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The effects of seasonal change on copepods and Euphausiids off the Western Antarctic Peninsula

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Title:
The effects of seasonal change on copepods and Euphausiids off the Western Antarctic Peninsula results from biochemical assays and respiration studies
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English
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Bellucci, Joël Laurent
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University of South Florida
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Subjects / Keywords:
diapause
globec
Paraeuchaeta
Metridia
enzyme
analysis
Euphasia
calanoides
Dissertations, Academic -- Marine Science -- Masters -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: We compared four metabolic indicators of nutritional state: citrate synthase activity (CS), malate dehydrogenase activity (MDH), lactate dehydrogenase activity (LDH) and percent body protein to each other and to respiration measurements. These comparisons were made for four species of copepods (Calanoides acutus, Metridia gerlachei, Paraeuchaeta antarctica and another form of Paraeuchaeta that was unidentifiable to species due to its early life stage), three species of Euphausids (Euphausia crystallorophias, Euphausia triacantha, Euphausia superba (including both F6-furcilial and adult stages)) and Thysonessa macrura which were collected off the Western Antarctic Peninsula (WAP) during Spring/Summer and Fall/Winter. Most species showed significant changes in one or more of the enzyme activities. In general, species that engage diapause during the Winter months showed a decrease in citrate synthase whereas those that actively feed throughout the year showed no significant changes. There was also evidence of correlations between citrate synthase activity and respiration as well as between malate dehydrogenase activity and respiration. The observed patterns are consistent with existing models of survival strategy for these Antarctic species.
Thesis:
Thesis (M.S.)--University of South Florida, 2004.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
Statement of Responsibility:
by Joël Laurent Bellucci.
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Title from PDF of title page.
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Document formatted into pages; contains 42 pages.

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notis - AJR1122
usfldc doi - E14-SFE0000270
usfldc handle - e14.270
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ABSTRACT: We compared four metabolic indicators of nutritional state: citrate synthase activity (CS), malate dehydrogenase activity (MDH), lactate dehydrogenase activity (LDH) and percent body protein to each other and to respiration measurements. These comparisons were made for four species of copepods (Calanoides acutus, Metridia gerlachei, Paraeuchaeta antarctica and another form of Paraeuchaeta that was unidentifiable to species due to its early life stage), three species of Euphausids (Euphausia crystallorophias, Euphausia triacantha, Euphausia superba (including both F6-furcilial and adult stages)) and Thysonessa macrura which were collected off the Western Antarctic Peninsula (WAP) during Spring/Summer and Fall/Winter. Most species showed significant changes in one or more of the enzyme activities. In general, species that engage diapause during the Winter months showed a decrease in citrate synthase whereas those that actively feed throughout the year showed no significant changes. There was also evidence of correlations between citrate synthase activity and respiration as well as between malate dehydrogenase activity and respiration. The observed patterns are consistent with existing models of survival strategy for these Antarctic species.
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The Effects of Seasonal Change on Copepods and Euphausiids off the Western Antarctic Peninsula: Results from Biochemical Assays and Respiration Studies by Jol Laurent Bellucci A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science College of Marine Science University of South Florida Major Professor: Joseph J. Torres, Ph.D. Edward S. Vanvleet, Ph.D. Stephen P. Geiger, Ph.D. Date of Approval: April 5, 2004 Keywords: enzyme, analysis, Euphasia, calanoides, Metridia, Paraeuchaeta, globec, diapause Copyright 2004, Jol Laurent Bellucci

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DEDICATION I would like to dedicate this thesis to my parents, Patrick and Jeanne-Marie Bellucci. They have been a constant source of love and support throughout my education and, of course, my life. I owe any of my successes and accomplishments to them.

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ACKNOWLEDGEMENTS I would like to acknowledge and thank my major professor, Dr. Jose Torres. He has been both an excellent advisor and an exemplary mentor, always knowing when to guide and when to let me take the reigns. Thanks, too, to the remaining members of my committee, Dr. Ted Vanvleet and Dr. Steve Ge iger, for their valued input and support. I would also like to acknowledge my lab mates Joe Donnelly, Michelle Grigsby, Chris Simoniello and Ester Quintana-Rizzo who have never failed to offer help and advice when I needed it. Theyre also pretty good for a laugh!

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TABLE OF CONTENTS LIST OF TABLES. ii LIST OF FIGURES... iii ABSTRACT...iv INTRODUCTION. 1 MATERIALS AND METHODS... 4 RESULTS. 13 Calanoides acutus 13 Euphausia crystallorophias. 14 Euphausia superba F6.. 14 Metridia gerlachei... 15 (Para)Euchaeta antarctica 15 Thysanoessa macrura... 16 Euphausia superba 16 Proxies for Respiration. 16 DISCUSSION... 20 Biochemical Indicators as a Proxy for Metabolism, Growth and Nutritional Condition ..20 Calanoides acutus Euphausia crystallorophias ...... 22 Euphausia superba F6....... 23 Metridia gerlachei. 23 (Para)Euchaeta antarctica...... 24 Thysanoessa macrura 26 Euphausia superba 27 Biochemical Indicators as a Proxy for Respiration... 29 CONCLUSIONS...31 REFERENCES. 32 i

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LIST OF TABLES Table 1. Summary of significant changes in biological indicators for C. acutus, E. superba, M. gerlachei, P. antarctica and Paraeuchaeta sp.. 14 Table 2. Comparison of correlations between CS, MDH and LDH to VO2 levels in Antarctic copepods. ..18 ii

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LIST OF FIGURES Figure 1. Location of 1993 cruise sample region relative to Antarctic peninsula .... 5 Figure 2a. Location of 2001 and 2002 GLOBEC cruises relative to the Antarctic Peninsula ... 7 Figure 2b. Closeup of L.M. Gould cruise tracks and stations..... 7 Figure 3. Closeup of N.B. Palmer cruise tracks and stations ... 8 Figure 4a. CS levels in E. superba, separated into size classes 4 through 7... 17 Figure 4b. MDH levels in E. superba, separated into size classes 4 through 7...... 17 Figure 4c. Percent protein in E. superba.... 18 iii

