Copepod Nauplii Thesis Defense
Transcript of Copepod Nauplii Thesis Defense
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The feeding ecology of copepod nauplii:the understudied life stage of copepods
Masters Thesis
Robert Vogt
March 11, 2013
Pseudodiaptomus marinus nauplii
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1. Background/motivation
2. Combined methods and results
3. Summary & future directions
- Study organisms
- Research question 1
- Research question 2
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P: Alexander Chullivan
Why investigate feeding?
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Small scale
Biology/physiology
Culturing
P: Alexander Chullivan
Why investigate feeding?
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Why investigate feeding?
Large scale
Trophic interactions
Invasive speciesimpacts
P: Alexander Chullivan
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Images: Pwfieldguide,FWS,nationalgeographic, wikipedia, tqn
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Img: WHOI
~1-2 mmadult
Copepods
Calanus pacificus
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nauplius stages (6)
copepodite stages (5)
Pseudodiaptomus marinus
metamorphosis
Life history
adult stage (1)
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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.
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The copepod diet
Kleppel 1993, Adrian and Frost 1993, Paffenhofer andKnowles 1980, Atkinson 1995, Berk et al. 1977, Heinle 1977
Microzooplankton
e.g., ciliates Bacteria Detritus Phytoplankton Other zoo.
Nauplii
?
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The copepod diet
Kleppel 1993, Adrian and Frost 1993, Paffenhofer andKnowles 1980, Atkinson 1995, Berk et al. 1977, Heinle 1977
Microzooplankton
e.g., ciliates Bacteria Detritus Phytoplankton Other zoo.
Nauplii
?
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~1mm
Pseudodiaptomus marinus
naupliusN5
adult femaleC6
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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
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Pseudodiaptomus marinus
nauplii adult
Videos: R. Vogt
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Oithona davisae
nauplii adult
Videos: R. Vogt
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size morphology feeding and swimming behavior
Due to differences in:
Hypothesis
Nauplii have reduced feeding
capabilities compared to adults
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1. Background/motivation
2. Combined methods and results
3. Summary & future directions
-Study organisms-Research question 1-Research question 2
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Google earth
Pseudodiaptomus marinus
Oithona davisae
~1400 m
~300 m
~500 m
~120 m
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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
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1. Background/motivation
2. Combined methods and results
3. Summary & future directions
-Study organisms-Research question 1-Research question 2
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Q1: What phytoplankton are nauplii and adults
capable of eating?
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Method 1: Inspection of guts using epifluorescence
Q1: What phytoplankton are nauplii and adults
capable of eating?
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Merrell & Stoecker 1998
(JPR)
Plingfactory.de
Eurytemoraaffinis
Strombidium sp.
CMFDA labeledprey
adult gut
nauplius gut
Calanoid copepod
Ciliate
Method 1: Inspection of guts using epifluorescence
Inspiration
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How it works Excitation~400-440
Emission~650-700
Copepod
Chlorophyll a
Image: suntrek.org
Excitation
response
800 nm550300
Algae
Chlorophyll a
Fluorescence
response
Method 1: Inspection of guts using epifluorescence
Stainedprey
CMFDA
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Method 1: Inspection of guts using epifluorescence
Experimental protocol
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Raw image
Gut pigment
Method 1: Inspection of guts using epifluorescence
Experimental protocol
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Raw image Color thresholdtool
(Photoshop CS)
Gut pigment
Method 1: Inspection of guts using epifluorescence
Experimental protocol
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Areameasurement tool
Raw image
e.g., 1000 pixels
Color thresholdtool
(Photoshop CS)
Gut pigment
Method 1: Inspection of guts using epifluorescence
Experimental protocol
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Digitized 2Darea of gut
extent
Area of gut pigment/area of gut extent= gut pigment index (GPI)
Method 1: Inspection of guts using epifluorescence
Experimental protocol
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Area of gut pigment/area of gut extent= gut pigment index (GPI)
Yes
No
None Moderate Extensive
0 100
%
Binary test Semi-quantitative
Method 1: Inspection of guts using epifluorescence
Experimental protocol
Digitized 2Darea of gut
extent
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Copepods staged, sortedStarved for 3 hours in filtered water
