copepod nauplii thesis defense

7/29/2019 Copepod Nauplii Thesis Defense

1/108

The feeding ecology of copepod nauplii:the understudied life stage of copepods

Masters Thesis

Robert Vogt

March 11, 2013

Pseudodiaptomus marinus nauplii


2/108

1. Background/motivation

2. Combined methods and results

3. Summary & future directions

- Study organisms

- Research question 1

- Research question 2


3/108

P: Alexander Chullivan

Why investigate feeding?


4/108

Small scale

Biology/physiology

Culturing




5/108


Large scale

Trophic interactions

Invasive speciesimpacts



6/108

Images: Pwfieldguide,FWS,nationalgeographic, wikipedia, tqn


7/108

Img: WHOI

~1-2 mmadult

Copepods

Calanus pacificus


8/108


9/108

nauplius stages (6)

copepodite stages (5)

Pseudodiaptomus marinus

metamorphosis

Life history

adult stage (1)


10/108

The copepod diet

Kleppel 1993, Adrian and Frost 1993, Paffenhofer andKnowles 1980, Atkinson 1995, Berk et al. 1977, Heinle 1977

1000s of studies

Microzooplankton

e.g., ciliates Bacteria Detritus Phytoplankton Other zoo.


11/108

The copepod diet


Microzooplankton


Nauplii

?


12/108

The copepod diet


Microzooplankton


Nauplii

?


13/108

~1mm


naupliusN5

adult femaleC6


14/108

1st Antenna

2nd Antenna

Mandible

1st

Antenna2nd Antenna

Mandible

1st Maxilla

5th leg

1st leg

Caudal ramus

Caudalsetae

Maxilliped

2nd Maxilla

Anterior

(Head end)

nauplius adult

Source: The Oceans,

Sverdrup, Johnson, Fleming


15/108


nauplii adult

Videos: R. Vogt


16/108

Oithona davisae

nauplii adult

Videos: R. Vogt


17/108

size morphology feeding and swimming behavior

Due to differences in:

Hypothesis

Nauplii have reduced feeding

capabilities compared to adults


18/108




-Study organisms-Research question 1-Research question 2


19/108

Google earth


Oithona davisae

~1400 m

~300 m

~500 m

~120 m


20/108

Image citations in order from top left: Leadingtec, Protist info server,MBL, Biomarks lifedesk, MD DNR, AlfredWegener Institute,CCMP, SMHI, jcoll.org

cryptomonadprasinophyte dinoflagellates

Not to scale1175 m

cyanobacteria haptophytes diatoms

Non-motile

MotileImage citations in order from left: CCMP, starcentral MBL, CCMP, TAMUG,LEPTON Wiki


21/108






22/108

Q1: What phytoplankton are nauplii and adults

capable of eating?


23/108

Method 1: Inspection of guts using epifluorescence

Q1: What phytoplankton are nauplii and adults

capable of eating?


24/108

Merrell & Stoecker 1998

(JPR)

Plingfactory.de

Eurytemoraaffinis

Strombidium sp.

CMFDA labeledprey

adult gut

nauplius gut

Calanoid copepod

Ciliate


Inspiration


25/108

How it works Excitation~400-440

Emission~650-700

Copepod

Chlorophyll a

Image: suntrek.org

Excitation

response

800 nm550300

Algae

Chlorophyll a

Fluorescence

response


Stainedprey

CMFDA


26/108


Experimental protocol


27/108

Raw image

Gut pigment




28/108

Raw image Color thresholdtool

(Photoshop CS)

Gut pigment




29/108

Areameasurement tool

Raw image

e.g., 1000 pixels

Color thresholdtool

(Photoshop CS)

Gut pigment




30/108

Digitized 2Darea of gut

extent

Area of gut pigment/area of gut extent= gut pigment index (GPI)




31/108

Area of gut pigment/area of gut extent= gut pigment index (GPI)

