal bloom effects on human health and wider implications can be addressed using eDPSEEA as both a theoretical model and stakeholder engagement process.

Conclusions - Using the example of harmful algal blooms, we highlight the need to account for second order effects on human health via ecosystem services. Furthermore, we show socio-economic context is vital to account for distributional effects and modifiers for exposure-effect-relationships. We also recognise the potential for positive health and well-being effects (e.g. through green or blue space; or blue carbon initiatives). Quantifications of effects which omit these positive aspects are potentially misleading, wrongly implying an overall reduction in societal welfare. Also it is vital to consider where measures to address an issue may create new problems.

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SLINKING HUMAN HEALTH AND WELLBEING WITH WEATHER, CLIMATE AND THE ENVIRONMENT WITH HARMFUL ALGAL BLOOMS (HABS)

Lora E Fleming1, Brian Golding2, Anthony Kessel4, Andy Haines3, Michael Depledge1, Anna Cichowska4, Shakoor Hajat3, Christophe Sarran2, Nick Osborne1, Clive Sabel1,5, Trevor Bailey5, Dan Bloomfield1,5

1European Centre for Environment and Human Health, University of Exeter Medical School, Truro, Cornwall, UK2UK Meteorological (Met) Office, Exeter, UK3London School of Hygiene and Tropical Medicine, London, UK4Public Health England, London, UK 5University of Exeter, Exeter, UK

A large part of the global disease burden can be linked to environmental factors, underpinned by unhealthy behaviours. However, research into these linkages suffers from the lack of common tools and databases for carrying out investigations across many different scientific disciplines to explore these complex associations. The MED MI Partnership brings together leading organisations and researchers in climate, weather, environment, and human health and wellbeing.

The main aim of the recently funded MED MI Project is to create a central data and analysis source as an internet-based Platform which will be a vital new common resource for medical and public health research in the UK and beyond. We will link and analyse complex meteorological, environmental, and epidemiological data. This is a vital step to translate this data and analysis resource into epidemiologic, clinical, and commercial collaborative applications, and thus, improved human health and wellbeing in a rapidly changing environment. Existing databases, currently stored in various locations/organizations, will be combined enabling climate, weather and environment data to be linked and analysed with human health and wellbeing data. With appropriate confidentiality and ethical safeguards, the Platform will be available to researchers.

The “Climate, Coastal & Ocean Dynamics, and HABs: Blue Sky Demonstration Project” will leverage new Met Office capabilities to link coastal and oceanographic processes with remote sensing data to explore the possible link between HABs and climate change. The aim will be to look at whether there is an association between recent climate variability and the observed occurrence of blooms of the specific species sampled in ongoing monitoring programmes. If associations are seen with this modelling, health endpoint data from a variety of sources will be linked to explore acute and chronic diseases possibly associated with HABs and residence relative to coasts and other water bodies using time series and case-crossover analyses. The expansive scale in time and space, as well as large sample sizes of the human health databases, will provide sufficient data to explore the climate change-HAB-human health hypothesis and other aspects (e.g. identification of potentially vulnerable populations, modelling for early HAB warnings).

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DEVELOPMENT AND EVALUATION OF A DIRECT LC-MS/MS METHOD FOR DETERMINATION OF DMSP IN BIOLOGICAL SAMPLES

Harold A. Flores Quintana1, Alison Robertson1 and Ronald P. Kiene2, 3

1 FDA, Gulf Coast Seafood Laboratory, Dauphin Island, AL 36528, USA2 Department of Marine Sciences, LSCB-25, University of South Alabama, Mobile, AL 36688, USA3 Dauphin Island Sea Lab, 101 Bienville Blvd, Dauphin Island, AL 36528, USA

Dimethylsulfoniopropionate (DMSP) is an organic sulfur zwitterionic compound produced by a variety of marine micro- and macro-algae, and a few higher plants. This compound is involved in oceanic carbon and sulfur cycling and is major precursor of marine dimethylsulfide (DMS). DMSP has been found in a variety of harmful algae including Alexandrium spp., Dinophysis spp., Prorocentrum spp., Gymnodinium spp., Karenia spp., Gambierdiscus spp., and may play an important role in toxin production.

Traditional quantification methods for DMSP are indirect and include base hydrolysis and measurement of the cleavage product DMS, using gas chromatography (GC). While sensitive, these methods may overestimate the DMSP concentration when other dimethylsulfonium compounds are present.

We developed a sensitive and specific ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for direct measurement of DMSP in a variety of biological matrices including seawater, phytoplankton cultures, macroalgae, plants, and fish tissues. Hydrophilic interaction chromatography (HILIC) was used to separate related dimethylsulfonium compounds including DMSP, 2-methyl-DMSP, dimethylsulfoxonium propionate (DMSOP), and gonyauline. Selected reaction monitoring (SRM) in positive ion mode with multiple transition ions was employed to unambiguously confirm and quantify DMSP and related compounds. The limit of quantification for DMSP was 0.37 nM and with a linear range of 0.37 – 745 nM (r2 > 0.998). Ionization suppression was observed in fish extracts but these matrix effects were eliminated using standard dilution methods. DMSP extraction methods were evaluated in fish tissue by spiking with 2-methyl DMSP as an internal standard. Maximum recoveries were obtained with 100% methanol and yielded > 90% recovery. Comparison of the UPLC-MS/MS method with the indirect GC method in phytoplankton, macro algae, higher plants, and fish samples confirmed that the latter, overestimates the DMSP concentrations in some instances. The highest levels of DMSP were found in higher plants and phytoplankton; concentrations in fish were much lower, and were species-specific.

This HILIC UPLC-MS/MS method is sensitive and provides a fast, direct, and specific analysis of four dimethylsulfonium compounds. It allows simultaneous determination of these compounds in a variety of biological matrices. Applications of this method may improve our understanding of the complex role of DMSP in the oceanic sulfur cycling and toxin production by harmful algal species.

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SA CHEMICAL ANALYSIS OF KAREnIA PAPILIOnACEA

Nicholas Fowler, Andrea Bourdelais, Carmelo Tomas, Elizabeth Elliott, Bob York, Daniel Baden.

Center for Marine Science, UNCW, 5600 Marvin K. Moss Lane, Wilmington NC 28409

Harmful algal blooms (HABs) occur worldwide and can produce devastating effects to marine life, such as massive fish kills, death to marine mammals or contamination of seafood for human consumption. One of the most widely studied organisms responsible for HABs is the marine dinoflagellate Karenia brevis. This organism produces the highly neurotoxic compounds known as brevetoxins. When ingested, these compounds produce severe gastrointestinal maladies, as well as other neurological disorders such as hot-cold sensorial reversal. A related dinoflagellate, Karenia papilionacea, is known to co-bloom with Karenia brevis. However, K. papilionacea has received little attention as a possible toxin producing species. Therefore, studies were undertaken, using a combination of liquid-liquid extraction techniques along with HPLC, Mass Spectrometry, NMR, and cytotoxicity assays to isolate compounds, test their toxicity and to perform structural identification of the compounds produced by two isolated strains of K. papilionacea

Results from these studies led to the isolation of a toxic extract from two isolated strains of K. papilionacea (New Zealand and Delaware). Comparison studies of this isolate with known compounds produced by K. brevis identified this compound as PbTx-2. These results reveal, for the first time, the identity and quantities of a toxin produced by K. papilionacea. This could provide an explanation for why the toxic bloom of 1993 in New Zealand showed the presence of brevetoxins, without the presence of a known brevetoxin producer. Full results of this research will be presented.

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TRANSLATIONAL RESPONSE TO HEAT STRESS IN THE FLORIDA RED TIDE DINOFLAGELLATE, KAREnIA bREvIS

Kelly A. Fridey1,2, Jeanine S. Morey2, Matthew R. Paul3, Paul E. Anderson3, Rosemary Jagus4, Allen R. Place4, and Frances M. Van Dolah2,1

1 Graduate Program in Marine Biology, College of Charleston, Charleston, SC 294122 NOAA Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC 29412 3 Bioinformatics Research Group, Department of Computer Science, College of Charleston, Charleston, SC 29424 4 Institute of Marine and Environmental Technology, University of Maryland, Baltimore, MD 21202

Karenia brevis is responsible for harmful algal blooms in the Gulf of Mexico that cause extensive marine animal mortalities and human illnesses. K. brevis blooms are particularly damaging when they persist at high density in coastal waters over long periods of time. However, it is not yet known how K. brevis cells adapt to stresses associated with changing coastal conditions. Previous work in our laboratory revealed a lack of transcriptional activation of stress genes under conditions that induced stress proteins. This is consistent with an emerging view that dinoflagellate gene expression is regulated predominantly at the post-transcriptional level, in part by differential rates of translation. The current project was undertaken to determine if stress responses are mounted at the translational level. Translational activity can be inferred from polysome profiles. Polysomes are mRNAs with multiple ribosomes attached and represent the mRNA pool being actively translated, whereas ribosome-free mRNAs are translationally inactive. In this study, triplicate cultures of K. brevis were exposed to a 5 °C heat shock for a short time course (0, 30, or 60 min) to determine their translational response to heat stress. Sucrose density gradient fractionation was used to separate polysomes from ribosome-free RNA, detected spectrometrically by absorbance of 254 nm. The abundance of polysomes decreased rapidly in response to heat shock, indicating a reduction of translation, with the lowest polysome abundance found at 60 min of exposure. RNA was isolated from the translationally active fractions at each time point and RNA-seq analysis was performed on an Illumina Hi-Seq2000 sequencer at a depth of 15 million reads per sample. A reference K. brevis transcriptome was assembled from 113 million Illumina reads (50 bp, paired ends) using Trinity. This assembly contains 127K unique contigs. Quantitative read mapping to the reference transcriptome, currently in progress, will allow the assessment of whether stress response gene transcripts are specifically recruited to the actively translated RNA pool following heat shock.

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SSTRUCTURE ACTIVITY RELATIONSHIP OF BREVENAL DERIVATIVES

Allan Goodman, Jennifer McCall, Karl Jacocks, Alysha Thompson, Daniel Baden and Andrea Bourdelais

Center for Marine Science, UNCW, 5600 Marvin K. Moss Lane, Wilmington, NC 28409

The marine dinoflagellate Karenia brevis is associated with the phenomenon known as Florida Red Tide and produces a variety of ladder frame polyether compounds, including the highly active neurotoxic brevetoxins. In addition to these toxins, K. brevis produces brevenal, a compound shown to possess antagonistic activity to the brevetoxins. In asthmatic sheep models, brevenal was able to both inhibit and reverse brevetoxin induced bronchoconstriction. Furthermore, when administered alone, brevenal was able to increase tracheal mucosal velocity. These findings have led to the development of brevenal as a potential treatment for cystic fibrosis and the compound is in late stage preclinical studies.

To further understand the mechanism of action of brevenal and potentially develop a second generation drug candidate, a series of brevenal derivatives were prepared through modification of the aldehyde moiety. These derivatives include aliphatic, aromatic and heteroaromatic derivatives. The brevenal derivatives were then tested in in vitro binding assays to determine the ability of the compounds to displace brevetoxins and brevenal from their native receptors. From the data generated, it was found that a large variety of functionalities were tolerated, but binding at the brevenal receptor remained mostly unchanged. However, compounds containing a meta-substituted phenylhydrazide led to ligands with the greatest ability to displace brevetoxin. For instance, in the competitive binding assay for brevetoxin, the 3-chloro- (1) and 3-methoxyphenylhydrazides (2) displaced the brevetoxin with 7 and 8 nM affinities respectively, compared to 1133 nM for brevenal in the same assay. Additionally, a biotin derivative was prepared to aid in a streptavidin mediated isolation of the brevenal receptor. Full findings from these studies will be presented.

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DETERMINING FUNCTIONAL EFFECTS OF BREVETOxIN AND BREVETOxIN ANTAGONISTS

Meghan Grandal1, Alison Taylor1, Daniel G. Baden2 and Andrea Bourdelais2

1Department of Biology and Marine Biology, University of North Carolina at Wilmington, Wilmington NC 28409 USA2Center for Marine Science, University of North Carolina at Wilmington, Wilmington NC 28409 USA

Brevetoxin (PbTx) is lipophilic polyether compound produced by the dinoflagellate Karenia brevis that is responsible for massive fish kills in the Gulf of Mexico and can result in neurotoxic shellfish poisoning in humans after consumption of shellfish contaminated with the toxins. The physiological effects of PbTx are due to its activity on neuronal sodium (Na+) channels where it binds selectively to site 5 of voltage gated sodium channels. Binding of brevetoxin to this receptor results in hyperexcitability by increasing the mean open time of the channel and inhibition of channel inactivation, resulting in a range of neuroexcitatory symptoms. The nontoxic brevetoxin derivative, brevenal, inhibits the icthyotoxic and cytotoxic effects of PbTx by competitively binding to Na+ channels. Attention has been drawn to brevenal as a potential therapy for specific channelopathies due to its affinity for Na+ channels and nontoxic nature. Specifically, brevenal has been shown to reduce bronchoconstriction and increase mucus clearance in an asthmatic sheep model more effectively than current marketed drugs for cystic fibrosis (CF). A number of other naturally and synthetically produced PbTx derivatives are currently being tested for pharmaceutical potential. In this study, cell assays have been developed to measure functional responses at the cellular level, using quantitative methods. Amperometry and mass spectrometry were used to quantify catecholamine release from PC12 cells; both brevetoxin and brevenal stimulate secretion of the neurotransmitter dopamine. This suggests that either both compounds affect Na+ dynamics and membrane potential upon binding to their receptor sites, or that entrance of the toxin through cell membrane (as shown by studies with fluorescently labeled PbTx derivatives) alters intracellular Ca2+ dynamics and cellular pathways leading to neurotransmitter secretion. Ion-sensitive dyes are currently being used to measure change in intracellular Na+ and Ca2+ after PbTx and brevenal treatment to characterize downstream signaling cascades and the mechanism by which these compounds stimulate secretion. The ability to stimulate secretion makes PbTx derivatives a compound of interest, not only for CF, but for a range of other neuro- and endocrine degenerative diseases.

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SELUCIDATING THE GENETIC DIVERSITY OF mICROCySTIS AERuGInOSA WITHIN A TOxIC BLOOM IN SOUTH CAROLINA COASTAL PONDS USING 454-PYROSEQUENCING TECHNOLOGY

Dianne I. Greenfield 1,2,3; William J. Jones 2,4; Sarah Hogan 3; Chuck Keppler 3

1Belle W. Baruch Institute for Marine and Coastal Sciences, University of South Carolina, Columbia SC 29208, USA 2Marine Sciences Program, University of South Carolina; Columbia SC 29208, US3Marine Resources Research Institute, Charleston SC 29412, USA 4Arnold School of Public Health, University of South Carolina, Columbia SC 29208, USA

Coastal South Carolina (SC) is currently undergoing some of most rapid development in the United States, including residential neighborhoods and recreational (golf course) construction with detention ponds as catchments for stormwater runoff. Due to their poor flushing, SC detention ponds accumulate nutrients and fertilizers, making them susceptible to eutrophication and harmful algal blooms (HABs). Cyanobacteria HABs are particularly common, with Microcystis aeruginosa being among the most abundant species. A particularly dense (> 6 x 105 cells/ml) and toxic multi-specific cyanobacteria bloom occurred during August-September of 2011 in one such pond, with M. aeruginosa as a dominant component. We evaluated the spatial and genetic diversity of a key gene critical for the production of the hepatoxin microcystin (mcyD) within this bloom. Relevant enviromental parameters (standard water quality, nitrogen, phosphorus, chlorophyll a, microcystin concentrations, etc.) were also evaluated from multiple sampling stations. To assess spatial variability within the bloom, each sample was individually barcorded using a unique multiplex identifier (MID) sequence, and DNA was amplified using primers specific to mcyD. Products were subsequently sequenced using Roche/454 pyrosequencing technology. Initial results revealed multiple and distinct clades, suggesting a high degree of intraspecific variability. Here we discuss our findings, the various challenges encountered while applying this sequencing approach to HAB research, and the environmental factors that may mediate cyanobacteria bloom development.

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SUCESSFUL OCEAN OBSERVATORIES AT WORK ON A HARMFUL ALGAL BLOOM ON THE WEST FLORIDA SHELF

Alan Hails1, Gary Kirkpatrick1, Chad Lembke2, Robert Currier1, L. Kellie Dixon1

1Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236, USA2University of South Florida, 140 7th Avenue South, St. Petersburg, FL 33701, USA

In October, 2012, during an evolving Red Tide bloom event, Mote Marine Laboratory (Mote) and University of Southwest Florida (USF) deployed multiple instruments and ocean platforms to gather data needed to characterize and study it. The autonomous underwater vehicle (AUV) portion of the resources for this collaborative project was sponsored by the Gulf of Mexico Coastal Ocean Observing System (GCOOS) and State of Florida Fish and Wildlife Conservation Commission (FWC). The GCOOS portion of the project had the additional objective of demonstrating real-time telemetering of multiple AUV and sensor data to the GCOOS data servers where visualizations of AUV data fused with satellite imagery and ocean models could be produced.

During the period from October 6 through October 23, 2012 the AUV Waldo operated by Mote and the AUV Bass, operated by USF, were concurrently “flown” through an area 15 nm offshore of Englewood, Florida to 50 nm offshore of Naples, Florida. Within that area a concentrated data collection effort took place 5-11 nm SW of Sanibel Island. Also concurrently, under other programs sponsored by FWC, manned surface research vessels were deployed by Mote on October 4, 9, and 10.

The Mote AUV was equipped with a flow-thru CTD and with the Mote-developed Optical Phytoplankton Discriminator. The OPD is a mobile water sampling spectrophotometer employed to quantify the many species of the Gulf phytoplankton community, including red tide. The USF AUV was equipped with a similar CTD as well as with Fluorometric sensors for chlorophyll, CDOM, and backscatter and with oxygen and irradiance sensors. The surface vessels took CTD measurements and obtained water samples returned to the lab for HPLC analysis of toxins and pigments and for direct cells/liter measurements of red tide. Ashore, remote imagery data and ocean model predictions were produced to guide the data collection efforts.

We describe the many resources employed during this event, the numerous types of data collected and how they were combined and compared to learn about the origins, 3-D location and progression and strength of the bloom, and how the data was ported to the Gulf regional observatory for presentation and sharing in real-time.

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SFACILITATING IMPLEMENTATION OF THE RECEPTOR BINDING ASSAY FOR PSP

Sherwood Hall1, Frances Van Dolah2, Gregg Langlois3, and Leanne J. Flewelling4

1U. S. Food and Drug Administration, 1US FDA Center for Food Safety and Applied Nutrition, 5100 Paint Branch Parkway, College Park, MD 20740, USA.2NOAA Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC 29412 3California Department of Public Health, Environmental Management Branch, 850 Marina Bay Parkway, Richmond, CA 948044Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 100 Eight Avenue SE, St. Petersburg, FL 33701, US

The receptor binding assay (RBA) for Paralytic Shellfish Poison (PSP) toxins offers excellent sensitivity, high throughput, and a reliable measure of potency. Several steps have been taken recently to facilitate its implementation. The RBA is now an AOAC International-validated method, OMA 2011.27. The radiolabeled saxitoxin required for the RBA is now commercially available. An exemption has been obtained from the Nuclear Regulatory Commission (NRC) so that labs wishing to use the RBA can now purchase, use, and dispose of the radiolabeled saxitoxin without an NRC license. The membrane suspension needed for the method is now also commercially available, frozen in vials, ready for use in the assay. Finally, it has been found that the multiwell plate counters needed for the assay are available used/reconditioned for about a third of the cost of a new counter, significantly lowering the cost of implementing the RBA.