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The Effects of Seasonal Change on Copepods and Euphausiids Off the Western Antarctic Peninsula: Results from Biochemical Assays and Respiration Studies Jol Laurent Bellucci ABSTRACT We compared four metabolic indicators of nutritional state: citrate synthase activity (CS), malate dehydrogenase activity (MDH), lactate dehydrogenase activity (LDH) and percent body protein to each other and to respiration measurements. These comparisons were made for four species of copepods (Calanoides acutus, Metridia gerlachei, Paraeuchaeta antarctica and another form of Paraeuchaeta that was unidentifiable to species due to its early life stage), three species of Euphausids (Euphausia crystallorophias, Euphausia triacantha, Euphausia superba (including both F6-furcilial and adult stages)) and Thysonessa macrura which were collected off the Western Antarctic Peninsula (WAP) during Spring/Summer and Fall/Winter. Most species showed significant changes in one or more of the enzyme activities. In general, species that engage diapause during the Winter months showed a decrease in citrate synthase whereas those that actively feed throughout the year showed no significant changes. There was also evidence of correlations between citrate synthase activity and respiration as well as between malate dehydrogenase activity and respiration. The observed patterns are consistent with existing models of survival strategy for these Antarctic species. iv

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1 I. Introduction Environmental extremes in the Southern Ocean have led to a variety of survival strategies in its inhabitants. Seasonal blooms of phytoplankton during the spring and summers followed by months of nearly absent primary production during the fall and winter make it necessary for zooplankton and micronekton to either practice carnivory or to slow their metabolic activity and live off of stored reserves (Quetin and Ross, 1991; Torres et al. 1994a; Torres et al. 1994b). Copepods represent a key node in the food web. As mesozooplankton, they provide a vital link in transporting nutrients from phytoplankton and micro zooplankton to higher trophic levels. The sheer volume of copepod biomass on the order of 50% of the mesozooplankton biomass in Antarctic seas (Atkinson and Peck, 1988; BoysenEnnen et al. 1991) – emphasizes their value to the ecology of the Southern Ocean. During 2001 and 2002, four cruises were conducted during the austral fall and winter along the Western Antarctic Peninsula (WAP) shelf. One of the goals of these Southern Ocean Global Ecosystems Dynamics [Southern GLOBEC] cruises was to shed light on the distribution and survivorship of krill and micronektonic zooplankton on the WAP shelf and to investigate their availability to higher trophic levels. Copepods, in particular, play an important role in coupling primary productivity to higher-trophic

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2 species. To extend the investigation of seasonal variation in the studied species, data from a 1993 cruise was included in this project’s analysis. This earlier cruise took place in November and December of that year, coinciding with the Austral spring/summer. This project examined 4 indicators of nutritional condition as well as data from respiration experiments in order to investigate the responses of the studied specimens to seasonal changes in their environment. The specific enzymes examined were citrate synthase (CS), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH). CS and MDH play important roles in the Kreb’s Cycle (aerobic metabolism) whereas LDH is the final step in anaerobic glycolysis. Examination of three enzymes makes it possible to estimate an organism’s ability to produce ATP and, thereby, to comment on their metabolic activity. In all, 3 species of order Euphausiacea and 3 species of order Calanoida of varying developmental stages were studied during the course of this project. The life histories of each are known to some degree and so it is possible to analyze the results of enzyme and protein analysis as well as respiration data to see if the results of the laboratory experiments reflect existing knowledge of the lives of these Antarctic invertebrates. A secondary goal of the project was to examine any relationships between the metabolic proxies (CS, LDH, MDH) and respiration data. Respiration experiments are unwieldy and limit the number of specimens that can be studied in a reasonable amount of time. Enzyme assays are relatively easy and inexpensive and allow for examination of frozen specimens, which can be collected for analysis in far greater numbers than live

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3 specimens. Hence, the isolation of a metabolic proxy that can double as a respiration proxy would be of great benefit.

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4 II. Materials and Methods Sample collection and processing Three species of zooplankton and three species of micronekton were collected in sufficient numbers to investigate the questions posed by this project. The zooplankton included three species of copepods: Calanoides acutus Metridia gerlachei Paraeuchaeta antarctica and Paraeuchaeta late stage copepodites identified to family. Also included were three species of Euphausiids: Euphausia crystallorophias Euphausia superba (including both F6-furcilial and adult stages) and Thysanoessa macrura Sampling was conducted during five separate research cruises. The first took place aboard the R/V Polar Duke in 1993 during November and December, coinciding with the Austral (southern hemisphere) fall/winter season. The sampling region ran along two transects, each composed of a series of sampling stations off the northeastern coast of the WAP (Fig. 1) (Geiger et al. 2001; Kawall et al. 2001). The latter four cruises occurred in 2001 and 2002 as part of Southern Ocean GLOBEC. Sampling aboard the R/V Laurence M. Gould took place in April and May of 2001 and 2002, coinciding with the Antarctic fall whereas winter sampling (July to September of 2001 and 2002) took place aboard the more ice-worthy RVIB Nathaniel B. Palmer Fall sampling took place at a

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5 Figure 1. Location of 1993 cruise sample region relative to Antarctic peninsula (lower left) Inset (A): 1993 cruise track.

