(4) Project description. (a) Topic being addressed by this proposal.

We propose to explore the fundamental mechanism(s) by which shellfish shells defendtheir owners from microbial and higher metazoan fouling and how those mechanisms may beaffected by progressive global warming, ocean acidification and other human factors that areconfounding and that are being imposed with particular vigor on the Gulf of Maine. We contendthat the basic antifouling mechanism used by shellfish is sensitive to both ocean acidificationand warming. The antifouling mechanism we focus on is based on the dissolution properties oflobster and mollusk CaCO3 shells that has been recently described (Kunkel et al. 2012; Kunkel& Jercinovic, 2013; Kunkel 2013). This is in stark contrast to some studies that suggest thatadult lobsters and mussels (Keppel et al., 2014) are not outside their pH and temperaturetolerances. This may be true for the factors they measured, which is questionable, but they didnot consider the shell disease issue and how ocean acidification and warming factored in there.

Most biofouling of natural and artificial surfaces is initiated by microbial attachment andmixed species biofilm formation on a surface. Further fouling by higher metazoans is based onattraction to and building on this biofilm. The majority of organisms in marine biofilms arebacteria and a few simple protists such as diatoms (Dobretsov et al. 2013). We need tounderstand the microbial biofilms and discrete colonies that exist on lobster and mollusk shellssince their microbes may contribute to disease and their association may be directly affected bychanging seawater qualities. We include mollusks here because they have a simpler structurethan lobster shells being for the most part devoid of phosphate mineral while lobster shellstructure depends heavily, based on our model which includes carbonate apatite (normally calledbone in vertebrate skeletons) (Kunkel et al. 2012; Kunkel & Jercinovic, 2013). Phosphate hasbeen generally ignored in modeling lobster cuticle (Bosselman et al. 2007) however we havecriticized that as being short sighted (Kunkel, 2013) given its apparent importance in the highresolution map of lobster cuticle minerals that we have established (Kunkel et al. 2012; Kunkel& Jercinovic, 2013). Since phosphate is a limiting resource in the North Atlantic we will beconcerned about its availability as a feature of seawater quality in our study and how that may beaffected by warming and acidification that is occurring and will continue to occur in the nearfuture.

Many organisms, including bacteria themselves, secrete substances that inhibit otherorganisms from settling and participating in a mixed biofilm. There is great competition forsurfaces in the marine environment. We will study the antifouling phenomena (Dobretsov et al.2013) associated with shellfish shells in which the CaCO3 in the shell structure slowly dissolvesthrough the overlying epicuticle in the lobster and periostracum in the mollusk (Kunkel et al.2012). The shell surface develops an unstirred layer of high pH (Kunkel & Jercinovic, 2013)which would make it incompatible with normal bacterial motility and growth, since bacteriallocomotion via the flagellum and much bacterial transport of nutrients are based on a suitablyhigh proton concentration (Button,1985). A high pH unstirred layer is described for the marinealga Ulva (Beer & Israel 1990). While this high pH unstirred layer phenomenon would apply toall shellfish it is particularly important in lobsters because any lesions on the lobster shell surfaceaffects its commercial value and, even more serious, if the lesions develop aggressively it cankill the lobster before it can shed its old lesioned cuticle. Epizootic Shell disease (ESD) hassubstantially reduced the lobster populations south of Cape Cod (Bouchard et al. 2012) and beenat least partially responsible for declines in lobster landings in Southern New England (ASMF,2014). ESD is particularly critical for female lobsters carrying eggs. So-called egger females

with ESD will delay their molting cycle for 6 months to allow eggs to mature and hatch, whichallows the shell disease to progress to kill the reproductive female. In the 2013 season, theincidence of ESD in egger females reached 34% (n= 56) in one survey of the deployed traps inCasco Bay (Tarbox 2013). Thus, as shell disease progresses in an area, the reproductive capacityof the population is degraded. It is therefore critical that we understand if the microbespopulating lobster shells are being encouraged by the warming and acidification of the Gulf ofMaine benthic environment.

In addition, lobsters are poikiolotherms and osmotic-conformers, that is, they operate atthe surrounding temperature and conform to the concentrations of local seawater. This has beenshown to apply to pH as well (Qadri et al. 2007). There is already evidence that lobsterpopulations have moved north, not surprisingly, correlated with an increase in southerly GoMtemperature. We as yet do not have adequate data to say what is happening to populationmovements related to prevailing pHs and the pH pattern of the Gulf of Maine is complex.

Lobster cuticle has been reported to be populated with a variety of bacteria however ingeneral these bacteria have been only recovered with vigorous treatment of the lobster surfaceand includes lobster shell disease lesions in which the lesions are quite advanced and infectedwith secondary infections in a modified lesion cuticle (Whitten et al., 2014). We believe thisvigorous treatment to retrieve samples is finding microbes that have little to do with the normallobster shell surface. Indeed the normal surface is not replete with abundant bacteria because ofthe high pH unstirred layer. We believe that what bacteria normally live in the unstirred layerare extremophiles, which are able to live in the high pH unstirred layer. None of the previouslyrecovered bacterial species extracted with vigorous abrasion have been selected or surveyed forthe extremophile behavior we suggest, i.e. being selected for populating a high pH surface. Wethus choose to focus on looking for bacteria that have chosen to live on the high pH surfaceof healthy lobsters as being in the highest probability of being associated with the initiationof shell disease. The search for extremophile marine bacteria that prefer to live on a lobster ormollusk shell surface is driven by several circumstantial evidences. The high pH unstirred layeris a marine environment that clearly exists but has been relatively ignored in the past (Beer &Israel 1990; Kunkel et al. 2012; Kunkel 2013). The bacteria that choose to live in a biofilm maybe phenotypically distinct from its conspecific planktonic relative by expression of large suitesof genes (An & Parsek, 2007). In studies with model bacteria it has been shown that flagellarand chemotaxis genes are generally repressed at pH 8.7 (Maurer et al. 2005) when bacterialflagellae cease to have sufficient protons to drive the flagellar motion. It is not clear howbacterial flagellae function in open ocean pH 8.2, benthos pHs closer to pH 7.8 or in shellfishunstirred layers which might reach pHs of 9 and 10. This however is countered by the existenceof bacterial flagellae which use a Na+ gradient as the motive force of flagellar propulsion (Sowa& Berry, 2007) rather than protons. Bacteria capable of using Na+ as a motive force for theirflagellae may populate the high pH shellfish surfaces. We hope to identify such a class ofextremophile bacteria.