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AN ExPERIMENTAL ASSESSMENT OF THE IMPORTANCE OF PROPAGULE PRESSURE AND COMMUNITY RESISTANCE ON THE INVASION SUCCESS OF THE TOxIGENIC HAPTOPHYTE PRymnESIum PARvum

Richard M. Zamor and K. David Hambright

Plankton Ecology and Limnology Laboratory and Program in Ecology and Evolutionary Biology, Department of Biology, University of Oklahoma, Norman, OK

In recent years, harmful algae have bloomed in habitats where they were previously unknown to exist, suggesting that they may be invasive species. Alternatively, harmful algae may have always been present in these systems, perhaps as members of the microbial rare biosphere, and these systems have changed environmentally (e.g., via nutrient pollution, water resource overexploitation, or climate change) to become more amenable to population explosions by these previously undetected species. The toxigenic and ecosystem disruptive algal bloom species, Prymnesium parvum, is one such species. Originally described in marine coastal environments, P. parvum has successfully established populations in inland brackish systems globally, causing major ecological devastation, exemplified by massive fish kills. Thus, P. parvum represents a model organism for studying potential invasion and range expansion in a microbial species. If P. parvum and other harmful algae can in fact be invasive, then we would expect that general ecological understanding and theory derived from the study of multi-cellular invaders should be applicable to microbes as well.

Two major factors implicated in the establishment success of invasive species are propagule pressure and community resistance to invasion. The former represents a game of numbers, with generally larger invading populations or populations with repetitive invasions having greater chances for successfully establishing reproducing populations in new habitats, while the latter is embodied in Hutchinson’s n-dimensional niche theory, where the number of niches available for invasion is dependent on the diversity of the community being invaded. In experimental mesocosms, we manipulated community diversity in a natural phytoplankton assemblage (isolated from an inland lake subject to frequent P. parvum blooms, and determined by high-throughput pyrosequencing) by altering resource availability. We then subjected the manipulated communities, in which P. parvum was not at detectable levels, to different levels of propagule pressure by P. parvum. Our results indicate that, in the environmentally suitable habitats of our experimental systems, neither community diversity nor resource availability had any effect on invasion success by P. parvum. However, our results documented a significant role for propagule pressure in the establishment of P. parvum populations in these experimental systems. Although it is difficult to extrapolate laboratory results to natural systems directly, our results indicate that general ecological principles thus far developed from the study of macrobial invasive species can provide a solid framework for better understanding what appear to be major range expansions in P. parvum and other harmful algae.

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SSEASONAL PATTERNS IN NUTRIENTS AND PHYTOPLANKTON IN QUARTERMASTER HARBOR, AN ENCLOSED PUGET SOUND EMBAYMENT WITH RECURRENT HARMFUL ALGAL BLOOMS.Gabriela Hannach1, Kimberle Stark2 and Cheryl Greengrove3

1Environmental Lab, King County Department of Natural Resources and Parks Seattle, WA, USA2King County Department of Natural Resources and Parks, Seattle, WA, USA3Department of Environmental Science, University of Washington, Tacoma, WA, USA

Quartermaster Harbor, an enclosed bay within the Puget Sound Central Basin, has poor tidal flushing and historically low levels of dissolved oxygen, leading King County to initiate a nitrogen management study with the goal to identify key factors affecting nitrogen loading in the bay. Additionally, recurrent blooms of the dinoflagellate Alexandrium catenella occurring in spring and fall have been shown to coincide with elevated levels of saxitoxin in shellfish and the frequent closure of shellfish beds to harvesting. Bottom surveys have revealed unusually high concentrations of A. catenella cysts in Quartermaster Harbor surface sediments, providing a ready source of motile cells under favorable conditions. Blooms of the toxigenic diatom Pseudo-nitzschia are also common in the bay. These combined findings have prompted a number of investigations aimed at improving our understanding of the relationship between the distinct environmental conditions present at Quartermaster Harbor and the seasonal dynamics of its phytoplankton populations.

Sampling of the water column at Quartermaster Harbor is ongoing and consists primarily of a combination of a) routine monthly or study-specific water samples for water quality parameters, chlorophyll-a fluorescence and nutrients, and b) moorings that provide continuous measurement of water quality parameters and chlorophyll-a fluorescence. Routine phytoplankton analysis is conducted bi-weekly from March to November.

A comparison with open water sites indicated that the bay is characterized by an extended bloom season, typically spanning from March or earlier and into October. The shallow inner harbor waters warm up earlier in the year and stay warmer longer, contributing to the extended bloom season. A sharp reduction in surface water nutrients follows the early spring blooms, and inorganic nitrogen tends to remain depleted throughout the summer. Large diatom blooms have also led to silica depletion. As is characteristic of many estuarine systems, flagellate populations increase in late summer/early fall in response to limited inorganic nutrients and warmer water temperatures. In contrast to other sites, where Alexandrium catenella rarely forms blooms, in Quartermaster Harbor it is often one of the dominant species during this time. Results from these water property and phytoplankton community studies with a focus on the occurrence of Alexandrium catenella and timing of shellfish bed closures will be presented.

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LINKING FLORIDA RED TIDES TO HUMAN HEALTH EFFECTS: DATA ISSUES AND PRELIMINARY SIGNALS

Porter Hoagland1, Di Jin1, Andy Beet1, Andy Reich2, Barbara Kirkpatrick3,4, Steve Ullmann6, Sharon Watkins2, Lora E Fleming4,5,7

1Marine Policy Center, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA2Aquatic Toxins Program, Environmental Health, Florida Department of Health, Tallahassee, FL 32399, USA3Mote Marine Laboratory, Sarasota, FL 34236, USA4Department of Epidemiology and Public Health, Miller School of Medicine, University of Miami, Miami, FL 33177 USA5Oceans and Human Health Center, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, Miami, FL 32399, USA6Programs and Center in Health Sector Management and Policy, University of Miami, Miami, FL 33177 USA7European Centre for Environment and Human Health, Peninsula College of Medicine and Dentistry, Truro, Cornwall, UK

Florida red tides due to harmful blooms of the dinoflagellate Karenia brevis are known to cause respiratory and gastro-intestinal illnesses in humans and marine mammals. In humans, these illnesses can lead to a range of health impacts as reflected in physician appointments, emergency department (ED) visits, and hospital admissions. Public health specialists would like to understand further the nature of the human health risks of Florida red tide across illness severities, but to date there is only a limited body of research on this topic. Kirkpatrick et al. (2006, 2009) found that respiratory and gastrointestinal ED visits in a Florida coastal community are associated with Florida red tides in Sarasota County. Hoagland et al. (2009) used an exposure-response framework to estimate the effects of Florida red tide on weekly ED visits at this hospital for respiratory illnesses during 2001-2006. These authors found that a local measure of in situ K. brevis cell counts lagged by one week could explain ED visits when controlling for temperature, influenza cases, pollen, and a measure of tourist visits. In our current study of the dynamics of HABS and human communities, we examine the human health risks of Florida red tide for both respiratory and gastrointestinal illnesses for both ED visits and hospitalizations over a longer period of time, across a wider geographic area of Florida, and stratified by age. Because of limitations in the geographic monitoring for K. brevis blooms, we have compiled panel data on the location and duration of closures due to Florida red tide for 43 shellfish harvesting areas (SHAs) along the Florida Gulf Coast. We discuss some of the difficulties in compiling data on exposures and responses, and we report on the preliminary results of the analysis. When controlling for resident population size, tourism, and seasonal effects, we find that ED visits for respiratory illnesses can be explained by the number of monthly closures in coastal counties of proximate SHAs as a measure of the occurrences of Florida red tide blooms. These findings are geographically restricted to four mid-coast counties (Pinellas, Hillsborough, Sarasota, and Lee), and further limited to persons ≥ 55 years. A relationship between the red tide measure and gastro-intestinal illnesses may exist, but is more ambiguous.

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SDEVELOPMENT OF AN LCMS ASSAY FOR DETERMINATION OF AMINO ACID CONCENTRATION IN KAREnIA bREvIS

Tanya Hogue, Nicholas Fowler, Daniel Baden and Andrea Bourdelais

Center for Marine Science, University of North Carolina at Wilmington, 5600 Marvin K. Moss Lane, Wilmington, NC 28409 USA

Karenia brevis is a marine dinoflagellate responsible for the production of brevetoxins and other ladder frame polyether (LFP) compounds. The brevetoxins are a class of neurotoxins and are associated with massive fish kills, marine mammal mortality and adversely affect tourism and seafood production, especially in the Florida coastal waters in the Gulf of Mexico. Previous research has shown that K. brevis exposed to sudden reduction in salinity levels, such as may be experienced when blooms approach estuarine environments, leads to a rapid increase in LFP production. Therefore, it has been hypothesized that the LFPs are utilized by K. brevis in an osmoregulatory fashion. An alternate hypothesis is that brevetoxins are not involved in acclimation to salinity changes, rather compounds such as: lipids, dimethylsulfoniopropionate, ions and amino acids may be the osmoregulators in phytoplankton

Therefore, studies were initiated to develop an LCMS based assay to determine intracellular concentrations of amino acids produced by K. brevis under salinity stress. One of the key challenges of this research has been development of a suitable method for isolating and detecting the very low concentrations of free amino acids found inside K. brevis cells. In particular, extraction and quantification of amino acids from K. brevis has required overcoming various obstacles such as: competition of intracellular amino acids and salts found in sea water with ion exchange columns, difficulty in separation of amino acids on traditional HPLC columns due to their polar properties, instability of amino acid conjugates and the inability to use non-volatile buffers with the LC-MS system available at UNCW. Various methods for isolation of amino acids from sea water, conjugation techniques to make the amino acids less polar and LC conditions were investigated. Development of methods for isolation and detection of amino acids produced by K. brevis will be presented.

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COMPETITIVE AND MUTUALISTIC INTERACTIONS BETWEEN TOxIC AND NON-TOxIC STRAINS OF THE HARMFUL ALGAL BLOOM-FORMING mICROCySTIS AERuGInOSA

Sarah M. Holmes1, Mark W. Silby1, and Pia H. Moisander1

1University of Massachusetts Dartmouth, Department of Biology, 285 Old Westport Rd., N. Dartmouth, MA 02747, USA

Microcystis aeruginosa is a common bloom-forming cyanobacterium that is found in eutrophic bodies of fresh or brackish water throughout the world. Both toxic and nontoxic strains can be found within the same bloom with composition changing over the course of the bloom. Some blooms are considered to be HABs as the toxic strain produces a hepatotoxin, microcystin, which can be lethal to livestock that accidentally drink contaminated water and has non-lethal effects on humans including gastrointestinal discomfort and contact dermatitis. The genetic mechanism by which some Microcystis strains are toxic is known but the ecophysiological reasons are not fully understood. The fact that toxic and nontoxic strains appear together and their ratios predictably change over time suggests the possibility of a specific interaction between the strains which may involve signaling by microcystin. In this ongoing study we are investigating the competitive and mutualistic interactions between the toxic and nontoxic Microcystis strains. We propose that the toxic strain is providing a benefit due to the toxin while the nontoxic strain saves energy by not participating in toxin production whereby that energy is directed toward reproduction while still gaining the benefit of the toxin. We are investigating variation in growth rate between mono- and co-cultures of the toxic PCC7806 strain and nontoxic KLA2 strain under varying light conditions in order to identify if the toxin increased fitness under high light. Initial experiments indicate that the co-culture has an increased growth rate in comparison to the mono-cultures which lends preliminary support to the concept of the increased benefit of the toxic strain to the nontoxic strain. Novel qPCR primer-probe sets were designed to target the two strains used in the co-culture experiments, using the published genome for PCC7806 and the draft genome for the non-toxic strain from our laboratory. Initial qPCR runs with the primer sets indicate that they will be useful in determining the abundance of toxic and nontoxic cells grown together since these strains are visually indistinguishable. In addition to growth rate data, we plan to look at the transcriptomes of mono- and co-cultures with the prediction that there will be differential transcription between these cultures. The results will improve our understanding of the role of the toxin while investigating communication between strains and influence of variations of their relationship under different environmental conditions. This knowledge will be useful in monitoring and managing cyanoHABs and protecting water resources.

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GEITLERInEmA SP. PRODUCES A NOVEL ICHTHYOTOxIC CYANOBACTERIAL TOxIN

I-Shuo Huang1, Peter Moeller2, Dale Casamatta3, Danielle Gutierrez1, and Paul V. Zimba1

1Texas A&M University—Corpus Christi, Center for Coastal Studies, 6300 Ocean Dr, Corpus Christi, TX 78412

2NOAA, Hollings Marine Laboratory, 331 Ft. Johnson Rd, Charleston, SC 29412

3University of North Florida, 1 UNF Dr, Jacksonville, FL 32224

Cyanobacterial toxins are best known in freshwater systems, with only two toxins known from marine systems. It is likely that many other marine cyanobacterial toxins are undescribed. A marine Geitlerinema sp. was isolated from an aquatic animal rearing facility having mysid mortality events. The cyanobacteria possibly originated from Chesapeake Bay, MD, or Corpus Christi Bay, TX. Unialgal bulk cultures were used to produce sufficient material to isolate the toxin. Isolation of the unknown toxin was accomplished by bioassay-guided fractionation using HPLC-MS. The morphology of the Geitlerinema isolate does not match other known marine taxa. Molecular phylogenetic analysis in addition to morphology will be used for species identification. The toxin has a unique mass-to-charge ratio of 475.3 amu that does not match any known toxins. Structural confirmation by TOF-MS and NMR is ongoing. Toxicity of fish, mysids, and copepods occur after toxin exposure; LD50 for copepods was 158 mg/L. The LD50 values are helpful for understanding the species response once exposed to the toxin and the relative potency of the toxin compared to other toxins.

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BIOASSAY-GUIDED PURIFICATION OF A BIOACTIVE CAROTENOID FROM THE FRESHWATER CYANOBACTERIUM, CyLIndROSPERmOPSIS RACIbORSKII

Asha Jaja-Chimedza1, Miroslav Gantar2, and John P. Berry1

1Department of Chemistry and Biochemistry, Florida International University, 3000 NE 151st Street, North Miami, FL 33181 USA2Department of Biological Sciences, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA

Cyanobacteria (“blue-green algae”) are recognized to produce a wide array of bioactive compounds. Cylindrospermopsis is a known toxigenic genus of freshwater cyanobacteria that produces a number of toxic metabolites, particularly including the water-soluble toxins cylindrospermopsin and saxitoxin, and is frequently associated with toxic cyanobacterial blooms. Lipophilic extracts from a strain of C. raciborskii that does not contain these known toxins, was previously shown to inhibit the development of zebrafish (Danio rerio) embryos as a relevant freshwater teleost fish species. Bioassay-guided fractionation using the zebrafish embryo, as a model of so-called “developmental toxicity,” was subsequently utilized in the purification of the apparent toxic metabolite. Using this approach, an apparent pigment was identified as the bioactive compound and partial chemical characterization suggested that this compound was a carotenoid. Carotenoids are widely distributed among cyanobacteria, and generally associated with harvesting light and protection against photooxidative damage, but they have not been previously associated with any bioactivity. Our studies have shown that this compound clearly inhibits vertebrate development in zebrafish embryos, and thus may, indeed, contribute to the toxicity of this species. Chemical structure and associated toxicology of this presumed carotenoid will be presented. Due to the frequent occurrence of C. raciborskii in association with cyanobacterial harmful algal blooms, these bioactive compounds may pose a previously unrecognized environmental and health risk.

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SINVOLVEMENT OF MULTIPLE EIF4ES IN MRNA RECRUITMENT IN DINOFLAGELLATES

Grant D. Jones1, 2, Tsvetan R. Bachvaroff2, Allen R. Place2 and Rosemary Jagus2

1University of Maryland, Baltimore, 620 W. Lexington St., Baltimore, MD 21201, USA

2Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science, 701 E. Pratt St., Baltimore, MD 21202, USA

Insight into the molecular mechanisms that control the growth and persistence of dinoflagellate blooms is critical for the development of mitigation strategies. Organization of the genome and gene expression in dinoflagellates has been shown to be distinct from other eukaryotes. A wide range of studies has implicated mRNA recruitment as a major site of regulating gene expression in dinoflagellates. However, relatively little is understood regarding this process in these organisms. It is known that a short 22-nucleotide Spliced Leader exon is spliced from a capped small nuclear RNA and transferred to pre-mRNA, thereby providing the 5’-terminal end and the cap structure to mature mRNAs. Given this modification of mRNAs and a non-traditional cap structure, we have focused our investigation on the cap-binding translational initiation factor eIF4E. Similar to plants and metazoans, dinoflagellates express a variety of eIF4E family members that have the potential to function in the recruitment of mRNA to the ribosome or in mRNA regulation. Our phylogenetic analysis of RNAseq data on multiple dinoflagellate species found three separate clades of eIF4E. Clade 1 eIF4Es displayed the highest expression and contained up to seven conserved subtypes. Clades 2 and 3 had lower expression and each contained two or three more divergent subtypes. To compare the functions of eIF4Es from each clade, we synthesized four different members of Clade 1, one member of Clade 2, and one member of Clade 3 from Karlodinium veneficum, and we cloned them into a mammalian expression vector for in vitro translation in rabbit reticulocyte lysate. Using binding to methyl7GTP-agarose beads as a measure of affinity to the 5’-mRNA cap, we demonstrated differential binding of the eIF4Es from different clades. Clade 1 eIF4Es bound to methyl7GTP beads. In contrast, members of Clades 2 and 3 did not bind to the methyl7GTP beads. This is consistent with Clade 1 eIF4Es representing the functional initiation factors. Clade 1 eIF4Es contain extended amino acid stretches between the structural units of the eIF4E core. Interestingly, these regions show marked heterogeneity between the Clade 1 subtypes. From this, we may anticipate differing functions between the subtypes, such as selectivity for specific mRNAs. Cap binding characteristics of eIF4Es from Clades 2 and 3 suggest they may fulfill regulatory functions. This work is establishing additional tools for investigating the post-transcriptional regulatory mechanisms in dinoflagellates.

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EFFECTS OF NUTRIENTS AND GRAZING MORTALITY ON THE ABUNDANCE OF AuREOumbRA LAGunEnSIS DURING A FLORIDA BROWN TIDE BLOOM IN 2012

Yoonja Kang, Florian Koch, and Christopher J. Gobler

School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY 11794-5000, USA

During the summer of 2012, a brown tide bloom caused by Aureoumbra lagunensis emerged in the Mosquito and Indian River Lagoons, Florida, USA. To assess contribution of top-down (e.g. grazing) and bottom-up (e.g. nutrients) controls in shaping phytoplankton community composition during the bloom, nutrient amendment and dilution-style zooplankton grazing experiments were conducted during this brown tide bloom in the southern Mosquito Lagoon. During the study, A. lagunensis represented from up to 90% of total phytoplankton biomass and cell densities exceeded 2 x 106 cells. The brown tide bloom was associated with high levels of dissolved organic nitrogen (DON, > 80 μM) and low levels of dissolved inorganic nitrogen (DIN, < 2 μM). The low levels of DIN and low DIN:DIP ratio were indicative of a inorganic nitrogen-limited system, while the high DON:DOP ratio indicated there was an abundant supply of organic nitrogen. Nutrient amendment experiments demonstrated that the addition of DIN and DIP favored the growth of non-brown tide eukaryotes and phycocyanin and phycoerythrin cyanobacteria population, whereas the growth of A. lagunensis was not significantly affected by the addition of the same nutrients. These results suggested that ambient nutrients were ideal for the growth of A. lagunensis during the blooms. Dilution experiments demonstrated that microzooplankton grazing rates on A. lagunensis were significantly lower than those on other major phytoplankton populations. Low grazing pressure likely allowed A. lagunensis to out-compete other phytoplankton populations. Given these findings, it is concluded that both top-down (selective microzooplankton grazing) and bottom-up (high DON and low DIN system) promoted A. lagunensis brown tides in the southern Mosquito Lagoon, FL, in 2012.