Yes

No

None Moderate Extensive

0 100

%

Binary test Semi-quantitative



Digitized 2Darea of gut

extent


32/108

Copepods staged, sortedStarved for 3 hours in filtered water




33/108


60 min incubation

Copepods were incubatedwith each algae (individually)+ controls




34/108


12 copepods processedper algae/stage

720 copepods imaged and processed



60 min incubation

Copepods were incubatedwith each algae (individually)+ controls


35/108

Motile

Non-motile

Results: Pseudodiaptomus marinus

C6adult

N5nauplius

Smallest1 m

Largest150 m

Gut pigment index

low high

Gut pigment index

low high


36/108

Motile

Non-motile


C6adult

N5nauplius

Smallest1 m

Largest150 m

Gut pigment index

low high

Gut pigment index

low high


37/108

Motile

Non-motile


C6adult

N5nauplius

Smallest1 m

Largest150 m

Gut pigment index

low high

Gut pigment index

low high


38/108

Motile

Non-motile


C6adult

N5nauplius

Smallest1 m

Largest150 m

Gut pigment index

low high

Gut pigment index

low high


39/108

Motile

Non-motile

Results: Oithona davisaeMotile

Non-motile

C6adult

N5nauplius

Smallest1 m

Largest150 m

Gut pigment index

low high

Gut pigment index

low high


40/108

Motile

Non-motile


Non-motile

C6adult

N5nauplius

Smallest1 m

Largest150 m

Gut pigment index

low high

Gut pigment index

low high


41/108

Motile

Non-motile


Non-motile

C6adult

N5nauplius

Smallest1 m

Largest150 m

Gut pigment index

low high

Gut pigment index

low high


42/108

Motile

Non-motile


Non-motile

C6adult

N5nauplius

Smallest1 m

Largest150 m

Gut pigment index

low high

Gut pigment index

low high


43/108

Motile

Non-motile


Non-motile

C6adult

N5nauplius

Smallest1 m

Largest150 m

Gut pigment index

low high

Gut pigment index

low high


44/108

Oithona eats diatoms?

Uses hydromechanical reception,so cannot detect non-motile food(Atkinson 1996; Uchima and Hirano 1986;

Nakamura and Turner 1997; Paffenhfer 1993)

No


45/108

Uses hydromechanical reception,so cannot detect non-motile food(Atkinson 1996; Uchima and Hirano 1986;

Nakamura and Turner 1997; Paffenhfer 1993)

Diatom frustules found in guts of

Oithonidae in Gulf of Mexico(Turner 1986)

Oithona similis feeds on pennate diatomsin the Arctic and North Atlantic(Atkinson 1996; Castellani et al. 2005; Castellani et al. 2008;Pond and Ward 2011).

No

Yes

Oithona eats diatoms?


46/108

Chaetoceros socialis

Anti-grazer defense?Buoyancy?

P.

marinusAdult

P.

marinusNauplii

O.

davisaeAdult

O.

davisaeNauplii

X X X X

Chaetoceros muelleri Same genus Similar morphology Chains, no social

colony size (5 m)

P.marinus

Adult

P.marinus

Nauplii

O.davisae

Adult

O.davisae

Nauplii

Chain diatom Colonial polymer mats Long siliceous spines

(10 m; cell only)


47/108

1 m 5 10 15 20 125 250

Feeding size range

P. marinus

O. davisae

nauplii

adults


48/108






49/108

Q2: How do feeding rates compare?


50/108

Method 2: Modified gut fluorescence method

Q2: How do feeding rates compare?


51/108

Flow cytometery

Microscopic counts

Zooplankton feeding rate estimationMethod 2: Modified gut fluorescence method

Images: soozial.com,

musc, soton


52/108

Flow cytometery

Microscopic counts



musc, soton


53/108

Flow cytometery

Microscopic counts



musc, soton


54/108


musc, soton



55/108

Mackas and Bohrer 1976 (JEMBE)

Imgs: NOAA, Water Research.net, Turner

Img: Albert Calbet

Centropages typicus


Inspiration


56/108

Adults

N requirements


20 - 200

Background

Fluorometer


57/108


58/108

Chlorophyll degradation

Leads to feeding rateunderestimates


Background


59/108

Gut fluorescence measurement using a microplate

reader

5 - 501 - 5

- Few individuals required -- Short incubations -

- Minimize degradation -


Background


60/108

Gut depuration time

Blank

Chosen depuration time

RawF

luorescence

Units

Time (min.

Whiskers:5-95%

percentile


Preliminary experiments


61/108

Gut saturation time

Blank

DefecationChosen incubationtimes

Time (min.)