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6 series of predetermined research stations along the western edge of the Antarctic Peninsula (Fig. 2a). The fall process cruises took place in water that was either completely open or, as was more often the case, partially ice-covered (Fig. 2b). Five to seven days of sampling were conducted at each of the fall research stations. Winter sampling was conducted along various points of a survey track in the same area (Fig 3.) On these cruises, sampling was conducted over a period of hours at the various station points along the winter survey track. The winter cruises generally operated in water either mostly covered or completely covered by the Antarctic ice pack. The Palmer ’s primary mission was to survey the hydrology and zooplankton and micronekton distributions within the study area. Although the Gould ’s primary function was scientific in nature during the GLOBEC cruises, it also served as courier and supplier for Palmer Station. Interaction between the two research vessels was frequent in the form of daily updates regarding ice conditions, wildlife sightings and general cruise progress. In addition, the Palmer sometimes served as icebreaker to the Gould when ice thickness exceeded the smaller vessel’s handling capacity. Sample specimens were collected primarily with a Tucker trawl (2.25m2 mouth area, 4mm mesh in the main body of the net, 1 mm mesh in the tail section and cod end) deployed obliquely from 0 to 500m. Additional samples were obtained using a 1 m2plummet net (163 m mesh) deployed vertically from 0 to 200m as well as a 10 m2Multiple Opening Closing Net and Environmental Sampling System (MOCNESS) with 3mm mesh in the main net and a 1 mm mesh cod end deployed from 0 to 1000m in discrete depth intervals. The six nets of the MOCNESS could be manually triggered

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7 Figure 2a. (left) Location of 2001 and 2002 GLOBEC cruises relative to the Antarctic Peninsula. Figure 2b. (below) Closeup of L.M. Gould cruise tracks and stations. Red indicates Fall 2001 (GLOBEC I) track. Green indicates Fall 2002 (GLOBEC III) track.

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8 Figure 3. Closeup of N.B. Palmer cruise tracks and stations. Yellow indicates Winter 2001 (GLOBEC II) track. Purple indicates Winter 2002 (GLOBEC IV) track.

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9 from the shipboard computer lab, allowing for precise depth-interval sampling. A typical 1000m trawl would see the first net open for the surface to depth deployment, then each of the remaining five nets opened sequentially in 200m depth ranges. For example, net 0 would typically sample from 0 to 1000m meters, net 1 from 1000m to 800m, net 2 from 800m to 600m, etc.. Sensors on the MOCNESS recorded depth, volume filtered, temperature and salinity for the duration of the trawl. Cod end samples were emptied into a container filled with coarsely filtered seawater, transferred to a shipboard laboratory and then removed from the container for respiration measurements. Copepods and furcilia used for respiration experiments were placed in 5 and 10 ml plastic and glass syringes along with 3 to 8 ml of seawater which had been finely filtered using 0.45 m membrane filters and treated with antibiotics (streptomycin sulfate and neomycin sulfate in concentration of 25mg/L each) to remove artifacts caused by bacterial respiration. The needle ends of the syringes were cut off and fitted with Clark style oxygen electrodes which recorded partial pressure of oxygen (PO2) every 15 seconds for at least 12 hours. The euphausiids were placed in airtight, water-jacketed chambers of varying volume (30mL to 400mL) filled with the filtered and treated seawater and fitted with the oxygen electrodes (Fig. 4). A constant temp of 0C was maintained in the jacketed chambers. The syringes were placed in water baths that also maintained a constant temperature of 0C. Upon completion of the experiments, syringe specimens were rinsed with 280mM sucrose solution to remove salt from the specimen surface (residual salt would cause inaccurate readings when the specimens would later be measured for mass), identified to species and

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10 Figure 4. Illustration of Clark style oxygen electrode. stage when possible, blotted dry, placed in individual containers and frozen on board at -80C. The euphausiids were identified as to species and sex, blotted dry, placed in individual containers and frozen on board at -80C. Additional specimens were sorted directly after the trawls and placed in frozen storage without gathering respiratory data. At the conclusion of the cruises, the frozen specimens were shipped on dry ice to St. Petersburg, Florida and placed in long-term storage at –80C. Joe Donnelly processed data collected during the respiration experiments at the home lab in St. Petersburg, FL.

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11 Relative metabolic rates and growth status of the specimens were determined using 4 different biochemical indices. Citrate synthase (CS) (Childress and Somero, 1979) activity was measured using modifications to (Sudgen and Newsholme, 1975) as outlined by (Geiger et al. 2001). Malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) activities were measured following (Walsh et al. 1989). Enzyme assays were run at 10C. The amount of protein per specimen, measured as percent protein per individual, was determined according to (Lowry et al. 1951). For these four assays, individual specimens were homogenized in at least 190 L of ice-cold, millipure water. Specific volumes of water were determined based on the mass of the specimen. In all but a few cases, there was sufficient homogenate to run all 4 assays on each specimen. To calculate water volumes, the mass of the specimen was simply multiplied by 24. For example, if the mass of the specimen was 0.00812g, then the calculation was 8.12mg x 24 (assuming a 25-fold dilution (1 part specimen to 24 parts water)) = 194.8 which was rounded up to 195 = 195 L. However, the minimum volume of water must be 190 l. Therefore, for smaller specimens, it became necessary to adjust the dilution. The dilution was calculated by dividing 190 by the mass of specimen. For example, if a specimen’s mass was 4.45 mg, the dilution calculation was 190 / 4.45mg = 42.69 which was rounded up to 42.7 (ratio of water) +1 (ratio of specimen) = 43.7 for the dilution. The resulting homogenates were divided into three portions based on their dilution ratios:

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12 € If the dilution was between 24:1 and 50:1, 3 aliquots of 10 L each were made for protein. € If the dilution was between 51:1 and 100:1, 2 aliquots of 20 L each were made for protein. € If the dilution was between 101:1 and 200:1, 1 aliquot of 40 L was made for protein. € If the dilution was greater than 200:1, 1 aliquot of 80 L was made for protein. In all cases, after aliquots were made for the protein assays, the remainder of the homogenate was used for the enzyme assays. Protein aliquots were combined with enough millipure water to bring the total in each sample tube to 80 L, sealed with parafilm and stored at –50C. Enzyme assays were run the same day the tissues were homogenized. 40 L of homogenate were used for the CS assays and 20 L were used for the MDH and LDH assays each. The results of the assays were sorted by species, stage and season and then further divided by date of collection. In doing so it was possible to analyze the data by the general season of collection and also to get a detailed view of the fluctuation of the indicators as a particular season(s) progressed. Where available, data from the current study was compared with data from the summer 1993 cruise. For each of these categories, average, standard deviation and ANOVA analysis were performed and 95% confidence errors bars were generated.