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SEFFECTS OF CHRONIC LOW-DOSE DOMOIC ACID ExPOSURE ON MITOCHONDRIAL FUNCTION IN MICE

Preston Kendrick1, Kathi Lefebvre2, David Marcinek1

1 Department of Radiology, University of Washington, Seattle WA 981092 Environmental and Fisheries Science Division, NOAA NMFS, Northwest Fisheries Science Center, Seattle, WA 98112

The neurobehavioral excitotoxic effects of acute exposure to domoic acid have been well documented in both field (marine mammal) and lab (rat, mouse) studies, but the impacts of long-term, sub-clinical exposure to domoic acid have not been addressed in a mammalian system. In zebrafish, repeated sub-clinical doses of domoic acid have been shown to impact mitochondrial function. These impacts included increased mitochondrial content in exposed fish, but decreased O2 flux per unit mitochondria, indicating that the mitochondria functioned less efficiently in exposed fish. Chronic sub-clinical exposure to domoic acid also induced an immune reaction in zebrafish, eliciting the production of a domoic acid specific antibody detectable in serum. Similar impacts from chronic exposure are expected in a mammalian system. To elucidate these responses, mice were exposed to sub-clinical doses of domoic acid once a week for six to twelve weeks. At the end of six weeks, samples of cerebellum were taken for testing of mitochondrial function and content. Additionally blood was collected after twelve weeks of exposure to test for the presence of a domoic acid specific antibody. Preliminary results indicate a decrease in mitochondrial function, however the presence of a domoic acid specific antibody was not detected.

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CHANS: PUBLIC PERCEPTION OF FERTILIZER ORDINANCES AND FLORIDA RED TIDES

Karen Scheller1,2, Barbara Kirkpatrick2,3,4, Kate Kohler2, Lora E Fleming3,4,5, Margaret Byrne3, Jamie Studts6, Andy Reich7, Gary Kirkpatrick2, Steve Ullman8, Gary Hitchcock4, Porter Hoagland9.

1University of Notre Dame, Notre Dame, Indiana2Mote Marine Laboratory, Sarasota, FL3Dept of Epidemiology, Miller School of Medicine, University of Miami, Miami, FL4Oceans and Human Health Center, RSMAS, University of Miami, Miami, FL5European Centre for Environment and Human Health, University Exeter Medical School, Truro, Cornwall, UK 6Department of Behavioral Science, University of Kentucky, Lexington, KY7Aquatic Toxins Program, Environmental Health, FL Dept of Health, Tallahassee, FL8Business School, University of Miami, Miami, FL9Marine Policy Center, Woods Hole Oceanographic Institute (WHOI), Woods Hole, MA

Background: Karenia brevis (Florida red tide), the most common marine HAB, produces natural toxins that causes morbidity and mortality in exposed animals and humans. A possible but controversial cause of Florida red tide blooms is coastal inputs of macronutrients, nitrate and phosphate, through runoff of fertilizers. Despite scientific uncertainty about causality, in 2007 some coastal communities instituted local nutrient-restriction policies (including a municipal ordinance regulating lawn fertilizer applications) in response to repeated and severe Florida red tide events.

Methods: In 2012, reported knowledge and behaviors around HABs and nutrients were evaluated in 305 permanent and seasonal (snowbirds) residents in a coastal community regularly affected by blooms of Florida red tide and with a municipal fertilizer ordinance.

Results: The overall study population was predominantly older with a mean age (±SD) of 56 ± 14.2 years (range: 19-86 years), female (57.9%), and highly educated. Only 34% of the study population had any knowledge of the fertilizer ordinance, yet 45.9% then stated that the purpose of the ordinance was to reduce red tide. 20% of participants reported applying fertilizer to their lawns themselves and another 25% reported that their lawn company applies fertilizer. There were no significant differences between the residents and snowbirds regarding their knowledge and behavior with regards to HABs, fertilizer use, or knowledge of policy.

Conclusions: This study demonstrates the gaps between public perceptions, and the implementation of public policy. With poor awareness of the fertilizer ordinance, the effectiveness of the ordinance is unknown. Given a scenario of unproven and controversial science, we recommend that local communities consider surveying stakeholder use and knowledge of potential impacts from fertilizer application prior to policy implementation.

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WHERE AND HOW DO INDIVIDUALS RECEIVE AND PREFER TO RECEIVE INFORMATION ON FLORIDA RED TIDE?

Kate Kohler1, Margaret M Byrne2, Jamie Studts4, Ben Whitenack1,5, and Barbara Kirkpatrick1,2,3

1Mote Marine Laboratory, Sarasota, FL2Dept of Public Health Studies, Miller School of Medicine, Univ of Miami, Miami, FL3Rosenstiel School of Marine and Atmospheric Sciences, Univ of Miami, Miami, FL4Department of Behavioral Science, Univ of Kentucky, Lexington, KY5University of Pennsylvania, Philadelphia, PA

Background: Harmful algal blooms (HABs) pose challenging issues for public health communication efforts. Many products such as brochures, pamphlets, websites and Facebook pages have been created over the years to dissemination information to interested parties. This is certainly true with Florida red tide, ie Karenia brevis, blooms. However, to our knowledge, those potentially affected by HABs have not been surveyed for their preferences regarding information delivery mechanisms and trusted information sources when seeking information regarding HABs.

Methods: We surveyed 185 residents and snowbirds (seasonal residents of 3-6 months) regarding where they want to receive their information regarding Florida red tide and ratings of trusted information sources regarding HAB information. This survey was a subset of a larger survey investigating people decision making during a Florida red tide (see Byrne and Studts abstracts). The survey was administered via SurveyMonkey in the summer, 2012.

Results: The overall study population was predominantly older with a mean age (±SD) of 58.8 ± 13.6 years (range: 21-88 years), female (66.5%), and highly educated (60% had at least a 4 year degree). When queried what 3 organizations people go to for information regarding Florida red tide, participants selected private organizations (75%), media (67%), and state agencies (65%). When queried about the type of information products people prefer, they preferred websites (70%), television (56%), and newspapers (54%) as the top three products. When queried about who they trust the most for Florida red tide information (0 = no trust, 1 = A little, 2= Some, and 3 = A lot), private organizations were trusted the most (2.84 + 0.45), with state and federal agencies the second and third choices (2.53 + 0.69 and 2.46 + 0.75 respectively).

Discussion: Although websites were selected as the top mechanism people seek for information regarding Florida, it should be noted that traditional sources such as media and newspaper were also highly preferred. Private organizations are the most trusted for information, with state and federal agencies also highly trusted. It should be noted that the survey respondents were older and well educated. We suspect that the lack of interest in social media such as Facebook and Twitter could be could be due to an age cohort effect. The Mote Marine Laboratory sent the survey recruitment email, and so could have caused positive bias toward private organizations such as Mote.

Conclusion: Our survey suggests that websites produced by private organizations are the preferred information source for HAB information such as Florida red tide.

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STIMULATION OF DOMOIC ACID PRODUCTION FROM TRANSIENT CHANGES IN NUTRIENTS

Raphael M. Kudela1, G. Jason Smith2, Kendra Hayashi1 and Clarissa Anderson1

1Ocean Sciences Department, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA 95064, USA 2Moss Landing Marine Laboratories, 8272 Moss Landing Rd, Moss Landing, CA 95039, USA

There is a wealth of evidence from both laboratory and field settings that domoic acid production in the genus Pseudo-nitzschia is linked to nutrient availability. Toxin production is generally linked to physiological stress and/or growth rate, suggesting that nutrient availability is simply modulating this response. Here we synthesize data from a range of experiments including semi-continuous batch cultures, xenic and axenic chemostats, and field studies (including both nutrient-amended grow-out experiments and natural perturbations) to examine the transient changes in domoic acid production resulting from changes in nutrients. While there is clear evidence for nutrient stress (slowing or cessation of growth) as a cause for increased toxicity, we also see evidence for rapid (days) changes in toxin cell quotas and particulate versus dissolved fractions, triggered by changes in nutrient type and concentration. These rapid responses are transient, indicative of physiological adjustments within the cellular biosynthetic pathways. Domoic acid production also reaches a “steady state” in the chemostat experiments after a greater delay than for other common metrics (cell density, pigment per cell, C:N ratio). These shifts in toxicity in response to a variety of conditions, not just nutrient stress leading to slower growth, perhaps explain the often contradictory reports in the literature regarding the triggers for toxin production. We use these data to test several of the recently proposed physiological models of domoic acid production that have been developed. Based on concomitant measurements of variable fluorescence (Fv/Fm), we suggest that Fv/Fm may be a universal indicator of cell toxicity, perhaps providing an easy to use and cost effective method for tracking toxin potential that could serve as a “trigger” for sampling using more conventional (but also more expensive and time-consuming) methods or in conjunction with automated detection systems such as the Environmental Sampling Processor (ESP).

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SSILICON CONTROL OF DIATOM COMPETITORS AGAINST THE RED TIDE DINOFLAGELLATE, KAREnIA bREvIS, WITHIN THE EASTERN GULF OF MExICO

Jason M. Lenes1, Brian P. Darrow1, John J. Walsh1, L. Kellie Dixon2, Gary J. Kirkpatrick2 and Margie R. Mulholland3

1 College of Marine Science, University of South Florida, 140 7th Avenue South, St. Petersburg, FL 33701, USA2 Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236, USA3 Department of Ocean, Earth and Atmospheric Sciences, Old Dominion University, 5115 Hampton Boulevard, Norfolk, VA 23529, USA

The role of dissolved silicon was explored numerically in 2001 as a potential control of annual phytoplankton succession, from diatoms to the toxic dinoflagellate, Karenia brevis, observed in the eastern Gulf of Mexico each year during these and other field samples of 1998-2001, 2003, and 2007-2011. Multivariate plots were constructed during some of the latter years of the percent in situ biomass of the total phytoplankton population attributed to each functional group of diatoms, cyanophytes, cholorophytes, haptophytes, prasinophytes, K. brevis, other dinoflagellates, cryptophytes, and prochlorophytes against each other and the dissolved inorganic and organic forms of silica, nitrogen, and phosphorus macronutrients. They suggested that diatoms and K. brevis were unlikely to co-exist. Diatom chlorophyll biomass off southwestern Florida, from Apalachee Bay to the Ten Thousand Islands, did not exceed 1 μg chl l-1, when silicate concentrations were <0.9 μmol SiO4 kg-1. Similarly, K. brevis abundances were <40% of the total micro-algal population at concentrations of >3 μmol SiO4 kg-l when K. brevis concentrations were <1 x 106 cells l-1. Within these niche constraints, the percentage of K. brevis was highest at total dissolved nitrogen concentrations of 6-14 μmol N kg-1 and at total dissolved phosphorus concentrations of 0.2-0.8 μmol P kg-1. The impact of Si-limitation was quantitatively studied with a one dimensional causal simulation model of the 2001 K. brevis harmful algal bloom (HAB) on the West Florida Shelf (WFS). These model results indicated that just alleviation of Si-limitation among diatoms reduced the depth-integrated biomass of competing HABs of K. brevis by 36% over the 20-m water column.

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GAmbIERdISCuS NUTRIENT UPTAKE KINETICS

Alexander K. Leynse and Michael L. Parsons

Florida Gulf Coast University, 10501 FGCU Boulevard South, Fort Myers, FL 33965, USA

Ciguatera fish poisoning (CFP) is the most wide-spread phycotoxin-borne illness in the world, responsible for tens of thousands of poisonings per year world-wide. The causative toxins are produced by epibenthic dinoflagellates of the genus Gambierdiscus. These toxins bio-accumulate and biomagnify in tropical fish causing these fishes to become toxic for human consumption.

CFP outbreaks have been difficult to predict, due in part to taxonomic ambiguities and an incomplete understanding of ecological aspects of Gambierdiscus. With recent revisions in Gambierdiscus taxonomy, it is important that future research focuses on ecophysiological, toxicological, and host preference variation among Gambierdiscus species and strains.

Many observed increases in harmful algal blooms appear to be related to anthropogenic nutrient loading. However, the influence of nutrient concentrations on Gambierdiscus blooms and toxin production are unclear. Several field studies have been conducted, but oftentimes Gambierdiscus cells densities did not correlate with inorganic nutrient concentrations. It is likely that species variation plays some role in this disconnection.

In this study, we measured the nitrate and ammonium uptake rates of multiple Gambierdiscus species in different concentrations of each nutrient. These data were then used to construct Michaelis-Menten kinetic equations. Species comparisons will be discussed as well as the future application of these data to ecological models for predicting ciguatera outbreaks.

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SCHANS: FRAMING THE RED TIDE STORY: DAILY NEWSPAPER COVERAGE IN SOUTHWEST FLORIDA

Z. Li1, B. Garrison1, and S. Ullmann2

1School of Communication, University of Miami, Coral Gables, FL 33146

2Programs and Center in Health Sector Management and Policy, University of Miami, Coral Gables, FL 33146

One of the central themes in media effects studies is the agenda-setting role of mass media, which argues that the mass media impact heavily on the dominant issues being discussed in the public agenda. Furthermore, as part of the efforts of agenda-setting research, scholars have argued over the “framing” effects of mass media, which draws attention to how particular news coverage attributes dominate – or frame – the way certain topics are addressed in storytelling. Based on framing theory, this study focuses on news media reporting about HABs (specifically Florida red tide) along the Southwest Coast of Florida during the past two decades.

The study examines how the red tide story has been framed by the media (e.g., as a human health risk issue, an environmental story focused on marine animal threats, a business story emphasizing tourism and recreation, and so forth). Approximately 20 years of data are content analyzed using the NewsBank and Lexis-Nexis databases from four local daily newspapers in three standard metropolitan statistical areas defined by the U.S. Census Bureau along the Florida Gulf Coast: Sarasota-Bradenton, Tampa Bay-St. Petersburg, and Bonita Springs-Naples. A coding scheme is designed based on previous literature and pretested for reliability. Two coders are used to collect data. The variables examined include the theme or frame of news storytelling, story type, the sources journalists use to report the story, and images used to supplement storytelling. Other newspaper story characteristics are also measured. We are also interested in how the health-related information has been communicated by the news coverage. Finally, the study looks at the coverage findings in the context of hospital occupancy, regional tourism, and timing of active red tide blooms. The study findings offer insights as to the long-term outcome of the public awareness and perceptions of Florida red tide.

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HABITAT MODELING OF PSEudO-nITZSCHIA DISTRIBUTION AND TOxICITY IN THE COASTAL WATERS OF THE NORTHWEST PACIFIC USING NON-PARAMETRIC MULTIPLICATIVE REGRESSION

Susan C. Lubetkin1 and Evelyn J. Lessard1

1University of Washington, School of Oceanography, Box 357940, Seattle, WA 98195, USA

It has been difficult to assess the environmental regulators of toxigenic Pseudo-nitzschia spp. blooms within the Juan de Fuca eddy and along the Pacific Northwest coast using traditional statistical methods, perhaps in part because of non-linear interactions between important variables. We are applying a new statistical habitat modeling method, non-parametric multiplicative regression (NPMR), to the rich dataset collected over ten ECOHAB and RISE cruises from 2003 to 2006 to isolate factors which best describe the abundance of Pseudo-nitzschia spp. and domoic acid (DA) production. NPMR combines predictor variables multiplicatively, such that the effect of one predictor variable can covary in a complex way with others, and requires no assumptions about the overall shape of the response surface. Using NPMR, we isolated several variables from a list of over 40 physical, chemical, and biological variables, which in combination are good predictors of Pseudo-nitzschia spp. and DA concentrations. The list of predictors varied across sampling subsets (i.e, season, cruises with extremely high Pseudo-nitzschia spp. concentrations), giving us new insights into the complex factors leading to toxigenic Pseudo-nitzschia spp. blooms in this region.

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SIDENTIFICATION OF CYANOBACTERIAL TOxINS INVOLVED IN THE APPARENT INTOxICATION OF DOLPHINS IN THE FLORIDA KEYS

Christina Lydon1, Shunmei Liu2, Larry Brand3 and John P. Berry1

1Department of Chemistry and Biochemistry, Marine Science Program, Florida International University, 3000 NE 151st Street, North Miami, FL 331812Department of Pharmaceutical and Biological Science, Weifang Medical University, 7166 West Baoton Street, Weifang, Shandong Province, 261053 3Division of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Sciences, University of Miami, 4600 Rickenbacker Causeway, Miami, FL 33146

Cyanobacteria produce a diverse repertoire of toxic or otherwise bioactive metabolites that have been linked to intoxication of humans and wildlife. Captive dolphins in the Florida Keys were recently observed to graze cyanobacteria, and perhaps other associated algae, occurring within enclosures followed by apparent signs of intoxication, and specifically seeming neurotoxicity. To identify possible toxic metabolites involved in these intoxication events, we have employed the zebrafish (Danio rerio) embryo, as a model of vertebrate toxicity, toward bioassay-guided fractionation/purification, and subsequent chemical and toxicological characterization, of relevant biologically active metabolites. Initial studies indicate that extracts, and several subsequent chemical fractions, derived from cyanobacteria are, indeed, toxic in the zebrafish model, and generally support these as potentially toxic algae. Current progress on the purification and characterization of these compounds will be presented.

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POTENTIAL HAB SPECIES FOUND IN THE CALOOSAHATCHEE ESTUARY,FLORIDA

Laura R. Markley and Michael L. Parsons

Coastal Watershed Institute, Florida Gulf Coast University, 10501 FGCU Blvd South, Fort Myers, FL, United States 33965

Anthropogenic activities over the last several decades are thought to play a role in the increased frequency, duration and intensity of harmful algal blooms (HABs) worldwide. While the dominant HAB species in the Gulf of Mexico, Karenia brevis, continues to be a focus of scientific study, research has been sparse on estuarine phytoplankton and other potential HAB species that may be present in inshore environments. In southwest Florida, the Caloosahatchee is a highly managed riverine system where freshwater inflow can be negligible in the dry season (winter-spring) and extremely high (>5000 cfs) in the wet season (summer-fall). As freshwater inflow is a primary controlling factor of both nutrient and light availability for phytoplankton in the Caloosahatchee estuary, phytoplankton growth can vary greatly throughout the year. We collected monthly plankton samples from 2008 to 2010 at 14 locations along the Caloosahatchee estuary from the Franklin lock and dam (S-79) to the river mouth. Phytoplankton were identified to the lowest taxonomic level possible and quantified as cells/L and cell volume/L. Several previously undocumented HAB species were found in the estuary (e.g., Akashiwo sanguinea and Pseudo-nitzschia pungens). Blooms were evident year-round, occurring upstream when freshwater inflows were low and further downstream as rates of discharge increased. As several species documented by this study have caused HABs in other areas, further studies should be conducted to determine if the strains of the species found in the estuary are toxic and what environmental conditions promote cell growth. These species also need to be considered in the management of freshwater inflow from S-79 in order to minimize the threat of HABs in the Caloosahatchee estuary.

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STIME SERIES ANALYSIS FOR PERIODS WITH HIGH CONCENTRATION VERSUS PERIODS WITH LOW CONCENTRATION OF KAREnIA bREvIS

Grace Maze1, Josefina Olascoaga1, Larry Brand1

1 University of Miami, RSMAS, 4600 Rickenbacker Causeway, Miami, FL, 33146

The West Florida Shelf is home to a toxic dinoflagellate, Karenia brevis. This dinoflagellate produces brevetoxins, a suite of polyether neurotoxins, which are hazardous to mammals and marine life. Since 1954, 28 K. brevis blooms with a concentration >104 cells/L have been observed off the coast of west Florida between Tampa Bay and Charlotte Harbor. There are many theories on what causes the onset of a bloom, however no theory currently accounts for all blooms.

Using Karenia brevis concentration between 26.5oN and 28oN and shoreward of 85oW periods of no bloom (<100 cells/L) and periods of large bloom (>105 cells/L) were identified. For each case the concentration levels were required to continue for at least 10 days with no deviations lasting more than 5 days. From 1992 to 2006, 15 instances were found with no bloom detected and 8 instances were detected with a large bloom. Values for alongshore wind, cross shore wind, Peace River discharge, Caloosahatchee River discharge, and the northern most position of the Loop Current were analyzed to compare the periods of no bloom versus the periods of large blooms. Using a two-sided student’s t-test the average values were compared and it was found that the only statistically significant difference between periods of large bloom and periods of no bloom was the freshwater discharge of the Peace River.

