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A comparative study of eucalanoid copepods residing in different oxygen environments in the eastern tropical north pacific :

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Title:
A comparative study of eucalanoid copepods residing in different oxygen environments in the eastern tropical north pacific : an emphasis on physiology and biochemistry
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English
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Cass, Christine J
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Body Composition
Ecological Adaptation
Lipid Biomarkers
Metabolism
Oxygen Minimum Zone
Dissertations, Academic -- Biological Oceanography Ecology Physiology -- Doctoral -- USF   ( lcsh )
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bibliography   ( marcgt )
non-fiction   ( marcgt )

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Summary:
ABSTRACT: The eastern tropical north Pacific (ETNP) is characterized by one of the ocean's most severe midwater oxygen minimum zones (OMZs), where oxygen levels are often less than 5 µM. The copepod family Eucalanidae is a numerically abundant and diverse zooplankton group in the ETNP, and displays a wide range of vertical distributions related to environmental oxygen concentrations. The goal of this dissertation was to develop a better understanding of the ecology, physiology, and biochemistry of closely related copepod species (family Eucalanidae) that inhabit the ETNP OMZ system. This was accomplished through examining different parameters relating to (1) metabolic rates, (2) detailed lipid composition and biomarkers, and (3) body composition, enzyme activity and survivorship in low oxygen water. Oxygen consumption, ammonium, urea, and phosphate excretion rates were generally highest in <italic>Subeucalanus subtenuis</italic>, a copepod primarily residing in the upper euphotic zone. <italic>Eucalanus inermis</italic>, typically found in the lowest oxygen environment of the species examined, showed significantly lower metabolic rates largely due to high water content. <italic>Rhincalanus rostrifrons</italic>, residing primarily in the upper oxycline, showed intermediate rates, likely relating to its higher reliance on lipid catabolism than <italic>S. subtenuis</italic> and <italic>E. inermis</italic>. Urea excretion rates showed a complicated relationship with temperature and oxygen, which calls for further study. Knowledge of such interactions is necessary for accurate modeling of nitrogen cycles in OMZ and other oceanic regions. Lipid biomarkers suggested that <italic>S. subtenuis, E. inermis</italic> and <italic>Pareucalanus attenuatus</italic> all fed primarily on particulates near the chlorophyll maximum region, while <italic>R. rostrifrons</italic> and <italic>R. nasutus</italic> likely fed on sinking particulates at depth. These results also emphasized the difference in lipid composition between wax esters and triacylglycerol components of storage lipids. This study suggested a much larger role of phylogeny in characterizing lipid contents than previously thought. Body composition, enzyme assays and survivorship studies suggested that <italic>E. inermis, S. subtenuis, P. attenuatus, R. nasutus</italic> and <italic>R. rostrifrons</italic> formed four separate ecological groups based on genus. <italic>E. inermis</italic> had low organic matter, moderate lactate dehydrogenase (LDH) activity, and high survivorship at oxygen concentrations < 20 µM. <italic>Rhincalanus</italic> spp. also had moderate LDH activity and high survivorship in low oxygen, but were unique in particularly low protein and high lipid content. <italic>S. subtenuis</italic> was characterized by high protein content, no measurable LDH activity and low survivorship in < 20 µM O2. <italic>P. attenuatus</italic> was similar to <italic>S. subtenuis</italic> in many respects, but had lower protein content and a different lipid accumulation strategy. In conclusion, eucalanoid copepods utilized many different ecological strategies in the ETNP OMZ system. Features of different ecological groups fit well with their observed vertical distributions in the water column. Understanding the ecology of organisms in OMZ systems will allow us better predictive capability for the effects of expanding OMZs in other regions.
Thesis:
Disseration (Ph.D.)--University of South Florida, 2011.
Bibliography:
Includes bibliographical references.
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by Christine J. Cass.
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Title from PDF of title page.
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Document formatted into pages; contains 165 pages.
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Includes vita.

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ABSTRACT: The eastern tropical north Pacific (ETNP) is characterized by one of the ocean's most severe midwater oxygen minimum zones (OMZs), where oxygen levels are often less than 5 M. The copepod family Eucalanidae is a numerically abundant and diverse zooplankton group in the ETNP, and displays a wide range of vertical distributions related to environmental oxygen concentrations. The goal of this dissertation was to develop a better understanding of the ecology, physiology, and biochemistry of closely related copepod species (family Eucalanidae) that inhabit the ETNP OMZ system. This was accomplished through examining different parameters relating to (1) metabolic rates, (2) detailed lipid composition and biomarkers, and (3) body composition, enzyme activity and survivorship in low oxygen water. Oxygen consumption, ammonium, urea, and phosphate excretion rates were generally highest in Subeucalanus subtenuis, a copepod primarily residing in the upper euphotic zone. Eucalanus inermis, typically found in the lowest oxygen environment of the species examined, showed significantly lower metabolic rates largely due to high water content. Rhincalanus rostrifrons, residing primarily in the upper oxycline, showed intermediate rates, likely relating to its higher reliance on lipid catabolism than S. subtenuis and E. inermis. Urea excretion rates showed a complicated relationship with temperature and oxygen, which calls for further study. Knowledge of such interactions is necessary for accurate modeling of nitrogen cycles in OMZ and other oceanic regions. Lipid biomarkers suggested that S. subtenuis, E. inermis and Pareucalanus attenuatus all fed primarily on particulates near the chlorophyll maximum region, while R. rostrifrons and R. nasutus likely fed on sinking particulates at depth. These results also emphasized the difference in lipid composition between wax esters and triacylglycerol components of storage lipids. This study suggested a much larger role of phylogeny in characterizing lipid contents than previously thought. Body composition, enzyme assays and survivorship studies suggested that E. inermis, S. subtenuis, P. attenuatus, R. nasutus and R. rostrifrons formed four separate ecological groups based on genus. E. inermis had low organic matter, moderate lactate dehydrogenase (LDH) activity, and high survivorship at oxygen concentrations < 20 M. Rhincalanus spp. also had moderate LDH activity and high survivorship in low oxygen, but were unique in particularly low protein and high lipid content. S. subtenuis was characterized by high protein content, no measurable LDH activity and low survivorship in < 20 M O2. P. attenuatus was similar to S. subtenuis in many respects, but had lower protein content and a different lipid accumulation strategy. In conclusion, eucalanoid copepods utilized many different ecological strategies in the ETNP OMZ system. Features of different ecological groups fit well with their observed vertical distributions in the water column. Understanding the ecology of organisms in OMZ systems will allow us better predictive capability for the effects of expanding OMZs in other regions.
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A Comparative Study of Eucalanoid Copepods Residing in Different Oxygen Environments in the East ern Tropical North Pacific: An Emphasis on Physiology and Biochemistry by Christine J. Cass A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy College of Marine Science University of South Florida Major Professor: Kendra L. Daly, Ph.D. Kent A. Fanning, Ph.D. Brad A. Seibel, Ph.D. Edward S. Van Vleet, Ph.D. Stuart G. Wakeham, Ph.D. Date of Approval: December 9, 2010 Keywords: Metabolism, Lipid Biomarke rs, Oxygen Minimum Zone, Ecological Adaptation, Body Composition Copyright 2011, Christine J. Cass

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ACKNOWLEDGEMENTS I would like to acknowledge the University of South Florida Graduate School, the College of Marine Science, a nd my advisor, Dr. Kendra Daly for financial support during the completion of my degree. Scientific funding for this project was provided by NSFOCE #0526545. I would also like to thank Dr. Brad Seibel for allowing me to collect samples during his work in the Gulf of California (NSF-OCE #0526493 ), and Dr. Joseph Torres for providing cruise time in the Gulf of Mexico. Thanks also to Dr. Stuart Wakeham and Dr. Edward Van Vleet for assist ance in processing lipid samples, Dr. Kent Fanning and his lab for processing of nutrient samples, and Dr. Andrew Remsen and Dr. Karen Wishner providing information on ver tical distributions of eucalanoid copepods during our cruises. I would al so like to thank all of thos e people who helped with the collection of my sample organisms during cr uises: Dr. M. Brady Olson, Sennai Habtes, Marianne Dietz, Leanne Birden, Amy Maas, Rebecca Williams, and Dawn Outram.

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TABLE OF CONTENTS LIST OF TABLES. iv LIST OF FIGURESvi ABSTRACT.viii CHAPTER 1: Introduction to the East ern Tropical North Pacific and Oxygen Minimum Zones. .1 CHAPTER 2: Variation in Metabolic Rates for Species of Eucalanoid Copepods in the Oxygen Minimum Zone of the Eastern Tropical North Pacific: Effects of Oxygen and Temperature.... 7 Introduction.7 Methods.12 Results17 Vertical Distribution of Cope pods in Relation to Environmental Parameters17 Oxygen Consumption Ammonium Excretion....20 Urea Excretion22 Percent Urea-Nitrogen Excretion23 Total Nitrogen Excretion25 Percent Nitrogen Body Turnover.. 25 Phosphate Excretion.. 28 Percent Phosphorus Body Turnover..29 Metabolic Ratios Q 10 Ratios.. 32 Discussion..34 Species Influences on Metabolic Rates. 34 Temperature.. 38 Oxygen Level.40 Implications of Oxygen and Temperature Interactions for Nitrogen Cycling in the ETNP.42 Year Depth..45 Conclusions i