The slow dissolution of the lobster shellsurface Ca/Mg calcite layer (Kunkel &Jercinovic, 2013) occurs at a generallyundersaturated pH for calcite and aragonite ofthe marine coastal shelf benthos which tendsto be several tenths of a pH unit below theopen ocean pH due to the active secretion ofprotons by respiring benthic organisms. Manystudies that have applied what they decidedwas highly acidified conditions relative tocurrent pH are doing so relative to the pH 8.2of open ocean pH (Keppel et al. 2012, 2014).When applied to pelagic living lobster larvaethe starting point for pH tolerance is perhapscorrectly set at pH 8.2, but the juvenile andadult lobsters are living in the benthos whichparticularly on the continental shelf and Gulfof Maine is substantially lower than 8.2, closerto 7.8. Our measurements of top and bottompHs on the continental shelf including theGulf of Maine suggest that much of theContinental Shelf water column is lower than

Figure 1. Spring 2009 continental shelfbottom pH contours measured from Niskinbottle samples taken in conjunction withCTD sampling on the NOAA Ship Bigelow(Kunkel 2014). Red dots are fig 2 sites.

Fig 2. Top to bottom pH profiles of select NE Shelf CTD stations collected by RosetteCarousel, Spring 2009 NE Groundfish Survey as determine by JG Kunkel (2014). Notice thatthe top pH somewhat reflects the bottom as we reported of the top/bottom contour plots.

pH 8.2 (Kunkel 2014). A typical pH profile in a transect down from the surface of the oceanusually reveals a starting pH at the ocean surface of pH 8.2 steadily declining to a pH of 7.6 inthe ocean at depths greater than 250 meters, which represents an historic reservoir of CO2

dissolution of the ocean basin carbonates established around 56 million years ago (Miller et al.1997; Katz et al, 1999). The current ocean top of pH 8.2 is thus recent, a result of the drawdownof atmospheric CO2 storage in the deep ocean, but now once again in the modern era we haverelatively rapidly rising CO2 in the atmosphere which is once again dissolving in the ocean.

However the idealized transect often does not hold for transects on the NE NorthAmerica continental shelf and the Gulf of Maine. Several transects top to bottom of thecontinental shelf were collected by NOAA Ship Bigelow technicians during the Spring 2009Groundfish Survey (Kunkel 2014). That study also provided general top and bottom Niskinbottle water samples analyzed by Joe Kunkel allowing a contour map of top and bottom pH to becreated for the continental shelf from southern New Jersey to the north of the Gulf of Maine, fig1. In addition 11 transects top to bottom using a Rosette Sampler were acquired providingdetails of pH in select water columns of the NE coastal shelf fig 2 (Kunkel 2014). The typicalargument is that CO2 is added at the ocean surface to varying extents depending on the oceansurface conditions, the more agitation at the surface the more CO2 is dissolved locally and themore acidic the very top is, an indication of current acidification being applied to the top of theocean. In shallow transects the difference between top and bottom are minimized likely byvigorous mixing such as on Nantucket Shoals, fig 2-6&7. In longer transects the acidificationevident at the surface leads to a open ocean pH 8.2 and then a water column that may vary in itsphotic and aphotic sections depending on algal metabolic and photosynthetic activity.

As variants on that theme, four transects, fig 2-1 through 2-4, bracket the Hudson canyonshowing approaches to abyssal pHs close to 7.6. In the Gulf of Maine two transects ofWilkinson's Basin show transects down to 7.7 but in the contours of top and bottom pH it is clearthat the top mirrors the bottom pH only a few tenths more basic. It is unlikely that this Gulf ofMaine basin is a relict of the 56 myr old acid bottom (Miller et al. 1997; Katz et al, 1999) sincethe age of the Gulf of Maine is only approximately 10,000 years old. The low pH is likely a partof a specific coastal phenomenon such as microbial metabolism of sediments from GoM riversthat persist from earlier logging days or are continuing based on current additions? It is clearthat we do not fully understand the coastal shelf phenomena of pH and CO2 based or pollutionbased ocean acidification on the continental shelf and methodology needs to be developed thatcan follow it. One perhaps encouraging observation is that the top pH measurements somewhatreflect the bottom measurements in both the top/bottom samples (Kunkel, 2014) and the 11Rosette sample transects (fig 2), which is possibly a general feature on the east coastal shelfwhere some type of mixing can be suspected. We are now poised to monitor the specialproperties of the Casco Bay seawater quality with our project and how that quality affectsshellfish health and the microbes that may participate. If the top pH can be an indicator ofbottom pH in our higher resolution study area and surrounds then that will be of greatconvenience for predictions in the future.

Ocean acidification is likely to have an earlier effect on the continental shelf including,gulfs, bays, inshore estuaries and nearshore benthic communities. Despite the alkaline design ofshellfish shells to dissuade biofilm formation, extremophile bacteria that have evolved to survivein high pH conditions are expected to populate the shell surface. The shell surface isconstructed and operates with an unstirred surface layer that we suggest is more like soilchemistry in that the thin unstirred layer of the ocean in direct contact with the shell is

dominated by dissolution chemistry of the CaCO3 that can result in pHs approaching 10, due tothe hydrolysis of water by dissolved CO3

-2 (Beer & Israel 1990; Sparks 2011). We will collectbacteria from different marine surfaces, identify several of the extremophile bacteria and studytheir properties as they relate to high pH surfaces. We have obtained a culture of a bacterium,genus Aquamarina (NS), which was derived from active lobster shell disease lesions (Quinn etal., 2012) and suggested to be associated with shell disease lesion formation. This organism is arepresentative of the heterogeneous population of 'normal' bacteria collected from ESD lobsters.We are proposing that a better approach to collecting candidates for causing shell disease wouldbe to search for extremophile bacteria that grow at a high pH. We believe that selecting for suchbacteria would enrich our chances of finding the problem bacteria. We have an establishedprotocol for collecting bacteria from healthy lobsters, trapped in the Casco Bay region (Tarbox,2013). This protocol was followed in collecting bacteria from outer Georges Bank healthylobster carapaces in Spring 2014 by Joe Kunkel on leg 3 of the Spring Groundfish Survey.These collections await barcoding to determine their phylogenetic relationships (Hebert et al.20013; Jermy, 2012). However we now propose that it may enrich our collections for relevantbacteria by selecting bacteria that will grow in elevated pH media and surfaces. Experimentswill be designed to test if swabs from lobster shells inoculating plates or media buffered to arange of higher pHs will find a different population of bacteria.