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DEVELOPMENT OF COMPETITIVE FLUORESCENCE-BASED BINDING ASSAYS FOR BREVETOxINS AND BREVENAL

Jennifer McCall, Karl Jacocks, Allan Goodman, Susan Niven, Alysha Thompson, Daniel Baden, and Andrea Bourdelais

Center for Marine Science, UNCW, 5600 Marvin K. Moss Lane, Wilmington, NC 28409

Brevetoxins are a family of ladder-frame polyether toxins produced during blooms of the marine dinoflagellate Karenia brevis. Consumption of fish exposed to K. brevis blooms can lead to the development of neurotoxic shellfish poisoning. The toxic effects of brevetoxins are due to activation of voltage-sensitive sodium channels (VSSCs) in cell membranes. K. brevis also produces a non-toxic antagonist to brevetoxin known as brevenal, which is able to inhibit and/or negate many of the detrimental effects of brevetoxins. Brevenal binding to VSSCs has yet to be fully characterized, in part due to the difficulty and expense of current techniques. Binding of toxins has historically been measured using a radioligand competition assay that is fraught with difficulty. In this study, we developed two novel fluorescence-based binding assays (FBAs) for the brevetoxin receptor and the brevenal receptor. Several fluorophores were conjugated to PbTx-2 or brevenal and used as the labeled ligands. Both assays were qualified against the standard radioligand receptor assay for the respective receptors and yielded comparable equilibrium constants. On the brevetoxin FBA, brevetoxin analogs were able to compete for binding with the fluorescent ligands. The FBA was used to determine relative concentrations of toxins in raw extracts of K. brevis culture, and to determine ciguatoxin affinity to site 5 of VSSCs. Using the brevenal FBA, in depth studies to characterize brevenal’s binding site on rat brain synaptosomes have shown that the fluorescent brevenal conjugate was not displaced by VSSC ligands for sites 1-5 or 7, or the sodium channel blocker amilioride, suggesting that brevenal does not bind to VSSCs at any of these locations. As such, brevenal may elicit its action through a novel mechanism and/or currently unknown receptor site on VSSCs. The FBAs are quicker, safer, far less expensive, and do not generate radioactive waste or need radioactive facilities. As such, these assays can be used to replace the current radioligand assays and will be a vital tool for future experiments examining the binding affinity of various ligands on sodium channels.

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SDEVELOPMENT OF PCNA AS A MOLECULAR BIOMARKER FOR GROWTH STATUS OF KAREnIA SPECIES IN GULF OF MExICO RED TIDES

Megan E. Meek1,2, Alina Corcoran3, and Frances M. Van Dolah2,1

1 Graduate Program in Marine Biology, College of Charleston, Charleston, SC 294122 NOAA Marine Biotoxins Program, Center for Coastal Environmental Health and Biomolecular Research, Charleston, SC 294123 Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, St. Petersburg, FL 33701

Blooms of the toxic dinoflagellate Karenia brevis have been documented in the Gulf of Mexico for more than 125 years. There have been many efforts to model K. brevis blooms to forecast impacts and improve management practices. Current models focus on physical drivers of large scale bloom dynamics, but do not provide insight into biological processes occurring within a given bloom patch that may be predictive of its fate. Moreover, models focus solely on the single species K. brevis, ignoring co-occurring Karenia mikimotoi, Karenia selliformis, and Karenia papilionacea, each of which has unique temporal and spatial distributions relative to K. brevis blooms. The purpose of this study is to develop a molecular biomarker for K. brevis growth rate and adapt it for use with existing Karenia probes to enhance monitoring and inform model development. The proliferating cell nuclear antigen (PCNA) protein is part of the DNA replication fork complex that occurs during the DNA synthesis (S) phase of the cell cycle. In K. brevis, the cell cycle is under circadian control. PCNA is present throughout the cell cycle in K. brevis, but its expression increases in actively growing cells, with the highest concentration occurring during S-phase. In laboratory cultures, a linear correlation is observed between growth rate and expression of PCNA (assessed by western blotting), suggesting a potential for PNCA as a biomarker for growth rate. Here we present preliminary data on the comparative circadian cell cycle behavior and PCNA expression in Karenia species from the Gulf of Mexico. We demonstrate that the anti-K. brevis PCNA antibody recognizes PCNA not only in K. brevis, but also in other Karenia species. We are currently adapting PCNA detection to a flow cytometric assay suitable for monitoring applications, with the application of species-specific rRNA probes in conjunction with anti-PCNA detection to enable the simultaneous determination of Karenia species composition and relative growth in mixed bloom populations.

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LOCALIZATION OF BREVISIN-DAUNORUBICIN CONJUGATE IN SJCRH30 CELLS

Nina E. Neill, Elizabeth A. Elliott, Allan J. Goodman, Daniel G. Baden and Andrea J. Bourdelais

Center for Marine Science University of North Carolina at Wilmington, Wilmington NC 28409

One of the most difficult challenges facing drug discovery is the issue of maximizing the bioavailability of active pharmaceutical agents, which is in part regulated by a compound’s ability to cross cellular membranes and overcome efflux mechanisms within the cell. In an effort to overcome these barriers, increasingly sophisticated methods to improve a drug’s effectiveness are being pursued, such as stimuli specific drug delivery systems and bio-engineered nanomolecular carriers. Research at UNCW’s Center for Marine Science on ladder frame polyethers (LFPs), including brevenal, brevisin and brevetoxins, produced by the dinoflagellate Karenia brevis, has led to another possible drug carrier with the capability of increasing the bioavailability of compounds that normally exhibit poor cellular uptake. LFPs demonstrate an ability to rapidly cross cell membranes. When conjugated to large compounds with low membrane permeability, such as the chemotherapeutic agent daunorubicin, LFPs retain the ability to quickly cross the cell membrane while concurrently escorting the drug into the cell. Once inside the cell, the LFP-drug conjugates localize in discrete subcellular compartments, the identity of which is critical to the complex’s function. By staining several cellular organelles and visualizing the uptake and localization of the naturally fluorescent brevisin-daunorubicin compounds, we were able to determine that the LFP conjugates primarily localize in the endoplasmic reticulum as well as the late endosomes. In contrast, daunorubicin alone has previously been shown to sequester primarily in lysosomes as well as the nucleus, to a lesser extent (Schindler 1996). Lysosomes are the enzyme containing organelles that are responsible for cellular waste degradation, leading to subsequent expulsion from the cell. Many basic drugs have been shown to accumulate within these organelles, which inhibits their efficacy. Our results have implications for future drug delivery developments as the property of LFP-conjugates to quickly and efficiently traverse cellular membranes and localize in new organelles could be capitalized upon to increase pharmaceutical bioavailability.

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SRESPONSES OF A NATURAL KAREnIA BLOOM AND OTHER TAxA OF THE WEST FLORIDA SHELF TO SINGLE NUTRIENT, MULTIPLE NUTRIENT, OR ESTUARINE WATER ADDITIONS

Ari Nissanka1, L. Kellie Dixon1, Susan Launay1, Lori J. Zaworski1, Patricia J. Minotti1, Camia M. Charniga1, Alexandria G. Hounshell1, Jennifer M. Vreeland1

1Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236, USA

While blooms of the toxic dinoflagellate, Karenia brevis, predominantly originate in oligotrophic waters offshore and while K. brevis is slow-growing relative to the diatoms that form a dominant community of the coastal phytoplankton on the West Florida Shelf, K. brevis blooms are carried inshore where they can outcompete other species and form nearly monospecific concentrations of many millions of cells per liter. The potential sources of nutrients to maintain such blooms are varied, but include estuarine outflow from the three major estuaries of SW Florida. A program was initiated to challenge and quantify the response of a naturally occurring K. brevis bloom with a variety of nutrients, including water from the three estuaries. A larger scale (~20 L per sample) bioassay was conducted with a natural bloom of 1.0-2.6 X 105 cell L-1. Treatments included three amino acids, inorganic nitrogen species (ammonia, nitrate), urea, inorganic phosphorus, silicate, urea plus inorganic phosphorus, and individually, the reduced salinity waters of the three major local estuaries. Reductions in salinity as a result of either reagent or estuarine water additions were carefully matched. Treatments were randomly prepared in triplicate, and samples incubated in ambient water at the surface, but under reduced light intensity (60%) for approximately 48 hours. Similar to prior work on coastal phytoplankton communities in the absence of Karenia, nitrogenous additions displayed responses in K. brevis growth and total biomass, elemental ratios, and other phytoplankton community components that differed in trajectory and endpoints from those of the other treatments. There was little response to the estuarine additions. The urea plus inorganic phosphorus resulted in the highest growth rates of Karenia, above that of either urea or phosphorus alone and with the lowest consumption of nitrogen. Among treatments, similar amounts of total fixed nitrogen were achieved with very different nitrogen uptakes due to the success of other taxonomic groups. In an interesting interaction between K. brevis and diatoms, the highest silica depletion was observed in the urea plus phosphorus treatment that also experienced the highest K. brevis growth rates. Additional combined treatments (N+Si, Urea+Si+P) should be investigated.

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ISOLATION AND CHARACTERIZATION OF A BREVENAL EPIMER FROM CULTURED KAREnIA bREvIS

Susan Niven1, Andrea Bourdelais1, Jen McCall1, Alysha Thompson1, Karl Jacocks1, Ned Martin2, Bill Abraham3, and Daniel Baden1

1Center for Marine Science, University of North Carolina at Wilmington, Wilmington NC 284092Department of Chemistry and Biochemistry, University of North Carolina at Wilmington, Wilmington NC 284033Division of Pulmonary and Critical Care Medicine, University of Miami at Mount Sinai Medical Center, Miami Beach, FL

Brevenal is a ladder frame polycyclic ether compound produced by Karenia brevis that was first described in 2004. Research has identified brevenal as a natural antagonist to the brevetoxins which can facilitate mucus clearance in mammals and block brevetoxin activity. Since its discovery, isolation methods for brevenal from K. brevis have been optimized to improve yield and increase purity. During the development of these methods, a second compound with a similar retention time and identical mass (MW=656.4) as brevenal was isolated. Comparison of 1H NMR spectra with brevenal showed large areas of similarity, but did not overlay exactly. A full suite of 1D and 2D NMR experiments were performed and the structure was elucidated. The new compounds was structurally similar to brevenal, but the chemical shifts of protons and carbons on the ladder frame were different starting at C18, and showed maximum differences in chemical shifts occurring at C27. Complete structure determination identified the new compound as an epimer of brevenal with different stereochemistry at C27. The other chemical shifts observed in the 1H NMR spectrum (C18-C26) are likely due to conformational changes induced by the different connectivity at C27. Molecular modeling comparisons of brevenal to epi-brevenal suggest that brevenal is in the lowest energy conformation with epi-brevenal at a slightly higher energy level.

Cell based, receptor binding and in vivo experiments have shown that the small difference in structure has a dramatic effect on the receptor binding to the brevenal and brevetoxin receptors. Epi-brevenal showed lower affinity for the brevenal receptor (Ki epi-brevenal = 2025 nM vs Ki brevenal = 146 nM ), however epi-brevenal had increased affinity for the brevetoxin receptor (Ki epi-brevenal = 3.1 nM vs Ki brevenal = 1133 nM). Both brevenal and epi-brevenal did not increase bronchoconstriction in sheep on their own and blocked brevetoxin induced bronchoconstriction significantly.

These results indicate that the stereochemistry at C27 is crucial for optimal affinity of brevenal to its receptor and suggest that the final ring closure is not enzymatic but chemical in nature, with the lowest energy conformer as the major product.

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SARE FATTY ACID AMIDES RESPONSIBLE FOR FISH MORTALITY DURING PRymnESIum PARvum BLOOMS?

Sean P. O’Mara1, Greg Southard2, Danielle Gutierrez1, and Paul V. Zimba1

1Texas A & M University – Corpus Christi, 6300 Ocean Drive, Corpus Christi, TX 78412, USA 2Texas Parks and Wildlife Department, Inland Fisheries-Analytical Services, 507 Staples Road, San Marcos, TX 78666, USA

The global frequency and distribution of harmful algal blooms has increased in recent decades. One species of concern is Prymnesium parvum - a microscopic, unicellular, flagellate from the phylum Haptophyceae, which has been associated with harmful algal blooms for nearly a century. The primary toxins produced by P. parvum have long been thought to be two metabolites, prymnesin 1 and 2. However, it was recently shown that toxic fatty acid amides (FAA) were undescribed toxic compounds produced by P. parvum. The purpose of this study will be to (1) determine a correlation of FAA concentration to P. parvum cell number using at least 5 strains isolated from sites distributed worldwide, and (2) perform fish dosing studies to determine the effect of pH on FAA toxicity. Fish dosing studies will be conducted in a full factorial design utilizing 2 FAA concentrations and 3 pH values. Unlike previous studies, preliminary findings suggest a strong correlation (r2=.87) between FAA concentration and cell number of field samples collected in Texas river systems, and a moderate correlation (r2=.33) between FAA concentration and ichthyotoxicity. A multiple regression analysis will be conducted to determine the environmental conditions (e.g. pH, salinity, temperature, etc.) that influence FAA concentration and ichthyotoxicity in Texas field samples. In addition, preliminary analysis of P. parvum strains supports previous findings that toxin production is reduced or halted during times of excess nutrients.

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PHOTOOxIDATION OF DOMOIC ACID

Sk Md Sazzad Hossain1, Jenna L. Logsdon1 Dionysios D. Dionysiou2, Kevin E. O’Shea1

1 Department of Chemistry and Biochemistry, Florida International University, Miami, FL, 33199, USA 2 Environmental Engineering and Science Program, University of Cincinnati, Cincinnati, OH 45221-0012, USA

Domoic acid, a structural analog of kainic acid, is a potent neurotoxin produced by the diatom species Pseudo-nitzschia during harmful algal blooms (HABs). Consumption of domoic acid tainted seafood leads to amnesic shellfish poisoning which causes short-term memory loss, brain damage and in severe cases, death. While photooxidation may play an important role in the transformation of domoic acid in the environment, the photooxidation of domoic acid has received little attention. In the presence of sunlight, oxygen and the colored pigments associated with HABs photodynamic action can lead to the formation of singlet oxygen and transformation of a variety of naturally occurring toxins. Domoic acid contains a number of functional groups, including a conjugated diene and carboxylic acids. Because of the high cost of domoic acid we have chosen to study the singlet oxygenation of sorbic acid, which also contains a conjugated diene and carboxylic acid functional groups as a model compound for domoic acid.

Domoic Acid Sorbic Acid

Our results demonstrate sorbic acid readily undergoes singlet oxygenation yielding a complex mixture of peroxide type products. We will report detailed product studies for the photooxidation of sorbic acid based on one and two-dimensional NMR techniques. The reaction kinetics and bimolecular rate constant for sorbic and domoic acids in aqueous media were evaluated using competition kinetics with furfuryl alcohol. The results of ongoing product studies and toxicity tests as a function of photooxidation of domoic acid will be reported.

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SBIOACCUMULATION OF BREVETOxINS AND MAJOR METABOLITES IN FILTER-FEEDING AND CARNIVOROUS MOLLUSKS ExPOSED TO NATURAL KAREnIA bREvIS HARMFUL ALGAL BLOOMS

R. Pierce1, M. Henry1, P. Blum1, R. Medvecky1, L. Flewelling2

1Mote Marine Laboratory, Sarasota, FL 34236, USA 2Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, 100 Eighth Avenue SE, St. Petersburg, FL 33701, USA

Harmful algal blooms (HABs) of the dinoflagellate Karenia brevis (Florida red tide) occur annually along the Florida Gulf coast. Neurotoxic compounds (brevetoxins, PbTxs) produced by K. brevis are ingested by filter-feeding mollusks as they filter K. brevis cells and toxins from the water. Human consumption of contaminated shellfish results in neurotoxic shellfish poisoning (NSP) that can cause serious neurological and gastrointestinal problems. Multiple cases of NSP have resulted from human consumption of various filter-feeding and well as carnivorous mollusks harvested non-commercially from unapproved areas. This study was undertaken to investigate the accumulation, transformation and persistence of brevetoxins and selected metabolites within various species of estuarine mollusks that are likely vectors of NSP. The most abundant metabolites observed to accumulate in filter-feeding mollusks (clams and oysters) were cysteine conjugates of brevetoxins. Parent brevetoxins produced by K. brevis (PbTx-1,-2,-3, etc.) were not detected in filter-feeding mollusks, except for low concentrations of PbTx-3. Filter-feeding mollusks retained the PbTx metabolites for several months after HAB termination, but did not retain NSP toxicity. The carnivorous mollusk (lightning whelk) exhibited NSP toxicity 6 months after HAB termination. Elevated concentrations of PbTx-3 and metabolites were still present when the study ended 7 months after HAB termination.

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GROWTH AND TOxIN PRODUCTION OF THE MIxOTROPHIC DINOFLAGELLATE ALExAndRIum CATEnELLA

Regina Radan1 and Raphael M. Kudela1

1University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA 95064 Despite the role of inorganic nutrients in maintaining toxic blooms of the dinoflagellate Alexandrium catenella, there is strong evidence for use of both particulate ingestion (mixotrophy) and direct utilization of organic compounds. A. catenella causes detrimental effects to humans, marine mammals, fish and seabirds from ingestion of contaminated fish or shellfish, and is routinely monitored in California because of the potential for negative impacts at low cell densities. Mixotrophy has the potential to provide A. catenella a competitive advantage over other phytoplankton when in nutrient-limited conditions. Another common strategy employed by dinoflagellates is to vertically migrate to acquire inorganic nutrients at depth. It is unclear whether A. catenella utilizes both strategies, and if so, whether these strategies are complementary, and how these nutritional modes might change growth and toxicity in California strains of A. catenella. Preliminary data has shown that cultures of A. catenella grown on a base medium of f/2 and then starved will ingest the haptophyte Isochrysis galbana. We will present results for growth, toxicity, and nutrient utilization using a laboratory-based vertical migration chamber to assess whether A. catenella exhibits a preference for vertical migration or mixotrophy when presented with both potential modes of nutrition. Cells in nutrient replete conditions should maintain faster growth rates then cells in nutrient limited conditions. If prey is present in nutrient-replete conditions A. catenella may ingest I. galbana as an additional nutrient source. However, in nutrient-limited conditions, I. galbana should allow A. catenella to maintain growth but won’t necessarily increase growth rate. Given the tradeoff between energy expenditure on vertical migration and mixotrophy, we expect there to be significant difference in cellular toxin quota due to changes in cellular stress and metabolic balance. These results will ultimately lead to a better understanding A. catenella growth dynamics and could be used to improve mechanistic models of growth and toxin production.

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SQUANTIFICATION OF DOMOIC AND OKADAIC ACID IN SHELLFISH AND ALGAE USING A LC/MS/MS (TRIPLE QUADRUPOLE) APPROACH

Carlton Rauschenberg1, Craig Burnell1 and Stephen Archer1

1 Bigelow Laboratory for Ocean Sciences

A series of test results are presented for the quantification of amnesic shellfish poisons (ASP) and diarrhetic shellfish poisons (DSP) in shellfish. The previously described LC/MS/MS approach (McNabb et al 2005 [M2005]) is practical for use in routine monitoring programs and the test results shown represent the initial steps towards single laboratory validation to quantify ASPs, DSPs and other lipophilic algal toxins in shellfish at Bigelow Laboratory. A comparison between the M2005 approach and traditional LC-UV approach is presented for domoic acid, addressing sensitivity and specificity of detection in shellfish matrix-fortified standards. The M2005 approach is applied to explore seasonal variations in both ASP and DSP content in natural shellfish populations in the Gulf of Maine.