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CHAPTER 3: Lipid Biomar kers of Eucalanoid Copepods in the Eastern Tropical North Pacific: Evidence fo r Differential use of Wax Ester and Triacylglycerol Storage Lipids 49 Introduction.. 49 Methods. 54 Copepod Collection and Measurement. 54 Particulate Matter Collection.55 Particulate Composition.56 Lipid Extraction and Analysis.. 56 Biomarkers.58 Statistics.58 Results Particulate Matter Composition and Lipids60 Copepod Storage Lipids 67 Copepod Phospholipids. 72 Copepod Fatty Alcohols and Sterol Fractions...72 Discussion..77 CHAPTER 4: Ecological Ch aracteristics of Eucala noid Copepods of the Eastern Tropical North Pacific: Ad aptations for Life Within a Low Oxygen System.... 94 Introduction... 94 Methods..98 Collection Site and Methods..98 Length-Weight Regressions...99 Body Composition101 Enzyme Analyses.102 Survivorship Studies Statistical Analyses..103 Results..104 Environmental Parameters and Copepod Distribution.104 Length-Weight Equations 105 Body CompositionSpecies Differences108 Body Composition Eucalanus inermis ..111 Body Composition Rhincalanus spp 113 Enzyme Activity.. 113 Survivorship Studies 115 Discussion 117 Eucalanus inermis Subeucalanus subtenuis ... 121 Rhincalanus rostrifrons and R. nasutus ...123 Pareucalanus attenuatus..126 Comparison with Other Eucalanoid Copepods Conclusions..134 CHAPTER 5: Summary of Major Conc lusions and Future Research.136 ii

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LIST OF REFERENCES.....139 APPENDICES.152 Appendix A: Extra Figures..153 iii

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LIST OF TABLES Table 2.1: Oxygen consumption rates for four eucalanoid copepod species at three temperatures........19 Table 2.2: Ammonium and urea excretion ra tes and percent urea of total nitrogen excretion for eucalanoid copepods...... 21 Table 2.3: Total nitrogen excretion ra tes and daily perc ent body nitrogen turnover for eucalanoid copepods... 26 Table 2.4: Adult female copepod weight and body composition for eucalanoid species......27 Table 2.5: Phosphate excretion rates a nd daily percent body phosphorus turnover for each eucalanoid species..29 Table 2.6: Metabolic ratio s for eucalanoid copepods.... 31 Table 2.7: Respiratory quotient data for Eucalanus inermis ..32 Table 2.8: Q 10 ratios for Eucalanus inermis and Subeucalanus subtenuis .33 Table 3.1: Shorthand, full and common names of sterol compounds Table 3.2: Particulate material at the Costa Rica Dome site..... 60 Table 3.3: Total fatty acid, alcohol, and st erol profiles for particulate samples Table 3.4: Summary of particulate lipids and biomarkers in molar percentages64 Table 3.5: Lipid classes and total lipids as percent of wet and dry weight of copepods.. 68 Table 3.6: Triacylglycerol profiles for copepods.. 70 Table 3.7: Wax ester fatty acid profiles for copepods.. 71 Table 3.8: Phospholipid fatty acid profiles for copepods..... 74 iv

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Table 3.9: Wax ester fatty alcohol profiles for Rhincalanus and Pareucalanus.. 75 Table 3.10: Sterol prof iles for copepods...76 Table 4.1: Length to weight rela tionship for eucalanoid copepods.... 107 Table 4.2: Body composition for eastern tr opical north Pacific adult female copepods Table 4.3: Body composition for ea stern tropical north Pacific Eucalanus inermis copepods...112 Table 4.4: Body composition for Rhincalanus cornutus from the Gulf of Mexico and R. nasutus from the Gulf of California...114 Table 4.5: LDH Activity for eastern tropical north Pacific copepods.115 v

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LIST OF FIGURES Figure 1.1: Oxygen concentrations (M) at minimal O 2 depth (m), indicating the extent of the OMZs (in red) ba sed on the WOA2005 climatology...2 Figure 1.2: Diagram of surface water masses and currents in the eastern tropical north Pacific Ocean....4 Figure 1.3: Sampling sites for the ea stern tropical north Pacific.6 Figure 2.1: The vertical dist ributions of adult female eucalanoid copepods in relation to temperature, fluorescence and oxygen concentrations during 2007 at the Costa Rica Dome site (9N, 90W).... 18 Figure 2.2: Percent urea-nitrogen for (a) Subeucalanus subtenuis and (b) Eucalanus inermis24 Figure 3.1: Map of eastern tropical north Pacific sampling sites..55 Figure 3.2: Cluster analysis comparing to tal fatty acid profiles for particulate material and storage lipid fatty acid profiles for copepods .... 62 Figure 3.3: Comparison of total fatty aci d profiles for particulates from the chlorophyll maxima and cope pod triacylglycerol fatty acid profiles for (A) 2007 and (B) 2008-2009......73 Figure 3.4: A comparison of (A) triacylg lycerol and (B) wax ester fatty acid profiles of Pareucalanus attenuatus ....82 Figure 4.1: Map of eastern tropical north Pacific sampling sites..99 Figure 4.2: Water column oxygen, temperatur e, and fluorescence profiles in the upper 500 m at two stations during cruises to the eastern tropi cal north Pacific...... 105 Figure 4.3: Percent survivorship of eucalanoid copepods during metabolic incubations.116 vi

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Figure 4.4: Oxygen consumption rates for Eucalanus inermis, Rhincalanus rostrifrons and Subeucalanus subtenuis at different temperatures and under conditions of oxygen saturation....118 Figure A1: Cluster analysis for the ster ol fraction of particulate samples..153 Figure A2: Cluster analysis for cope pod phospholipid fatty acid profiles.154 Figure A3: Cluster analysis for copepod sterol profiles..... 154 vii

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ABSTRACT The eastern tropical north Pacific (ETNP) is characterized by one of the oceans most severe midwater oxyge n minimum zones (OMZs), where oxygen levels are often less than 5 M. The copepod fa mily Eucalanidae is a numeric ally abundant and diverse zooplankton group in the ETNP, and displays a wide range of vertical distributions related to environmental oxygen concentrations. The goal of this dissertation was to develop a better understanding of the ecology, physiology, and biochemistry of closely related copepod species (family Eucalanidae) that inhabit the ETNP OMZ system. This was accomplished through examining different pa rameters relating to (1) metabolic rates, (2) detailed lipid composition and biomarkers, and (3) body composition, enzyme activity and survivorship in low oxygen water. Oxygen consumption, ammonium, urea, and phosphate excretion rates were generally highest in Subeucalanus subtenuis, a copepod primarily residing in the upper euphotic zone. Eucalanus inermis, typically found in the lowest oxygen environment of the species examined, showed significantly lower metabolic rates largely due to high water content. Rhincalanus rostrifrons residing primarily in the upper oxycline, showed intermediate rates, likely relating to its higher reliance on lipid catabolism than S. subtenuis and E. inermis Urea excretion rates showed a complicated relationship with temperature and oxygen, which calls for further study. Knowledge of su ch interactions is necessary for accurate modeling of nitrogen cycles in OMZ and other oceanic regions. viii

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Lipid biomarkers suggested that S. subtenuis E. inermis and Pareucalanus attenuatus all fed primarily on particulates near the chlorophyll maximum region, while R. rostrifrons and R. nasutus likely fed on sinking particulates at depth. These results also emphasized the difference in lipid composition between wax esters and triacylglycerol components of storage lipids. This study suggested a much larger role of phylogeny in characterizing lipid co ntents than previously thought. Body composition, enzyme assays and survivorship studies suggested that E. inermis, S. subtenuis, P. attenuatus, R. nasutus and R. rostrifrons formed four separate ecological groups based on genus. E. inermis had low organic matter, moderate lactate dehydrogenase (LDH) activity, and high survivor ship at oxygen concentrations < 20 M. Rhincalanus spp. also had moderate LDH activity and high survivorship in low oxygen, but were unique in particularly lo w protein and high lipid content. S. subtenuis was characterized by high protein c ontent, no measurable LDH ac tivity and low survivorship in < 20 M O 2 P. attenuatus was similar to S. subtenuis in many respects, but had lower protein content and a different lipid accumulation strategy. In conclusion, eucalanoid copepods utili zed many different ecological strategies in the ETNP OMZ system. Features of diffe rent ecological groups fit well with their observed vertical distributions in the water column. Un derstanding the ecology of organisms in OMZ systems will allow us bette r predictive capability for the effects of expanding OMZs in other regions. ix

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CHAPTER ONE Introduction to the Eastern Tropical North Pacific and Oxygen Minimum Zones Midwater oxygen minimum layers are features which exist in all of the worlds oceans. They are characterized by decr eased dissolved oxygen at inte rmediate depths below the thermocline (usually 1000-1500 m), as comp ared to more highl y oxygenated waters above the thermocline and in the deep ocean. These common oxygen minimum layers are distinct from oxygen minimum zones (OMZs) which are more severe in their oxygen depletion and often found at shallower depths (Paulmier & Ruiz-Pino 2009). Particularly prominent OMZs are found in several areas, including the eastern tropical north and south Pacific, Arabian Sea, and the Bay of Bengal, where oxygen levels drop to less than 20 M (Paulmier & Ruiz-Pino 2009). The ea stern tropical north Pacific (ETNP), in particular, has one of the shallowest OMZs a nd is highly depleted in oxygen (Figure 1.1). These suboxic regions in open ocean water columns are typically maintained as a result of poor ventilation, sluggish circulation, oxyge n-poor source waters, and decomposition of sinking particles (Wyrtki 1962). In addi tion, layers of permanent hypoxic or anoxic waters are found in some basins and trench es, including the Black Sea, Red Sea, Santa Barbara Basin, Santa Monica Basin, and th e Cariaco Basin (Kamykowski & Zentara 1990). 1

