The feeding ecology of copepod nauplii:

the understudied life stage of copepods

Pseudodiaptomus marinus nauplii

Masters Thesis Robert Vogt March 11, 2013

1. Background/motivation

2. Combined methods and results

- Study organisms - Research question 1 - Research question 2

3. Summary & future directions

Why investigate feeding?

P: Alexander Chullivan

Why investigate feeding?

Small scale Biology/physiology Culturing
P: Alexander Chullivan

Why investigate feeding?

Large scale Trophic interactions Invasive species impacts
P: Alexander Chullivan

Images: Pwfieldguide,FWS,nationalgeographic, wikipedia, tqn

Copepods

~1-2 mm adult

Calanus pacificus
Img: WHOI

Life history

nauplius stages (6)

metamorphosis
Pseudodiaptomus marinus

copepodite stages (5)

adult stage (1)

The copepod diet

1000s of studies
Microzooplankton e.g., ciliates

Bacteria

Detritus

Phytoplankton

Other zoo.

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

The copepod diet

Nauplii

? of studies <25
Microzooplankton e.g., ciliates Bacteria Detritus Phytoplankton Other zoo.

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

The copepod diet

Nauplii

? of studies <25
Microzooplankton e.g., ciliates Bacteria Detritus Phytoplankton Other zoo.

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

~1 mm

Pseudodiaptomus marinus

nauplius N5

adult female C6

Anterior (Head end)

1st Antenna 2nd Antenna Mandible 1st Maxilla 2nd Maxilla Maxilliped 1st leg 5th leg Caudal ramus Caudal setae

1st Antenna 2nd Antenna Mandible

nauplius

adult
Source: The Oceans, Sverdrup, Johnson, Fleming

Pseudodiaptomus marinus

nauplii

adult
Videos: R. Vogt

Oithona davisae

nauplii

adult
Videos: R. Vogt

Hypothesis
Due to differences in:
morphology capabilities compared to adults feeding and swimming behavior

Nauplii have reduced feeding size

1. Background/motivation

2. Combined methods and results

- Study organisms - Research question 1 - Research question 2

3. Summary & future directions

~120 m

~500 m

Oithona davisae
Google earth ~300 m

~1400 m

Pseudodiaptomus marinus

Not to scale 1175 m

cyanobacteria

haptophytes

diatoms

Non-motile

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

prasinophyte

cryptomonad

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

Motile

1. Background/motivation

2. Combined methods and results

- Study organisms - Research question 1 - Research question 2

3. Summary & future directions

Q1: What phytoplankton are nauplii and adults capable of eating?

Q1: What phytoplankton are nauplii and adults capable of eating? Method 1: Inspection of guts using epifluorescence

Method 1: Inspection of guts using epifluorescence Inspiration

adult gut

Merrell & Stoecker 1998 (JPR)

Calanoid copepod Eurytemora affinis

nauplius gut

CMFDA labeled prey

Ciliate Strombidium sp.

Plingfactory.de

Method 1: Inspection of guts using epifluorescence Emission Excitation How it works

~400-440 ~650-700

Copepod

Chlorophyll a

CMFDA

Chlorophyll a

Image: suntrek.org

300

550

800 nm

Fluorescence response

Excitation response

Stained prey

Algae

Method 1: Inspection of guts using epifluorescence Experimental protocol

Method 1: Inspection of guts using epifluorescence Experimental protocol

Gut pigment

Raw image

Method 1: Inspection of guts using epifluorescence Experimental protocol

Gut pigment

Raw image

Color threshold tool (Photoshop CS)

Method 1: Inspection of guts using epifluorescence Experimental protocol

e.g., 1000 pixels

Gut pigment

Raw image

Color threshold tool (Photoshop CS)

Area measurement tool

Method 1: Inspection of guts using epifluorescence Experimental protocol

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

Digitized 2D area of gut extent

Method 1: Inspection of guts using epifluorescence Experimental protocol

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

Digitized 2D area of gut extent

Binary test

Semi-quantitative None Moderate Extensive

Yes No

100

Method 1: Inspection of guts using epifluorescence Experimental protocol

Copepods staged, sorted Starved for 3 hours in filtered water

Method 1: Inspection of guts using epifluorescence Experimental protocol

Copepods staged, sorted Starved for 3 hours in filtered water

Copepods were incubated with each algae (individually) + controls 60 min incubation

Method 1: Inspection of guts using epifluorescence Experimental protocol

Copepods staged, sorted Starved for 3 hours in filtered water

Copepods were incubated with each algae (individually) + controls 60 min incubation