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USE OF DISSOLVED INORGANIC AND ORGANIC NITROGEN BY THE TOxIC DINOFLAGELLATES, KAREnIA bREvIS AND KAREnIA mIKImOTOI (DINOPHYCEAE)

Bill Richardson

Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, 100 8th Avenue SE, Saint Petersburg, FL 33701, USA

Dissolved inorganic nitrogen (DIN) and dissolved organic nitrogen (DON) compounds are important sources of nitrogen (N) for large harmful blooms of Karenia brevis and Karenia mikimotoi. The ability to use different DIN and DON compounds as sole sources of N for growth was evaluated in the laboratory using two Karenia brevis clones (CCFWC 257, CCMP 2281) and a single Karenia mikimotoi clone (CCFWC 67) grown axenically and non-axenically on forty-nine different N-containing compounds. DIN compounds included NH4

+, NO3-, and NO2

- and DON compounds included amino acids, peptides, a protein, nucleic acids, nucleotides, purines, a polyamine, and an amino sugar. Both axenic and non-axenic cultures grew well on all DIN compounds, but differed in their ability to use DON. Axenic cultures of K. brevis and K. mikimotoi were unable to grow on any DON compound other than urea. In contrast, non-axenic cultures of K. brevis and K. mikimotoi grew well on urea and most of the other DON compounds. Among the N compounds that supported growth, no single compound provided exceptionally superior growth for either axenic or non-axenic Karenia cultures. Overall, growth was better on DIN compounds than on DON compounds. Differences in growth between the axenic and non-axenic cultures indicated that K. brevis and K. mikimotoi were able to directly use all DIN forms and urea for growth, while the use of other forms of DON for growth was conditional upon prior bacterial mineralization. The results suggest a potentially beneficial relationship in which N-rich components of the DON pool not directly accessible to either K. brevis or K. mikimotoi are mineralized by co-occurring bacterial communities into N forms that can then be used by these Karenia species to meet their cellular N growth demands. This relationship may become increasingly important for both Karenia species when DIN and urea are in short supply.

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STHE EFFECTS OF FIELD ExPOSURE OF CRASSOSTREA vIRGInICA TO KAREnIA bREvIS: HISTOPATHOLOGY AND BREVETOxIN ACCUMULATION IN GAMETES

Anne Rolton1,2, Julien Vignier1,2, Audrey Barbe1, Philippe Soudant2, Richard Pierce3, Sandra Shumway4, Monica Bricelj5 and Aswani Volety1.

1Florida Gulf Coast University, College of Arts and Sciences, 10501 FGCU Blvd South, Fort Myers, Florida 33965; 2Universitéde Bretagne Occidentale—IUEM, LEMAR CNRS UMR 6539, Place Nicolas Copernic, Technopôle Brest Iroise, 29280 Plouzané, France; 3Mote Marine Laboratory, 1600 Ken Thompson Parkway, Sarasota, FL 34236; 4Department of Marine Sciences, University of Connecticut, 1080 Shennecossett Road, Groton, CT 06340; 5Institute of Marine and Coastal Sciences, Rutgers University, 71 Dudley Road, New Brunswick, NJ 0890.

The most prevalent HAB species in the Gulf of Mexico is the red-tide dinoflagellate, Karenia brevis, a producer of lipophilic brevetoxins (PbTxs). As well as impacting commercially and ecologically important shellfish species, K. brevis is also a risk to human health, with consumption of contaminated shellfish leading to neurotoxic shellfish poisoning (NSP). The specific effects of K. brevis on shellfish reproduction remain poorly understood; however, it is known to cause recruitment failure in some shellfish species. The frequency of Karenia blooms is increasing and for certain shellfish species, periods of ripening/spawning overlap with red-tide blooms. It is therefore important to determine effects on reproduction and recruitment.

In Lee county, FL, the 2012-2013 red-tide began in September/October 2012 and has continued through April 2013. The linear distance of the bloom has extended beyond Lee county borders, about 100 miles from Lee county to Pinellas, FL. Throughout this time, the eastern oyster, Crassostrea virginica, which contributes markedly to the ecology of the region, has been exposed to the bloom during a time of gamete ripening, spawning and larval recruitment. Although C. virginica can accumulate brevetoxins, it is not known if there is maternal transfer of brevetoxin to the gametes. Previous laboratory exposures of C. virginica to cultured K. brevis have shown increased histopathological lesions in exposed oysters.

In this study, ripe adult eastern oysters were collected from a site exposed to K. brevis (Iona cove, Caloosahatchee river, FL) and a site 200 miles north with no previous exposure to K. brevis (Shellfish Harvesting Area 3402, Cedar key, FL). Oysters from exposed and non-exposed sites were examined for histopathology and brevetoxin content was assessed on stripped, filtered and concentrated gametes using LC-MS/MS analysis.

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KAREnIA bREvIS REFERENCE TRANSCRIPOME ASSEMBLY AND TRANSMEMBRANE PROTEIN SEQUENCE DISCOVERY

Darcie Ryan1 and Lisa Campbell1

1Texas A&M University, 3146 TAMU Dept. Oceanography, College Station, TX 77843

Karenia brevis, a brevetoxin-producing dinoflagellate, blooms nearly annually in the Gulf of Mexico and is linked to marine animal mortalities and human health threats. Though the biological function of brevetoxins is unknown, elevated brevetoxin levels in K. brevis cells have been measured during laboratory osmotic stress treatments. To investigate the mechanisms underlying K. brevis toxin production, we assembled reference transcriptomes for three clones: Wilson CCFWC268, SP3, and SP1, a low toxin producing variant. These are among the first dinoflagellate transcriptomes assembled. Each contained ≈90,000 contigs.

Complete core eukaryotic genes were identified in the transcriptomes to assess completeness of assembly without a reference genome, and approximately 40% of the contigs produced a significant hit (E value < 1.00E-6) with a sequence in the NCBI non-redundant (nr) protein database with a BLASTX search. Of the non-hits, more than 85% contained a predicted complete open reading frame (ORF) that was ≥ 100 amino acids long. These contigs represent potential unique K. brevis proteins.

Putative aquaporin and voltage-gated Na+, Ca2+, or K+ channel sequences were discovered in the transcriptomes using a variety of techniques, including conserved domain searches and transmembrane region prediction. The putative aquaporin sequence was 405 aa long and highly conserved among clones. The sequence very significantly (E value < 1.00E-20) matched over 70 aquaporin or potential aquaporin entries in the nr database. However, the top three hits were hypothetical proteins from Aureococcus anophagefferen, Emiliania huxleyi, and Guillardia theta. This indicates that similar major intrinsic proteins are present in algae other than K. brevis, but have not yet been annotated.

Of the 32 putative ion channels, 16 were Na+ channels, 4 were Ca2+ channels, and 12 were K+ channels, based on BLASTx top hit results and conserved amino acid motifs. They were identified with strict search criteria: each first hit one or more proteins in a custom multi-species voltage-gated Na+ channel α subunit database, contained a complete ORF and six predicted transmembrane regions, and finally significantly (E value < 1.00E-6) contained one of the Pfam Ion_trans families during a conserved domain search. The 32 putative ion channel sequences therefore represent a conservative set that was chosen based on similarity to annotated voltage-gated Na+ channel units.

The identification of ion channels and aquaporins suggests that the concentrations of ions and water within K. brevis cells are regulated by transmembrane proteins. Further, K. brevis osmoacclimatory and osmoregulatory processes may be sensitive to voltage gradients.

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SBIOFOULING TUNICATES ON AQUACULTURE GEAR AS POTENTIAL VECTORS OF HARMFUL ALGAL INTRODUCTIONS

M. Rosa1, S.E. Shumway1, B.A.Holohan1, S.G. Bullard2, G.H. Wikfors3, S. Morton4, and T. Getchis5

1Department of Marine Sciences, University of Connecticut, 1080 Shennecosset Road, Groton, CT, 06340 USA 2University of Hartford, Hillyer College, 200 Bloomfield Ave., West Hartford, CT 06117, USA 3NOAA, National Marine Fisheries Service, Northeast Fisheries Science Center, 212 Rogers Avenue, Milford, CT 06460, USA 4NOAA, National Ocean Service, Marine Biotoxins Program, 219 Fort Johnson Rd., Charleston, SC 29412, USA 5CT Sea Grant College Program, Department of Marine Sciences, University of Connecticut, 1080 Shennecosset Road, Groton, CT 06340, USA

Biofouling ascidians are ubiquitous in coastal ecosystems and are among the main colonizers of aquaculture gear. Our study tested the hypothesis that the transport, removal, and transfer of fouling ascidian species by aquaculturists provide a mechanism for concentration and distribution of harmful-algal cells to new areas. Wild-caught specimens of common, biofouling ascidian species (Styela clava, Ciona intestinalis, Molgula manhattensis, Bottryloides violaceus, Didemnum vexillum, and Botryllus schlosseri) were exposed individually to cultured strains of co-occuring harmful algae (Prorocentrum minimum, Alexandrium fundyense, Alexandrium monilatum, Karenia brevis, Aureococcus anophagefferens, or Heterosigma akashiwo) at simulated bloom cell densities of each HAB species. After feeding, ascidians were transferred to ultrafiltered seawater. Immediately after exposure, and after 24 and 48 h in ultrafiltered seawater, biodeposits were collected and observed microscopically for the presence of intact, potentially-viable cells. Subsamples of biodeposits were transferred into culture tubes with ultrafiltered seawater and monitored for algal growth during 8 weeks. Cells of all HAB species were found to pass intact through the ascidian digestive system, remained viable, and in many cases were capable of re-establishing populations at least 48 h post-ingestion. The results of our study will inform industry and managers as to the potential threat and ecological impact of spreading biofouling ascidians, and practices to mitigate adverse impacts. Additionally, these management practices have been formally incorporated into a new cost-share program developed to help shellfish producers implement management practices to prevent the further spread of ascidians and associated HAB species.

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THE INFLUENCE OF NUTRIENT AND LIGHT PERTURBATIONS ON NATURAL PHYTOPLANKTON ASSEMBLAGES CONTAINING THE TOxIN PRODUCING DIATOM PSEudO-nITZSCHIA

Jayme Smith1, Alyssa Gellene1, Paige Conne1l1, Victoria Campbell1, Erica Seubert1 and David A. Caron1

1 University of Southern California, 3616 Trousdale Parkway, AHF 301, Los Angeles, California 90089, USA

In March 2013, a multi-institutional ECOHAB field study was organized to characterize the physical and biological parameters that are thought to influence bloom formation of the toxin-producing diatom, Pseudo-nitszchia in the Southern California Bight. Pseudo-nitszchia blooms now occur with some regularity in the springtime in this region, an observation that informed the timing of the field study. The principal hypothesis of this study was that seed populations of Pseudo-nitszchia cells are advected from a subsurface deep chlorophyll maximum to surface waters during upwelling events, leading to a toxic event. Environmental parameters leading up to a bloom were characterized, via ship based sampling and remote sensing.

In addition to field sampling efforts, three community incubation experiments were conducted to test the effects of irradiance and nutrient availability on the growth and toxin production of Pseudo-nitzschia cells presents in communities collected at different time points. Each experiment was incubated for six days in ambient light and temperature with a suite of samples collected, including samples for particulate domoic acid, chlorophyll a, and preserved whole seawater at five discrete time points.

Incubations 1 and 2 were conducted with seawater collected at the deep chlorophyll maxima prior to obvious environmental indicators of a bloom event. A third incubation was conducted with surface water collected during a minor Pseudo-nitszchia bloom event as indicated by a regional shore based sampling effort. In each experiment, communities were incubated with an array of nutrient and vitamin treatments, including treatments with nitrate, ammonia, urea, and high and low light levels. Chlorophyll a values indicate a general increase in biomass for incubations 1 and 2 with particular growth observed in treatments containing nitrogen and vitamins. Incubation 3 showed a general decrease in biomass except in the treatment containing vitamins. Overall particulate domoic acid increased incrementally from below detectable levels for all treatments in incubation 1. Domoic acid per cell in incubation 1 increased marginally through the duration of the experiment with the increase occurring most obviously in the high light urea treatment. A difference in domoic acid per cell in high light and low light urea treatments was also observed. Domoic acid production remained below detection in incubation 2, despite the stimulation of Pseudo-nitzschia cell grow in some of the treatments. Domoic acid was down regulated in all treatments of incubation 3 despite measurable domoic acid per cell concentrations at the beginning of the experiment.

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SUNPRECEDENTED mICROCySTIS BLOOM IN NORTHERN CALIFORNIA’S BAY-DELTA ESTUARY AND POSSIBLE IMPACT ON ZOOPLANKTON ABUNDANCE

William T Stringfellow1,3, Chelsea L Spier1, Jeremy Hanlon1,3, Mark Brunell1,2, Monica Estiandan1,2, Teemu Koski4, and Juha Kääriä4

1Ecological Engineering Research Program, School of Engineering & Computer Science, University of the Pacific, 3601 Pacific Avenue, Stockton, CA 95211 2Department of Biological Sciences, University of the Pacific, 3601 Pacific Avenue, Stockton, CA 95211 3Lawrence Berkeley National Laboratory Earth Sciences Division, 1 Cyclotron Road, Berkeley, CA 94720 4Turku University of Applied Sciences, Sepänkatu 1, Fin-20700 Turku, Finland

California’s Sacramento-San Joaquin Delta is an expansive fresh-water tidal estuary that is part of the San Francisco Estuary, the largest estuary on the US Pacific coast. Recently, there has been a collapse in the pelagic fish community in the San Francisco Estuary, known as the pelagic organism decline (POD). Blooms of the cyanobacteria Microcystis, which often produce the cyanotoxin microcystin, were first documented in the Sacramento-San Joaquin Delta in 1999. Cyanotoxins have been suspected as one contributing factor to POD. It has been proposed that microcystin could be contributing to POD directly though poisoning of fish or indirectly by affecting zooplankton and other food sources. In this study, we documented the spatial and temporal extent of cyanobacteria blooms in the Southern Bay-Delta Estuary (south Delta), an area further east and upstream in the San Francisco Estuary than previously studied. Copepod, rotifer and total zooplankton abundance was examined in comparison to the spatial distribution of microcystin. The environmental factors associated with cyanobacteria blooms in the south Delta were investigated. Microcystis and other cyanobacteria were common in the south Delta and a large, persistent Microcystis bloom was observed in the summer of 2012, but not in 2009 or 2011. There was a strong relationship between microcystin and cyanobacteria biomass (r2=0.74). In July and August of 2012, microcystin concentrations were measured above California EPA recreational advisory limits (0.8 µg/L) and World Health Organization drinking water limits (1.0 µg/L), with a maximum observed concentration of 2.1 µg/L. In previous years, maximum observed microcystin concentrations were below 0.1 µg/L in this study and in studies by others (2004, 2005, 2007, and 2008). This bloom was associated significantly with lower density and mass of zooplankton, including species which are important to a functioning food-web in the Delta. We compared water quality, flow and climate conditions of 2012, when the persistent bloom occurred, to 2009 and 2011. Significantly higher phosphorus concentrations and temperatures occurred in 2012 corresponding to higher cyanotoxins concentrations, and timing of Spring flows differed in 2012 compared 2009, both low flow years, but cause and effect cannot yet be established. The implications of these results in the context of the POD and climate change will be discussed.

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EPOxIDE HYDROLASES IN KAREnIA bREvIS: KEY ENZYMES IN BREVETOxIN BIOSYNTHESIS

Pengfei Sun and Kathleen S. Rein


Epoxide hydrolases have been proposed to be key enzymes in the biosynthesis of polyether ladder (PE) compounds such as the brevetoxins. These enzymes have the potential to catalyze kinetically disfavored endo-tet cyclization reactions. As such, they could have enourmous potential if applied toward semi-enzymatic syntheses. An epoxide hydrolase which was identified in a Karenia brevis EST library has been cloned and expressed for characterization. Kinetic parameters were evaluated using a variety of epoxide substrates to assess substrate selectivity and enantioselectivity as well as its potential to catalyze the critical endo-tet cyclization of epoxy alcohols.

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SCOMPARING THE DIEL VERTICAL MIGRATIONS OF SYMPATRIC KARLOdInIum vEnEFICum (DINOPHYCEAE) AND CHATTOnELLA SubSALSA (RAPHIDOPHYCEAE) IN LABORATORY COLUMNS

Charles L. Tilney1 and Mark E. Warner1

1 University of Delaware, 700 Pilottown Road, Lewes, DE 19958, USA

In a wide range of organisms, adaptation to the daily rise and fall of the sun has led to diel rhythms in physiology and behavior. Diel rhythms, particularly those controlled by molecular clocks (=endogenous circadian rhythms), are thought to enhance an individual’s fitness by allowing organisms to utilize and preempt, cyclical changes (temporal and spatial) in the biotic and abiotic environment. Factors such as light and temperature vary predictably throughout the day, and in marine environments, factors such as: light, temperature, nutrients, predators, and competitors can also be vertically segregated in the water column. Many motile marine plankton have adapted to these temporal and spatial gradients by performing diel vertical migrations (DVM) in addition to exhibiting diel rhythms in photosynthesis and other metabolic processes. Here we compare the diel rhythms in vertical migration and photophysiology in a mixotrophic dinoflagellate (Karlodinium veneficum) and a mixotrophic raphidophyte (Chattonella subsalsa) in a variety of artificial water column conditions. No comprehensive assessment of DVM has yet been published for K. veneficum, and the comparison with a sympatric raphidophyte may yield clues about these species’ relative reliance on DVM, and on conditions conducive to affording a competitive advantage to one or the other. Water columns were held in transparent acrylic columns (1.8m tall, holding 31L), each illuminated from above by ramped-intensity LEDs. In-vivo chlorophyll fluorescence was used to monitor the vertical distribution of cells, and active chlorophyll fluorescence was used to assess PSII functionality with water column depth. Measurements were made every 3h for 39h at 11 depths. In nutrient replete columns under 14:10 L:D cycles, both species underwent diel vertical migrations, rising toward the surface during the daytime, and receding to depth at night. During the day, C. subsalsa showed a single well-defined near-surface peak, whereas K. veneficum migrated to a bi-modal distribution with 1 broad mid-depth peak, and 1 narrow surface peak. Migration to the surface or near-surface in both species resulted in reductions in the maximum quantum yield of PSII (Fv/Fm), likely due to photoprotective mechanisms or photodamage. K. veneficum exhibited larger declines in Fv/Fm, that occurred earlier in the day than C. subsalsa, perhaps explaining the formation of a secondary mid-depth biomass peak. Following several days of L:D cycles, columns were shifted into continual darkness (D:D). Diel and depth dependent periodicity of photochemistry noted under L:D cycles, was immediately lost under continuous darkness in both species, wherein K. veneficum photochemistry remained at the pre-dawn minimum level, while C. subsalsa photochemistry matched night-time values. Furthermore, DVM in K. veneficum ceased in constant darkness, while C. subsalsa continued DVM, suggesting that DVM is under endogenous circadian control in C. subsalsa, but not in K. veneficum. The effect of continuous light and nutriclines will be presented, with an assessment of the relative gains in carbon fixation from DVM in both species.

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ENVIRONMENTAL FACTORS AFFECTING THE RELEASE OF DOPAMINE AND OxIDANTS BY THE BLOOM-FORMING GREEN SEAWEED uLvARIA ObSCuRA

Kathryn L. Van Alstyne, Katie J. Anderson, and Daniel H. van Hees

Shannon Point Marine Center, Western Washington University, 1900 Shannon Point Road, Anacortes, WA, USA

In Washington State, ulvoid macroalgae can form large blooms that have detrimental effects on other organisms in the vicinity. A prominent component of these blooms is the seaweed Ulvaria obscura, which grows in the lower intertidal and subtidal zones. During spring tides, Ulvaria can be stranded on the shore during low tide were it may be subjected to several stresses, including desiccation, high light levels, and high temperatures. This alga can release a variety of compounds including dopamine and oxidants, which are known to be toxic to other marine organisms. Using a series of experiments that simulated conditions that might be encountered by low intertidal algae, we quantified the release of dopamine and oxidants. Dopamine was released when Ulvaria was desiccated then rehydrated, but was not released in response to high water temperatures or different light conditions. It also was not released in response to the presence of dopamine or exudates from other individuals. Oxidant release was significantly lower in desiccated than undesiccated plants, regardless of the duration of desiccation. This contrasts with results from another bloom-forming ulvoid alga, Ulva lactuca, in which similar amounts of oxidants were released by undesiccated plants and plants that had been desiccated for 1, 2, or 4 hours prior to rehydration. Our results demonstrate that that the release of toxins by Ulvaria is affected by desiccation at low tides in a species-specific manner.