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Figure 1.1. Oxygen concentrations (M) at minimal O 2 depth (m), indicating the extent of the OMZs (in red) based on the WOA2005 climatology. The color bar scale corresponds to a 1 2 M interval between 0 and 20 M, and a 20 2 M interval between 20 and 340 M. Isolines indicate the upper depth (m) limit of the OMZ core (where O 2 < 20 M), with a 100-m contour interval. ENP is eastern north Pacific. The black box surrounds the eastern tropical north Pacific region. Figure after Paulmier and Ruiz-Pino (2009). In the ETNPs OMZ, dissolved oxygen concentrations usually are less than 4.5 M (Brinton 1979, Levin et al. 1991, Vinogradov et al. 1991, Saltzman & Wishner 1997a), with some oxygen levels reported below 0.5 M (Sameoto 1986, Levin et al. 1991). The minimum oxygen concentration ca n occur anywhere be tween about 300 and 1000 m, with an overall thickness of the OM Z of 200 to over 1000 m (Fiedler & Talley 2006). The ETNP is characterized by a str ong, shallow pycnocline and a pronounced 2

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oxycline (Fiedler & Talley 2006), where ch lorophyll, primary production, and copepod maxima occur (approximately 40-50 m dept h) (Herman 1989). Overall, primary productivity and zooplankton biomass vary grea tly throughout the regi on, with the Costa Rica Dome (a wind-driven upwelling area) having 5-6 times higher primary productivity and 1.7-2.1 times greater total zooplankton/micronekton biomass than a non-upwelling region (Sameoto 1986). Wind-driven upwelling o ccurs in several different areas near the coast of Central America, due to focused jets of wind across the Central American isthmus (Figure 1.2) (Amador et al. 2006). Wh ile upwelling is observed to some extent throughout the year, the strongest upwelling norm ally occurs in the late fall and early winter (Pennington et al. 2006) Circulation in the ETNP area is complicated (Figure 1.2), as this region lies at th e end of two major western boun dary currents (California and Peru Currents) and also near two subtr opical gyres (Fiedler & Talley 2006). Studies examining the vertical distribut ion of organisms within the ETNP have found that all taxa, from zooplankton to micr onekton to benthic fauna, seem to have distinct layers of peak abundance often re lated to oxygen concentrations (Brinton 1979, Chen 1986, Sameoto 1986, Sameoto et al. 1987, Levin et al. 1991, Vinogradov et al. 1991, Wishner et al. 1995, Saltzman & Wi shner 1997a, Saltzman & Wishner 1997b). Members of the copepod family Eucalanidae ( Eucalanus, Rhincalanus, Subeucalanus, Pareucalanus) are well represented in the ETNP. They also have variable vertical distributions, with S. subtenuis, S. subcrassus, S. pileatus, and P. attenuatus residing primarily in the euphotic zone and R. rostrifrons and R. nasutus concentrated above and below the OMZ, without representation in the OMZ core. In contrast, E. inermis individuals are found throughout the water colu mn to about 1200 m, but are concentrated 3

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in the mixed layer, upper OMZ interface, and lower OMZ interface. The high abundances and variation in ve rtical distributions for euca lanoid copepods in the ETNP makes these ideal organisms for the study of copepod adaptations to low oxygen systems. Figure 1.2. Diagram of surface water masses and currents in the eastern tropical Pacific Ocean. STSW is Subtropical Surface Water; TSW is Tropical Surface Water; ESW is Equatorial Surface Water. Shading repres ents mean sea surface temperature, where darker is cooler. Solid arrows represent ap proximate locations of strong wind jets which contribute towards regional upw elling. Figure modified from Fiedler and Talley (2006). OMZ regions are important ecologically for many reasons. The vertical oxygen gradients in OMZs structure biological assemblages and biogeochemical processes. Habitats of organisms intolerant to low oxygen may be compressed into the shallow, near-surface oxygenated waters (Prince & G oodyear 2006). OMZs also are areas of complex nitrogen cycling and, therefore, it is especially important to understand the sources and cycling of the various forms of nitrogen in these regions. Due to the near 4

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anoxic conditions encountered in OMZs, anaerobic nitrogen pathways, including denitrification and anammox (anaerobic ammonium oxidation) are prominent features. Via these pathways, OMZ regions are thought to contribute up to 50% of the total nitrogen lost from the oceans to the atmosphere (Gruber & Sarmiento 1997, Codispoti et al. 2001). OMZ systems are expanding and will likely continue to do so in the future (Stramma et al. 2008, Stramma et al. 2010), maki ng it essential for scientists to develop a greater understanding of these regions. The goal of this dissertation was to asse ss the impact of the OMZ on a dominant group of zooplankton, the eucalanoid copepods, and to advance understanding of the ecology and physiology of thes e closely related copepod species that inhabit the ETNP OMZ system (Figure 1.3). These copepods display a wide variety of vertical distributions, providing a co mparative study of behavioral strategies. Eucalanoid copepods also are widespread throughout th e oceans, allowing for comparisons between species or populations inhabiting OMZ regi ons with those in higher oxygen areas. Chapter Two assesses the variability of metabolic parameters within and among eucalanoid species in relation to different temperatures and oxygen concentrations. Chapter Three examines food sources for fi ve species of eucalanoid copepods under different environmental condi tions using lipid biomarkers Chapter Four combines information on body composition, enzyme activity, and survivorship at low oxygen concentrations to determine the dominant eco logical strategies for different eucalanoid copepods of the ETNP. A summary of major c onclusions is provided in Chapter Five, as well as suggestions for further research. 5

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Figure 1.3. Sampling sites for the eastern tropi cal north Pacific. A cruise transect and station locations are superimposed onto a MODIS image from November, 2001. Red circles emcompass the two major sampling locati ons for this dissertation: the Costa Rica Dome (9N, 90W) and the Tehua ntepec Bowl (13N, 105W). 6

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CHAPTER TWO Variation in Metabolic Rates for Sp ecies of Eucalanoid Copepods in the Oxygen Minimum Zone of the Eastern Tropical North Pacific: Effects of Oxygen and Temperature INTRODUCTION Recent reports indicate that the oceans are decreasing in oxygen in response to global warming, primarily through surface hea ting and stratificati on (Keeling & Garcia 2002, Emerson et al. 2004). In addition, regions of the ocean having oxygen minimum zones (OMZs), which are characterized by ox ygen deficient waters at intermediate depths, appear to be expanding (Stramma et al. 2008, Stramma et al. 2010). The suboxic regions in open water OMZs are typically maintained as a resu lt of poor ventilation, sluggish circulation, oxygen-poor sources wate rs, and decomposition of sinking particles (Wyrtki 1962). The extent of low oxygen or hypoxic waters (usually defined as < 2 mg/L or < 60 M) in coastal regions also has increa sed in the last three decades due to natural and human activities (Helly & Levin 2004, Rabalais et al. 2009). Little is known, however, about the effects of OMZs and hypoxic coastal regi ons on carbon and nitrogen cycles, marine biota, and the effi ciency of the biological pump. Metabolic rates of marine organisms, in particular, will be se nsitive to changing ocean conditions. Increasing wate r temperatures and decreasing O 2 and pH levels will exceed physiological tolerances of many mari ne organisms and eventually limit suitable 7

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habitats (Prince & Goodyear 2006 ). Metabolic rate s of marine zooplankton are known to be influenced by a number of different fact ors. Oxygen consumption rates, which are most directly related to metabolism, are st rongly influenced by temperature (Childress 1977, Barber & Blake 1985, Hirche 1987, Donne lly & Torres 1988, Ikeda et al. 2001). Other factors can include body mass (Conove r & Gustavson 1999, Ikeda et al. 2001), salinity (Barber & Blake 1985), season (Conover 1959, Torres et al. 1994, Conover & Gustavson 1999), pressure (Childress 1977), depth of occurrence (Childress 1975, Torres et al. 1994, Seibel & Drazen 2007), life strategy (Company & Sard 1998), feeding activity or feeding history (Ikeda 1971, Mayzaud 1976, Ikeda 1977, Bohrer & Lampert 1988), swimming activity (Childress 1968, Torre s & Childress 1983, Swadling et al. 2005), and in situ oxygen concentrations (Childress 1975, Childress 1977, Donnelly & Torres 1988, Cowles et al. 1991). Other metabolic parameters, such as ammonia, urea, and phosphate excretion rates also may be influenced by many of the same factors, including temperature (Qua rmby 1985, den Oude & Gulati 1988, Aarset & Aunaas 1990, Ikeda et al. 2001), salinity (Barber & Blake 1985), body mass (Conover & Gustavson 1999, Ikeda et al. 2001) and feeding hi story (Mayzaud 1976, Ikeda 1977, Miller & Roman 2008, Saba et al. 2009). Metabolic ratios (O:N, N:P and O:P) are useful as indicators of metabolic substr ate catabolized during respira tion, and also may vary with season (Snow & Williams 1971, Hatcher 1991, Gaudy et al. 2003), timing in reproductive cycle (Barber & Blak e 1985), dry weight (Ikeda et al. 2001), feeding history (Ikeda 1977, Quetin et al. 1980, Mayzaud & Conover 1988, Hatcher 1991), general taxa (Ikeda & Skjoldal 1989) and occasionally temperature (Aarset & Aunaas 1990). 8

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However, excretion rates and metabolic ratios have rarely been examined in relation to variable oxygen concentrations. The lethal and sublethal effects of co astal hypoxic oxygen concen trations are well documented for some benthic organisms (V aquer-Sunyer & Duarte 2008). However, little work has been done to examine the de leterious effects of low oxygen levels on pelagic crustaceans. Crustacean studies on effects of low oxygen have largely concentrated on changes in oxygen consumption rates, egg production, growth, development, activity rates and survival (Svetlichny et al. 2000, Svetlichny & Hubareva 2002, Auel & Verheye 2007). Few studies, if any, have examined the effects of low oxygen conditions on ammonia, urea or phospha te excretion rates or on metabolic substrate use. One study on the white shrimp ( Penaeus setiferus ) found that protein catabolism dominated at low oxygen, whereas s ubstrate use switched to a combination of lipid and protein catabolism at higher oxygen levels (Rosas et al. 1999). Thus, low in situ oxygen concentrations have the potential to influence other metabolic parameters besides respiration rates, and changes in metabolic pathways could influence the composition of excreted by-products. The eastern tropical north Pacific (ETNP) is the largest low oxygen oceanic biome (Paulmier & Ruiz-Pino 2009). The ETN P is characterized by a strong, shallow pycnocline and a pronounced oxycline (Fiedler & Talley 2006), where chlorophyll, primary production, and copepod maxima occu r (Herman 1989). Oxygen concentrations of less than 50 M occur as shallow as 40 m and often re ach less than 4.5 M in the OMZ core (Brinton 1979, Levin et al. 1991, Vi nogradov et al. 1991, Saltzman & Wishner 1997a). Some oxygen levels have been reported below 0.5 M (Chen 1986, Sameoto 9