RawF

luorescence

Units

Whiskers:5-95%

percentile


Preliminary experiments


62/108


1. Stage and sort copepods



63/108

2. Allow copepods tostarve for3 hours




64/108


3. Add algae(~500 g C/L-1)




65/108


3. Add algae(~500 g C/L-1) 4. Incubate (30-60 min)




66/108



5. Process copepods place in centrifuge tubes




67/108




6. Flash freeze on dry ice



h d 2 difi d fl h d


68/108


3. Add algae(~500 g C/L-1)

4. Incubate (30-60 min)


7. Extract samplesovernight in 95% ethanol

@ -20 C

6. Flash freeze on dry ice



M h d 2 M difi d fl h d


69/108

8. Measure concentrationof standard using

spectrophotometer



M th d 2 M difi d t fl th d


70/108


spectrophotometer

9. Prepare dilution seriesof stock standards





71/108


spectrophotometer

9. Prepare dilution seriesof stock standards

10. Pipette samples,standards, and blanks

into microplate





72/108

8. Prepare standard 9. Prepare dilution seriesof standard


into microplate

11. Read samples on amicroplate reader (Tecan

infinite)





73/108

8. Prepare standard 9. Prepare dilution seriesof standard


into microplate

11. Read samples on amicroplate reader (Tecan

infinite)



Chlorophyll is degraded by:

HeatLight

Oxidation

Dirty looks*

* source: Julian Herndon

Chl a



74/108

30-60 min incubation

3 species of algae

Fed 500 g/C L-1 of algae


Treatment groups



75/108


3 species of algae


T0 control Tf control Rinsecontrol


T0: Not fed, sampled beforeincubationTf: Not fed, sampled after incubation

Rinse: Fed, incubation terminatedimmediately

Treatment groups

Control groups



76/108


3 species of algae


T0 control Tf control Rinsecontrol


T0: Not fed, sampled beforeincubationTf: Not fed, sampled after incubation

Rinse: Fed, incubation terminatedimmediately

Treatment groups

Control groups

Prepared/read on microplate reader



77/108

Algae species

ngchlain

d.

-1


Whiskers:5-95% percentile

Feeding rate of nauplii: ~7-10% of adult

Oithona davisae


78/108

Algae species

ngchlain

d.

-1 0.08

0.03

Oithona davisae

Feeding rate of nauplii: ~40% of adulton Tetraselmis suecica

Oithona davisae grows


79/108

Oxyrrhis marinaImg: NHS

Tetraselmis suecicaImg: NHS

Oithona davisae growson larger food


80/108

Rhodomonas salina

P.marinus

Adult

P.marinus

Nauplii

O.davisae

Adult

O.davisae

Nauplii

P.marinus

Adult

P.marinus

Nauplii

O.davisae

Adult

O.davisae

Nauplii

X X

Feeding trial experiment

Grazing rate experiment

Cryptomonad

Flagellate

Known good food for calanoidcopepods

GPI


81/108

Oithona davisaevs. Rhodomonas salina

Saiz et al. 2003 reported feeding by O.davisae on R. salina was minimal

Could not keep O. davisae alive on it(Personal comm. w/ E. Saiz - Institut de Cincies del Mar)


82/108

Oithona davisaevs. Rhodomonas salina

Saiz et al. 2003 reported feeding by O.davisae on R. salina was minimal

Could not keep O. davisae alive on it(Personal comm. w/ E. Saiz - Institut de Cincies del Mar)

Below lower detection limit? (~5-6 cells) Lower density effect? Active rejection; poor taste or nutrition?

Comparisons with other studies


83/108

Estimated daily ration %

(prey carbon predator carbon-1 d-1)

Pseudodiaptomus marinus Oithona davisae

Nauplii Adult Nauplii Adult

N/A 50a 85b; 100c 428d

24-29 5-15 90 45


Uye 1986a; Almeda et al. 2010b; Henriskenet al. 2007c; Saiz et al. 2003d



84/108

Estimated daily ration %

(prey carbon predator carbon-1 d-1)

Pseudodiaptomus marinus Oithona davisae

Nauplii Adult Nauplii Adult

N/A 50a 85b; 100c 428d

24-29 5-15 90 45

Uye 1986a; Almeda et al. 2010b; Henriskenet al. 2007c; Saiz et al.2003d


1 Background/motivation


85/108






86/108

Summary: naupliar feeding

Broad feeding capability

f


87/108


High feeding rate biomass


S li f di


88/108



Underestimated impact? vs.