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13 III. Results Measurable levels of all 4 biochemical indices were found in all species studied. In general, there was a decreasing trend in levels of metabolic enzymes as summer progressed to fall progressed to winter in species known to engage diapause as an overwintering strategy. The results for all species except E. superba adults aare summarized in Table 1. E. superba results are presented in Figures 4a, b and c. Calanoides acutus Citrate synthase decreased significantly in C. acutus CV copepods as well as adults. CS levels in C. acutus CV copepods dropped from a high of 1.42 mol/min/g wet mass in fall to a low of 0.47 mol/min/g wet mass in winter (F=32.6, P= 3.46E-07). C. acutus adults had a CS high of 2.72 mol/min/g wet mass in the summer and reached a low of 0.84 mol/min/g wet mass in the late winter (F=6.7, P=0.02). MDH levels in late stage copepodites dropped from a value of 9.99 mol/min/g wet mass fall to 4.99 mol/ min/g wet mass in winter (F=37.2, P=6.77E-08) and in females the levels dropped from 12.6 mol/min/g wet mass in summer to 5.2 mol/min/g wet mass in winter (F=5.4, P=0.03). Percent protein levels in CV copepodites remained fairly constant from summer

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14 Table 1. Summary of significant changes in biological indicators for C. acutus E. superba M. gerlachei P. antarctica and Paraeuchaeta sp. Information includes summer data from Geiger (2001). to winter whereas adult females’ percent protein increased from 7.41% to 12.95% from summer to late winter (F=7.44, P=0.01). Euphausia crystallorophias The four measures of condition (CS, LDH, MDH and protein) did not show a significant increase or decrease from fall to winter. Euphausia superba F6 The specimens sampled showed a significant drop in both CS and LDH. E. superba F6 showed a decrease in CS from 2.72 mol/min/g wet mass in the fall to 1.48 mol/min/g wet mass in the winter (F=97.2, P=3.3E-08). LDH dropped from 10.95

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15 mol/min/g wet mass in fall to 3.03 mol/min/g wet mass in winter (F=32.3, P=3.4E-05). Percent protein levels from fall to winter (levels that are similar even between early fall and late winter) remained stable. Metridia gerlachei CS shows a decrease in M. gerlachei females, dropping from 2.54 mol/min/g wet mass in the summer to 1.43 mol/min/g wet mass in the winter (F=14.0, P=.00056). The CV copepodite samples from this species showed stability in all 4 indicators from fall to winter. (Para) Euchaeta antarctica P. antarctica adult females showed a drop in CS levels from summer to winter, decreasing from 1.68 to 1.01 mol/min/g wet mass (F=5.2, P=0.03). LDH values also dropped from 8.3 to 1.98 mol/min/g wet mass from summer to fall and winter (F=12.4, P=0.002) as did MDH which dropped from 6.10 to 4.33 mol/min/g wet mass (F=5.1, P=0.03). Continuing with this downward trend, the percent protein levels of P. antarctica females dropped from 11.7% to 8.27% during the same time period (F=4.8, P=0.04). Paraeuchaeta sp. CV copepodites showed decreases in both CS and MDH. From fall to winter, CS dropped from 1.64 to 1.35 mol/min/g wet mass (F=17.2, P=0.0007). MDH dropped from 6.7 to 4.34 mol/min/g wet mass during the same time period (F=17.1, P=0.0007).

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16 Thysanoessa macrura This species showed no significant changes in any of the 4 indicators. Euphausia superba Summer CS and MDH data from Kawall (2001) were combined with the fall and winter CS and MDH data of this study to get a broader view of the strategies E. superba employs from season to season. For these two enzyme analyses, specimens were separated according to 4 different size classes (Kawall et al. 2001) and then sorted by season. Across all size classes, there is a marked drop in CS activity from summer to fall and then a small rise from fall to winter (Fig. 4a). The same trend is present in MDH levels, though not as clearly (Fig. 4b). This is accompanied by a decrease in protein from fall to winter (Fig. 4c). Proxies for Respiration One of the project’s goals was to investigate any relationships between respiration levels and the more easily measured metabolic proxies. When examining only copepod species – both copepodites and adults – CS showed a correlation during the fall with an R2 value of 0.69. LDH showed a somewhat lower correlation with an R2 of 0.52 (Table 2). Winter values showed no correlation between any of the metabolic enzymes and VO2 levels. Combining both fall and winter seasonal values shows that,

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17 0 0.5 1 1.5 2 2.5 3 3.5 4 4.54567Figure 4a. CS levels in E. superba separated into size classes 4 through 7. Error bars indicate 95% confidence intervals. Summer Fall Winter 0 20 40 60 80 100 1204567Figure 4b. MDH levels in E. superba separated into size classes 4 through 7. Error bars indicate 95% confidence intervals. Summer data from Kawall (2001). mol/min/g wet mass mol/min/g wet mass

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18 0.00% 2.00% 4.00% 6.00% 8.00% 10.00% 12.00% 14.00% 1 Fall WinterFigure 4c. Percent protein in E. superba Error bars indicate 95% confidence intervals.percent protein Table 2. Comparison of correlations between CS, MDH and LDH to VO2 levels in Antarctic copepods. Bolded R2 values indicate the highest R2 value (ergo the most significant) for each of the season/stage categories.

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19 again, CS has the strongest relationship with an R2 of 0.55, as compared to 0.45 and 0.36 for LDH and MDH, respectively. Narrowing the data set further crystallizes this relationship. When looking at both fall and winter just for adult copepod species, the R2values increase for all three enzymes to 0.87, 0.88 and 0.98 for CS, LDH and MDH, respectively. This was the only instance in which CS did not show the strongest correlation with respiration. There are similar, though not as pronounced, increases in the R2 values when looking at combined seasonal data only for copepodite stages of the studied species. The R2 values for CS, LDH and MDH in this case were 0.84, 0.46 and 0.29, respectively. These relationships are summarized in Table 2.