12 copepods processed per algae/stage 720 copepods imaged and processed

Motile Nonmotile

Results: Pseudodiaptomus marinus

N5 nauplius C6 adult

Smallest 1 m

Largest 150 m low high Gut pigment index low high Gut pigment index

Motile Nonmotile

Results: Pseudodiaptomus marinus

N5 nauplius C6 adult

Smallest 1 m

Largest 150 m low high Gut pigment index low high Gut pigment index

Motile Nonmotile

Results: Pseudodiaptomus marinus

N5 nauplius C6 adult

Smallest 1 m

Largest 150 m low high Gut pigment index low high Gut pigment index

Motile Nonmotile

Results: Pseudodiaptomus marinus

N5 nauplius C6 adult

Smallest 1 m

Largest 150 m low high Gut pigment index low high Gut pigment index

Motile NonMotile motile Nonmotile

Smallest 1 m

Results: Oithona davisae

N5 nauplius C6 adult

Largest 150 m low high Gut pigment index low high Gut pigment index

Motile NonMotile motile Nonmotile

Smallest 1 m

Results: Oithona davisae

N5 nauplius C6 adult

Largest 150 m low high Gut pigment index low high Gut pigment index

Motile NonMotile motile Nonmotile

Smallest 1 m

Results: Oithona davisae

N5 nauplius C6 adult

Largest 150 m low high Gut pigment index low high Gut pigment index

Motile NonMotile motile Nonmotile

Smallest 1 m

Results: Oithona davisae

N5 nauplius C6 adult

Largest 150 m low high Gut pigment index low high Gut pigment index

Motile NonMotile motile Nonmotile

Smallest 1 m

Results: Oithona davisae

N5 nauplius C6 adult

Largest 150 m low high Gut pigment index low high Gut pigment index

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

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

Yes

Diatom frustules found in guts of Oithonidae in Gulf of Mexico

(Turner 1986)

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

Chaetoceros socialis
P. P. O. marinus marinus davisae Adult Nauplii Adult O. davisae Nauplii

Chain diatom Colonial polymer mats Long siliceous spines (10 m; cell only)

Chaetoceros muelleri Same genus Similar morphology Chains, no social colony size (5 m) P. P. O. marinus marinus davisae Adult Nauplii Adult O. davisae Nauplii

Anti-grazer defense? Buoyancy?

Feeding size range

1 m

10

15

20

125

250

nauplii adults
P. marinus O. davisae

1. Background/motivation

2. Combined methods and results

- Study organisms - Research question 1 - Research question 2

3. Summary & future directions

Q2: How do feeding rates compare?

Q2: How do feeding rates compare? Method 2: Modified gut fluorescence method

Method 2: Modified gut fluorescence method Zooplankton feeding rate estimation

Microscopic counts

Flow cytometery
Images: soozial.com, musc, soton

Method 2: Modified gut fluorescence method Zooplankton feeding rate estimation

Microscopic counts

Flow cytometery
Images: soozial.com, musc, soton

Method 2: Modified gut fluorescence method Zooplankton feeding rate estimation

Microscopic counts

Flow cytometery
Images: soozial.com, musc, soton

Method 2: Modified gut fluorescence method Zooplankton feeding rate estimation

Images: soozial.com, musc, soton

Method 2: Modified gut fluorescence method Inspiration

Mackas and Bohrer 1976 (JEMBE)

Centropages typicus
Img: Albert Calbet
Imgs: NOAA, Water Research.net, Turner

Method 2: Modified gut fluorescence method Background

N requirements

Adults

Fluorometer 20 - 200

Method 2: Modified gut fluorescence method Background

Chlorophyll degradation

Leads to feeding rate underestimates

Method 2: Modified gut fluorescence method Background Gut fluorescence measurement using a microplate reader

- Few individuals required - Short incubations - Minimize degradation -

1-5

5 - 50

Method 2: Modified gut fluorescence method Preliminary experiments

Gut depuration time

Chosen depuration time Raw Fluorescence Units

Whiskers: 5-95% percentile

Blank

Time (min.)

Method 2: Modified gut fluorescence method Preliminary experiments

Gut saturation time

Chosen incubation times Raw Fluorescence Units Defecation

Whiskers: 5-95% percentile

Blank

Time (min.)