It is additionally possible that the bacteria isolated from shell disease lesions are alsoextremophiles, and thus we will include testing the lesions of Casco Bay shell diseased lobstersfor their pH dependent growth properties and compatibilities. Bacterial resistance to high pHenvironments may allow them to be more aggressive in the microenvironment of the lesionwhere the dissolution of amorphous CaCO3 creates an added microenvironment of high pH(Kunkel & Jercinovic, 2013) approaching the high alkalinity pH of CaCO3 dissolving in theabsence of a ready source of CO2. The dynamic reality of this environment operates in therestricted space with dimensions in the 10s and 100s of microns in which bacteria live on theshell surface or in a small crack in the cuticle surface. While the physical chemistry ofcarbonates may be predictable in many respects, the surfaces of decapod and mollusk shells arepart of living organisms in which the properties of the secretions and protective layers of shellsmust be examined and factored into the protective role of the shell. The recent elaboration of thefine structural mineralogy of the lobster shell has suggested mechanisms of defense that were notconsidered previously. As suggested by a recent review (Behringer, 2012) “...work (Kunkel etal. 2012) on mineralization and the structural defense of the exoskeleton has taken us a leapforward in understanding how the cuticle structure itself might moderate susceptibility to shelldisease, and opened up numerous new channels to explore”. We propose to examine a few ofthe most consequential of those new channels of exploration in populations of Gulf of Mainelobsters which are experiencing increased levels of Epizootic Shell Disease (ESD).

(b) Proposed scientific objectives and research activities. Our objective is to use a three pronged approach to study the vulnerability of the lobster andpotential reservoir populations to ocean acidification focusing on their shell's defenses againstmicrobial attack:

(1) We will study the population ecology of lobster ESD incidence in the Casco Bay areaand include the monitoring and analysis of water quality and bottom conditions surrounding thedeployed lobster traps. PI Brian Tarbox will survey the incidence of ESD using the establishedscale 0-4 of ESD severity (Smolowitz et al. 2005). Roxanne Smolowitz has agreed to be on our

Board of Advisors, which will provide us her expert knowledge of the disease as well as provideconfirmations of questionable diagnoses if they arise. Water quality analyses will include, pH,total carbonate, silicate, and nitrate & nitrite. These will allow us to interleave our measures ofseawater quality with databases on the wider Gulf of Maine, surrounding shelf and ocean(Rebuck et al. 2012). We will also be recording the bottom type and take bottom samples in oursurveys. Past foci of ESD have been shown to include titers of alkylphenols in lobster tissue aswell as in bottom substrate samples. Alkylphenols are known to interfere with crustacean shelldevelopment (Laufer et al. 2012, 2013). Alkylphenols as persistent industrial chemicals havebeen identified as an unknown factor in the Gulf of Maine seawater and sediment chemistry(Wells, 2010). We see them as a potential indicator of industrial affects that could be importantfactors in vulnerability of the lobster shell to microbes in our transect of Casco Bay. Therefore,we will include analysis of alkylphenols in the Casco Bay transect lobster tissue and control areabottom sample analysis. We will monitor lobster health factors including the carapace length,weight, sex, egg status and ESD presence and stage continuing the recording protocol that hasbeen established (Tarbox, 2013). In addition to the established scale of ESD severity, we willtake images on board our lobster boat for select lobsters of the fine detail of the lobster carapacein efforts to establish criteria for the very early stages of ESD corresponding to the earliesterosion of the epicuticle and calcite layer which do not appear as melanizations. That data willbe correlated with the ancillary bottom type and physical chemical data on seawater qualitywhich will be collected coincident with the ESD monitoring. The latitude and longitude of probitESD incidence data will be related to the hypothetical predictor data using the Linear AdditiveModel (LAM) as implemented in the R mgcv function library. A selection of the identifiedepicuticle and calcite-layer-erosion individuals will be tagged and held in containment cagesnear to the collection site to check for any progress toward the established scale of ESDdiagnosis using periodic digital images of the cuticle surface. We will experiment with vital dyewashes of the cuticle surface that may identify foci of acid producing bacteria by visualizing thepre- and post-dye washed surface, which may require using digital-difference viewing softwarewe have in our tool kit.(2) For all microbiological analyses, we will collect microbial and shell specimens fromcarapace surfaces of lobsters in the following treatment groups: A- healthy cuticle from non-ESD lobsters; B- healthy cuticle from stage-1-ESD lobsters (Tarbox 2013); C- calcite-layer-erosion shell areas of apparently healthy lobsters; D- digital difference foci of acid productionfrom healthy lobsters; E- shell disease lesions from ESD lobsters. For each individual sampled,triplicate sterile cotton swabs will be used to collect specimens; two of the swabs will bepreserved in 95% ethanol at -20°C (Whitten et al., 2014) for 16S rRNA and qPCR analysis(described below), and the third swab will be placed into a tube containing 3 ml filter-sterilizedseawater and vigorously vortexed to release associated microbes (Gram et al., 2010). Theresulting cell-sea water suspension will be stored at 4°C until microbiological analysis(described below) is performed (no more than 12 h post-sampling).

We will use 16s rRNA analysis to compare the shell-associated microbial communitiesbetween treatment groups and to look for presence of alkaliphilic bacteria. Microbial genomicDNA will be extracted from a single ethanol-preserved swab per individual and used as thetemplate in a subsequent polymerase chain reaction (PCR) to amplify bacterial 16S rRNA, usingestablished primer sequences (Whitten et al. 2014). Denaturing gradient gel electrophoresis(DGGE) will be used to separate the resulting 16S rRNA amplicons. DGGE is a well-documented method of resolving nearly single-base differences between 16S rRNA of different

bacterial species, and is well-documented as a useful tool for evaluating species diversity withincomplex microbial communities (Burkholder et al., 2008). Bands of interest will be selectedfrom DGGE gels based on abundance (band intensity) and frequency or uniqueness withinindividual treatment groups, and these bands will be excised and used for subsequent cloninginto E. coli, using the pGEMTeasy cloning vector (Promega). Plasmid DNA will be isolatedfrom individual clones and the 16S rDNA will be sequenced via the University of Maine DNAsequencing facility. Resulting sequences will be aligned with 16S rRNA sequences within theNCBI GenBank database using programs available within the Ribosomal Database Project II(RDP II, http://rep.cme.msu.edu) (Cole et al., 2005). Multiple sequence alignments will beperformed using the ClustalW program and similarity percentages between treatment groups willbe calculated in the ClustalW program (Ma et al., 2004).