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1986, Levin et al. 1991). Other tropical and subtr opical water regions in the Atlantic and western Pacific oceans have less intense OMZs with minimum oxygen le vels of 60 to 80 M (Paulmier & Ruiz-Pino 2009). The vertic al oxygen gradients in OMZs structure biological assemblages and biogeochemical proce sses. Habitats of organisms intolerant to low oxygen may be compressed into the shallow, near-surface oxygenated waters (Prince & Goodyear 2006). The goal of this study was to assess re spiration and excret ion rates of the eucalanoid copepods Subeucalanus subtenuis S. pileatus, Rhincalanus rostrifrons and Eucalanus inermis, in order to investigate the metabo lic responses of these four closely related species to low oxygen concentrations in ETNP OMZ system. Herein, we present results on oxygen consumption, carbon dioxide production, and ammonium, phosphate, and urea excretion rates, as well as O:N, N: P and O:P metabolic ratios and respiratory quotients (RQs). Metabolic ratios and RQ va lues indicate preferred substrate utilization (lipid, carbohydrate or protein) during the c ourse of the experiment (Kleiber 1961, Ikeda 1977, Mayzaud & Conover 1988). Measurements we re obtained at high (saturation) and low (15-20% saturation) oxygen concentrations at representative temperatures for this study site: 10C for the upper oxycline, 17C for chlorophyl l maximum depths, and 23C for near-surface waters. The low oxygen treatm ent (15-20% saturation, or approximately 50 M) was representative of conditions at the chlorophyll maximum, which is a region of maximum abundance for many species of copepods (Longhurst 1985, Chen 1986, Sameoto 1986, Herman 1989, Saltzman & Wishner 1997b) Members of the family Eucalanidae ar e among the dominant zooplankton in this region and adult females vary in their rang e of vertical distri butions (Longhurst 1985, 10

PAGE 22

Chen 1986, Sameoto 1986, Saltzman & Wishner 1997b). S. subtenuis and S. pileatus (formerly Eucalanus subtenuis and E. pileatus (Geletin 1976)) are found in highest abundance in the shallo w euphotic zone, while R. rostrifrons has maximum abundances in the upper oxycline. E. inermis adult females have a verti cal range which spans much of the upper 1000 m, with peaks in abundan ce near the chlorophy ll maximum, the upper oxycline and the lower oxycline. This spec ies is also present in small numbers throughout the core of the OMZ, where oxygen levels are nearly zero. Such differences in vertical distributions s hould allow us to examine the metabolic response of closely related species to low in situ oxygen concentrations in the open ocean. 11

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METHODS Copepods were collected during two cruise s to the eastern tropical north Pacific (ETNP); from 18 October 17 No vember 2007 aboard the R/V Seward Johnson and 8 December 2008 6 January 2009 aboard the R/V Knorr Sampling occurred at two stations: 9N, 90W (Costa Rica Dome) a nd 13N, 105W (Tehuantepec Bowl) using bongo tows, Tucker trawls, and MOCNESS (Multiple Opening/ Closing Net and Environmental Sampling System) tows. Subeucalanus subtenuis and S. pileatus were collected from the upper 50 m and Rhincalanus rostrifrons in the 200-300 m range. Eucalanus inermis was collected from both depths. Immediately after capture, adult female copepods were sorted and individuals of each species were separated into small containers containing 0.2 m filtered seawater at in situ temperature. Copepods were kept at in situ temperatures for approximately 3-12 hours to allow them to empty their guts. End point metabolic experiments were performed using 60 and 300 ml BOD bottles, containing filtered s eawater with antibiotics (25 mg/l each of streptomycin and ampicillin). Typically 2-15 individuals were used for the 60 ml bottles and 20-30 individuals for the 300 ml bottles, depending on size of species and temperature. This number was optimal to achieve a measurab le drawdown of oxygen, while trying to avoid crowding effects. Eucalanoid copepod densities in thin layers within the water column could be up to 4 per 60 ml in the ETNP (A. Remsen, personal communication), which were similar to or slightly lower than dens ities used in incubations during this study. Experiment duration was typically 12-24 hrs. Bottles were kept in the dark and in water baths to maintain the desired temperature. Samples were taken immediately before and 12

PAGE 24

after each experiment for oxygen, phosphate, ammonium, and urea concentrations. Carbon dioxide samples also were taken for a limited number of experiments. Experiments were run at 10 and 23C duri ng the 2007 cruise and 10 and 17C during the 2008-2009 cruise. During both years, experiments were run at high (100% air saturation) and low (15-20% air saturation) initial oxyge n concentrations. Low oxygen conditions were obtained by bubbling nitrogen or a lo w-oxygen gas mixture into the filtered seawater. Control bottles without any copepods also were run under the same conditions. Only experimental runs where all copepods su rvived the incubation were used for rate measurements. The experimental temperatures were repr esentative of different depths: 10C for the oxycline, 17C for chlorophyll maximum dept hs, and 23C for near-surface waters (see Figure 2.1). The high (100% saturation; 201 325 M) oxygen treatment was representative of surface wate rs above the pycnocline and the low (15-20% saturation; 36 78 M) oxygen treatment was representative of conditions in the upper oxycline near the chlorophyll maximum, which is a regi on of maximum abundance for many species of copepods (Longhurst 1985, Herman 1989, Saltzman & Wishner 1997b). A few experiments were run at lowe r oxygen concentrations (5% saturation; 8 15 M) at 10 C, which represented the lowest oxygen levels able to be used accurately for end point experiments. However, only changes in oxygen were large enough to be measured accurately using the techniques outlined below. Oxygen samples were analyzed shipboard using a Clark-type oxygen electrode (Strathkelvin instruments). Preand post-e xperiment, a 1.5 ml gas ti ght syringe was used to collect water from the BOD bottles and in ject it into a small chamber surrounding the 13

PAGE 25

electrode to obtain a reading. Electrodes were calibrated da ily to high and low points using air saturated and nitrogen bubbled water, resp ectively. Carbon dioxide levels were assessed during 2008-2009 onboard the ship us ing the flow-through MICA instrument system (Wang et al. 2007). Air tight 250 ml glass syringes were used to remove water from the 300 ml BOD bottles and transfer it into the CO 2 system. Ammonium and phosphate concentrations were determined using a Technicon Autoan alyzer II (Gordon et al. 2000). Urea was measured by the manual spectrophotometric method using diacetylmonoxime without deproteinizatio n (Rahmatullah & Boyd 1980, Whitledge et al. 1981). At the end of each experiment, copepods were frozen at -80C in cryovials containing a small amount of filtered seawat er. In the lab, individuals from each BOD bottle were thawed, briefly rinsed in DI water to remove adhering salts, blotted dry, placed in an aluminum capsule, and weighe d on a Mettler Toledo UMX2 microbalance to obtain wet weight (WW). Samples were then dried in an oven at 60C for several days and weighed again to measure dry weight (DW). Metabolic ratios were calculated using the changes in oxygen, phosphate, urea and ammonium concentrations. N:P and O: N ratios were calculated twice, once using ammonium excretion values and a second time using total nitrogen excretion (urea and ammonium nitrogen excretion combined). Theoretical ratios are derived assuming only ammonium excretion (Mayzaud & Conover 1988), although total nitrogen values may be more accurate for comparison purposes when urea excretion is a major component. The respiratory quotient (RQ) was determined for the limited number of data points where good dissolved inorganic carbon (DIC) readings were obtained. With this information, 14

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the equations of Lauff and W ood (1996) were used to calcul ate the exact percentages of each metabolic substrate catabolized during the experiment. Factors that may influence metabolism were examined, including temperature, species, starting oxygen concentration, in terannual variability, and depth. S. subtenuis E. inermis and R. rostrifrons were run at 10 and 17C and S. subtenuis, E. inermis, and S. pileatus were run at 23C. Two oxygen levels we re used (100% and 15% saturation) at each temperature for each species, to assess th e effect of starting oxygen concentration. The sole exception was S. pileatus which was present in very low numbers, and thus could only be examined at 100% air saturation. A few experiments for R. rostrifrons, E. inermis and S. subtenuis utilized conditions of 5% sa turation at 10C. Interannual variability was examined by comparing similar treatments between 2007 and 2008-2009. As 10C was the only temperature run during both years, all interannual variability was assessed at this temperature. The e ffect of depth was also examined in E. inermis. Here, adult females were either classified as sh allow (collected in the upper 50 m) or deep (collected at 200-300 m depth). The majority of the deep data points were generated during the 2008-2009 cruise. Q 10 values were calculat ed for 10-23C using values from 2007 and 10-17C using values from 2008-2009. When no signifi cant difference was seen between oxygen treatment levels, values were combined. When differences existed, only the high oxygen data points were used. Only shallow E. inermis individuals were used for Q 10 comparisons. Prior to statistical analyses data were tested for nor mality, and then appropriate parametric or non-parametric tests were used (Zar 1984). Often, by converting to ranked 15