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20 IV. Discussion Biochemical Indicators as a Proxy for Metabolism, Growth and Nutritional Condition Calanoides acutus The importance of krill in the Antarctic food chain has received a great deal of emphasis due to the link krill provide between the phytoand zooplanktonic biomass and that of larger species such as whales and seals. Krill species may dominate the zooplankton biomass in coastal Antarctic waters, but recent studies have revealed that calanoid copepods are more important offshore (Atkinson and Peck, 1988; Boysen-Ennen et al. 1991). Among the Antarctic calanoid species, Calanoides acutus is among the most numerically dominant (Hopkins and Torres, 1988). C. acutus ’ life history is fairly well established. Late stage copepodites overwinter at depths of 500 meters or more where they enter a state of diapause (Marin, 1988; Burghart et al. 1999). The low metabolic rate characteristic of diapause requires little use of lipid reserves that were built up during the spring and summer. However, the reserves likely come into play when the late stage copepodites molt to the adult stage and mate (Marin, 1988). As winter turns to spring, the females ascend in order to spawn. This allows their offspring the chance to take advantage of the feeding opportunities

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21 provided by the spring bloom’s high productivity (Marin, 1988; Hagen, 1996; Geiger et al. 2001). During the spring and summer, CIV and CV copepodites feed and develop while also building up lipid reserves in the form of wax esters (Voronina, 1970; Marin, 1988; Kawall et al. 2001). As adults die off, juveniles become numerically dominant, and with the onset of autumn and the lower primary productivity that accompanies it, they descend to deeper waters to begin the cycle again (Marin, 1988). The overwintering strategy of C. acutus is reflected in the enzyme analysis results. CS and MDH levels decline from summer to winter as the adults sink to depth during diapause. Concurrently, CS and MDH levels in late stage copepodites drop over the same time period. As winter deepens, feeding activity has ceased, but the wax ester depots built up during high productivity fuel the CV’s spring development into adults as well as providing energy for mating and egg production. As expected, protein levels in adult females show a sharp increase from summer to winter. Summer females will have recently spawned and, likely, are not far from death. During the winter, however, mating and egg production occur. Higher protein levels during this season could reflect the growth of fertilized eggs within the females. Copepodite stages are essentially in a constant state of growth, either working off of stored reserves during the winter or from newly acquired energy from feeding. Hence, one would expect protein levels in these copepodite stages to be fairly constant. Indeed, the data show this to be the case.

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22 Euphausia crystallorophias There is some debate concerning this species’ overwintering strategy. Analysis from Littlepage (1964) and Kittel and Ligowski (1980) suggest that E. crystallorophias relies on phytoplankton and therefore is dependent on the Antarctic spring bloom. However, Hopkins (1987) found zooplankton within the gut contents of E. crystallorophias specimens collected in March. Additionally, they were observed feeding in open water several meters beneath the ice pack where suspended phytoplankton was likely too sparse to support their energy needs (O’Brien, 1987; Marschall, 1988). This suggests that E. crystallorophias is an omnivore that maintains a steady intake of nutrients, at least some of which is allocated into a lipid reserve (Hagen, 1996). The species endures a number of energy demands: development into the adult stage from fall into winter (Hagen, 1996), mating in (most likely) late winter (Stepnik, 1982), spawning in spring (Littlepage, 1964; Hempel et al. 1979; Makarov, 1979). All of the measures of condition (CS, LDH, MDH and protein) remained stable from fall to winter, a period of time during which the euphausiids were developing and then mating and, presumably, when fertilized females carried developing eggs. The stability of their metabolism and growth indicators suggests only that they were surviving off of large lipid reserves, that they continued actively feeding, or that they utilized a combination of the two strategies. However, this data set does not provide direct insight into which of these strategies is dominant.

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23 Euphausia superba F6 The last stage in the furcilia’s development into adulthood is marked by growth, as are the stages that precede it. The specimens sampled show a significant drop in both CS and LDH, suggesting a lower activity rate, possibly to compensate for the lack of available phytoplankton during the lean winter months. Marr (1962) and Daly (1990) found continued development of larval krill in the field, however. The constant protein levels from fall to winter (levels that remain stable even between early fall and late winter) support their observations. Additionally, Quetin and Ross (1991) concluded that grazing of under-ice algae was probably an essential practice to larval krill considering their low lipid stores. Therefore, it is likely that feeding continued albeit at a lower rate than in spring/summer through winter in order to maintain growth and development. During under-ice SCUBA dives in the fall of 2001, furcilia were observed and videotaped in dense swarms under the then still developing ice field. It is possible that they were grazing on under-ice algae to maintain their energy supply, a practice that has been observed in adults of the species (Kottmeier and Sullivan, 1987; O’Brien, 1987; Marschall, 1988; Stretch et al. 1988). Metridia gerlachei The omnivorous Metridia gerlachei is far less dependent on the spring bloom than its calanoid cousins and appears to feed throughout the year on both zooplankton and phytoplankton to sustain several cycles of egg production annually (Huntley and Escritor, 1992; Schnack-Schiel and Hagen, 1994; Metz and Schnack-Schiel, 1995;

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24 Burghart et al. 1999). By opportunistically feeding on diatoms, dinoflagellates and ciliates, as well as drawing upon lipid stores, M. gerlachei can sustain fairly constant, low rates of egg production throughout the year (Atkinson et al. 1996; Calbet and Irigoien, 1997). The specimens collected in summer and spring reflect some of these expected trends. There is a drop in CS levels in females from summer to fall and winter, suggesting that activity may have been curtailed somewhat as the bloom receded. It is possible that the transition from an herbivorous to an omnivorous diet is not immediate, perhaps because the zooplankton that contribute to M. gerlachei ’s diet during the winter are not so easily found as diatoms are during the spring/summer bloom. The females may have to reduce their activity, living off of lipid reserves, until fresh supplies of zooplankton are discovered. Percent protein levels from summer to winter in the sampled females remained fairly constant. CV copepodites were also sampled during the fall and winter cruises. Their metabolic activity appears to have been even more stable through those seasons than the females of the species. There were no significant increases or decreases in any of indicators. (Para) Euchaeta antarctica (Para) Euchaeta antarctica maintains full carnivory (Littlepage, 1964; Hopkins, 1987; Hopkins and Torres, 1989) and remains reproductively active throughout the year (Littlepage, 1964; Clarke, 1984; Oresland, 1995; Geiger et al. 2001). Its most intense reproductive efforts take place in late summer and into winter (Ward and Robins, 1987).