Both targeted and classical approaches will be used to determine the proportion ofextremophiles to non-extremophiles in distinct lobster populations. In the targeted approach, wewill select alkaliphilic and non-alkaliphic species of interest from the 16S rRNA sequencing data(above) and will use quantitative PCR (qPCR) to determine the relative abundance of thespecific microbial species within each treatment group. Microbial DNA will be isolated from asingle ethanol-preserved swab specimen per individual and qPCR primers specific for 16S rRNAand bacterial housekeeping gene DNA gyrase (Cassler et al., 2008). For a more classicalapproach, shell swabs will be suspended and vortexed in sterile seawater (sample collectiondescribed above), and aliquots of the resulting microbial suspension will be plated on marineagar at pH ranging from pH 7.6 to pH 12 and incubated at 20°C until visible colony growth (3-7days). Resulting colony forming units (CFU) will be enumerated and used to calculate theproportion of alkaliphiles (growth at pH 9-12) to non-alkaliphiles (growth at pH 7.6 and pH 8).

To evaluate susceptibility of shell surfaces to microbial colonization and growth, shellfragments will be excised from lobsters within all treatment groups. A spectrofluorometric plateassay will be used to quantify bacterial growth on these natural shells as well as mollusk shellsurfaces (blue mussel) and artificial surfaces (modified calcite blocks, and fired ceramicsurfaces). Briefly, excised shells or control surfaces will be placed in individual wells of a 96-well plate and inoculated with a minimal growth medium containing individual cultures offluorescent-labeled alkaliphilic and non-alkaliphilic bacteria isolated from marine agar atvarying pH (described above), or Aquamarina homaria, a tentatively-named bacterium (Whittenet al. 2014) known to colonize lobster shells and shell disease lesions. Shell/calcite cultures willbe maintained in minimum growth medium for a 2 week period, and spectrofluorometricreadings will be taken at 24-hour intervals to evaluate microbial growth on the shell surfaces.Relative fluorescence intensity, reflecting microbial load, will be compared between shellsobtained from lobsters of different treatment groups.

A strategy of the shell defense against microbes is to create a zone (the unstirred layer ofhigh pH) in which bacteria are restricted in their motility and ability to settle, metabolize andnavigate on the shell surface or through the bacterial film on the shell surface. We propose that aselect group of bacteria will be able to colonize the shell surface as discrete colonies or in mixedbiofilms, which will classify them as pH extremophiles. Our first objective is to explore theextremophile concept by comparing the lobster shell populations with control surfacepopulations and populations on natural high pH unstirred layer surfaces such as Ulva (Beer &Israel 1990). We will deploy the same control surfaces with the traps we deploy and samplethose surfaces for developing microbial populations. There are published mechanisms thatbacteria could use to populate the high pH unstirred layer of shellfish (Sowa & Berry 2008). It

might be expected that the marine bacteria living on the high pH surfaces of shellfish would bepowered by Na+ rather than protons. It is also possible that motility on the shell surface is Na+

driven while motility in the general benthos is proton based since it is established that Na+ drivenrotors of Bacillus species are favored in higher pH conditions (Terahara et al. 2008). Thisambivalant capability to pH could be an important difference to establish for shellfish microbesas it might be a general property by which to recognize shell surface living bacterial forms.Flexibility in this phenotype might also be important to understand in terms of the microbialstrategy to enter the shellfish surface unstirred layer, near which they might need to deploy theirNa+ based flagellar rotor power. Establishing the ambivalence mechanism(s) might be anobjective for additional work after we have established a shellfish surface residence and growthtype. We will also fix selected natural and control surfaces for examination by high resolutionlight microscopy and Scanning Electron Microscopy (SEM) in an attempt to identify themembership of bacterial associations in films or discrete colonies.

(3) We will examine the shells of lobsters during their intermolt and molt cycles todetermine their defense mechanisms and potential vulnerability to acidification. Measurementson lobster shells will be examined on live animals as described (Kunkel et al., 2012) because weconsider the shell to be a living part of this arthropod (Wigglesworth 1948). We will do so usinginstantaneous measures of Ca2+ and H+ flux and their computed effects on shell chemistryknowing the ecology of the site that the lobster came from. We can measure and then predict theeffect on acidification on shell structure and function by measurements made in the laboratory.Long term experiments creating the appropriate pH by adjusting the pH by controlling CO2

composition of air used to aerate culture conditions require large-scale and long term effort. Wewill rather approximate the conditions needed to measure the instantaneous effects of pH bycontrolling pH using so called Good buffers (Kunkel et al. 2001, Porterfield et al. 2009), whichwill force the available total CO3

2- to approximate conditions we wish to impose whilemeasuring the resultant instantaneous fluxes. This approach will avoid unrealistic conditions inwhich adjusting dissolved CO2 results in abnormally low oxygen levels. Temperature during thismeasurement of instantaneous flux will be maintained by Peltier controlled circulation ofseawater such that differences in temperature can be measured instantaneously as it reacts withcontrolled pH. The ability to measure interactions between pH and temperature on the shell ofeach live lobster (cf fig 2 in Kunkel et al, 2012) is a powerful approach that eliminates some ofthe distracting variability among groups of lobsters when they are cultured for long treatmenttimes and the average effects of the treatments measured to test a hypothesis. Directlymeasuring interactions of pH and temperature in several individuals from each benthic conditionis a better approach to understanding the effects of pH and temperature and relating them toprevailing conditions.

There is published evidence (Kunkel et al. 2012) that the surface calcite layer of lobstercuticle is ~10% Mg calcite and that during the early phase following a molt and establishment ofthe calcite layer, the Mg calcite preferentially dissolves, which might function to give the surfacea higher pH unstirred layer than if the dissolving entity were pure Ca calcite. In a few timedHomarus gammarus sampled shortly (1-4 days) after molting we seen a level Mg level ~10%throughout the calcite layer (Kunkel unpublished), which prompts or hypothesis that the outerMg calcite dissolves preferentially early post molt. This early preferential Mg calcitedissolution behavior of the lobster cuticle would lead to our observed steep declining gradient ofMg calcite toward the shell surface observed using Electron Microprobe (EMP) analysis aspublished fig 7 in Kunkel and coworkers (2012). Having access to fresh lobster samples that can

be quickly transported to our Marine Science centers and have their surface flux of Ca2+ andprotons measured and then shell is prepared for EMP, FTIR and Raman Spectrometry analysis.This will be a unique opportunity to observe the shell defense mechanisms during the criticalstages when the soft cuticle individuals appear and when it is thought that shell disease has itsbest opportunity of being established.

This project will also give us the opportunity to study the earliest stages of shell diseasein which, we propose, the thin epicuticle is first eroded and then the calcite layer is progressivelyeroded before any melanizations are recognizable as ESD lesions. This subliminal stage has beenseen and suspected visually but rarely explored experimentally. These erosions of the epicuticlemust allow a more rapid dissolution of the calcite which we should be able to recognize with ourion probe since it should result in a higher pH in the unstirred layer than truly normal cuticle.Once we have identified suspect areas with the ion probe we will be able to more thoughtfullyprepare select areas to be studied more structurally with EMP, FTIR and Raman Spectrometrywhich establish the chemistry that is happening in those areas of active ion flux. We can as wellrecover extremophile bacteria from such hot spots of high pH which may be the as yetunidentified primary cause of ESD. We will be able to measure the progress of the defensiveaction using our ion probe equipment and test its ability to withstand the acidification processwhich will perhaps have its most serious effects on our benthic environments.