PAGE 27

data, normality and equal variance assumptions were met. Comparisons between species and treatment groups were made using ANO VAs and parametric and non-parametric pair-wise comparison tests in SigmaPlot 11.0. T-tests assuming unequal variances and Mann-Whitney rank sum tests also were used for some comparisons Significance was assessed at = 0.05. Due to the non-normality of many of the rate data sets, central values here will be reported as medians and quartile ranges, rather than averages and standard deviations or errors. Unless otherw ise noted, the statistics reported are based on rates per unit WW. 16

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RESULTS Vertical Distribution of Copepods in Relation to Environmental Parameters An example of the vertical abundance and distribution of adult female eucalanoid copepods is shown for the Costa Rica Dome site (9N, 90W) during 2007 in relation to temperature (T), fluorescence and oxygen concen trations (Figure 2.1). Subeucalanus subtenuis occurred primarily in near-surface waters, with a maximum abundance at 25 m (T = 16C; 60 M O 2 ), mid-thermocline, in the vicin ity of the chlorophyll maximum. Rhincalanus rostrifrons was largely observed at th e base of the upper oxycline, just above the core of the OMZ at 275 m (T = 11 C; 6 M O 2 ). Eucalanus inermis had a peak of maximum abundance near the base of the upper oxycline at 325 m (T = 10C; 2 M O 2 ), just within the low oxygen core of the OMZ, and low conc entrations of indivi duals extending down to the lower oxycline. In addition, this species had a secondary peak in the thermocline near the chlorophyll maximum at 35 m (T = 15C; 35 M O 2 ). S. pileatus abundances are not depicted, but they generally inhabite d the upper 15-20 m of the water column (T = 26-27C; > 80 M O 2 ). Detailed distributions of copepods from the 2008-2009 cruise or from the Tehuantepec Bowl site (13N, 105W) in 2007 were not available for comparison. Oxygen Consumption The variation in oxygen consum ption rates was primarily a function of species-specific differences an d temperature (Table 2.1). Adult female E. inermis had significantly lower ra tes at 10 and 17C than S. subtenuis and R. rostrifrons S. subtenuis and R. rostrifrons however, were not significantl y different from each other 17

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at those temperatures. At 23C, S. subtenuis, E. inermis, and S. pileatus were all significantly different from each other. Abundance (Number per 100 m3) 0 1000 2000 3000 4000Depth (m) 0 100 200 300 400 500 600 700 Oxygen Concentration (M) 0255075100125150175200225 Temperature (C) 051015202530 Fluorescence (mg/m3) 0.00.20.40.60.81.01.21.41.61.82.0 S. subtenuis E. inermis R. rostrifrons Oxygen Concentration Temperature Fluorescence Figure 2.1. The vertical distribu tions of adult female eucala noid copepods in relation to temperature, fluorescence and oxygen con centrations during 2007 at the Costa Rica Dome site (9N, 90W). Abundance data for Subeucalanus subtenuis Eucalanus inermis and Rhincalanus rostrifrons are from day tows (courtesy of Karen Wishner and Dawn Outram). 18

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Table 2.1. Oxygen consumption rates for four eucalanoid copepod species at three temperatures. Values are medians (25 th quartile-75 th quartile) and number of replicates. Rates per mg wet weight (WW) and dry weight (DW) are shown at all temperatures. Following some temperatures, interannua l rates are provided when copepod consumption rates ar e significantly different between years at those specific temperatures. and denote the 2007 and the 2008-2009 cruises. High O 2 and Low O 2 refer to rates obtained at 100% and 15% air saturation oxygen tr eatments, respectively. NS denotes data that was not significantly different from other treatments. ND (not determined) indicates no data was available Species O 2 Consumption (nmol (mg WW) -1 hr -1 ) O 2 Consumption (nmol (mg DW) -1 hr -1 ) E. inermis 10C 2007 2008 17C 23C 1.71 (1.28-2.27) 45 1.15 (0.49-1.43) 18 1.95 (1.68-2.42) 27 2.38 (2.30-2.60) 9 5.55 (5.31-7.09) 5 27.8 (21.3-35.8) 45 19.7 (8.1-25.7) 18 32.0 (27.3-37.2) 27 43.9 (37.1-45.7) 9 90.5 (89.8-140.9) 5 R. rostrifrons 10C 17C 6.06 (3.75-6.59) 9 7.01 (6.18-7.85) 2 40.3 (24.8-51.3) 9 55.8 (48.8-62.8) 2 S. subtenuis 10C 17C High O 2 Low O 2 23C 7.58 (5.65-10.22) 22 11.49 (7.66-12.31) 11 12.31 (12.14-12.56) 6 7.54 (7.35-7.78) 5 16.01 (15.73-17.82) 9 56.8 (43.0-77.2) 22 85.2 (55.8-91.2) 11 91.2 (90.6-92.9) 6 53.2 (50.7-58.4) 5 144.9 (121.9-166.5) 9 S. pileatus 23C 48.16 (42.27-55.36) 3 436.4 (386.5-506.7) 3 Within species, temperature was the prim ary factor influencing variability among respiration rates. E. inermis had significantly differe nt oxygen consumption rates between 10 and 23C, as well as 10 and 17C treatments. S. subtenuis had significantly different rates between 23C and the other two temperature groups, but not between 10 and 17C. R. rostrifrons did not show any significant differences in respiration between temperatures. 19

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20 Starting oxygen concentrations (100% vers us 15 20% saturati on) only affected S. subtenuis at higher temperatures (17C). S. subtenuis had significantly lower respiration rates (about a factor of 2/3) in the low oxygen treatment compared to that in the 100% air saturation experiment. Slightly lowe r rates also were observed for the low oxygen treatment at 23C, but this difference was not significant. The few experiments carried out at approximately 5% saturation showed significantly lower oxygen consum ption rates (0.83, 1.04, 1.06 nmol O 2 (mg WW) -1 hr -1 ) for E. inermis at 10C versus rates measured at 15-20% and 100% saturation (median of 1.71 nmol O 2 (mg WW) -1 hr -1 with 25 th to 75 th quartile ranges of 1.28 to 2.27). R. rostrifrons whose sole oxygen consumption measurement at 5% saturation was 1.62 nmol O 2 (mg WW) -1 hr -1 showed a substantially lower rate at 5% saturation than those obtained at the higher oxygen concen trations (median value of 6.06 O 2 nmol (mg WW) -1 hr -1 with 25 th to 75 th quartile ranges of 3.75 to 6.59) Due to high mortality of S. subtenuis (Chapter 4), no measurements were su ccessfully obtained for this species. Interannual variability (assessed at 10C) only was observed for E. inermis where 2008-2009 rates were significantly higher than those in 2007. No differences in respiration rates were observed between E. inermis collected from the upper 50 m and those collected between 200-300 m when experiments having the same temperature and oxygen saturation levels were compared. Ammonium Excretion Similar to oxygen consumption rates, ammonium excretion rates were largely controlled by temper ature and species (Table 2.2). S. subtenuis had significantly higher rates at bot h 10 and 17C treatments than E. inermis and R.

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21 Table 2.2. Ammonium and urea excretion rates and percent ur ea of total nitrogen excret ion for eucalanoid copepods. Notations described in Table 1. Excre tion rates are in nm ol urea or ammonium Species NH 4 + Excretion (nmol (mg WW) -1 hr -1 ) NH 4 + Excretion (nmol (mg DW) -1 hr -1 ) Urea Excretion (nmol (mg WW) -1 hr -1 ) Urea Excretion (nmol (mg DW) -1 hr -1 ) % Urea N (nmol Urea-N (nmol Total N) -1 ) E. inermis 10C High O 2 Low O 2 2007 2008 17C High O 2 Low O 2 23C 0.23 (0.16-0.34) 42 NS NS 0.34 (0.26-0.49) 16 0.19 (0.14-0.31) 26 0.40 (0.32-0.60) 9 NS NS 0.77 (0.60-1.00) 4 3.9 (2.7-5.6) 42 NS NS 5.7 (4.8-8.2) 16 3.1 (2.2-4.9) 26 6.6 (5.1-9.4) 9 NS NS 14.4 (10.2-19.2) 4 0.03 (0.00-0.06) 39 0.00 (0.00-0.02) 20 0.06 (0.03-0.08) 19 NS NS 0.01 (0.00-0.06) 9 0.06 (0.04-0.09) 4 0.00 (0.00-0.00) 5 0.01 1 0.4 (0.1-1.1) 39 0.1 (0.0-0.2) 20 0.9 (0.4-1.3) 19 NS NS 0.1 (0.0-0.1) 9 1.0 (0.7-1.5) 4 0.0 (0.0-0.0) 5 0.2 1 14.8 (2.1-39.0) 38 2.4 (0.0-12.9) 20 34.7 (20.6-40.6) 18 NS NS 1.9 (0.0-17.5) 9 19.9 (13.6-30.1) 4 0.0 (0.0-0.0) 5 2.4 1 R. rostrifrons 10C High O 2 Low O 2 17C 0.21 (0.00-0.43) 9 NS NS 0.29 (0.23-0.35) 2 1.3 (0.0-3.5) 9 NS NS 2.3 (1.9-28) 2 0.19 (0.01-0.72) 5 0.01 (0.00-0.01) 2 0.72 (0.46-1.96) 3 0.08 (0.04-0.12) 2 1.9 (0.1-4.1) 5 0.1 (0.0-0.1) 2 4.1 (3.0-12.3) 3 0.7 (0.3-1.0) 2 77.5 (5.6-100.0) 5 2.8 (1.4-4.2) 2 100.0 (88.8-100.0) 3 22.3 (11.1-33.4) 2 S. subtenuis 10C 17C High O 2 Low O 2 23C 0.94 (0.66-1.22) 22 0.99 (0.77-1.30 ) 11 NS NS 3.16 (2.37-3.40) 7 8.0 (4.4-10.5) 22 7.8 (5.1-9.7) 11 NS NS 26.1 (20.3-30.8) 7 0.29 (0.19-0.45) 19 0.27 (0.11-0.53) 11 0.40 (0.29-0.57) 6 0.08 (0.00-0.14) 5 0.30 (0.28-0.30) 3 2.0 (1.3-3.7) 19 2.0 (0.7-3.9) 11 3.0 (2.1-4.2) 6 0.6 (0.0-0.8) 5 2.7 (2.5-2.8) 3 43.9 (34.2-62.6) 19 39.6 (14.9-50.0) 11 42.4 (39.7-52.7) 6 13.0 (0.0-22.0) 5 14.4 (13.0-15.4) 3 S. pileatus 23C 5.37 (5.14-5.88) 3 48.6 (46.9-53.9) 3 1.20 (1.05-2.13) 3 11.1 (9.6-19.7) 3 32.9 (28.9-40.9) 3