Method 1: Inspection of guts using epifluorescence
Experimental protocol
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Copepods staged, sortedStarved for 3 hours in filtered water
60 min incubation
Copepods were incubatedwith each algae (individually)+ controls
Method 1: Inspection of guts using epifluorescence
Experimental protocol
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Copepods staged, sortedStarved for 3 hours in filtered water
12 copepods processedper algae/stage
720 copepods imaged and processed
Method 1: Inspection of guts using epifluorescence
Experimental protocol
60 min incubation
Copepods were incubatedwith each algae (individually)+ controls
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Motile
Non-motile
Results: Pseudodiaptomus marinus
C6adult
N5nauplius
Smallest1 m
Largest150 m
Gut pigment index
low high
Gut pigment index
low high
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Motile
Non-motile
Results: Pseudodiaptomus marinus
C6adult
N5nauplius
Smallest1 m
Largest150 m
Gut pigment index
low high
Gut pigment index
low high
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Motile
Non-motile
Results: Pseudodiaptomus marinus
C6adult
N5nauplius
Smallest1 m
Largest150 m
Gut pigment index
low high
Gut pigment index
low high
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Motile
Non-motile
Results: Pseudodiaptomus marinus
C6adult
N5nauplius
Smallest1 m
Largest150 m
Gut pigment index
low high
Gut pigment index
low high
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Motile
Non-motile
Results: Oithona davisaeMotile
Non-motile
C6adult
N5nauplius
Smallest1 m
Largest150 m
Gut pigment index
low high
Gut pigment index
low high
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Motile
Non-motile
Results: Oithona davisaeMotile
Non-motile
C6adult
N5nauplius
Smallest1 m
Largest150 m
Gut pigment index
low high
Gut pigment index
low high
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Motile
Non-motile
Results: Oithona davisaeMotile
Non-motile
C6adult
N5nauplius
Smallest1 m
Largest150 m
Gut pigment index
low high
Gut pigment index
low high
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Motile
Non-motile
Results: Oithona davisaeMotile
Non-motile
C6adult
N5nauplius
Smallest1 m
Largest150 m
Gut pigment index
low high
Gut pigment index
low high
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Motile
Non-motile
Results: Oithona davisaeMotile
Non-motile
C6adult
N5nauplius
Smallest1 m
Largest150 m
Gut pigment index
low high
Gut pigment index
low high
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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
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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?
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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)
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1 m 5 10 15 20 125 250
Feeding size range
P. marinus
O. davisae
nauplii
adults
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1. Background/motivation
2. Combined methods and results
3. Summary & future directions
-Study organisms-Research question 1-Research question 2
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Q2: How do feeding rates compare?
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Method 2: Modified gut fluorescence method
Q2: How do feeding rates compare?
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Flow cytometery
Microscopic counts
Zooplankton feeding rate estimationMethod 2: Modified gut fluorescence method
Images: soozial.com,
musc, soton
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Flow cytometery
Microscopic counts
Zooplankton feeding rate estimationMethod 2: Modified gut fluorescence method
Images: soozial.com,
musc, soton
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Flow cytometery
Microscopic counts
Zooplankton feeding rate estimationMethod 2: Modified gut fluorescence method
Images: soozial.com,
musc, soton
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Images: soozial.com,
musc, soton
Zooplankton feeding rate estimationMethod 2: Modified gut fluorescence method
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Mackas and Bohrer 1976 (JEMBE)
Imgs: NOAA, Water Research.net, Turner
Img: Albert Calbet
Centropages typicus
Method 2: Modified gut fluorescence method
Inspiration
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Adults
N requirements
Method 2: Modified gut fluorescence method
20 - 200
Background
Fluorometer
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Chlorophyll degradation
Leads to feeding rateunderestimates
Method 2: Modified gut fluorescence method
Background
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Gut fluorescence measurement using a microplate
reader
5 - 501 - 5
- Few individuals required -- Short incubations -
- Minimize degradation -
Method 2: Modified gut fluorescence method
Background
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Gut depuration time
Blank
Chosen depuration time
RawF
luorescence
Units
Time (min.
Whiskers:5-95%
percentile
Method 2: Modified gut fluorescence method
Preliminary experiments
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Gut saturation time
Blank
DefecationChosen incubationtimes
Time (min.)