Our combined ecological, microbial and physiological approach should allow us toobserve the microbes in benthic conditions in a perhaps key environmental ecosystem, CascoBay, which may be the first waters in the Gulf of Maine to experience the changes associatedwith a substantially increased incidence of ESD. We ask why in Casco Bay? We will have asample of microbes, benthos bottom and water conditions from that locale and then recreate theobserved conditions in the lab and measure the instantaneous effects on ionic flux from thelobster shell, which we hypothesize create the protective high pH unstirred layer. In such casesthe individual live-lobster shell will be further challenged with pHs outside the prevailingconditions at that sampling site. Understanding this complex situation will perhaps allow us topredict its repercussions to the health of the lobster population. Current models of oceanacidification do not include the phenomenon of high pH unstirred layers on marine organismsdespite the fact that they are known to exist on algae (Beer & Israel 1990) and we have reportedthem on lobsters and mollusks (Kunkel et al. 2012). Controls of note:

Samples of seawater and bottom will be taken from each trap location we sample.Seawater quality will be analyzed for total carbonate, alkalinity and pH plus silicate, NO2 &NO3, PO4 and the bottom and lobster tissue for alkylphenols. Control samples from inside andoutside the Casco Bay area of lobster traps will also be requested from several cooperatingprograms to provide the extended background for a smoother fit of the seawater quality: (1)Bottom and top seawater Niskin bottle samples from NOAA Ship Bigelow, Fall and SpringGroundfish Survey will be requested from routine CTD trawl locations. (2) Bottom and topNiskin bottle samples from Ventless Trap Survey sites in coastal Maine will be requested usingUNE or SMCC student research assistants in order to complete the nearshore seawater qualitysampling. (3) We will also join cooperating lobstermen in the NOAA lobster trap monitorprogram (Manning and Sheremet 2014) using sensors that are deployed on the lobster traps. (4)If possible we will also establish cooperation with a deployable pH monitoring project to obtaincontinuous pH sampling at our trap sites. Our work with non-invasive ion probes (Kunkel et al.2005) and developments of new laboratory micro-optrode technology for oxygen and pH sensors

make us particularly aware of developing technology in this area, despite the fact that it is notready for routine deployment on the ocean floor.

Contrasting surfaces to lobster shell will include ceramic tile, resin embedded calcitecrystals, blue mussel shells, Cancer crab shells, Razor Clam shells and Ulva surfaces each ofwhich will have an unstirred pH consistent with its composition and solubility properties.Ceramic porcelain tile is a null control which exhibits no unstirred layer pH modification.

Contrasting Lobster populations: Casco BayTransect, will be compared to Outer Georges Bankand Scotian Shelf lobsters, which currently havelow ESD incidence, and will be sampled for theirmicrobial populations.

3-Dimensional Landmark analysis () of thelobster carapace shape of lobster populations willbe characterized as a control response variable andalso related to recorded benthic conditions.Recently we applied Landmark Analysis to theshape of the carapace between offshore (GeorgesBank and Scotian Shelf) and inshore (mainlyCasco Bay) lobsters which indicated a sizeindependent shape difference between the offshoreand inshore populations (fig 3). The distinctdifference in an abstraction of shape plotted in fig

3 can be visualized in a 3-D stereo plot of the data which some people can view directly butothers may need a stereo-viewer to see (fig 4). Volunteer scientist time on the Groundfish Surveywill collect shape data on the trawl collected samples of the Survey. These will be compared totrapped lobster shape data from Brian Tarbox's Casco Bay string of traps. Currently ourobservations of carapace shape is based on trawl capture lobsters off shore but trap collectedlobsters near shore. Our null hypothesis in this case is that trawl collected shape data isindistinguishable from trap collected shape data. For the inshore population we can comparetrap collected shape data with trawl collected lobsters from the NOAA Groundfish Survey. For

the offshore populations we will also need tocompare the NOAA trawl collected lobster shapeswith commercial trap collected lobsters which willbe negotiated with cooperating lobstermen.

The data on which these differences arebased can be the basis of a discriminant functionthat can allow prediction of what population anindividual lobster came from. This type ofprediction could be valuable to a monitoringagency to test a sample of landed or marketedlobsters for where they were derived from. At themoment we are able to reliably predict themembership to near-shore vs offshore populations,however by extending these studies we may beable to provide an inexpensive and predictive toolto the lobster industry which might become more

Fig 3. Allometric Shape Difference of inshore vsoffshore American lobsters.

Fig 4. Stereo pair of vectors of inshore vs offshoreshape difference of lobster carapace landmarks.

critical if a predictable zone develops higher ESD prevalence. The vectoral differences plottedin fig 4 which give rise to the abstraction of shape difference plotted in fig 3 involve severalhomologous muscle attachment sites on the carapace which may be an indication that themuscles of different lobster populations are specialized for differences in the offshore vs inshorebottom environment or associated behaviors such as migration. We will be working assuggested with carapace shape as a response variable to establish a correlation between thisproperty and the environmental variables we measure. This shape study is an experimentalcontrol which we know shows a difference between populations. It will be applied so that wehave a constant reference in which we can see a significant difference in multivariate data to becompared to our other data on ESD and microbial population differences about which we will betesting contrasts for differences in similar ways to carapace shape. Contrasts of note to be tested:

(1) Contrasts of extremophile bacteria will be measured from lobster surfaces fromdifferent lobster bottom habitats. Casco Bay mud and rocky bottoms trap locations will be usedas factors for comparison.

(2) Offshore lobsters of distinct carapace shape from outer canyons of Georges Bank andScotian Shelf via trawl and trap collection. (3) Collection of water samples and bottom grabs from all trap and trawl sites. (4) Seasonal contrasts to seek Vulnerable Phases for ESD. (5) Comparison of trap vs otter trawl lobster microbe surveillance. It is possible that thetrawl caught lobsters would be contaminated by being in the net with groundfish of all sorts.The trawl record of the NOAA Groundfish survey may be helpful in discriminating thatpossibility. Light vs heavy trawls may allow cross-contamination to be reduced toindistinguishable from trapped microbe population statistics.