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rostrifrons R. rostrifrons had the lowest excretion ra tes. In the 23C treatment, S. pileatus, S. subtenuis, and E. inermis were all significantly different from each other, with S. pileatus having the highest rate and E. inermis the lowest. Temperature strongly influenced ammonium excretion in E. inermis. Excretion rates at 10C were significantly lower than at the other two temperatures. S. subtenuis excretion rates also signifi cantly decreased at lower temp eratures. No temperature variation was observed for R. rostrifrons Initial oxygen concentration did not appear to affect ammonium excretion rates for any species at any temperature. In terannual variability only was observed in E. inermis, where excretion rates in 2007 were higher than those in 2008-2009. However, when only shallow data were considered, no interannual variation was seen. Deep E. inermis collected in 2008-2009 had a bout half the ammonium excretion rate of shallow individuals collected in the same year (median: 0.16 v. 0.31 nmol (mg WW) -1 hr -1 ). While this difference itself is not statistica lly significant, it likely contributed to the interannual variability observed. Urea Excretion Species was an important factor a ffecting urea excretion rates (Table 2.2). At 10C, S. subtenuis had significantly higher rates than either R. rostrifrons or E. inermis. However, at 17C, only S. subtenuis and E. inermis were significantly different from each other. S. subtenuis urea rates were an order of ma gnitude higher than that of E. inermis at 23C; however, these rates could not be tested for significant differences owing to the limited number of experiments. 22

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Unlike many of the other metabolic para meters, urea excretion rates were not influenced by temperature. Urea excretion, however, was affected by an interaction of starting oxygen concentration and temperature, although not in a consistent manner. For example, both R. rostrifrons and E. inermis had higher excretion rates in lower oxygen concentrations at low temperature (10C). In contrast, S. subtenuis and E. inermis had a 5-6 fold decrease in rates when exposed to lower oxygen concentrations at intermediate temperatures (17C). There did not appear to be any effect of year or the depth of individuals collected on urea excretion rates. Percent Urea-Nitrogen Excretion This parameter quantified the proportion of total nitrogen (N) excreted by an organism that was in the form of urea. Species differences only were observed at 10C a nd 23C (Table 2.2). At 10C, S. subtenuis had significantly higher values than E. inermis R. rostrifrons rates were too variable (25 th -75 th percentile range of 5.6-100%) to be significantly different from the other species. S. pileatus had significantly higher % urea-N excretion rates at 23C than S. subtenuis While the median value of % urea-N excreted increased with decreasing temperature, the overall trend was not statis tically significant owing to the considerable variation in rates. As with urea excretion rates, R. rostrifrons and E. inermis excreted a higher % of urea-N in low oxygen treatments at the low temperat ure (10C) (Figure 2.2). At 17C, E. inermis and S. subtenuis also excreted a higher % of urea-N in the high oxygen treatment. There did not appear to be any interannual variability for any species or variability between shallow and deep E. inermis 23

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0 10 20 30 40 50 60 70 80 10 17 23Temperature (C)% Urea N Total High Oxygen Low Oxygen 19 9 10 11 6 5 3 3 (a) 0 5 10 15 20 25 30 35 40 45 10 17 23Temperature (C)% Urea N Total High Oxygen Low Oxygen 38 20 18 9 4 5 1 1 (b) Figure 2.2: Percent urea-nitrogen for (a) Subeucalanus subtenuis and (b) Eucalanus inermis. Columns mark the median value and error bars denote 25 th -75 th quartiles. Values above error bars ar e the number of replicates. 24

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Total Nitrogen Excretion Total nitrogen excretion rates were influenced by species. At 10C, E. inermis had significantly lower excretion rates than R. rostrifrons and S. subtenuis (Table 2.3). In the 17C treatment, S. subtenuis had a significantly higher excretion rate than the other two. No differences were seen at 23C. Temperature only appeared to influence S. subtenuis total N excretion rates. Here the 23C treatment had significantly higher rates than 17 or 10C treatments. Oxygen level did not influence total N excretion rates. However, some interannual variability was seen within E. inermis Similar to ammonium excretion rates, 2007 rates were higher than 2008-2009 rates. Depth also was a significant factor governing total nitrogen excret ion, with deeper living E. inermis having significantly lower rates than the shallow living i ndividuals. Since most of the deep E. inermis individuals were collected during 2008-2009, this also helped to explain the observed interannual variabilit y. When shallow E. inermis were compared between the two years, no significant differe nce was detected. Percent Nitrogen Body Turnover The percent daily turnover of body nitrogen was determined by comparing the total nitrogen ex cretion rate (Table 2.3) to the average amount of body nitrogen (Chapter 4, see Table 2.4). S. subtenuis had the highest protein content, % body N, and % body phosphorus, whereas E. inermis had the lowest. When total nitrogen excretion rates were norma lized to body nitrogen, no differences between species were observed at any of the temperatures. Temperature differences, however, were still observed within some species. S. subtenuis had significantly higher turnover rate s at 23C, compared to the lower 25

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26 temperatures E. inermis also had the highest body turnove r rate at 23C, but since there was only one experiment, no statistical significance could be ascertained. R. rostrifrons showed no temperature variability. Table 2.3. Total nitrogen excretion rates and daily percent body nitrogen turnover for eucalanoid copepods. Notation described in Table 1 Species Total N Excretion (nmol (mg WW) -1 hr -1 ) Total N Excretion (nmol (mg DW) -1 hr -1 ) % N Turnover (nmol Total N (nmol Body N) -1 day -1 ) E. inermis 10C 2007 Shallow Deep 2008 Shallow Deep 17C 23C 0.34 (0.21-0.43) 38 0.43 (0.34-0.53) 14 0.41 (0.34-0.51) 13 2.05 1 0.28 (0.18-0.37) 24 0.33 (0.31-0.39) 8 0.20 (0.16-0.37) 16 0.51 (0.25-0.66) 9 0.90 1 5.4 (3.4-7.2) 38 7.9 (5.6-8.5) 14 7.7 (5.6-8.4) 13 31.1 1 4.6 (2.9-5.8) 24 5.2 (4.8-6.1) 8 3.2 (2.6-5.6) 16 8.6 (5.4-11.4) 9 17.8 1 3.6 (2.2-4.6) 38 4.5 (3.6-5.6) 14 4.4 (3.3-5.2) 13 20.6 1 3.2 (1.8-3.7) 24 3.8 (3.5-4.4) 8 2.0 (1.6-3.7) 16 5.9 (2.9-7.5) 9 9.6 1 R. rostrifrons 10C 17C 0.54 (0.45-1.87) 5 0.46 (0.32-0.60) 2 3.8 (3.7-10.7) 5 3.7 (2.5-4.8) 2 2.1 (1.8-7.3) 5 1.8 (1.2-2.3) 2 S. subtenuis 10C 17C 23C 1.35 (1.08-2.25) 19 1.23 (1.06-2.49) 11 4.13 (3.96-4.31) 3 10.0 (8.2-18.9) 19 9.1 (7.7-18.6) 11 37.5 (36.4-38.3) 3 3.3 (2.8-6.0) 19 3.0 (2.6-6.1) 11 11.0 (10.5-11.5) 3 S. pileatus 23C 7.31 (7.23-9.90) 3 67.4 (66.1-91.6) 3 ND

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27 Table 2.4. Adult female copepod weight and body compos ition for eucalanoid species. Weights are in mg per individual. Percent water, nitrogen, protein, a nd phosphorus are in terms of wet weight. Data are means standard deviation (number of replicates). ND (not determined) means no data was available Species Wet Weight (mg) Dry Weight (mg) % Water % Nitrogen 1 % Protein 1 % Phosphorus 1 E. inermis 5.41.79 (100) 0.33.06 (100) 93.90.5 (100) 0.320.04 (34) 1.630.27 (51) 0.0270.007 (59) R. rostrifrons 0.690.05 (20) 0.090.02 (20) 86.72.3 (20) 0.810.07 (5) 3.230.57 (14) 0.0480.013 (18) S. subtenuis 0.94.11 (52) 0.12.02 (52) 87.2.4 (52) 1.34 0.13 (12) 7.04.48 (32) 0.1250.019 (33) S. pileatus 0.290.01 (3) 0.030.00 (3) 89.10.1 (3) ND ND ND 1 Body composition from copepods collected at the same time as the metabolic experimental animals (Chapter 4)

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No effect of oxygen level was determined for any species or temperature. Body N turnover in E. inermis was interannually variable with 2008-2009 having lower turnover rates. A significant e ffect of depth of collection al so was observed, with deeper E. inermis individuals having lower turnover rates than shallower individuals. Comparisons of shallow individuals from both years showed no significant differences. Therefore, interannual variability in E. inermis was primarily due to differences in turnover rate with depth rather than between years, as deep individuals were mainly sampled in 2008-2009. Phosphate Excretion Species was a significant factor influencing the variability of phosphate excretion rates (Tab le 2.5). In general, S. subtenuis had the highest excretion rate, while E inermis had a significantly lower rate. No species differences were detected at 17C. Within species, temperature significantly influenced phosphate excretion rates for E. inermis and S. subtenuis. E. inermis had significantly higher excretion rates at 23C than at 10C. S. subtenuis also had significantly higher rates at 23 and 17C compared to that at 10C. No temperature effect was seen for R. rostrifrons No interannual or depth variabilit y was observed. The only oxygen level differences observed were for E. inermis at 10C, where the low oxygen group had significantly higher ex cretion rates than the high oxygen group. 28