Method 2: Modified gut fluorescence method Experimental protocol

1. Stage and sort copepods

Method 2: Modified gut fluorescence method Experimental protocol

2. Allow copepods to starve for 3 hours

Method 2: Modified gut fluorescence method Experimental protocol

2. Allow copepods to starve for 3 hours

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

Method 2: Modified gut fluorescence method Experimental protocol

2. Allow copepods to starve for 3 hours

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

4. Incubate (30-60 min)

Method 2: Modified gut fluorescence method Experimental protocol

2. Allow copepods to starve for 3 hours

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

4. Incubate (30-60 min)

5. Process copepods place in centrifuge tubes

Method 2: Modified gut fluorescence method Experimental protocol

2. Allow copepods to starve for 3 hours

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

4. Incubate (30-60 min)

5. Process copepods 6. Flash freeze on dry ice place in centrifuge tubes

Method 2: Modified gut fluorescence method Experimental protocol

2. Allow copepods to starve for 3 hours

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

4. Incubate (30-60 min)

5. Process copepods 6. Flash freeze on dry ice 7. Extract samples place in centrifuge tubes overnight in 95% ethanol @ -20 C

Method 2: Modified gut fluorescence method Experimental protocol

8. Measure concentration of standard using spectrophotometer

Method 2: Modified gut fluorescence method Experimental protocol

8. Measure concentration of standard using spectrophotometer

9. Prepare dilution series of stock standards

Method 2: Modified gut fluorescence method Experimental protocol

8. Measure concentration of standard using spectrophotometer

9. Prepare dilution series of stock standards

10. Pipette samples, standards, and blanks into microplate

Method 2: Modified gut fluorescence method Experimental protocol

8. Prepare standard

9. Prepare dilution series of standard

10. Pipette samples, standards, and blanks into microplate

11. Read samples on a microplate reader (Tecan infinite)

Method 2: Modified gut fluorescence method Experimental protocol Chlorophyll is degraded by: Heat Light Oxidation 8. Prepare standard 9. Prepare dilution series Dirty looks* of standard

Chl a 10. Pipette samples, standards, and blanks into microplate 11. Read samples on a microplate reader (Tecan * source:infinite) Julian Herndon

Method 2: Modified gut fluorescence method

Treatment groups 3 species of algae Fed 500 g/C L-1 of algae 30-60 min incubation

Method 2: Modified gut fluorescence method

Treatment groups 3 species of algae Fed 500 g/C L-1 of algae 30-60 min incubation Control groups T0: Not fed, sampled before incubation Tf: Not fed, sampled after incubation T0 control Tf control Rinse control Rinse: Fed, incubation terminated immediately

Method 2: Modified gut fluorescence method

Treatment groups 3 species of algae Fed 500 g/C L-1 of algae 30-60 min incubation Control groups T0: Not fed, sampled before incubation Tf: Not fed, sampled after incubation T0 control Tf control Rinse control Rinse: Fed, incubation terminated immediately

Prepared/read on microplate reader

Pseudodiaptomus marinus

Whiskers: 5-95% percentile

ng chl a ind.

-1

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

Oithona davisae

ng chl a ind.

-1

0.08

0.03

Algae species Feeding rate of nauplii: ~40% of adult on Tetraselmis suecica

Oithona davisae grows on larger food

Oxyrrhis marina
Img: NHS

Tetraselmis suecica
Img: NHS

Rhodomonas salina Feeding trial experiment

P. P. O. marinus marinus davisae Adult Nauplii Adult O. davisae Nauplii

GPI <25%

Grazing rate experiment

Cryptomonad Flagellate Known good food for calanoid copepods P. P. O. marinus marinus davisae Adult Nauplii Adult O. davisae Nauplii

Oithona davisae vs. 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)

Oithona davisae vs. 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

Estimated daily ration % (prey carbon predator carbon-1 d-1)
Pseudodiaptomus marinus Nauplii Adult N/A 24-29 50a 5-15 Oithona davisae Nauplii Adult 85b; 100c 90 428d 45

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

Comparisons with other studies

Estimated daily ration % (prey carbon predator carbon-1 d-1)
Pseudodiaptomus marinus Nauplii Adult N/A 24-29 50a 5-15 Oithona davisae Nauplii Adult 85b; 100c 90 428d 45

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

1. Background/motivation

2. Combined methods and results

- Study organisms - Research question 1 - Research question 2

3. Summary & future directions

Summary: naupliar feeding

Broad feeding capability

Summary: naupliar feeding

Broad feeding capability

High feeding rate biomass

Summary: naupliar feeding

Broad feeding capability

High feeding rate biomass

Underestimated impact?

vs.