(c) Discussing how the proposed project lends value to the program goalsOur project examines the American lobster, an economically vital species to the states

which border on and access the Gulf of Maine and Georges Bank. We are focusing on aneconomically vital area, Casco Bay, which is currently undergoing alarming increase in lobsterESD. Even if the shift in lobster peak population is to the north in the Gulf of Maine, thephenomena of increasing ESD needs to be understood where it is now happening to allow forpotential mitigation or avoidance in other locations. The increase in ESD with particularprevalence in egg-bearing females (Tarbox, 2013) resembles in some degree the experiencesouth of Cape Cod and needs to be monitored. Our Advisory Board includes Kathy Castro whowas the Lead PI of the RI-Seagrant Lobster Health Initiative who co-authored the report which isthe most comprehensive study of the increase in ESD we have so far (Bouchard et al. 2012).Comparison of the pH and carbonate chemistry in our study area with that of the remainder ofthe Gulf of Maine is vital given that we are now seeing increased ESD in the Casco Bay studyarea. Another of our Advisory Board members is James Manning who is the NOAA NMFSperson in charge of the deployable sensors on lobster traps (Manning and Sheremet 2014).Understanding the relationship of ESD prevalence to seawater conditions and bottom type needto be established now if we are to be able to relate them to future conditions in other locations.We will be developing techniques which may allow us and lobstermen to detect the early signsof ESD or predictive environmental correlates in areas where ESD is currently rare or at timeswhen ESD is rare in an affected area during a phase (e.g. shortly after molting) when ESD issubliminal and hard to detect. Early detection techniques will be investigated. This early

detection is vital to the marketability of lobsters, particularly those that are to be held in poundsawaiting ideal market conditions. Another of our Board of Advisor members is Carry Byronwho has the pulse of the data modeling community and will help us relate our objectives andfindings to the current modeling community to argue the inclusion of the concept of high pHunstirred layers on marine organisms into the models of ocean vulnerability to acidification.

It is also important to establish any population differences between the inshore, nearshoreand offshore populations of lobsters. Several adult tagging studies (Fogarty et al. 1980; Cowan2012) have established that the majority of inshore adult lobsters move only modest distances (2-6 miles) during their seasonal migrations with notable exceptions that can travel long distances.On the other hand the offshore lobsters on the outer rim of the continental shelf and Georgesbank have been shown to make substantial shoalward migrations in the summer (Cooper andUzmann, 1971). Whether this difference between the inshore and offshore behavior representsdistinct populations or accommodations between differences in the environment does not seemto have been resolved yet but may be important in future epidemiology of ESD.

(d) Identifying the function of each PI and research associate.Joseph Kunkel is the lead PI and physiologist on the project. His elaboration of the fine

resolution model of the American lobster cuticle (Kunkel et al. 2012; Kunkel & Jercinovic,2013; Kunkel 2013) has provided new avenues of understanding of the role of lobster cuticlemineral structure in resistance to microbial attack. His role is to corroborate the predictions ofhis cuticle model in how it predicts the ionic fluxes that participate in maintaining the shell'shigh-pH unstirred layer, which interact with the bacteria that impinge on the shell surface. Hisstrength in measuring ionic fluxes and measuring the mineral content of the cuticle at highresolution are attested to by his publication record in those areas. To accomplish these functionshe maintains a laboratory which includes two ion-probe rigs, one an upright zoom-scopemicroscope devoted to measuring ionic fluxes from model and artificial surfaces of lobster ,mollusks and algal surfaces. A second rig uses an inverted microscope (Zeis IM-35) which is tobe used to study the ionic flux behavior of bacterial colonies and biofilms (McLamore, 2008)living on surfaces. These two ion probe rigs under Kunkel's supervision are essential toestablish the consequences of the increases in ocean acidity which have a two target additive andperhaps multiplicative effect, one on the acid dependent rate of dissolution of the calcite layer ofthe shell (measured on one probe rig) and another on the capacity for acid secretion by microbialcolonies on the surface of shells (measured by the second probe rig). He will coordinate UNEand SMCC Marine Science researchers working on the lobster and mollusk shells as well as theUNE Microbiology researchers working on ion flux of microbial colonies. With no courseworkduties at UNE Kunkel is free to participation in the NOAA FSCS Groundfish Survey as avolunteer scientist covering the Gulf of Maine and Georges Bank regions of interest as well asthe state Ventless Trap Program surveys which survey the inshore waters of Maine and NewHampshire. In those cooperating programs Kunkel and the students he supervises will collectmicrobial and lobster samples and measurements for the project.

Brian Tarbox is a PI and Marine Ecologist on our project. He is an active observer ofthe developing ESD in the population of lobsters he traps using his lobster boat and string of 70lobster traps on various bottom types in a transect away from Portland Harbor in Casco Bay(Tarbox 2013). He will also supervise carrying out the protocols for monitoring the seawaterquality and ancillary data including bottom type and sampling in the trap environments. Theancillary data Brian collects includes the ESD status of different lobster groups including molt-

stage and reproductive-stage of the lobster. His SMCC students will be participating in allaspects of the live lobster research including the microbial sampling, water quality sampling,imaging of the lobster cuticle surface for extending the ESD scale to subliminal shell conditions.The bottom sample and tissue sample analysis for alkylphenols would be outsourced to acommercial mass spectrometry laboratory under the supervision of expert advisor on the topic,Hans Laufer. Brian Tarbox will also collaborate with Kristin Burkholder in deploying thecontrol surfaces attached to his lobster traps and storage cages from which our collection ofcontrol-surface microbes will be characterized.

Kristin Burkholder is a PI and Microbiologist on our project. Dr. Burkholder’s researchfocuses on bacterial pathogenesis and host-pathogen interactions (Burkholder et al., 2011;Burkholder and Bhunia, 2010; Burkholder et al., 2009), and she also has experience in usinggenomic methods to characterize complex microbial communities from biological specimens(Burkholder et al., 2008). She will supervise the study of marine microbes isolated from themarine ecology operations of the project. Her basic objective will be to compare the relativeproportion of alkaliphilic to non-alkaliphilic microbes in healthy and shell-diseased lobsters tocontrol surfaces. Her role will include supervising the identification and study of growth andmotility properties of bacterial cultures we isolate from marine surfaces comparing inert controlsurfaces exposed in the lobster trap setting to lobster shell isolates from various lobster shellsurfaces. The Burkholder lab will also cooperate with the Kunkel lab in using cell biologicaland physical techniques to evaluate the pH-dependent motility of select bacterial species.

Both classical and targeted approaches will be used to determine the proportion ofextremophiles to non-extremophiles in distinct lobster populations. In the classical approach,scrapings obtained from shells of healthy animals and diseased shells will be cultured on marineagar at pH ranging from pH 7.6 - 11, and colony forming units (CFU) will be enumerated andused to calculate the proportion of alkaliphiles to non-alkaliphiles.