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STHE DEVELOPMENT AND VALIDATION OF A MULTIPLEx QPCR ASSAY FOR THE DETECTION OF TOxIN PRODUCING CYANOBACTERIA

Mark Van Asten1 Brett Neilan2 Jamal Al-Tebrineh2

1 Diagnostic Technology Pty Ltd, Suite 45, 7 Narabang Way, Belrose, NSW, 2085, Australia2 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney NSW 2052, Australia

We now have a good understanding of the genetic basis for toxin production by a number of groups of microorganisms. The discovery of these toxin biosynthetic pathways has enabled the development of genetic screening of environmental samples.

Cyanotoxins pose a direct threat to public health. Molecular genetics underlying cyanotoxin production in fresh and brackish water environments, but more specifically, the toxin biosynthesis genes have been used to develop a multiplex quantitative PCR assay for the detection and quantitation of micocystin, nodularin, cylindrospermopsin and saxitoxin producing genes. Along with the copy number of the relevant cyanotoxin biosynthesis gene an internal cyanobacteria-specific 16S rDNA control target is used as a reference target. Validation and test data of the assay will be presented along with details of the development of standards to each of the relevant target toxin genes to which these sequences are quantitated against.

This multiplex quantitative PCR assay should become a very important addition to the resources available to laboratories and authorities for better surveillance, detection, prediction and monitoring of harmful algal blooms.

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IMMUNE FUNCTION IN RESCUED MANATEES ExPOSED TO BREVETOxINS

Catherine J. Walsha, Jennifer E. Yordya, Matthew B. A. Butawana, Courtney L. Bennetta, Leanne Flewellingb, Ray Ballc, Martine De Witb, Katarina Englisha, Robert K. Bonded

aMarine Immunology Program, Mote Marine Laboratory, Sarasota, FL, USA bFish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission, St. Petersburg, FL, USA cLowry Park Zoo, Tampa, FL, USAdSirenia Project, Southeast Ecological Science Center, United States Geological Survey, Gainesville, FL, USA

The health of many Florida manatees (Trichechus manatus latirostris) is adversely affected by exposure to blooms of the toxic dinoflagellate, Karenia brevis. K. brevis blooms are common in manatee habitat areas off the southwestern coast of Florida and produce a group of cyclic polyether toxins collectively referred to as red tide toxins, or brevetoxins. Although the majority of manatees exposed to significant levels of red tide toxins die, many manatees are also rescued as a result of sublethal exposure and are successfully treated and returned to the wild. Sublethal exposure to brevetoxins may potentially impact the manatee immune system. Lymphocyte proliferative responses and a suite of immune function parameters in the plasma were used to evaluate effects of brevetoxin exposure on manatee health in manatees rescued as a result of natural exposure in their habitat. Healthy, unexposed manatees from Crystal River, FL, were used for comparison of immune function parameters. Peripheral blood leukocytes (PBL) isolated from whole blood of rescued manatees were stimulated with T-cell mitogens, ConA and PHA. Significant decreases (P < 0.05) in lymphocyte proliferation were observed in ConA or PHA stimulated lymphocytes collected from rescued animals compared to unexposed animals. A suite of plasma parameters, including plasma protein electrophoresis profiles, lysozyme activity, superoxide dismutase activity, and reactive oxygen/nitrogen species, were also used to assess manatee health. A significant correlation between plasma brevetoxin concentration and plasma ROS/RNS concentrations was observed. No significant correlations were observed among other immune function parameters measured in manatee plasma. To summarize, manatees rescued from red tide toxin exposure demonstrate reduced lymphocyte proliferation responses compared to healthy free-ranging manatees not exposed to brevetoxins. A potential for increased oxidative stress in rescued manatees exists, with a positive correlation between ROS/RNS activity and brevetoxin levels in the plasma. Sublethal exposure to brevetoxins in the wild has the potential to impact immune function, and thus overall health, in the Florida manatee.

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SMONITORING NUTRIENTS IN SODUS BAY NEW YORK

Samantha Weber1 and Gregory L. Boyer2

1State University of New York – Environmental Science and Forestry School, 1 Forestry Drive, NY 132102 State University of New York – Environmental Science and Forestry School, 1 Forestry Drive, NY 13210

Located on the southern shore of Lake Ontario, Sodus Bay is an ecologically diverse watershed that is also a popular tourist destination during the summer months. Blue-green algal blooms are naturally occurring in Sodus Bay, but beginning in 2010 toxin-producing harmful algal blooms began occurring in concentrations high enough to cause closures in some parts of the Bay. These blooms are likely to be driven by phosphorus entering the Bay through the surrounding creeks and watershed. To help determine the phosphorus loads to Sodus Bay from Sodus Creek East (the main creek entering Sodus Bay), a continuous soluble reactive phosphorus monitoring system (WET Labs Cycle-P) will be deployed just before the creek enters the Bay. This system should allow us to determine the episodic phosphorus concentrations entering the bay from the creek. This information will help us identify potential management options that may help reduce the intensity and occurrence of these algal blooms in the future.

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ASSESSING THE STRENGTHS AND WEAKNESSES OF EACH FLORIDA HAB REPORTING SYSTEM

Ben Whitenack1,2, Kate Kohler1, and Barbara Kirkpatrick1,3,4

1Mote Marine Laboratory, Sarasota, FL2University of Pennsylvania, Philadelphia, PA3Dept of Public Health Studies, Miller School of Medicine, Univ of Miami, Miami, FL4Rosenstiel School of Marine and Atmospheric Sciences, Univ of Miami, Miami, FL

The 2012-2013 Karenia brevis bloom impacting the west coast of Florida started in late September, with approximately six months of human health impacts at the beach Also in the Fall of 2013, he National Weather Service (NWS) instituted their new Harmful Algal Bloom (HAB) reporting system, a “beach hazard statement”. The beach hazards statements are pushed directly to various public instant weather alerting systems. With this new alert system, there are now five HAB reporting systems in operation that cover the Gulf coast of Florida; the Mote Marine Laboratory (MML) Beach Conditions Reporting System, the Florida Fish and Wildlife Conservation Commission/Fish and Wildlife Research Institute’s (FWC/FWRI) Red Tide status report, the National Oceanic and Atmospheric Administration’s (NOAA) HAB Bulletin, and Florida Department of Health in Sarasota County’s Healthy Beaches reports

A mixed method approach to assessing the Florida HAB reporting systems was used to measure the intended usage of these five systems. A qualitative approach using interviews of the administrators and creators of each of the systems was used to understand the history of the system; the intended and actual audience; evaluations; and desired prospective improvements. The quantitative method followed, comparing Sarasota lifeguarded beach visitor counts at six beaches with the Karenia cell counts, the usage numbers of the various HAB systems and temperature.

Conclusions support a need for better evaluations from the user groups as well as the intended stakeholders of each HAB reporting system in order to identify desired prospective improvements to each system. Additionally, in order to increase the marketability and usage of all of the systems, as all five systems collaborate with each other, messaging of the benefits and weaknesses of each system needs more prominent attention.

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SROLE OF ROS SIGNALING IN THE DOMOIC ACID BIOSYNTHETIC PATHWAY

April L. Woods1 and G. Jason Smith1

1Moss Landing Marine Labs, 8272 Moss Landing Rd Moss Landing, CA 95039

This study investigates the role of cellular chemical signaling during initiation of Domoic Acid (DA) biosynthesis in the HAB forming diatom Pseudo nitzschia. Environmental parameters promoting DA production include silica limitation, source of nitrogen supply and the availability of redox active metals (particularly iron and copper). This study explores the role of nutrient stress and metal stress in the generation of Reactive Oxygen Species (ROS), both biotically (intracellular) and abiotically (extracellular). When present at levels higher than can be neutralized by cellular machinery, ROS cause damage to cells; sometimes resulting in cell death. Conversely, low levels of ROS operate as a normal part of healthy cell signaling, cueing the cell to generate a response.

An assay has been optimized to quantify the ROS generating potential of the cell free environmental sample based on dithiothrietol oxidation. This assay is employed to evaluate the correlation of ambient ROS activity with DA accumulation and potential mitigation effects of DA on ROS active compounds. Profiles of ROS generating potential in natural seawater, assessed from cruises taken off the Southern CA Bight and the Monterey Peninsula conducted in Spring and Fall of 2013, respectively will be presented. Variation in intracellular (pDA) vs extracellular (dDA) pools will be evaluated with respect to patterns of ROS signaling observed. The ROS dependent fluorescence probe Carboxyl-H2DCFDA (CHDCFDA), evaluated by flow cytometry, is used to elucidate the magnitude and dynamics of intracellular ROS signaling during stress challenge. Preliminary results using fluorescence show that an immediate ROS burst is issued upon challenge with a ROS generating reagent, but little ROS signal is seen if the culture is first allowed to acclimate to the challenge before addition of the probe. Taken together, patterns of response to extra and intracellular ROS challenges will help elucidate the role of ROS signaling in regulation of DA biosynthesis by Pseudo-nitzschia.

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DETERMINATION OF PHYTOPLANKTON GROUPS USING TURNER DESIGNS’ PHYTOFIND

Lawrence Younan

Turner Designs Turner Designs, Inc., 845 W. Maude Ave., Sunnyvale, CA 94085 USA

Turner Designs’ PhytoFind is configured with multiple sensors, each with different excitation/emission wavelengths designed to distinguish among phytoplankton groups using group specific fluorescence characteristics. Preliminary lab data show positive results for differentiating among phytoplankton groups, which is desirable for monitoring efforts of most all aquatic habitats because it provides researchers with valuable data that can be used for: predicting algal blooms, determining whether phytoplankton communities have the potential to be harmful, monitoring algal stock used for feeding aquaculture farms, detailed estimates of productivity and system turnover rates. The problem with this type of measurement are interference factors, primarily turbidity and dissolved organic material, that convolute the fluorescence response from various algal groups making it difficult to accurately determine percent group contribution in a mixed phytoplankton community. Empirically derived correction factors used for correcting such interferences are disadvantageous because they don’t encompass the various types of materials that exist, both as dissolved organics and suspended sediments. The PhytoFind uses additional sensors configured to measure scattering and dissolved organic materials, along with algal fluorescence, simultaneously. This results in the generation of a correction factor per data point, increasing accuracy of measurement greatly. The PhytoFind’s ability to accurately determine percent algal group contribution and provide phytoplankton concentration estimates makes it a powerful research tool for monitoring or characterizing algae in any type of system.

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DEVELOPMENT OF CAPILLARY ELECTROPHORESIS AS PART OF A MICRO-TOTAL ANALYTICAL SYSTEM FOR MICROCYSTINS

Bingxue Zheng, Bruce McCord and John P. Berry

Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA

The presence of “harmful algal blooms” (HAB) is a growing concern in aquatic environments worldwide. Among HAB organisms, cyanobacteria are of special concern because they have been reported worldwide to cause environmental and human health problem through contamination of drinking water, and possibly bioaccumulation in freshwater food-webs. Although several analytical approaches have been applied to monitoring cyanobacterial toxins, conventional methods (e.g. ELISA, HPLC-MS) are both costly and time-consuming such that analyses take weeks for field sampling and subsequent lab analysis. The results obtained from the field sample, thus, reflect the outdated data, but not the current status. Deployable micro total analytical system (µTAS) for in situ detection of bloom toxins is emerging as a promising approach to monitor these toxins. As part of on-going research, a deployable µTAS for real-time and continuous detection of cyanotoxins, and specifically the microcystin (MC) family, is currently being developed. Toward this end, capillary electrophoresis (CE) is a particularly suitable method of analytical separation that can couple very small samples (~1 nL) and rapid separations (≤2-5 min) to a wide range of selective and sensitive detection techniques (e.g. UV, LIF, MS, ECD). Moreover, with respect to this on-going research, CE is scalable to existing µTAS instrumentation, particularly including so-called “lab-on-a-chip” (LOC) platforms. We will present current research into the optimization of CE-based analysis of MCs, in relation to the future adaptation to previously developed LOC-based µTAS, including investigation of analytical separation of MC variants (i.e. modifications of buffer/solvent and pseudo-stationary phases) and subsequent detection (e.g. highly sensitive laser-induced fluorescence versus UV detection).

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aAbbott, Margaret _____________________ 45, 72Abraham, Ann __________________________ 124 Abraham, Bill ___________________________ 175 Adams, Jillian __________________________ 110 Adams, Rachel M ________________________ 78 Allen, Allison L. __________________________ 86 Al-Tebrineh, Jamal ______________________ 190Anderson, Clarissa R. _________________ 31, 163 Anderson, Donald M. ____________ 36, 39, 52, 59, 61, 65, 106, 110, 120, 140Anderson, Katie J. _______________________ 189 Anderson, Paul E. _______________________ 145 Anglin, Scott B __________________________ 125Archer, Stephen ________________________ 180Ardjmand, Ehsan _________________________ 64Asong, Jinkeng ___________________________ 39Atkinson, Joseph _________________________ 35Austen, Melanie ________________________ 141Avila, Christian _________________________ 135

BBachvaroff, Tsvetan R. ________________ 69, 158Baden, Daniel G_________________ 127, 137, 144, 146, 147, 154, 171, 173, 175 Bailey, Trevor __________________________ 142Baker, David _____________________________ 80Ball, Ray ______________________________ 191Banas, Neil S. _________________________ 49, 65Barbe, Audrey _________________________ 182Barbieri, Michelle ________________________ 71Bargu, Sibel _____________________________ 77Barron, Heather W. _______________________ 32Bartleson, Richard D. __________________ 32, 33Beck, Sheila ___________________________ 141Becker, Richard __________________________ 35Beet, Andrew ______________________ 108, 153Belisle, B. Shafer _________________________ 78Bennett, Courtney L _________________ 131, 191Berry, Dianna L. __________________________ 56Berry, John P _________________ 34, 54, 116, 157, 168, 196Bianco, Colleen __________________________ 37Bill, Brian D __________________________ 46, 65Blair, Stephen __________________________ 135Bloomfield, Dan ________________________ 142

Blum, Patricia __________________________ 178Boase, Nick ____________________________ 126Bonde, Robert K. _______________________ 191Borchert , Jerry __________________________ 46Bossart, Gregory ________________________ 131Bottein, Marie-Yasmine ____________________ 71 Bourbonniere, Richard A ___________________ 78Bourdelais, Andrea ____________ 41, 89, 127, 137,

144, 146, 147, 154, 171, 173, 175Bowers, Holly A. __________________ 73, 87, 115Boyer, Gregory L ______________ 35, 78, 105, 107,

112, 113, 192Boyes, A.J. _____________________________ 128Brame, Julie ___________________________ 103Brand, Larry ________________________ 168, 170Brandt, Ashley _________________________ 138Bricelj, Monica _________________________ 182Briggs, Travis _______________________ 119, 136Bronk, Deborah __________________________ 47Brooks, Christopher _____________________ 103Brosnahan, Michael L. _________________ 36, 106Brunell, Mark __________________________ 186Bullard, S.G. ____________________________ 184Bumpus, Liz ____________________________ 76Burks, Robert ______________________ 119, 136Burnell, Craig __________________________ 180Burson, Amanda ________________________ 129Butawan, Matthew B.A ___________________ 191Byrne, Jon ______________________________ 57Byrne, Margaret M ____________ 79, 88, 161, 162

CCampagna, Shawn R. _____________________ 78Campbell, Katrina ________________________ 39Campbell, Lisa _____________ 41, 48, 89, 118, 183Campbell, Victoria ______________________ 185Canfield, Dan __________________________ 119Carmichael, Wayne ______________________ 53Caron, David A. ___________________ 51, 75, 185Casamatta, Dale ________________________ 156Cassell, Ryan ________________________ 90, 130Chao, Yi ________________________________ 51Charniga, Camia M. _____________________ 174Chauan, Vinita S. _______________________ 127Chen, Fei ______________________________ 118Chen, Wei _____________________________ 130Choi, C.J. ______________________________ 139

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Christman, Mary C _______________________ 91Cichowska, Anna _______________________ 142Clark, Quintin ___________________________ 76Clement, Amy ___________________________ 95Clemente, Jennifer _______________________ 76Cocilova, Courtney ______________________ 131Cody, Theresa ___________________________ 99Collado-Vides, Ligia _____________________ 135Connell, Laurie __________________________ 68Connell, Paige ___________________________ 75Connolly, Thomas ________________________ 49Corcoran, Alina _________________________ 172Cox, Annie _____________________________ 94Coyne, Kathryn J _______________ 37, 70, 83, 102Cray, Carolyn ____________________________ 32Crespo, Bibiana G. ______________________ 110Crump, Byron C. _________________________ 63Currier, Robert _________________________ 149

dDaly, Kendra L. __________________________ 92Darrow, Brian P. ________________________ 164Davis, Edward ___________________ 98, 132, 133Davis, Kristen ____________________________ 49de Bruijne, Wilco _______________________ 129De Wit, Martine ________________________ 191Dearth, Stephen P. _______________________ 78Deeds, Jonathan R. _______________________ 38Delwiche, Charles F. ______________________ 69Depledge, Michael H _________________ 141, 142Derner, Katherine ____________________ 98, 133Dickey, Robert W ________________________ 124Dill, Brian D. ____________________________ 78Dionysiou, Dionysios D ________________ 97, 177Disney, Jane _____________________________ 52Dixon, L. Kellie _______________ 47, 121, 134, 149,

164, 174Doll, Cameron ___________________________ 37Dou, Natalie ___________________________ 135Doucette, Gregory J ________________ 39, 73, 87Dreher, Theo W. _________________________ 66Dupuy, Danielle ____________________ 119, 136

eEberhart, Bich-Thuy L. _____________________ 46Edwards, Christopher A ___________________ 31

Effler, T. Chad ___________________________ 78El Said, Kathleen R ______________________ 124Eldred, K. C. _____________________________ 65Elliott, Christopher T. ______________________ 39Elliott, Elizabeth A ___________ 127, 137, 144, 173Ellsworth, Amanda ______________________ 138Engene, Niclas ___________________________ 40English, Katarina ________________________ 191Erdner, Deana L. ____________________ 110, 139Errera, Reagan M. _____________________ 41, 89Estenik, John F. _________________________ 113Estiandan, Monica ______________________ 186

fFahnenstiel, Gary L. ______________________ 64Fahnenstiel, John A. ______________________ 64Faris, Jeremy ___________________________ 108Fernandez, Facundo M. ___________________ 57Fine, Isaac ______________________________ 49Fischer, Alexis D. ________________________ 140Fleming, Lora E ______________ 50, 126, 141, 142,

153, 161 Flewelling, Leanne J __________ 52, 103, 117, 131, 150, 178, 191Flores Quintana, Harold A ____________ 52, 59, 72,

143Foreman, Michael ________________________ 49Fortuin, Anne __________________________ 129Fowler, Nicholas ____________________ 144, 154Frame, Elizabeth _________________________ 58Frenzel, Hartmut _________________________ 51Fridey, Kelly A. _________________________ 145Fries, David _____________________________ 93Fu, Feixue _____________________________ 117Fulton, Rolland _____________________ 119, 136Fuquay, Jennifer _________________________ 71

gGantar, Miroslav __________________ 34, 54, 157Gao, Yonghui ____________________________ 74Garcia, Ana C. ________________________ 59, 72Garrett, Matthew J. ___________________ 47, 92Garrison, B. ____________________________ 166Gaze, William __________________________ 126Geier, Susan _____________________________ 49Gellene, Alyssa G. _________________ 51, 75, 185

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Gerssen, Arjen _________________________ 129Getchis, T. _____________________________ 184Gibble, Corinne M. _______________________ 42Gibbs, Patrick D. L. ____________________ 34, 54Giddings, Sarah _________________________ 49Gilmore, K. ____________________________ 128Glibert, Patricia __________________________ 43Gobler, Christopher J _______________ 44, 56, 159Goebel, Nicole L _________________________ 31Golding, Brian __________________________ 142Goodman, Allan ________________ 127, 137, 146,