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25 There is some debate over the importance of lipids to this species. P. antarctica has been found to have high lipid levels throughout the year (Clarke, 1984; Donnelly et al. 1990; Hagen, 1996). Sargent et al (1981) suggest that low levels of lipid are used for sustenance while overwintering, but Hagen (1996) concludes that lipid reserves are used by adults to foster egg production and are not used as a source of nutrition. Similarly, Geiger et al. (2001) found a drop in condition in females prior to spawning, suggesting that energy was allocated to egg production. Furthermore, Kawall et al. (2001) found lower metabolic rates in P. antarctica sampled during the winter season and in the pack ice. The data from the current study concur with previous findings. All three metabolic proxies for P. antarctica females showed a marked decrease from summer to fall and winter. All available data also support the observation that these carnivores, though yearround feeders, are likely affected by the lack of food supply characteristic of the Antarctic winter and compensate by reducing activity while allocating energy from lipid reserves into egg production. While egg production – and the allocation of lipid reserves to it may well take place year-round, metabolism remains high in the summer months as the adults can sustain themselves solely from feeding, a luxury not possible during the fall and winter. Undoubtedly there are some zooplanktonic sources of energy available during the lean times and consequently P. antarctica need not engage a true diapause state like C. acutus but the collective data make it clear that a reduction in metabolic activity is indeed occurring. Since egg production is constant and therefore represents a constant draw upon lipid reserves, the adults must reduce overall activity in order to sustain egg production.

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26 Further evidence of these conclusions can be found in the protein levels of the species. Protein levels in P. antarctica females showed a small but significant decrease from summer to fall and winter, suggesting that egg production may slow somewhat, but does not cease. The slower production coincides with a lowering of metabolism as well as with low food availability. As egg production proceeds and food availability decreases, the females must reduce metabolic activity. In addition to females, Paraeuchaeta (identified to family) in late copepodite stages were also analyzed. Their CS and MDH levels dropped from fall to winter, indicating a lowering of metabolism as winter set in. Protein levels in the copepodites did not fluctuate significantly from fall to winter. This is consistent with the assumption that the late stage copepodites were in a state of growth and development, perhaps relying heavily on stored lipid reserves to fuel the developmental process. Thysanoessa macrura Another Antarctic resident that has adapted to an omnivorous diet is the euphausiid Thysanoessa macrura (Hopkins, 1985; Mayzaud et al. 1985; Hopkins, 1987; Hopkins and Torres, 1989; Rau et al. 1991). Hagen (1996) found very high lipid levels in overwintering T. macrura despite the fact that they were in the process of reproducing. This suggested that they were relatively unaffected by the low phytoplankton availability as a result of their omnivorous diet. The data from this project support that conclusion. None of the biochemical indicators fluctuated significantly from fall to winter. However, Torres et al. (1994a) and Torres et al. (1994b) found a decrease in metabolic rate from

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27 fall to winter in T. macrura It should be noted that the data set for fall biochemical indicators for this species in this project carries an n value of 4, and so the results must be regarded accordingly. Euphausia superba Because of its importance in transferring energy from lower to higher trophic levels, and due to an impressive biomass on the order of hundreds of millions of tons (Miller and Hampton, 1989), Euphausia superba has been the subject of a great deal of study and, was, in fact, at the heart of all the research conducted during the GLOBEC cruises. Together, the Calanoid family of copepods and E. superba make up the bulk of the Antarctic zooplankton community. However, whereas the Calanoid overwintering strategies are fairly well understood – at least for the biomass dominant species – there is still debate as to how E. superba manages to live through the nutrient-poor Antarctic winter. Omnivory may be one solution. Krill have been observed along the undersurface of the ice pack grazing on the algae that clings to the frigid topography (Hempel, 1987; Kottmeier and Sullivan, 1987; Marschall, 1988; Smetacek et al. 1990). It has also been suggested that they ingest non-phytoplanktonic sources of food to supplement their overwintering diet (Ikeda and Dixon, 1982; Daly and Macaulay, 1991; Huntley et al. 1994). Hagen (1996) found evidence of the use of lipid reserves by E. superba during winter. He observed that the species “exhibits pronounced seasonal lipid dynamics with an extensive accumulation of energy reserves during the productive season and their

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28 mobilization during the dark period.” This suggests that ingestion of under-ice algae may not be sufficient to sustain adults through the winter. In addition to living off of stored reserves, E. superba may enter a state of depressed metabolic activity accompanied by a slowing or halting of sexual development (Quetin and Ross, 1991). Torres et al. (1994) observed lower metabolic and protein levels in overwintering krill. Beyond simply slowing themselves down, starvation experiments indicate that E. superba have the option to feed off their own bodies, resulting in a reduction in size and even reversion to an earlier life stage (Ikeda and Dixon, 1982). This is undoubtedly a “last ditch” effort on the part of the species to preserve itself when all other options (algae feeding, lower metabolism and lipid reserves) have been exhausted. The E. superba adults studied for this project seem to follow previously observed trends. Across all size classes, there is a marked drop in CS activity from summer to fall and then a small rise from fall to winter. The same trend is present in MDH levels, though not as clearly. This is accompanied by a significant decrease in protein from fall to winter. The increase in metabolic activity from Fall to Winter suggests that these specimens may have fed off the energy reserves of their own body tissues to maintain and even increase metabolic activity as they left a state of semi-dormancy. Given the number of different overwintering strategies employed by E. superba and the varying results obtained during different studies, it is fair to guess that this a species whose behavior varies with the varying conditions it encounters during the Antarctic winter. Under-ice algae and zooplankton are not uniformly distributed through

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29 the Southern Ocean. Therefore, any species that remains even partially active and actively travels through the winter months must necessarily adapt to the changing conditions it will encounter. Biochemical Indicators as a Proxy for Respiration Respiration experiments are relatively difficult to set up and maintain, requiring either digital or analog data recorders, water baths, airtight chambers of varying volume and a significant amount of time to run. Because of the complex interplay of equipment, respiration experiments are unwieldy and require a high level of expertise. One is also limited by the number of experiments that can be run before one’s specimens begin dying or become too acclimated to the captive environment, thus producing results that may not reflect their condition in the wild. Nevertheless, respiration experiments have been conducted successfully (Torres et al. 1994a; Kawall et al. 2001). On the other hand, enzyme assays are relatively easy and inexpensive to perform, require minimal training time and, perhaps most importantly, work on frozen specimens. This allows for a much greater volume of data collection than is possible when running experiments on live specimens. Because of the simpler nature of enzyme assays, they are much easier to replicate and more apt to provide consistent results than respiration experiments. Hence, one of the goals of this project was to establish which, if any, of the biochemical indicators served as a proxy for respiration so that the enzyme’s assay might one day be used in lieu of, or at least in conjunction with, respiration experiments.