For a more targeted approach, we will select alkaliphilic and non-alkaliphilic species ofinterest from the DNA sequencing data obtained in part 1A and will use quantitative PCR(qPCR) to determine the relative abundance of each microbial species in healthy and diseasedshellfish.

The Burkholder lab will cooperate with the Kunkel lab using cell biological techniques tomeasure the microbial motility of select bacterial strains found to occupy the lobster shellsurface. The ionic basis of the motility will be determined by analyzing the sensitivity ofmotility to proton or Na concentrations. It might be expected that the marine bacteria living onthe high pH surfaces of shellfish would be powered by Na rather than protons. It is also possiblethat motility on the shell surface is Na driven while motility in the medium is proton based sinceit is established that Na driven rotors of Bacillus species are favored in higher pH conditions(Terahara et al. 2008). This would be an important difference to establish for shellfish as itmight be a general property by which to recognize shell surface living bacterial forms.Flexibility in this phenotype might also be important to understand in terms of the microbialstrategy to attack shellfish surfaces at which they might need to deploy their Na based flagellarrotor power.

Katja Huemer, is a polymer scientist about to get her Ph.D. studying lobster and cancercrab shell structure using Raman Spectrometry and is also familiar with the EMP and FTIRmethodology, which we propose to use to examine the fine resolution carbonate and phosphatemineralogy of the lobster cuticle shell. Her role will be to establish the mineral basis of theestablishment of the high pH unstirred layer during the early post molt hardening phase of the

shell. She will spend the six months post molt when that process is happening in the Kunkel labidentifying the best specimens of shell to fix and process for EMP, FTIR and Raman analysis. (e) Detailed data management plan (1) Articulating the coordination with one or more management entities

The Kunkel Lab database is maintained by the IT Staff in the Biology Department atUMass Amherst, where Kunkel remains Emeritus Professor with all the benefits that derive. Heis the manager of his own LabWiki, maintained on the Biology Department Sparcstation internetserver, which consists of an instance of MediaWiki and provides a password protected Wikienvironment for recording all the results of the Kunkel lab's research since 2007. The Wikiallows for all research group members to have password access to storage, annotation andretrieval of data archived there as images (GIF, JPEG, PNG, AVI) or text, plain (TXT) orformatted (PDF), as well as embedded in a simple MediaWiki grammar in which formatted text,tables and images can be provided in a readable, browser accessible, manner. Select images anddata sets in the Wiki Archive are directly publishable to the public as URLs.

The decision to collect data in a new and separate database from the extant databases(e.g. NERACOOS) is based on the experience that our data is primarily outside the current datatypes and information and would be difficult to shoehorn into the current databases. Where datatypes are comparable we will communicate with the Databases and provide compatible fileswhere possible. We have a Board of Advisors member, Steven Brewer, who is fluent in relationdatabase structure and accession.

A separate instance of MediaWiki will be established on the UMass Amherst BiologyDepartment computer system authorized by the Director of Computing and Networking, Collegeof Natural Science, UMass Amherst. Our Advisory Board member, Steve Brewer, is theDirector of the Biological Computer Resource Center who will be advising us on setting up ourMediaWiki instance to collect, manage and share our study data as well as how our data relatesto the Gulf-of-Maine issues and priorities.

A relational database management system (RDBMS) will be established utilizing mysql,the open source database system used by MediaWiki, to house our observations on lobsters,

locations and microbial samples. Eachlobster in the study will be represented by arecord. Each microbial sample will berepresented by a record and each lobster trapand NOAA trawl survey site in the studywill be represented by a record. All data inthe study will be identifiable by itsassociation with a specific lobster, trap andmicrobial sample. All recorded results ofthe study will inform us about a lobster, trapor trawl location and microbial samplecollected. The database will be accessed bystructured query language (SQL). We willlikely use Navicat software to help usadminister and navigate the databasestructure. We will use the R computationlanguage which includes a library offunctions for accessing various databases

Fig 5. Chlorophyl feature in Wilkerson Basinat depth >40m and < 160m from Rebuck et al.(2012) data, which correlates with Kunkelacidic bottom/top feature from Spring 2009.

including MySQL, producing so-called data frames of data extracted from our database. Accessto other databases such as the Rebucks and coworker (2012) Gulf of Maine Region Nutrient andHydrographic Database has already been achieved.

PI Kunkel has accessed the Rebuck and coworker (2012) Gulf of Maine Region Nutrientand Hydrographic Database, which is warned to be larger than Excel capacity, requiring Rlibraries of functions designed for large data sets: bigmemory, biganalytics and bigtabulate.These R libraries have allowed us to correlate our data on the Wilkinson's Basin acidic bottomand top seawater samples with the Rebuck Gulf of Maine big data on chlorophyll at differentdepths, fig 5. Several open access global databases are significant to our study area (e.g. theNERACOOS study area). Being prepared to read and process Big Data is an important option incooperating with ancillary data collection research programs. The MediaWiki instance to becreated specifically for this project is based on eight years of experience of managing the KunkelLabWiki. Our new Gulf_of_Maine_Wiki will house access to all the data and processingalgorithms and make them available to the research public. The current plan is to use the GnuPublic R Computation Environment as the basic computation environment and engine in ourresearch group, which has been sufficient for all aspects of our analysis to date. This use of aGnuPublic software mechanism will also facilitate us sharing analyses and analytic methods foraccessing our data with the research and broader public. We are also experimenting with thenew Julia computation environment which may afford a faster environment but would currentlyrequire individuals more experienced in large computation models (Bezanson et al, 2012).

Particular interest will begiven to available data from theNOAA buoys (NOAA 44007, 44005,44030, 44032) which are near oradjacent to our Casco Bay traps, fig6. Data from these buoys can berecorded at approximately the sametimes as we are sampling from ourtraps in Casco Bay. We areparticularly interested in usingAnalysis of Dispersion and the Testof Additional Information (Rao,1965), which have beenimplemented in R (Kunkel 2011) andwould allow asking such questionsas: A. What fraction of theconcordance in top vs bottom pHs

associated with deployed lobster traps that are observed in Casco Bay is ascribable to thephysical measures of turbulence measurable by the NOAA buoys in and surrounding CascoBay? B. Is there any significant additional information about bottom_vs_top_pH, associatedwith deployed traps, that is explainable by NOAA buoys 44005, 44030, 44032 above andbeyond that explained by the nearby NOAA buoy 44007 in Casco Bay? This type ofmultivariate-test-of-covariance has been used in other aspects of PI Kunkel's research design(McKenna et al. 2009). Here the nearness of buoy NOAA 44007 may provide informationwhich may explain some of the variance seen between sampling times at the traps. The fartheraway buoys may provide no additional information and be ignored. The near buoy may also

Fig 6. PI Tarbox deployed lobster traps for 2014 season.

provide no additional information beyond that provided by the Manning lobster trap sensors. Ifthe near NOAA buoy is useful, will it provide as much or significantly more that the Manningsensors on the traps? These are all useful questions. The expertise of PI Kunkel with this typeof multivariate analysis, posing meaningful questions of the data, and his ability to access andprocess available database information related to this proposal is key to our learning more aboutthe benthic processes in Casco Bay. Those proceses may be a model for things happeningalready elsewhere currently or in the future in the Gulf of Maine in general. (2) Discussing the expected significance of the project to resource management priorities.