171, 173Grandal, Meghan _______________________ 147Granholm, April A. ______________________ 117Grattan, Lynn M. ___________________ 45, 58, 72Greenfield, Dianne I. __________________ 37, 148Greengrove, Cheryl L. _________________ 65, 152Gregson, Brian __________________________ 93Gunasekera, Sarath P. _____________________ 40Gutierrez, Danielle __________________ 156, 176

hHails, Alan _____________________________ 149Haines, Andy __________________________ 142Hajat, Shakoor _________________________ 142Hall, Lauren _____________________________ 56Hall, Nathan S. ___________________________ 67Hall, Sherwood _________________________ 150Hallam, Steven __________________________ 94Hambright, K. David _____________________ 151Hanlon, Jeremy ________________________ 186Hannach, Gabriela ______________________ 152Hardison, D. Ransom ______________________ 60Harrington, Neil _________________________ 46Harvey, Elizabeth L. ______________________ 62Harvey, J. B. J. ___________________________ 73Hatch, Marco B.A. _______________________ 94Hattenrath-Lehmann, T.K. __________________ 44Hauser, Loren J. _________________________ 78Hay, L. R. _______________________________ 65Hayashi, Kendra__________________ 51, 115, 163Heil, Cynthia ____________________________ 47Hendrickson, John __________________ 119, 136Henrichs, Darren W. ______________________ 48Henry, Chuck ___________________________ 76Henry, Michael _________________________ 178

Henschen, Karen _______________________ 103Hetland, Robert D. ___________________ 48, 118Hickerson, Emma L. _______________________ 81Hickey, Barbara __________________________ 49Higginbotham, Tom ______________________ 76Hiolski, Emma ___________________________ 58Hitchcock, Gary _________________ 47, 50, 92, 95,

101, 161Hoagland, Porter ____________ 50, 101, 108, 111,

153, 161 Hogan, Sarah __________________________ 148Hogue, Tanya __________________________ 154Holland, William C. _______________________ 81Holmes, Sarah M. _______________________ 155Holohan, B.A ___________________________ 184Hooe-Rollman, Jennifer I. __________________ 72Hoogenboom, Ron ______________________ 129Hossain, Sk Md Sazzad ___________________ 177Hounshell, Alexandria G. _________________ 174Howard, Meredith D.A. ____________________ 51Hoyer, Mark ___________________________ 119Huang, I-Shuo __________________________ 156Hubbard, Katherine A. _____________ 52, 93, 103Hudnell, H. Kenneth ______________________ 53Hudson, Derrick S. ______________________ 101Huisman, Jef ___________________________ 129Hutchins, David A. ______________________ 117

iIngraham, Helen L. _______________________ 32

JJacocks, Karl _______________ 127, 146, 171, 175Jagus, Rosemary ____________________ 145, 158Jaja-Chimedza, Asha ______________ 54, 116, 157Janecek, Paul ___________________________ 93Jayroe, David S. _________________________ 96 Jester, Edward L.E. ______________________ 124Jiang, Wenjun ___________________________ 97Jin, Di ________________________________ 153Jochens, Ann E. _________________________ 55Johnstone, J. A. __________________________ 65Jones, Burton ___________________________ 51Jones, Carrie ___________________________ 103Jones, Christina __________________________ 57

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Jones, Grant D. _________________________ 158Jones, William J. ________________________ 148

KKääriä, Juha ___________________________ 186Kachel, Nancy ___________________________ 49Kang, Yoonja ________________________ 56, 159Kashinsky, Liz ___________________________ 71Kaufman, Angie _________________________ 71Kavanaugh, Karen ________________ 98, 132, 133Keafer, Bruce A. ___________________ 39, 61, 110Kendrick, Preston ____________________ 58, 160Keppler, Chuck _________________________ 148Kessel, Anthony ________________________ 142Khidekel, Roman _______________________ 113Kibler, Steven R. _________________________ 81Kiene, Ronald P. ________________________ 143Killberg-Thorsen, Lynn ____________________ 47Kirkpatrick, Barbara _____________ 50, 55, 79, 88,

95, 101, 111, 153, 161, 162, 193Kirkpatrick, Gary _______________ 47, 50, 95, 101,

121, 134, 149, 161, 164Kleindinst, Judith L. _______________________ 52Knight, Christopher _____________________ 103Koch, Florian ________________________ 56, 159Kohler, Kate ___________________ 50, 79, 88, 95,

111, 161, 162, 193, Koski, Teemu __________________________ 186Krausfeldt, Lauren E. ____________________ 107Kubanek, Julia __________________________ 57Kudela, Raphael M ______________ 31, 42, 49, 51,

87, 115, 163, 179, Kulis, David M. _________________________ 120

lLandsberg, Jan H. _____________________ 91, 99Langlois, Gregg _________________________ 150Lasi, Margaret ___________________________ 56Launay, Susan ______________________ 121, 174Lazensky, Rebecca ______________________ 108LeCleir, Gary R. _________________________ 107Ledreux, Aurelie _________________________ 71Lefebvre, Kathi ______________________ 58, 160Leliaert, Frederik _______________________ 135Lembke, Chad __________________________ 149Lenes, Jason M. __________________ 47, 134, 164

Lessard, Evelyn J. ____________________ 49, 167Leynse, Alexander K. ____________________ 165Li, Zhou ________________________________ 78Li, Zongchao ___________________________ 166Liefer, Justin D. _______________________ 59, 72Linton, Brian ____________________________ 91Lirman, Diego __________________________ 135Litaker, R. Wayne _____________________ 60, 81Littan, Charles ___________________________ 71Liu, Shunmei ___________________________ 168Loeffler, Christopher R. __________________ 100Logsdon, Jenna L. _______________________ 177Lovko, Vincent J. ______________ 50, 95, 101, 121Lubetkin, Susan C. ___________________ 49, 167Luther, Mark E. __________________________ 92Lydon, Christina ________________________ 168

mMacCready, Parker _______________________ 49MacLeod, B. ___________________________ 128Main, Christopher R. _________________ 37, 102Makarewicz, Joseph ______________________ 35Mantua, N. J. ____________________________ 65Marcinek, David _____________________ 58, 160Marin, Roman ________________________ 39, 87Markley, Laura R. _______________________ 169Marriott, Ian ___________________________ 127Martin, Ned ___________________________ 175Martin, Robbie M. ______________________ 107Marzolf, Erich ______________________ 119, 136Masson, Diane __________________________ 49Masura, J. E. ____________________________ 65Matthijs, Hans C.P. ______________________ 129May, Amanda L. _________________________ 78Maze, Grace ___________________________ 170McCabe, Ryan ___________________________ 49McCall, Jennifer ________________ 146, 171, 175McCord, Bruce _________________________ 196McGillicuddy, Dennis J. _________________ 52, 61 McInnis, Katherine B. _____________________ 32McLaughlin, Karen _______________________ 51McLean, Timothy I. ___________________ 96, 125Medvecky, Rebecca _____________________ 178Meek, Megan E. ________________________ 172Menden-Deuer, Susanne __________________ 62Mendez, Hector _________________________ 76

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Mengel, Michael J. _______________________ 51Meyer, Kevin A. _______________________ 47, 63Mihuc, Timothy __________________________ 35Mikulski, Christina M. _______________ 39, 73, 87Milbrandt, Eric C. ________________________ 33Miller, Jan D. ____________________________ 56Millie, David F. __________________________ 64Milton, Sarah __________________________ 131Minotti, Patricia J. ______________________ 174Moeller, Peter __________________________ 156Moisander, Pia H. _______________________ 155Moore, Leslie____________________________ 46Moore, Stephanie K. ___________________ 65, 94Moran, N. _____________________________ 128Morey, Jeanine S. ____________________ 82, 145Morris, George _________________________ 141Morris, J. Glenn ____________________ 45, 58, 72Morton, Steve _______________________ 52, 184Mueller, Karl ____________________________ 94Mulholland, Margaret R. _______________ 47, 164

nNeilan, Brett ___________________________ 190Neill, Nina E. ___________________________ 173Nezlin, Nikolay ___________________________ 51Nilsson, William _________________________ 94Nissanka, Ari ___________________ 121, 134, 174Niven, Susan ____________________ 41, 171, 175Nunn, Brook ____________________________ 57

oO’Dea, Sheila N. _____________________ 52, 103O’Mara, Sean P. ________________________ 176O’Neil, Judy M. _______________________ 47, 63O’Neill, Charles __________________________ 35O’Shea, Kevin E. _____________________ 97, 177Oesch, Carl ____________________________ 113Ogle, Marcus T. _________________________ 118Olascoaga, Josefina _____________________ 170Olsen, David A. _________________________ 100Olson, Robert J. __________________________ 36Osborne, Nick __________________________ 142Otten, Timothy G. _____________________ 66, 67Owen, Jenna L. _________________________ 104Owen, Daniel P. ________________________ 104Owen, Kevin C. _________________________ 104

pPaerl, Hans W. ________________________ 66, 67Palubok, Val ___________________________ 121Paolisso, Michael _________________________ 74Parsons, Michael L. _____________ 138, 165, 169Paul, Matthew R. _______________________ 145Paul, Valerie J. ___________________________ 40Perri, Katherine A. ______________________ 105Phillips, Jennifer _________________________ 68Pierce, Richard _____________________ 178, 182Pitz, Kathleen __________________________ 106Place, Allen R. ________________ 69, 74, 145, 158Plakas, Steven M. _______________________ 124Pokrzywinski, Kaytee L. ___________________ 70Poulson-Ellestad, Kelsey ___________________ 57Pound, Helena L. _______________________ 107Procise, Leo _____________________________ 47

QQin, Boqiang ____________________________ 67

rRadan, Regina __________________________ 179Radke, Elizabeth G. ____________________ 45, 72Ralston, David K. _________________________ 36Ramsdell, John __________________________ 71Rauschenberg, Carlton ___________________ 180Redshaw, Clare _________________________ 126Reich, Andrew _______________ 50, 108, 153, 161Rein, Kathleen S. _________________ 90, 130, 187Reis, Stefan ____________________________ 141Rhodes, Linda ___________________________ 94Richards, R. Peter ________________________ 80Richardson, Bill __________________ 47, 109, 181Richlen, Mindy L. _____________________ 59, 110Roberts, Sparkle M. ________________ 45, 58, 72Robertson, Alison _______________ 45, 52, 59, 72,

100, 143, Robertson, George ____________________ 51, 75Rodriguez, Dení ________________________ 135Rolton, Anne ___________________________ 182Rosa, M. ______________________________ 184Roy, Jessie ______________________________ 57Rudge, Katrin __________________________ 111Ryan, Darcie ________________________ 89, 183Ryan, John P. _________________________ 73, 87

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SSabel, Clive ____________________________ 142Salathé, E. P. ____________________________ 65Sandifer, Paul A. _________________________ 86Sarran, Christophe ______________________ 142Savage, Marci L. ________________________ 112Savage, Thomas J. _______________________ 115Sayers, Michael J. ________________________ 64Scheller, Karen _________________________ 161Schmale, Michael C. ______________________ 34Schmidt, Justine R. __________________ 107, 113Schneck-Scott, Adria ______________________ 98Schneider, Sabrina ______________________ 135Schnetzer, Astrid _________________________ 75Scholin, Christopher A____________ 39, 73, 87, 94Scott, Paula ____________________________ 103Sellner, Kevin G. _________________________ 74Senqupta, Ashmita _______________________ 51Seubert, Erica L. __________________ 51, 75, 185Shankar, Sugandha ______________________ 114Shaskus, Mylynda _______________________ 113Shawvner-Karnitz, A. _____________________ 128Shea, Damian ___________________________ 60Shipley, Dianne __________________________ 76Shoesmith, Zoe __________________________ 95Shuchman, Robert A. _____________________ 64Shumway, Sandra E. _________________ 182, 184Siedlecki, Samantha ______________________ 49Silby, Mark W. __________________________ 155Sipler, Rachel ____________________________ 47Sirois, Alison ____________________________ 52Smith, Don______________________________ 58Smith, Emily A. __________________________ 77Smith, G. Jason ______________ 87, 115, 163, 194Smith, Jayme ________________________ 75, 185Smith, Juliette L. _____________________ 39, 120Smith, T. ________________________________ 59Sosik, Heidi M. ___________________________ 36Soudant, Philippe _______________________ 182Southard, Greg _________________________ 176Spier, Chelsea L. ________________________ 186Stark, Kimberle _________________________ 152Steele, Dalton D. ________________________ 116Steffen, Morgan M. ______________________ 78Steidinger, Karen A. ____________________ 27, 91Stein, J. E. ______________________________ 65

Steur, Kees ____________________________ 129Stomp, Maayke ________________________ 129Stringfellow, William T. ___________________ 186Strom, Mark ____________________________ 94Studts, Jamie L. _______________ 79, 88, 161, 162Stumpf, Richard P. ______________ 80, 86, 91, 101,

119, 136Sultan, Mohammed ______________________ 35Sun, Pengfei ___________________________ 187Sunda, William G. ________________________ 60Sutula, Martha __________________________ 51Sweeney, Pamela _______________________ 135

tTalens, Renee __________________________ 129Tang, Ying Zhong _________________________ 56Tatters, Avery O. ________________________ 117Taylor, Alison __________________________ 147Taylor, Tim ____________________________ 141Tester, Patricia A. ______________________ 60, 81Thomas, Maura. A. ___________________ 61, 114Thompson, Alysha ______________ 146, 171, 175Thompson, Mark A. ______________________ 33Thomson, Richard ________________________ 49Thyng, Kristen M. _______________________ 118Tiedeken, Jessica ________________________ 71Tilney, Charles L. _____________________ 83, 188Tomas, Carmelo ________________________ 144Tomlinson, Michelle C. ____________ 80, 119, 136Tong, Mengmeng _______________________ 120Townsend, David W. __________________ 61, 114Trainer, Vera L. ____________________ 46, 65, 94Tustison, Jacob __________________________ 91

uUllmann, Steve __________________ 50, 153, 166Urizar, Cristina _____________________ 132, 133

vVan Alstyne, Kathryn L. __________________ 189Van Asten, Mark _______________________ 190Van Dolah, Frances M _____________ 82, 145, 150,

172van Hees, Daniel H. _____________________ 189van Scheppingen, Yvonne ________________ 129Vandersea, Mark W. ______________________ 81

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VanDolah, Elizabeth ______________________ 74VerBerkmoes, Nathan C. __________________ 78Viant, Mark _____________________________ 57Vignier, Julien __________________________ 182Visser, Petra M. ________________________ 129Volety, Aswani _________________________ 182Vreeland, Jennifer M ____________ 121, 134, 174,

WWallace, Ryan B. _________________________ 44Walsh, Breanne __________________________ 47Walsh, Catherine J. __________________ 131, 191Walsh, John J. _______________________ 47, 164Walter, John ____________________________ 91Walters, Linda ___________________________ 56Walton, Katherine ________________________ 34Wang, Zhihong __________________________ 71Warner, Mark E. __________________ 70, 83, 188Watkins, Sharon ________________________ 153Watson, Sue B. _______________________ 35, 78Watzin, Mary ____________________________ 35Weber, Samantha _______________________ 192Weckman, Gary R. _______________________ 64Weidert, Chase __________________________ 77Weisberg, Robert ________________________ 47White, Mathew ____________________ 126, 141Whitenack, Ben ____________________ 162, 193Wikfors, G.H. ___________________________ 184

Wilhelm, Steven W. _______________ 35, 78, 107Williams, Ernest ______________________ 69, 74Wilson, Patrick __________________________ 99Wolfe, Steven H. _________________________ 55Wolny, Jennifer _________________________ 103Woods, April L. _________________________ 194Wren, Jeff _____________________________ 103Wynne, Timothy T. _______________________ 80

xXu, Hai _________________________________ 67

yYordy, Jennifer E. ___________________ 131, 191York, Bob _____________________________ 144Younan, Lawrence _______________________ 195Young, William A., II ______________________ 64

zZamor, Richard M. ______________________ 151Zaworski, Lori J. ________________________ 174Zhang, Xiaoqian ________________________ 118Zhang, Y. _______________________________ 73Zheng, Bingxue _________________________ 196, Zhu, Guangwei __________________________ 67Zimba, Paul V. ______________________ 156, 176

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ann abrahamUS Food and Drug Administration P.O. Box 158, 1 Iberville DriveDauphin Island, AL [email protected]

Clarissa andersonUniversity of California Santa Cruz1156 High StreetSanta Cruz, CA [email protected]

don andersonWoods Hole Oceanographic InstitutionMS #32Woods Hole, MA [email protected]

Scott anglinThe University of Southern Mississippi118 College Drive, #5018 Hattiesburg, MS [email protected]

dan ayresWashington Department of Fish and Wildlife119 Brady Loop Road EastMontesano, WA [email protected]

lorraine C. BackerCenters for Disease ControlNational Center for Environmental Health4770 Buford Highway NEMS F-60Chamblee, GA [email protected]

heather Barron, dvmCROW, Inc.PO Box 150Sanibel, FL [email protected]

richard BartlesonSanibel-Captiva Conservation Foundation Marine Laboratory900 A Tarpon Bay RoadSanibel, FL [email protected]

John BerryFlorida International University3000 NE 151st Street North Miami, FL [email protected]

patricia BlumMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

Jerry BorchertWashington State Department of HealthP.O. Box 47824Olympia, WA [email protected]

andrea BourdelaisUniversity of North Carolina WilmingtonCenter for Marine Science5600 Marvin K. Moss LaneWilmington, NC [email protected]

holly BowersMonterey Bay Aquarium Research Institute7700 Sandholdt RoadMoss Landing, CA [email protected]

greg BoyerState University of New York - ESF1 Forestry DriveSyracuse, NY [email protected]

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anamari BoyesManatee County Water Treatment Plant QC Lab17915 Waterline RoadBradenton, FL [email protected]

ashley BrandtFlorida Gulf Coast University10501 FGCU Blvd. SouthFort Myers, FL [email protected]

michael BrosnahanWoods Hole Oceanographic InstitutionRedfield 3-30, MS 32Woods Hole, MA [email protected]

amanda BursonUniversity of Amsterdam904 Science Park Amsterdam, the Netherlands 1098 [email protected]

meridith ByrdTexas Parks and Wildlife2805 North Navarro, Suite 600AVictoria, TX [email protected]

margaret ByrneUniversity of MiamiDepartment of Public Health ServicesMiller School of Medicine 1120 NW 14th Street, Suite 912Miami, FL [email protected]

lisa CampbellTexas A&M University3146 TAMUCollege Station, TX [email protected]

Wayne CarmichaelDepartment of Biological Sciences Wright State UniversityDayton, OH [email protected]

ryan CassellFlorida International University875 NW 2nd StreetMiami, FL [email protected]

Camia CharnigaMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

Wei ChenFlorida International University11200 SW 8th StreetMiami, FL [email protected]

mary ChristmanMCC Statistical Consulting2219 NW 23rd TerraceGainesville, FL [email protected]

Courtney CocilovaFlorida Atlantic University777 Glades Road Boca Raton, FL [email protected]

Jaime CookFlorida Department of Health in Collier CountyEnvironmental Health and Engineering3339 Tamiami Trail East, Suite 145Naples, FL [email protected]

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alina CorcoranFlorida Fish and Wildlife Conservation CommissionFish and Wildlife Research Institute100 8th Avenue SESt Petersburg, FL [email protected]

Kathryn CoyneUniversity of DelawareSchool of Marine Science and Policy 700 Pilottown RoadLewes, DE [email protected]

edward davisNOAA National Ocean Service 1305 East West Highway Silver Spring, MD [email protected]

Jonathan deedsUS Food and Drug Administration5100 Paint Branch ParkwayCollege Park, MD [email protected]