High feeding rate biomass

F di i


89/108

Future directions

Food limitation

F t di ti


90/108

Future directions

?

Food limitation

Other food resources?Prey selection?

protists bacteria detritus otherzoo.

F t di ti


91/108

Future directions

Food limitation

Other food resources?Prey selection?

Different food:

growth and survival

?

protists bacteria detritus otherzoo.


92/108

Oceanographerschoice.com, doubleklawnandlandscape.com, Richard Kirby, microscopy-uk.org.uk, bio.miami.edu,, wikimedia.or

Manuscript coauthors

Wim Kimmerer, Lead PI


93/108

Thanks! Wim Kimmerer, Lead PI Toni Ignoffo Lindsay Sullivan Julian Herndon Jonathon StillmanKimmerer lab

Anne Slaughter Rita Dumais Jessica Donald Karen Kayfetz Aaron Johnson

Julien Moderan Caroline KostekiStudent interns

Sean Rohtla Annie De LancieCochlan lab

Chris Ikeda Bill CochlanStillman lab Nate MillerMoral supporters

Olivia De Lancie The Vogt family RTC Community


94/108



95/108

0.93

Takatsuji 1997

(JPR)

g

Trial experiment

0.83



96/108

Before acidification reading

After acidification reading

Total chlorophyll

Rb

Ra

r = Rb/RaResponse ratios of calibration standards

Chlorophyll a concentration

Pheophytin a concentration

Response factors of calibration standardsFs = Cs/Rs

Fs(r/r-1)(Rb-Ra)

Fs(r/r-1)(rRa-Rb)

Chloro + Pheo

g

Data analysis


97/108

12individuals

Eachcopepod

EachstageAveraged



98/108

Field

Pros Cons Pros Cons

Laboratory

Justification



99/108

Field

Pros Cons

Field organisms

Pros Cons

Cultured

organisms

Laboratory

Justification



100/108

Pros Cons

Field organisms

Natural prey/abundances

Pros Cons

Cultured

organisms

Cultured prey

Field Laboratory

Justification



101/108

Pros Cons

Field organisms


Natural

environment

Pros Cons

Cultured

organisms

Cultured prey

Controlled

environment

Field Laboratory

Justification



102/108

Pros Cons

Field organisms


Natural

environment

No need toincubate

Pros Cons

Cultured

organisms

Cultured prey

Controlled

environment

Lengthyincubations*

Field Laboratory

Justification



103/108

Pros Cons

Field organismsPigment

degradation


Natural

environment

No need toincubate

Pros Cons

Pigment

degradationminimized

Cultured

organisms

Cultured prey

Controlled

environment

Lengthyincubations*

Field Laboratory

Justification



104/108

Pros Cons


degradation


Grazing ratemodeled bydigestion

Natural

environment

No need toincubate

Pros Cons

Pigment


Cultured

organisms

Grazing ratemodeled byingestion

Cultured prey

Controlled

environment

Lengthyincubations*

Field Laboratory

Justification



105/108

Pros Cons


degradation



Naturalenvironment

Difficult to

determinewhats beeneaten

No need toincubate

Pros Cons

Pigment


Cultured

organisms


Cultured prey

Control what

can be eaten

Controlled

environment

Lengthyincubations*

Field Laboratory

Justification



106/108

Pros Cons


degradation



Naturalenvironment

Difficult to

determinewhats beeneaten

No need toincubate

Limited #individuals/difficult ID

Pros Cons

Pigment


Cultured

organisms


Cultured prey

Control what

can be eaten

Controlled

environment

Unlimited #individuals/

easy ID

Lengthyincubations*

Field Laboratory

Justification


l


107/108

Cell count with hemocytometer (400-1000 Cells)

Chlorophyll extracted from algae culture, measured w/fluorometer

Cell count interpolated from chlorophyll measuremente.g.,.010 ng chl a cell-1

1 ng chl a in copepod incubated for 1 hr

100 cells ingested/hr

Data analysis


P B A l i l


108/108

Pure chlorophyll fromAnacystis

Nidulans cyanobacteria

Extracted in 95% Ethanol

Preserved with 99.9%argon gasand flame-sealed

Part B: Analysis protocol

copepod nauplii thesis defense

Documents