Of particular significance is the priority of the valued fisheries of the Gulf of Maine,particularly the American lobster. We do not know the cause of ESD even as we see itincreasing in frequency alarmingly in Casco Bay. We have the unique opportunity at this time toperhaps see the earliest stages of the disease and identify the organism(s) that initiate it and howthey are associated with the seawater conditions and lobster and microbe physiology surroundingthe infection.

The incidence of ESD will be fit to various predictor variables principally seawaterproperties, seasonal timing factors and microbial properties, including the microbial-dye-bindingmeasures, using the General Additive Model. It is expected that some of the predictor variables,including various aspects of water quality will be natural predictors of shell disease. It is furtherexpected that some of these predictor variables would be useful in estimating the incidence andoutcomes of incipient shell disease in regions which are approaching the seawater qualityconditions observed in Casco Bay. The role of alkylphenols in the incidence of ESD is ofparticular interest because it is one parameter that could possibly be controlled by effectivelegislation. This factor may be of particular interest to states in which lobster provides asubstantial part of the economy.

In particular the ability to predict future lobster health, based on seawater qualities orsubliminal properties of ESD discoverable with our methods, will be of value to both lobstermenand marketing groups. The decision about when lobster, as product, should be switched fromthe live market to canning processing depends on whether subliminal clues of ESD can be reliedupon to predict the quality of the lobsters in the public's hands. (3) Describing specific activities, such as workshops or development of outreach ...

It is expected that the collected information on Casco Bay lobster health and its relationto the health of lobsters in the remainder of the Gulf of Maine and the microbes of the shellfishsurface will be of interest to the lobstermen, marketing representatives and the public. As wecarry out this project the outcomes will be shared and discussed within the research groupthrough our project Wiki which will also be developed as an avenue for sharing of data andanalytic tools with the broader research community as well as with the public. Data sets storedin the MySQL database tables will be extracted into the R computation environment by theresearch group and analysed routinely by R-scripts. These R-scripts will be posted on the Wikiin password protected areas until they are used and validated by research group members anddiscussed in Research Group Meetings. Validated exemplar R-scripts that produce output orgraphics of general interest will be posted to the public windows of the Wiki.

Our research group in its broader context will consist of the PIs and their researchassistants and colleagues plus a Board of Advisors drawn from lobster research, state agencies,fishing industry, NOAA expertise and the public interest groups. Meetings of these groups willdecide the outreach which is appropriate. Our experimentation with imaging and vital dying oflobsters to reveal subliminal ESD may turn into a practical assay similar to vital dye use in the

dental office to indicate gum disease (Chetrus 2014). In the event of such a development we willcertainly confer with our Advisory Board to determine the most efficient method promulgatingthe method to the fishermen and marketing groups with greatest interest.

Our protocols for collecting water quality will be made to conform as closely as possibleto standards that would allow inclusion in databases of ocean chemistry. We will coordinate ourdata sharing based on our advisories and with prior communication with regional organizationssuch as NERACOOS and the NE Groundfish Survey which can provide abundant CTD data onthe NE Coastal Shelf including the Gulf of Maine. Kunkel has experience accessing severallarge databases on world eco-physiological data, has graduate and postdoctoral training inbiometry and experience teaching biometry at UMass Amherst to undergraduate and graduatestudents as well as advising and collaborating with researchers in need of programming andanalysis skills. Having experience programming initially in fortran in biometrical training atCase-Western Reserve University, Kunkel proceeded though learning Pascal, C, APL and nowR. The fortran package that he developed in his dissertation to implement multivariate Analysisof Dispersion (Rao 1965) is now translated by him into R and will be used for analysis needed inthis project (Kunkel, 2011). Kunkel is aware of the math anxiety of the general public and willattempt to present the available tools of R and how they can allow us to look at environmentaldata to the listening audience. His skills with data analysis and presentation will be used inoutreach to public and professional groups who may be interested in the data we collect and howit relates to other data resources. One type of outreach workshop that Kunkel could participatein would be one on Big Data in Studying the Gulf of Maine which could help facilitate thebroader access of the research and industry community to the data being accumulated on theGulf of Maine and its relation to the larger world databases. On a local scale SMCC has aScience Department Seminar Series which provides outreach to students and citizens of SouthPortland and southern Maine in general. PI Joe Kunkel is an invited speaker to that series thiscoming Feb 5, 2015. His chosen topic will be “Landmark Analysis of Drosophila,Dragonflies and Lobsters”.

UNE Biddeford and SMCC have a cooperative agreement that was recently instituted inwhich Marine Science Majors with a 2 year AA degree in Marine Science can continue at UNEBiddeford and obtain a BS degree in one of UNE's Marine Science Majors with credit given fortheir SMCC courses. This will be an important and economic avenue for students to enter thefield of Marine Science. We will be drawing our undergraduate participants in our project fromthat pool of students.

Our current confirmed Board of Advisors includes:A. Roxanna Smolowitz, D.V.M, Visiting Professor of Biology, Roger Williams U., Bristol, RI B. Kathleen M. Castro, PhD, University of RI, Fisheries Center, East Farm, Kingston, RIC. Hans Laufer, Research Professor, U Connecticut, Dept of Molecular and Cell BiologyD. James Manning, NOAA NMFS,

http://www.nefsc.noaa.gov/epd/ocean/MainPage/lob/resume.htmE. Carrie Byron, Research Assistant Professor, UNE Biddeford, ME.F. Steven Brewer, Senior Lecturer, Biology Department, UMass Amherst, MA.

The Board of Advisors will meet together with the research group in joint meetings andbreakout groups once a year on the UNE Biddeford Campus. Oral reports will be given to theBoard and their comments and advice solicited during and after the meetings. We willencourage active communication between the research team and Board via Email and the ProjectWiki and comments of the Board will be posted on the Wiki for public access.