RawF
luorescence
Units
Whiskers:5-95%
percentile
Method 2: Modified gut fluorescence method
Preliminary experiments
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Method 2: Modified gut fluorescence method
1. Stage and sort copepods
Experimental protocol
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2. Allow copepods tostarve for3 hours
Method 2: Modified gut fluorescence method
Experimental protocol
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2. Allow copepods tostarve for3 hours
3. Add algae(~500 g C/L-1)
Method 2: Modified gut fluorescence method
Experimental protocol
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2. Allow copepods tostarve for3 hours
3. Add algae(~500 g C/L-1) 4. Incubate (30-60 min)
Method 2: Modified gut fluorescence method
Experimental protocol
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2. Allow copepods tostarve for3 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
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2. Allow copepods tostarve for3 hours
3. Add algae(~500 g C/L-1) 4. Incubate (30-60 min)
5. Process copepods place in centrifuge tubes
6. Flash freeze on dry ice
Method 2: Modified gut fluorescence method
Experimental protocol
h d 2 difi d fl h d
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2. Allow copepods tostarve for3 hours
3. Add algae(~500 g C/L-1)
4. Incubate (30-60 min)
5. Process copepods place in centrifuge tubes
7. Extract samplesovernight in 95% ethanol
@ -20 C
6. Flash freeze on dry ice
Method 2: Modified gut fluorescence method
Experimental protocol
M h d 2 M difi d fl h d
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8. Measure concentrationof standard using
spectrophotometer
Method 2: Modified gut fluorescence method
Experimental protocol
M th d 2 M difi d t fl th d
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8. Measure concentrationof standard using
spectrophotometer
9. Prepare dilution seriesof stock standards
Method 2: Modified gut fluorescence method
Experimental protocol
M th d 2 M difi d t fl th d
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8. Measure concentrationof standard using
spectrophotometer
9. Prepare dilution seriesof stock standards
10. Pipette samples,standards, and blanks
into microplate
Method 2: Modified gut fluorescence method
Experimental protocol
M th d 2 M difi d t fl th d
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8. Prepare standard 9. Prepare dilution seriesof standard
10. Pipette samples,standards, and blanks
into microplate
11. Read samples on amicroplate reader (Tecan
infinite)
Method 2: Modified gut fluorescence method
Experimental protocol
M th d 2 M difi d t fl th d
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8. Prepare standard 9. Prepare dilution seriesof standard
10. Pipette samples,standards, and blanks
into microplate
11. Read samples on amicroplate reader (Tecan
infinite)
Method 2: Modified gut fluorescence method
Experimental protocol
Chlorophyll is degraded by:
HeatLight
Oxidation
Dirty looks*
* source: Julian Herndon
Chl a
Method 2: Modified gut fluorescence method
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30-60 min incubation
3 species of algae
Fed 500 g/C L-1 of algae
Method 2: Modified gut fluorescence method
Treatment groups
Method 2: Modified gut fluorescence method
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30-60 min incubation
3 species of algae
Fed 500 g/C L-1 of algae
T0 control Tf control Rinsecontrol
Method 2: Modified gut fluorescence method
T0: Not fed, sampled beforeincubationTf: Not fed, sampled after incubation
Rinse: Fed, incubation terminatedimmediately
Treatment groups
Control groups
Method 2: Modified gut fluorescence method
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30-60 min incubation
3 species of algae
Fed 500 g/C L-1 of algae
T0 control Tf control Rinsecontrol
Method 2: Modified gut fluorescence method
T0: Not fed, sampled beforeincubationTf: Not fed, sampled after incubation
Rinse: Fed, incubation terminatedimmediately
Treatment groups
Control groups
Prepared/read on microplate reader
Pseudodiaptomus marinus
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Algae species
ngchlain
d.
-1
Pseudodiaptomus marinus
Whiskers:5-95% percentile
Feeding rate of nauplii: ~7-10% of adult
Oithona davisae
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Algae species
ngchlain
d.
-1 0.08
0.03
Oithona davisae
Feeding rate of nauplii: ~40% of adulton Tetraselmis suecica
Oithona davisae grows
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Oxyrrhis marinaImg: NHS
Tetraselmis suecicaImg: NHS
Oithona davisae growson larger food
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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
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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)
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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
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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
Comparisons with other studies
Uye 1986a; Almeda et al. 2010b; Henriskenet al. 2007c; Saiz et al. 2003d
Comparisons with other studies
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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
Comparisons with other studies
1 Background/motivation
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1. Background/motivation
2. Combined methods and results
3. Summary & future directions
-Study organisms-Research question 1-Research question 2
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Summary: naupliar feeding
Broad feeding capability
f
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Summary: naupliar feeding
High feeding rate biomass
Broad feeding capability
S li f di
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Summary: naupliar feeding
Broad feeding capability
Underestimated impact? vs.