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30 In general, citrate synthase (CS) and lactate dehydrogenase (LDH) both correlated well with oxygen consumption rates, although CS proved the better predictor (Table 2). CS catalyzes the first reaction in the Krebs cycle. This first reaction is also the ratelimiting one and so CS is an indicator of aerobic respiration. LDH catalyzes the interconversion of pyruvate and lactate and, as one of the rate-limiting steps in this reaction, is an indicator of anaerobic respiration. Kawall et al. (2001) found malate dehydrogenase (MDH) to be the best proxy for respiration, although they also found that CS provided a good fit to the data. The differences between their findings and those of this study are likely due to the difference in species examined. Kawall focused on euphausiids and amphipods whereas this project’s respiration comparisons examined copepods. This suggests that the best proxy for respiration may be family-dependent. Interestingly, though, the best fit in the current data set did indeed come from MDH. Specifically, when comparing metabolic indicators to routine oxygen consumption (VO2) results in adult copepods for both fall and winter, it was MDH that produced the best R2 value. However, this was the only instance in which MDH had the best fit. In fact, it was the only instance in which MDH fit the data at all.

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31 V. Conclusions 1.In general, the metabolic enzymes served as good proxies for activity level and nutritional condition. Results from data analysis were consistent with what is currently known about the life histories of the species that were studied for this project. 2.There was a general trend of herbivores to slow their metabolisms as fall and winter set in and for omnivores and carnivores to maintain activity levels during this same time period. 3.CS proved to be the best proxy for respiration. LDH also showed significant, positive correlations with respiration.

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32 VI. References Atkinson, A. and J. M. Peck (1988). “A Summer-Winter Comparison of Zooplankton in the Oceanic Area Around South Georgia.” Polar Biology 8 : 463-473. Atkinson, A., P. Ward and E. J. Murphy (1996). “Diel periodicity of Subantarctic copepods: relationships between vertical migration, gut fullness and gut evacuation rate.” Journal of Plankton Research 18 : 1387-1405. Boysen-Ennen, E., W. Hagen, G. Hubold and U. Piatkowski (1991). “Zooplankton biomass in the ice-covered Weddell Sea, Antarctica.” Marine Biology 111 : 227-235. Burghart, S. E., T. L. Hopkins, G. A. Vargo and J. J. Torres (1999). “Effects of a rapidly receding ice edge on the abundance, age structure and feeding of three dominant calanoid copepods in the Weddell Sea, Antarctica.” Polar Biology 22 : 279-288. Calbet, A. and X. Irigoien (1997). “Egg and fecal pellet production rates of the marine copepod Metridia gerlachei northwest of the Antarctic Peninsula.” Polar Biology 18 : 273-279. Childress, J. J. and G. N. Somero (1979). “Depth-related enzymic activities in muscle, brain and heart of deep-living pelagic marine teleosts.” Marine Biology 52 : 273-283. Clarke, A. (1984). “The lipid content and composition of some Antarctic macrozooplankton.” Bulletin of the British Antarctic Survey 63 : 57-70. Daly, K. L. (1990). “Overwintering development, growth, and feeding of larval Euphausia superba in the Antarctic marginal ice zone.” Limnology and Oceanography 35 : 1564-1576. Daly, K. L. and M. C. Macaulay (1991). “Influence of physical and biological mesoscale dynamics on the seasonal distribution and behavior of Euphausia superba in the antarctic marginal ice zone.” Marine Ecology Progress Series 79 : 37-66. Donnelly, J., J. J. Torres, T. L. Hopkins and T. M. Lancraft (1990). “Proximate composition of Antarctic mesopelagic fishes.” Marine Biology 106 : 13-23.

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33 Geiger, S. P., H. G. Kawall and J. J. Torres (2001). “The effect of the receding ice edge on the condition of copepods in the northwestern Weddell Sea: results from biochemical assays.” Hydrobiologia 453/454 : 79-90. Hagen, W. (1996). The Role of Lipids in the Ecology of Polar Plankton and Nekton a Synopsis. Als Habilitationsschrift der Mathematisch-Naturwissenschaftlichen Fakultt der Christian-Albrechts-Universitt zu Kiel Hempel, G. (1987). “The krill-dominated pelagic system of the Southern Ocean.” Environm. Internat. 13 : 33-36. Hempel, I., G. Hempel and A. d. C. Baker (1979). “Early life history stages of krill ( Euphausia superba ) in Bransfield Strait and Weddell Sea.” Meeresforsch 27 : 267-281. Hopkins, T. L. (1985). “Food web of an Antarctic midwater ecosystem.” Marine Biology 89 : 197-212. Hopkins, T. L. (1987). “Midwater food web in McMurdo Sound, Ross Sea, Antarctica.” Marine Biology 96 : 93-106. Hopkins, T. L. and J. J. Torres (1988). “The zooplankton community in the vicinity of the ice edge, western Weddell Sea.” Polar Biology 9 : 79-87. Hopkins, T. L. and J. J. Torres (1989). “Midwater food web in the vicinity of a marginal ice zone in western Weddell Sea.” Deep Sea Research 36 : 543-560. Huntley, M. E. and F. Escritor (1992). “Ecology of Metridia gerlachei Giesbrecht in the western Bransfield Strait, Antarctica.” Deep Sea Research 39 : 1027-1055. Huntley, M. E., W. Nordhausen and M. D. G. Lopez (1994). “Elemental composition, metabolic activity and growth of Antarctic krill Euphausia superba during winter.” Marine Ecology Progress Series 107 : 23-40. Ikeda, T. and P. Dixon (1982). “Body shrinkage as a possible over-wintering mechanism of the Antarctic krill Euphausia superba Dana.” Journal of Experimental Marine Biology and Ecology 62 : 143-151. Kawall, H. G., J. J. Torres and S. P. Geiger (2001). “Effects of the ice-edge bloom and season on the metabolism of copepods in the Weddell Sea, Antarctica.” Hydrobiologia 453/454 : 67-77. Kittel, W. and R. Ligowski (1980). “Algae found in the food of Euphausia crystallorophias (Crustacea).” Polish Polar Res. 1 : 129-137.