Katherine dernerNOAA National Ocean Service 672 Independence ParkwayChesapeake, VA 23320 [email protected]

l. Kellie dixonMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

Quay dortchNOAA NCOS1305 East West HighwayBuilding IV, Room 8218Silver Spring, MD [email protected]

natalie doucetteFlorida International University11200 SW 8th Street, OE 167Miami, FL [email protected]

greg doucetteNOAA National Ocean Service219 Fort Johnson RoadCharleston, SC [email protected]

danielle dupuyNOAA/NCCOS & CSS Dynamic10301 Democracy Lane, Suite 300Fairfax, VA [email protected]

elizabeth elliottUniversity of North Carolina WilmingtonCenter for Marine Science5600 Marvin K. Moss LaneWilmington, NC [email protected]

amanda ellsworthFlorida Gulf Coast University10501 FGCU Blvd South Fort Myers, FL [email protected]

niclas engeneSmithsonian Marine Station at Ft. Pierce701 Seaway DriveFort Pierce, FL [email protected]

ivory engstromMcClane Research Labs121 Bernard Saint Jean DriveEast Flamouth, MA [email protected]

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deana erdnerUniversity of Texas Marine Science Institute750 Channel View DrivePort Aransas, TX [email protected]

reagan erreraTexas A&M University3146 TAMUCollege Station, TX [email protected]

alexis fischerWoods Hole Oceanographic Institution266 Woods Hole RoadRedfield 332 (MS #32)Woods Hole, MA [email protected]

timothy fitzpatrickSaigene Biotech1660 Race StreetDenver, CO [email protected]

lora flemingEuropean Centre for Environment and Human Health University of Exeter Medical SchoolTruro Cornwall, United KingdomTR1 [email protected]

leanne flewellingFlorida Fish and Wildlife Conservation CommissionFish and Wildlife Research Institute100 8th Ave SESt Petersburg, FL [email protected]

harold flores QuintanaUS Food and Drug Administration P.O. Box 158, 1 Iberville DriveDauphin Island, AL [email protected]

nicholas fowlerUniversity of North Carolina Wilmington5600 Marvin K. Moss LaneWilmington, NC [email protected]

hugo freudenthalLong Island University (Retired)1524 Fairway DriveDunedin, FL [email protected]

anita freudenthalNassau County Dept. of Health (Retired)1524 Fairway Dr.Dunedin, FL [email protected]

Kelly frideyCollege of Charleston219 Fort Johnson RoadCharleston, SC [email protected]

matthew garrettFlorida Fish and Wildlife Conservation CommissionFish and Wildlife Research Institute100 8th Avenue SESt Petersburg, FL 33701matt.garrett@ myfwc.com

Corinne gibbleUniversity of California, Santa Cruz 217 Highland Ave. Apt. 1Santa Cruz, CA [email protected]

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pat glibertUniversity of Maryland Center for Environmental ScienceP.O. Box 775 Cambridge, MD [email protected]

Christopher goblerSchool of Marine and Atmospheric Sciences Stony Brook UniversityStony Brook, NY [email protected]

allan goodmanUniversity of North Carolina WilmingtonCenter for Marine Science5600 Marvin K. Moss LaneWilmington, NC [email protected]

meghan grandalUniversity of North Carolina Wilmington601 S. College RoadWilmington, NC [email protected]

lynn grattanUniversity of Maryland School of Medicine110 S. Paca Street Baltimore, MD [email protected]

dianne greenfieldUniversity of South CarolinaHollings Marine Laboratory331 Fort Johnson RoadCharleston, SC [email protected]

Brian gregsonSpyglass Technologies, Inc.101 16th Avenue South, Suite 4A St. Petersburg, FL [email protected]

andrew griffithStony Brook UniversityStony Brook, NY [email protected]

alan hailsMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

K. david hambrightDepartment of Biology University of Oklahoma730 Van Vleet Oval304 Sutton Hall Norman, OK [email protected]

gabriela hannachKing County Environmental Lab322 West Ewing StreetSeattle, WA [email protected]

Clara hardWashington State Dept of HealthP.O. Box 47824 Olympia, WA [email protected]

Samantha harlowMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

neil harringtonJamestown S’Klallam Tribe1033 Old Blyn Hwy.Sequim, WA [email protected]

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marco hatchNorthwest Indian College2522 Kwina RoadBellingham, WA [email protected]

Cynthia heilBigelow Laboratory for Ocean Sciences60 Bigelow Dr., P.O. Box 380East Boothbay, ME [email protected]

John hendricksonSt. Johns River Water Management District4049 Reid StreetPalatka, FL [email protected]

darren henrichsTexas A&M University3146 TAMU, Eller O&M BldgCollege Station, TX [email protected]

michael henryMote Marine Laboratory1600 Ken Thompson PkySarasota, FL [email protected]

Barbara hickeyUniversity of WashingtonSchool of OceanographyBox 355351Seattle, WA [email protected]

gary hitchcockUniversity of Miami4600 Rickenbacker Causeway Miami, FL [email protected]

porter hoaglandWoods Hole Oceanographic InstitutionMS#41, Marine Policy CenterWoods Hole, MA [email protected]

tanya hogueUniversity of North Carolina WilmingtonCenter for Marine Science5600 Marvin K. Moss LaneWilmington, NC [email protected]

Sarah holmesUniversity of Massachusetts Dartmouth1399 Phillips Rd New Bedford, MA [email protected]

alexandria hounshellMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

meredith howardSouthern California Coastal Water Reserach Project3535 Harbor Blvd. Suite 110Costa Mesa, CA 92626 [email protected]

i-Shuo huangTexas A&M University-Corpus Christi, Center for Coastal Studies7350 McArdle Rd.Corpus Christi, TX [email protected]

Kate hubbardFlorida Fish and Wildlife Conservation CommissionFish and Wildlife Research Institute100 8th Avenue SESt Petersburg, FL [email protected]

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h. Kenneth hudnellMedora Corp. & UNC-Chapel Hill105 Serrano Way Chapel Hill, NC [email protected]

derrick hudsonMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

asha Jaja-ChimedzaFlorida International University3000 N.E. 151st Street, MSB 332North Miami, FL [email protected]

david JayroeUniversity of Southen Mississippi118 College Drive, #5018Hattiesburg, MS [email protected]

Wenjun JiangFlorida International University11200 SW 8th Street Miami, FL [email protected]

ann JochensGCOOSTexas A&M University 3146 TAMU College Station, TX [email protected]

heather JohnsonU.S. Geological Survey 6520 Mercantile WaySuite 5Lansing, MI [email protected]

grant JonesUniversity of MarylandCenter for Environmental Sciences701 East Pratt StreetBaltimore, MD [email protected]

Sonia Joseph Joshi NOAA Center of Excellence for Great Lakes and Human Health 4840 South State Road Ann Arbor, MI 48108 [email protected]

yoonja KangStony Brook University239 Montauk Highway.Southampton, NY [email protected]

david KarlenEnvironmental Protection Commissionof Hillsborough CountyP.O. Box 1110Tampa, FL [email protected]

Karen KavanaughNOAA National Ocean Service1305 East-West Highway Silver Springs, MD [email protected]

preston KendrickNOAA Fisheries2725 Montlake Blvd EastSeattle, WA [email protected]

Barb KirkpatrickMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

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gary KirkpatrickMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

Judy KleindinstWoods Hole Oceanographic InstitutionMS #32, Redfield 332Woods Hole, MA [email protected]

florian KochStony Brook UniversitySchool of Marine and Atmospheric Sciences 239 Montauk Highway Southampton, NY [email protected]

Kate KohlerMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

Julia KubanekGeorgia Institute of TechnologySchool of Biology, 310 Ferst DriveAtlanta, GA [email protected]

raphael KudelaOcean Sciences DepartmentUniversity of California Santa Cruz1156 High StreetSanta Cruz, CA [email protected]

Jan landsbergFlorida Fish and Wildlife Conservation CommissionFish and Wildlife Research Institute100 8th Ave SESt Petersburg, FL [email protected]

Brian lapointeHarbor Branch Oceanographic InstituteFlorida Atlantic University777 Glades RoadBoca Raton, FL [email protected]

Susan launayMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

Stephanie learMote Marine Lab1600 Ken Thompson ParkwaySarasota, FL [email protected]

Kathi lefebvreNOAA FisheriesNorthwest Fisheries Science Center2725 Montlake Boulevard EastSeattle, WA 98112 [email protected]

Jason lenesUniversity of South Florida140 7th Avenue SouthSt Petersburg, FL [email protected]

alex leynseFlorida Gulf Coast University10501 FGCU Blvd South Fort Myers, FL [email protected]

zangchao (Cathy) liUniversity of Miami School of Communication5150 Brunson Drive Coral Gables, FL [email protected]

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Justin lieferUS Food and Drug Administration P.O. Box 158, 1 Iberville DriveDauphin Island, AL [email protected]

Wayne litakerNOAA101 Pivers Island Dr.Beaufort, NC [email protected]

Christopher loefflerUS Food and Drug AdministrationP.O. Box 158, 1 Iberville DriveDauphin Island, AL [email protected]

Keith loftinU.S. Geological Survey4821 Quail Crest PlaceLawrence, KS [email protected]

vince lovkoMote Marine Laboratories1600 Ken Thompson PkwySarasota, Fl [email protected]

Susan lubetkinUniversity of WashingtonBox 357940Seattle, WA [email protected]

Christina lydonFlorida International University3000 NE 151st Street North Miami, FL [email protected]

rob magnienDOC/NOAA/NOS/CSCOR1305 East West HighwaySilver Spring, MD [email protected]

Christopher mainUniversity of Delaware700 Pilottown RoadLewes, DE [email protected]

laura markleyFlorida Fish and Wildlife Conservation CommissionFish and Wildlife Research Institute100 8th Ave SESt Petersburg, FL [email protected]

grace mazeRSMAS, University of Miami3894 Grand Ave Miami, FL [email protected]

Jennifer mcCallUniversity of North Carolina WilmingtonCenter for Marine Science5600 Marvin K. Moss LaneWilmington, NC [email protected]

dennis mcgillicuddyWoods Hole Oceanographic InstitutionBigelow 209 - MS 11Woods Hole, MA [email protected]

megan meekGraduate Program in Marine Biology, College of Charleston219 Fort Johnson RoadCharleston, SC [email protected]

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Susanne menden-deuerUniversity of Rhode IslandGraduate School of OceanographySouth Ferry RoadNarragansett, RI [email protected]

Kevin meyerUniversity of Maryland Center for Environmental Science2020 Horns Point RoadCambridge, MD [email protected]

elizabeth millerFlorida Department of Environmental Protection2600 Blairstone Road, MS6515Tallahassee, FL [email protected]

david milliePalm Island Enviro-Informatics/ Michigan Technological University4645 Stone Ridge TrailSarasota, FL [email protected]

Sarah miltonFlorida Atlantic University777 Glades Road, SC 288Boca Raton, FL [email protected]

Stephanie mooreNOAA FisheriesNorthwest Fisheries Science Center2725 Montlake Boulevard EastSeattle, WA 98112 [email protected]

nina neillUniversity of North Carolina WilmingtonCenter for Marine Science5600 Marvin K. Moss LaneWilmington, NC [email protected]

harry nelsonFluid Imaging Technologies65 Forest Falls DriveYarmouth, Maine [email protected]

ari nissankaMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

Susan nivenUniversity of North Carolina WilmingtonCenter for Marine Science5600 Marvin K. Moss LaneWilmington, NC [email protected]

Sheila o’deaFlorida Fish and Wildlife Conservation CommissionFish and Wildlife Research Institute100 8th Ave SESt Petersburg, FL [email protected]

Sean o’maraTexas A&M University - Corpus Christi6300 Ocean Drive, Unit 5866Corpus Christi, TX [email protected]

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Kevin o’SheaFlorida International University11200 SW 8th StreetMiami, FL [email protected]

timothy ottenOregon State University220 Nash HallCorvallis, OR [email protected]

Kevin owenPort Dolphin Energy, LLCHöegh LNG AS400 North Tampa Street, Suite 1015Tampa, FL [email protected]

hans paerlUniversity of North Carolina at Chapel Hill Institute of Marine Sciences3431 Arendell StreetMorehead City, NC [email protected]

valeriy palubokMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

michael parsonsFlorida Gulf Coast University10501 FGCU Blvd SouthFort Myers, FL [email protected]

Katherine perriState University of New York - College of Environmental Science and Forestry1 Forestry Drive, 121 Jahn Lab Syracuse, NY [email protected]

Kevin petrusFlorida Department of Environmental Protection2600 Bairstone RoadTallahassee, FL [email protected]

Jennifer phillipsUniversity of Maine5735 Hitchner Hall, Rm. 297Orono, ME [email protected]

richard pierceMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

Kathleen pitzWoods Hole Oceanographic Institution266 Woods Hole Road Woods Hole, MA [email protected]

allen placeUMCES-IMET701 East Pratt StreetBaltimore, MD [email protected]

Kaytee pokrzywinskiUniversity of Delaware700 Pilottown RdLewes, DE [email protected]

helena poundUniversity of Tennessee KnoxvilleM409 Walters Life Sciences Knoxville, TN [email protected]

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pete QuasiusCollier County Audubon4523 E Riverside DriveFort Myers, FL [email protected]

regina radanUniversity of California Santa Cruz1156 High StreetSanta Cruz, CA [email protected]

John ramsdellNOAA-National Ocean Service219 Fort Johnson RoadCharleston, SC [email protected]

Carlton rauschenbergBigelow LaboratoryP.O. Box 380East Boothbay, ME [email protected]

andrew reichFlorida Department of Health4052 Bald Cypress WayBin A12 Tallahassee, FL [email protected]

Kelly reinFlorida International University11200 SW 8th StreetMiami, FL [email protected]

Bill richardsonFlorida Fish and Wildlife Conservation CommissionFish and Wildlife Research Institute100 8th Ave SESt Petersburg, FL [email protected]

tammi richardsonUniversity of South CarolinaDept. of Biological Sciences and Marine Science ProgramColumbia, SC [email protected]

mindy richlenWoods Hole Oceanographic Institution266 Woods Hole Road, MS32Woods Hole, MA [email protected]

alison robertsonUniversity of South AlabamaDepartment of Marine SciencesMobile, AL [email protected]

anne roltonFlorida Gulf Coast University10501 FGCU Blvd, SouthFort Myers, FL [email protected]

Katrin rudgeRiverview High School1 Ram Way Sarasota, FL [email protected]

John ryanMonterey Bay Aquarium Research Institute7700 Sandholdt RoadMoss Landing, CA [email protected]

darcie ryanTexas A&M UniversityCollege Station, TX [email protected]

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marci SavageState University of New York Environmental Science and Forestry School155 Hood AveSyracuse, NY [email protected]

Justine SchmidtState University of New YorkCollege of Environmental Science and Forestry341 Jahn Lab, 1 Forestry Drive Syracuse, NY [email protected]

Julius SchneiderEast Georgia State College131 College CircleSwainsboro, GA [email protected]

Kevin SellnerChesapeake Research Consortium645 Contees Wharf RoadEdgewater, MD [email protected]

erica SeubertUniversity of Southern California3616 Trousdale ParkwayLos Angeles, CA [email protected]

Sugandha ShankarUniversity of MaineSchool of Marine Sciences5706 Aubert HallOrono, ME [email protected]

Justin ShapiroMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

dianne ShipleyFlorida Department of Health in Sarasota County2200 Ringling Boulevard Sarasota, FL [email protected]

Sandra ShumwayUniversity of ConnecticutDepartment of Marine Sciences1080 Shennecossett RoadGroton, CT [email protected]

Chris SimonielloGCOOS140 7th Avenue SouthSt Petersburg, FL [email protected]

emily SmithLouisiana State University1239 Energy, Coast and Environment Building Baton Rouge, LA [email protected]

g. Jason SmithMoss Landing Marine Laboratories8272 Moss Landing Road Moss Landing, CA [email protected]

Jayme SmithUniversity of Southern California3616 Trousdale Parkway, AHF 301Los Angeles, CA [email protected]

Juliette SmithWoods Hole Oceanographic Institution266 Woods Hole RoadWoods Hole, MA [email protected]

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Stephanie SmithBeagle Bioproducts, Inc.959 Schrock RoadColumbus, OH [email protected]

dalton SteeleFlorida International University3000 N.E. 151st Street North Miami, FL [email protected]

morgan SteffenUniversity of TennesseeM409 WLS Knoxville, TN [email protected]

Karen SteidingerFlorida Fish and Wildlife Conservation CommissionFish and Wildlife Research Institute100 8th Ave SESt Petersburg, FL [email protected]

William StringfellowUniversity of the Pacific-Ecological Engineering Research Program3601 Pacific AvenueChambers Tech Center Rm 224Stockton, CA [email protected]

Jamie StudtsDepartment of Behavioral Science University of Kentucky127 Medical Behavioral Science Building Lexington, KY [email protected]

rick StumpfNOAA NCOS1305 East-West HighwaySilver Spring, MS [email protected]

marc SuddlesonDOC/NOAA/NOS/CSCOR1305 East-West Highway, Rm 8254Silver Spring, md [email protected]

pengfei SunFlorida International University11200 SW 8th Street Miami, Fl [email protected]

Karen SwajianUS Food and Drug Administration5100 Paint Branch ParkwayCollege Park, MD [email protected]

avery tattersUniversity of Southern California3616 Trousdale ParkwayLos Angeles, CA [email protected]

pat testerNOAA National Ocean Service101 Pivers Island RoadBeaufort, NC [email protected]

Kristen thyngTexas A&M University 3146 TAMU College Station, TX [email protected]

Charles tilneyUniversity of Delaware700 Pilottown Rd.Lewes, DE [email protected]

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mengmeng tongZhejiang University/Woods Hole Oceanographic Institution266 Woods Hole Road, MS32Woods Hole, MA [email protected]

Kevin tyreFlorida Gulf Coast University10501 FGCU Blvd SouthFort Myers, NY [email protected]

Steven ullmannThe University of Miami Health Sector Management and PolicyP.O. Box 248442Coral Gables, FL [email protected]

Cristina urizarNOAA/NOS/CO-OPS1305 East West Highway, Station 7124Silver Spring, MD [email protected]

Kathryn van alstyneWestern Washington University1900 Shannon Point RoadAnacortes, WA [email protected]

mark van astenDiagnostic Technology Pty Ltd.Suite 45, 7 Narabang WayBelrose, NSW, 2085 [email protected]

frances van dolahNOAA Center for Coastal Environmental Health and Biomolecular Research219 Fort Johnson RoadCharleston, SC [email protected]

Jennifer vreelandMote Marine Laboratories1600 Ken Thompson ParkwaySarasota, FL [email protected]

Cathy WalshMote Marine Laboratory1600 Ken Thompson ParkwaySarasota, FL [email protected]

mark WarnerUniversity of Delaware700 Pilottown RoadLewes, DE [email protected]

rhonda WatkinsCollier County Governemnt3327 Tamiami Trail EastNaples, FL [email protected]

Cathy WazniakMaryland Dept of Natural Resources580 Taylor Ave, D2Annapolis, MD [email protected]

Samantha WeberState University of New York College of Environmental Science and Forestry816 Maryland AveSyracuse, NY [email protected]

Ben WhitenackMote Marine Lab1600 Ken Thompson ParkwaySarasota, FL [email protected]

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Steven WilhelmDepartment of MicrobiologyThe University of TennesseeKnoxville, TN [email protected]

april WoodsMoss Landing Marine Labs8272 Moss Landing RoadMoss Landing, CA [email protected]

Jennifer yordyMote Marine Laboratory1600 Ken Thompson PkwySarasota, FL [email protected]

lawrence younanTurner Designs, Inc.845 West Maude AvenueSunnyvale, CA [email protected]

rich zamorUniversity of Oklahoma1033 W. Boyd St.Norman, OK [email protected]

lori zaworskiMote Marine Laboratory1600 Ken Thompson Pkwy.Sarasota, FL [email protected]

Bingxue zhengFlorida International University11200 SW 8th Street Miami, FL [email protected]

paul zimbaCenter for Coastal StudiesTexas A&M University6300 Ocean Drive Unit 5866Corpus Christi, TX [email protected]

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PhycologicalSociety of

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