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Table 2.5. Phosphate excretion rate s and daily percent body phosphorus turnover for each eucalanoid species. Notation described in Table 1 Species PO 4 3Excretion (nmol (mg WW) -1 hr -1 ) PO 4 3Excretion (nmol (mg DW) -1 hr -1 ) %P Turnover (nmol PO 4 3P (nmol Body P) -1 day -1 ) E. inermis 10C High O 2 Low O 2 17C 23C 0.006 (0.001-0.011) 36 0.001 (0.000-0.007) 16 0.009 (0.002-0.015) 20 0.053 (0.027-0.082) 3 0.041 (0.036-0.052) 4 0.09 (0.01-0.19) 36 0.02 (0.00-0.11) 16 0.15 (0.04-0.24) 20 0.93 (0.47-1.23) 3 0.77 (0.61-1.00) 4 1.4 (0.2-2.8) 36 0.3 (0.1-1.8) 16 2.5 (0.7-4.5) 20 18.0 (9.0-27.7) 3 9.3 (8.1-11.8) 4 R. rostrifrons 10C 17C 0.06 (0.03-0.09) 8 0.48 1 0.4 (0.2-0.8) 8 3.8 1 8.7 (4.6-13.3) 8 68.8 1 S. subtenuis 10C 17C 23C 0.08 (0.02-0.12) 16 0.17 (0.12-0.23) 10 0.39 (0.25-0.42) 7 0.6 (0.2-1.0) 16 1.3 (0.8-1.6) 10 3.5 (2.0-3.9) 7 4.2 (0.4-7.3) 16 9.5 (7.0-13.0) 10 24.1 (15.8-26.4) 7 S. pileatus 23C 0.23 (0.21-0.35) 3 2.2 (1.9-3.2) 3 ND Percent Phosphorus Body Turnover The percent daily tu rnover of body phosphorus (P) was determined by comparing the daily phosphat e excretion rate rela tive to total body P (Table 2.5). Species differences only were onl y observed at 10C. At this temperature, E. inermis had a significantly lower turnover rate than R. rostrifrons and S. subtenuis No differences were observed at 17 or 23C. Both E. inermis and S. subtenuis had significantly higher turnover rates at 23C than at 10C. There was no effect of temperature for R. rostrifrons No depth or interannual variability was observed. Similar to phosphate excretion rates, E. inermis phosphorus body turnover rates were higher in the low oxygen treatments at 10C. This trend was detected primarily for the deep E. inermis individuals. Metabolic Ratios Due to the large variation in the metabolic ratios, very few significant differences were seen for a given ratio (Table 2.6). Statistical test s were done using the 29

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30 O:N and N:P ratios that were ge nerated from the total N excretion data, as this was likely the most accurate representation of these ratios. O:N ratios showed no significan t effect between different temperatures or species, although R. rostrifrons appeared to have slightly higher ratios than S. subtenuis or E. inermis. Interannual variab ility was observed for E. inermis with 2008-2009 having higher ratios than 2007. Both shallow and deep E. inermis likely contributed towards this trend, with both groups having somewh at higher values in 2008-2009 than 2007. The N:P ratio at 10C was influenced by species. E. inermis had significantly higher N:P ratios than either S. subtenuis or R. rostrifrons Temperature did not seem to be a major factor affecting N:P ratios within species, with S. subtenuis showing the only significant difference between 10 and 17C tr eatment groups. No interannual or depth variability was observed. O:P ratios did not show many significant differences, likely due to the large variability in these values. Species differences only were observed at 23C, where S. pileatus showed a significantly higher ratio than S. subtenuis No effect of temperature, year, or depth was observed. Respiratory quotients (RQs), or the mola r ratio of carbon dioxide produced to oxygen consumed, were calculated for E. inermis collected from deep water and run in two experiments at 10C and full oxygen sa turation. The RQ values were 0.85 and 0.77 (Table 2.7).

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31 Table 2.6. Metabolic ratios for eucalanoid co pepods. Notation described in Table 1 O:N N:P Species NH 4 + N Only Total N NH 4 + N Only Total N O:P E. inermis 10C 2007 2008 17C 23C 13.6 (8.6-23.5) 42 8.2 (2.6-12.1) 16 21.6 (14.2-33.1) 26 11.5 (7.9-18.3) 9 16.0 (13.4-19.1) 4 11.1 (7.3-20.2) 38 5.6 (1.7-7.8) 14 14.2 (10.7-22.8) 24 9.0 (7.8-14.0) 9 15.8 1 46.9 (15.4-98.4) 27 NS NS 6.1 (5.8-351.4) 3 17.0 (16.8-17.9) 4 64.3 (27.6-107.3) 26 NS NS 13.0 (6.6-354.9) 3 20.9 1 433.3 (198.7-1870.1) 29 NS NS 116.9 (59.8-18692.2) 3 297.9 (226.0-362.8) 4 R. rostrifrons 10C 17C 30.7 (22.3-42.7) 5 51.6 (47.0-56.2) 2 22.3 (9.6-29.0) 5 42.2 (32.8-51.5) 2 8.2 (3.2-10.2) 5 0.4 1 4.7 (2.7-13.6) 4 0.4 1 177.6 (108.6-291.3) 8 22.2 1 S. subtenuis 10C 17C 23C 22.6 (9.1-27.0) 20 16.9 (15.2-23.3) 11 11.3 (9.6-13.8) 7 12.7 (4.3-16.6) 19 11.5 (9.1-19.1) 11 8.0 (6.2-8.7) 3 11.5 (8.3-13.5) 11 4.5 (4.0-7.3) 9 8.5 (7.5-9.4) 7 16.8 (11.3-26.6) 11 8.4 (4.5-9.3) 9 9.6 (9.6-10.1) 3 206.7 (93.1-305.0) 12 107.1 (56.3-120.1) 8 90.0 (78.3-127.8) 7 S. pileatus 23C 17.9 (14.7-21.7) 3 13.5 (9.6-15.3) 3 27.4 (19.4-27.6) 3 41.2 (28.2-47.4) 3 312.2 (258.8-508.9) 3

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32 Table 2.7. Respiratory quotient data for Eucalanus inermis. Summary of E. inermis metabolic rates from 2008/2009 used to calculate the respiratory quotient (RQ) and to determine the percent substrate (lipid, carbohydrate, or protein) used during metabolism based on the equations of Lauff and Wood (1996). Units for metabolic rates are in nmol (mg WW) -1 hr -1 ND (not determined) indicates missing data Experiment 1 Experiment 2 O 2 Consumption 2.19 2.12 CO 2 Production 1.85 1.64 Total N Excretion 0.06 0.03 PO 4 3Excretion 0.001 ND RQ 0.85 0.77 % Lipid 49 77 % Carbohydrate 41 18 % Protein 10 5 O:N 75.8 152.3 N:P 50.3 ND O:P 3815.5 ND Q 10 Ratios Q 10 ratios represent the factor of a rates increase over a 10 degree Celsius temperature range, indicating the sensitivity of certain rates to temperature changes (Hochachka & Somero 2002). Q 10 values were generally in the 1.5-2.5 range, except for phosphate excretion rates, which were approxi mately 3-4 (Table 2.8). Values generally were similar between 2007 and 2008-2009, with the exception of oxygen consumption and phosphate excretion rates for E. inermis Oxygen consumption rates for this species had a higher Q 10 in 2007 that 2008-2009 (4.10 v. 1.26), while phosphate excretion rates had a lower Q 10 (2.94 v. 3.99). No values are reported for R. rostrifrons as no significant temperature differences existed. Urea excretio n rates also were not examined, as there was no significant temperature vari ation within any species.

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33 Table 2.8. Q 10 ratios for Eucalanus inermis and Subeucalanus subtenuis. Metabolic rates from 2007 ( 10 v. 23C) and 2008-2009 (10 v. 17C) E. inermis S. subtenuis 10 v. 17C 10 v. 23C 10 v. 17C 10 v. 23C O 2 Consumption 1.26 4.10 1.69 2.12 NH 4 + Excretion 2.20 2.03 1.43 2.04 Total N Excretion 1.54 1.75 1.35 1.21 PO 4 3Excretion 3.99 2.94 3.20 3.49

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34 DISCUSSION Species Influences on Metabolic Rates Eucalanus inermis had significantly lower weight-specific oxygen consumption and amm onium, urea, and phosphate excretion rates than Subeucalanus subtenuis, particularly at 10C, althoug h this trend between species also occurred at 17 and 23C. This was not surprising, as E. inermis had been reported to be a jelly-bodied copepod, having meta bolic rates and a body composition per wet mass that were more similar to gelatinous plankton than calanoid copepods (Flint et al. 1991). This was supported by percent water meas urements taken of the individuals used for incubations (Table 2.4). E. inermis had a percent water content of about 94% WW, while the other three species contained about 87-89% water. Water content for S. subtenuis, Rhincalanus rostrifrons, and S. pileatus was more typical for crustacean plankton, while E. inermis had water content more similar to chaetognaths, ctenophores, and gelatinous medusae (Beers 1966, Child ress & Nygaard 1974, Bailey et al. 1995). Rates generated by this study were similar to those reported by Flint et al. (1991). Flint et al. (1991) found an average oxygen c onsumption rate of approximately 2.5 nmol mg WW -1 hr -1 at 15-16C, while this studys rate was about 2.4 nmol mg WW -1 hr -1 at 17C. These rates were even more consistent when considering that wet weights of copepods in Flint et al. were generated usi ng a general equation for conversion of length and width measurements to WW (Kuzmichova 1985). Comparisons of measured WWs of copepods from this study with those estimated by the ge neral equation demonstrated that Kuzmichovas equation underestimates WW of E. inermis, S. subtenuis and R. rostrifrons on average by 11, 25, and 34%, respectively. This suggests that Kuzmichovas equation is not generally ap plicable to members of the family