High feeding rate biomass
F di i
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Future directions
Food limitation
F t di ti
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Future directions
?
Food limitation
Other food resources?Prey selection?
protists bacteria detritus otherzoo.
F t di ti
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Future directions
Food limitation
Other food resources?Prey selection?
Different food:
growth and survival
?
protists bacteria detritus otherzoo.
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Oceanographerschoice.com, doubleklawnandlandscape.com, Richard Kirby, microscopy-uk.org.uk, bio.miami.edu,, wikimedia.or
Manuscript coauthors
Wim Kimmerer, Lead PI
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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
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Method 2: Modified gut fluorescence method
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0.93
Takatsuji 1997
(JPR)
g
Trial experiment
0.83
Method 2: Modified gut fluorescence method
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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
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12individuals
Eachcopepod
EachstageAveraged
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Field
Pros Cons Pros Cons
Laboratory
Justification
Method 2: Modified gut fluorescence method
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Field
Pros Cons
Field organisms
Pros Cons
Cultured
organisms
Laboratory
Justification
Method 2: Modified gut fluorescence method
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Pros Cons
Field organisms
Natural prey/abundances
Pros Cons
Cultured
organisms
Cultured prey
Field Laboratory
Justification
Method 2: Modified gut fluorescence method
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Pros Cons
Field organisms
Natural prey/abundances
Natural
environment
Pros Cons
Cultured
organisms
Cultured prey
Controlled
environment
Field Laboratory
Justification
Method 2: Modified gut fluorescence method
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7/29/2019 Copepod Nauplii Thesis Defense
102/108
Pros Cons
Field organisms
Natural prey/abundances
Natural
environment
No need toincubate
Pros Cons
Cultured
organisms
Cultured prey
Controlled
environment
Lengthyincubations*
Field Laboratory
Justification
Method 2: Modified gut fluorescence method
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7/29/2019 Copepod Nauplii Thesis Defense
103/108
Pros Cons
Field organismsPigment
degradation
Natural prey/abundances
Natural
environment
No need toincubate
Pros Cons
Pigment
degradationminimized
Cultured
organisms
Cultured prey
Controlled
environment
Lengthyincubations*
Field Laboratory
Justification
Method 2: Modified gut fluorescence method
-
7/29/2019 Copepod Nauplii Thesis Defense
104/108
Pros Cons
Field organismsPigment
degradation
Natural prey/abundances
Grazing ratemodeled bydigestion
Natural
environment
No need toincubate
Pros Cons
Pigment
degradationminimized
Cultured
organisms
Grazing ratemodeled byingestion
Cultured prey
Controlled
environment
Lengthyincubations*
Field Laboratory
Justification
Method 2: Modified gut fluorescence method
-
7/29/2019 Copepod Nauplii Thesis Defense
105/108
Pros Cons
Field organismsPigment
degradation
Natural prey/abundances
Grazing ratemodeled bydigestion
Naturalenvironment
Difficult to
determinewhats beeneaten
No need toincubate
Pros Cons
Pigment
degradationminimized
Cultured
organisms
Grazing ratemodeled byingestion
Cultured prey
Control what
can be eaten
Controlled
environment
Lengthyincubations*
Field Laboratory
Justification
Method 2: Modified gut fluorescence method
-
7/29/2019 Copepod Nauplii Thesis Defense
106/108
Pros Cons
Field organismsPigment
degradation
Natural prey/abundances
Grazing ratemodeled bydigestion
Naturalenvironment
Difficult to
determinewhats beeneaten
No need toincubate
Limited #individuals/difficult ID
Pros Cons
Pigment
degradationminimized
Cultured
organisms
Grazing ratemodeled byingestion
Cultured prey
Control what
can be eaten
Controlled
environment
Unlimited #individuals/
easy ID
Lengthyincubations*
Field Laboratory
Justification
Method 2: Modified gut fluorescence method
l
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7/29/2019 Copepod Nauplii Thesis Defense
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
Method 2: Modified gut fluorescence method
P B A l i l
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108/108
Pure chlorophyll fromAnacystis
Nidulans cyanobacteria
Extracted in 95% Ethanol
Preserved with 99.9%argon gasand flame-sealed
Part B: Analysis protocol