Future directions Food limitation

Future directions Food limitation

Other food resources? Prey selection?

protists bacteria detritus

?
other zoo.

Future directions Food limitation

Other food resources? Prey selection?

protists bacteria detritus

?
other zoo.

Different food: growth and survival

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

Manuscript coauthors Wim Kimmerer, Lead PI Toni Ignoffo Lindsay Sullivan Julian Herndon Jonathon Stillman Kimmerer lab Anne Slaughter Rita Dumais Jessica Donald Karen Kayfetz Aaron Johnson Julien Moderan Caroline Kosteki Student interns Sean Rohtla Annie De Lancie Cochlan lab Chris Ikeda Bill Cochlan Stillman lab Nate Miller Moral supporters Olivia De Lancie The Vogt family RTC Community

Thanks!

Method 2: Modified gut fluorescence method Trial experiment

Takatsuji 1997 (JPR) 0.93 0.83

Method 2: Modified gut fluorescence method Data analysis

Before acidification reading After acidification reading

Rb Ra Cs/Rs

Response factors of calibration standards = Fs Response ratios of calibration standards = r Chlorophyll a concentration Pheophytin a concentration Total chlorophyll

Rb/Ra

Fs(r/r-1)(Rb-Ra) Fs(r/r-1)(rRa-Rb) Chloro + Pheo

12 individuals Each copepod Each stage Averaged

Method 2: Modified gut fluorescence method Justification

Field
Pros Cons

Laboratory
Pros Cons

Method 2: Modified gut fluorescence method Justification

Field
Pros
Field organisms

Laboratory
Cons Pros Cons
Cultured organisms

Method 2: Modified gut fluorescence method Justification

Field
Pros
Field organisms Natural prey/ abundances

Laboratory
Cons Pros Cons
Cultured organisms Cultured prey

Method 2: Modified gut fluorescence method Justification

Field
Pros
Field organisms Natural prey/ abundances Natural environment

Laboratory
Cons Pros Cons
Cultured organisms Cultured prey Controlled environment

Method 2: Modified gut fluorescence method Justification

Field
Pros
Field organisms Natural prey/ abundances Natural environment No need to incubate

Laboratory
Cons Pros Cons
Cultured organisms Cultured prey Controlled environment Lengthy incubations*

Method 2: Modified gut fluorescence method Justification

Field
Pros
Field organisms Natural prey/ abundances Natural environment No need to incubate

Laboratory
Cons Pros
Pigment degradation minimized

Cons
Cultured organisms Cultured prey Controlled environment Lengthy incubations*

Pigment degradation

Method 2: Modified gut fluorescence method Justification

Field
Pros
Field organisms Natural prey/ abundances Natural environment No need to incubate

Laboratory
Cons Pros
Pigment degradation minimized Grazing rate modeled by ingestion

Cons
Cultured organisms Cultured prey Controlled environment Lengthy incubations*

Pigment degradation Grazing rate modeled by digestion

Method 2: Modified gut fluorescence method Justification

Field
Pros
Field organisms Natural prey/ abundances Natural environment No need to incubate

Laboratory
Cons Pros
Pigment degradation minimized Grazing rate modeled by ingestion Control what can be eaten

Cons
Cultured organisms Cultured prey Controlled environment Lengthy incubations*

Pigment degradation Grazing rate modeled by digestion Difficult to determine whats been eaten

Method 2: Modified gut fluorescence method Justification

Field
Pros
Field organisms Natural prey/ abundances Natural environment No need to incubate

Laboratory
Cons Pros
Pigment degradation minimized Grazing rate modeled by ingestion Control what can be eaten Unlimited # individuals/ easy ID

Cons
Cultured organisms Cultured prey Controlled environment Lengthy incubations*

Pigment degradation Grazing rate modeled by digestion Difficult to determine whats been eaten Limited # individuals/ difficult ID

Method 2: Modified gut fluorescence method Data analysis

Cell count with hemocytometer (400-1000 Cells)

Chlorophyll extracted from algae culture, measured w/ fluorometer

Cell count interpolated from chlorophyll measurement e.g., .010 ng chl a cell-1 1 ng chl a in copepod incubated for 1 hr 100 cells ingested/hr

Method 2: Modified gut fluorescence method Part B: Analysis protocol

Pure chlorophyll from Anacystis Nidulans cyanobacteria Extracted in 95% Ethanol Preserved with 99.9% argon gas and flame-sealed