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34 Kottmeier, S. T. and C. W. Sullivan (1987). “Late winter primary and bacterial production in sea ice and seawater west of the Antarctic peninsula.” Marine Ecology Progress Series 36 : 287-298. Littlepage, J. L. (1964). “Seasonal variation in lipid content of two Antarctic marine Crustacea. In: Carrick, R.; Holdgate. M. W.; Prevost, J.” Biologie antarctique, 1er SCAR Symp. : 463-470. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall (1951). “Protein measurement with the Folin phenol reagent.” Journal of Biological Chemistry 193 : 265275. Makarov, R. R. (1979). “Larval distribution and reproductive ecology of Thysanoessa macrura (Crustacea: Euphausiacea) in the South Sea.” Marine Biology 52 : 377-386. Marin, V. (1988). “Qualitative models of the life cycle of Calanoides acutus Calanus propinquus and Rhincalanus gigas .” Polar Biology 8 : 439-446. Marr, J. W. S. (1962). “The natural history and geography of the Antarctic krill ( Euphausia superba ).” Discovery Report 32 : 33-464. Marschall, H.-P. (1988). “The overwintering strategy of Antarctic krill under the pack-ice of the Weddell Sea.” Polar Biology 9 : 129-135. Mayzaud, P., J. Farber-Lorda and M. C. Corre, Eds. (1985). Aspects of nutritional metabolism of two Antarctic euphausiids:Euphausia superba and Thysonessa macrura. Antarctic nutrient cycles and food webs. Berlin, Springer. Metz, C. and S. B. Schnack-Schiel (1995). “Observations on carnivorous feeding in Antarctic calanoid copepods.” Marine Ecology Progress Series 129 : 71-75. Miller, D. G. M. and I. Hampton (1989). “The biology and ecology of the Antarctic krill ( Euphausia superba Dana): a review.” BIOMASS Sci. Ser. 9 : 1-166. O’Brien, D. P. (1987). “Direct observations of the behavior of Euphausia superba and Euphausia crystallorophias (Crustacea: Euphausiacea) under pack ice during the Antarctic spring of 1985.” Journal of Crustacean Biology 7 : 437-448. Oresland, V. (1995). “Winter population structure and feeding of the chaetognath Eukrohnia hamata and the copepod Euchaeta antarctica in Gerlache Strait, Antarctic Peninsula.” Marine Ecology Progress Series 119 : 77-86. Quetin, L. B. and R. M. Ross (1991). “Behavioral and physiological characteristics of the Antarctic krill, Euphasia Superba .” American Zoology 31 : 49-63.

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35 Rau, G. H., T. L. Hopkins and J. J. Torres (1991). “15N/14N and 13C /12C in Weddell Sea invertebrates: implications for feeding diversity.” Marine Ecology Progress Series 77 : 16. Sargent, J. R., R. R. Gatten and R. J. Henderson (1981). “Lipid biochemistry of zooplankton from high latitudes.” Oceanis 7 : 623-632. Schnack-Schiel, S. B. and W. Hagen (1994). “Life cycle strategies and seasonal variations in distribution and population structure of four dominant calanoid copepod species in the eastern Weddell Sea, Antarctica.” J Plankton Res 16 : 1543-1566. Smetacek, V., R. Scharek and E. M. Nothig, Eds. (1990). Seasonal and regional variation in the pelagial and its relationship to the life history cycle of krill. Antarctic Ecosystems. Ecological change and conservation. Berlin, Heidelberg, Springer. Stepnik, R. (1982). “All-year populational studies of Euphausiacea (Crustacea) in the Admiralty Bay (King George Island, South Shetland Islands, Antarctic).” Polish Polar Res. 3 : 49-68. Stretch, J. J., P. P. Hamner, W. M. Hamner, W. C. Michel, J. Cook and C. W. Sullivan (1988). “Foraging behavior of Antarctic krill Euphausia superba on sea ice microalgae.” Marine Ecology Progress Series 44 : 131-139. Sudgen, P. H. and E. A. Newsholme (1975). “Activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenases, glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase in nervous tissues from vertebrates and invertebrates.” Biochemistry Journal 150 : 105-111. Torres, J. J., A. V. Aarset, J. Donnelly, T. L. Hopkins, T. M. Lancraft and D. G. Ainley (1994). “Metabolism of Antarctic micronektonic Crustacea as a function of depth of ocurrence and season.” Marine Ecology Progress Series 113 : 207-219. Torres, J. J., J. Donnelly, T. L. Hopkins, T. M. Lancraft, A. V. Aarset and D. G. Ainley (1994). “Proximate composition and overwintering strategies of Antarctic micronekton Crustacea.” Marine Ecology Progress Series 113 : 221-232. Voronina, N. M. (1970). “Seasonal cycles of some common Antarctic copepod species. In: Holdgate, M. W. (ed.).” Antarctic Ecology I : 162-172. Walsh, P. J., C. Bedolla and T. P. Mommsen (1989). “Scaling and sex-related differences in toadfish ( Opsanus beta ) sonic muscle enzyme activities.” Bulletin of Marine Science 45 : 68-75. Ward, P. and D. B. Robins (1987). “The reproductive biology of Euchaeta antarctica Giesbrecht (Copepoda: Calanoida) at South Georgia.” Journal of Experimental Marine Biology and Ecology 108 : 127-145.