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35 Eucalanidae. When taking into account that WW of E. inermis was probably underestimated by 10% in Flint et al. (1991), th eir average rate drops to slightly lower than the rate measured at 17C in this study. E. inermis oxygen consumption rates also compared well with that reported by Dagg et al. (1980). Both rates are about 43-44 nmol (mg DW) -1 hr -1 at 17C. Dagg et al. (1980) also measured nitrogen excretion rates of E. inermis from the Peru Current, which were substantially high er than those obtained by this study (27% versus 6-10% body N daily). This was mos tly due to differences in the body nitrogen contents of E. inermis, as levels in this study were 2-2.5 times higher than Dagg et al. (1980) reported. Additionally, these authors measured amine excretion, which was about 5-15% of total N excretion. After corr ecting for amine excr etion and body content differences between the studi es, the rates of ammonium and urea excretion found by Dagg et al. (1980) appeared to be similar to this study per unit wet or dry weight. Nitrogen excretion rates have also been investigated for S. pileatus from the Atlantic (Gardener & Paffenhfer 1982). Gardener and Pa ffenhfer measured rates at 20C of approximately 30 nmol ammonium excret ed [mg ash-free dry weight (AFDW)] -1 hr -1 When converted to AFDW, my rates were cl oser to 55-60 nmol ammonium [mg ash-free dry weight (AFDW)] -1 hr -1 at 23C. These differences may partially be due to temperature, or perhaps reflect variati on in metabolism between individuals from different locations or seasons. Unfortunate ly, no metabolic rates were available for comparison for S. subtenuis or R. rostrifrons R. rostrifrons rates appeared to be intermediate between E. inermis and S. subtenuis. For oxygen consumption and phosphate excretion, R. rostrifrons rates at both

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36 10 and 17C were more similar to that of S. subtenuis (higher). Ammonium, urea, and total N excretion rates, on the other hand, tended to be more similar to E. inermis rates (lower). Higher oxygen consumption and phosphate excretion rates relative to nitrogen excretion were consistent with a tendency toward lipid catabolism, which was supported by the metabolic ratios. The median O:N ra tio was higher than the other species while the N:P ratio was lower, again suggesting more of a relative reliance on lipid catabolism (Mayzaud & Conover 1988). This was not surp rising, given that there were extremely large lipid sacs present in individual R. rostrifrons from the incubation experiments that were sometimes large enough to fill the entire body cavity. While the other species also had visible lipid sacs, they were much smaller in relation to body cavity volume. E. inermis and S. subtenuis tended more towards protein catabolism than R. rostrifrons. Unfortunately R. rostrifrons was not nearly as abundant as E. inermis and S. subtenuis and, therefore, fewer experiment al replicates were completed. A larger data set would be interesting to aid in further examination of the differences among these three species. Given that R. rostrifrons resides almost exclusively at depth (Figure 2.1), where oxygen levels are much lower than the low oxygen treatments, it is noteworthy that its observed oxygen consumption rate was similar to that of an active surface species ( S. subtenuis ). One possible explanation for this was the observed variability in behavior and activity rates observed be tween species. These experiments were conducted in the dark with no food present; therefore, the rate measurements are representative of routine metabolic rates, which include minimal activity. During sorting, S. subtenuis and S. pileatus were by far the most active. R. rostrifrons, on the other hand, was the least active. Many times, it was difficult to tell if these individuals were even alive.

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37 Sometimes, little to no escape response was e licited by gentle touching with forceps and a heartbeat was the only indicat or of life. Thus, while both species had similar rates when there was minimal activity and no feeding, R. rostrifrons likely continued this low activity lifestyle in situ giving this species an advantage in oxygen-limited environments. This was supported by its low protein content relative to S. subtenuis (Table 2.4), indicating less muscle mass per weight. E. inermis also appeared to have adapted to a low-oxygen environment by having little activel y metabolizing tissue for its size. Compared to the other species of this study, the activity level of E. inermis was probably intermediate. Percent urea-N excreted was variable among species; S. subtenuis excreted the highest percentage at 10C, while the maximal rates at 23C were seen in S. pileatus. Overall levels for E. inermis were variable, ranging fr om 0-63% across the three temperatures, with median values of 2-15%. This was consistent w ith Dagg et al. (1980), who found average urea excretion rates of 8-25% of total N excretion for E. inermis. Overall, rates for the four species were comp arable with previously reported values. In general, % urea-N rates were highly variable in the literatu re, with averages between 560% for tropical to temperate copepods (E ppley et al. 1973, Smith & Whitledge 1977, Mitamura & Saijo 1980, Steinberg et al. 2002, Miller & Roman 2008, Saba et al. 2009). Even though nitrogen and phosphate excre tion rates showed species differences, the daily percent N and P body turnover showed little variation. The only difference observed was for E. inermis at 10C, which had lower % P body turnover rates than the other species. This suggests that even t hough rates are variable in terms of body mass,

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38 once they are standardized to elemental co mposition, there is little functional metabolic difference between species. Temperature Temperature was a major factor in almost all of the measured rates (oxygen consumption, ammonium excretion, phos phate excretion, total N excretion, %N and %P turnover). Th e highest temperature (23C) ha d the highest metabolic rates, which was expected from previous stud ies (e.g., Barber & Bl ake 1985, Donnelly & Torres 1988, Ikeda et al. 2001). Although th e 23C and 10C treatment groups were almost always significantly different from each other, the 17C treatment group was not always significantly different fr om either 23 or 10C. Therefore, temperature differences of 6-7C were not always large enough to se e significant temperatur e effects for these species. Q 10 ratios were primarily in the 1.5-2.5 range similar to previously determined values for crustacean metabolic rates (e.g., Aarset & Aun aas 1990, Mauchline 1998, Irwin et al. 2007). Phosphate excretion rates for both E. inermis and S. subtenuis had Q 10 ratios in the 3-4 range, suggesting highe r sensitivity of phosphate excretion to temperature changes (Childress 1977). In contrast, urea excretion rates did not vary significantly with temperature for any species, and median values did not show any trend. One other study using prawns showed that urea excretion ra tes may either increase or decrease with temperature (Quarmby 1985), depending on the sex of the prawn. Thus, there does not appear to a standard response of urea excr etion rate to temperature.

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39 Since urea excretion rates were inde pendent of temperature and ammonium excretion rates increased with temperature, the % urea-N values tended to decrease as temperature increased. However, this trend wa s not statistically significant, likely due to the large variation in % urea-N within tr eatment groups. Decreasing % urea-N as temperature increased was also seen in th e spot prawn, but only for large males and females (Quarmby 1985). Small males showed the opposite trend, suggesting again that there is not a standard effect of te mperature on % urea-N within Crustacea. Metabolic ratios also did not appear to be influenced by temperature, indicating that substrate usage was not temperature de pendent. This was consistent with most previous work comparing O:N, N:P and O:P ratios and temp erature (Ikeda 1985, Ikeda et al. 2001). O:N ratios, which are suggested to be the most useful in discerning protein versus lipid and carbohydrate usage (Ikeda 1977, Ikeda & Skjoldal 1989), suggested that protein catabolism was most dominant for all four species. However, O:N ratios for R rostrifrons were slightly higher than the other sp ecies, indicating more lipid utilization than the E. inermis, S. subtenuis and S. pileatus This was not surprising, given the large storage lipid stores observed in R. rostrifrons in this region (Chapter 4). RQ values and O:N ratios for E. inermis during the two CO 2 production experiments indicated that lipid catabolism accounted for about ha lf to two-thirds of the s ubstrate catabolized. This deviated from the average O:N values observe d during the other experimental replicates, which were substantially lower and indicated almost entirely protein catabolism (11.1 versus 75-150) (Mayzaud & C onover 1988). These differen ces may represent natural variability in substrate catabolism found within a species. Howe ver, within the CO 2 production experiments, RQ and O:N ratio s showed similar results, suggesting a

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40 promising future for the us e of the on-board MICA CO 2 system for metabolism work. Accurate CO 2 production measurements are difficult to attain, but would be useful for improved understanding of metabolic processes. Oxygen Level At the concentrations tested, oxyge n level did not appear to influence many metabolic parameters. At higher temperatures, S. subtenuis had lower oxygen consumption rates in the lo w oxygen than high oxygen treatme nt (although this was only significant at 17C). This suggest ed that at lower temperatures, S. subtenuis did not consume oxygen at a high enough rate to be affected by the low oxygen treatment. However, as temperature increased and routine metabolic rates increased, oxygen started to become limiting. This was interesting, as the in-situ condition of 17C and 15-20% air saturation fell near the chlorophyll/fluorescence maximum in this region (Daly, unpublished data; Figure 2.1), which was the approximate depth of maximum abundance for S. subtenuis. Thus, this species appeared to be functioning at a depth where they may be slightly stressed metabolically. Add itionally, based on their high mortality during several 5% saturation treatments, it seems unlikely that S. subtenuis would be able to function at depths much below the chlorophyll maximum. Neither E. inermis nor R. rostrifrons showed differences in oxygen consumption rates between the low and high oxygen treatmen ts at any temperature, suggesting that oxygen was not limiting for them at 15-20% air saturation. However, both species showed decreases in oxygen consumption rate s at 5% air saturation based on the few replicates carried out at this oxygen level. This indicates that the critical partial pressure of oxygen (or the minimum concentration of o xygen in which an organism can maintain

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41 its normal oxygen consumption rate (With ers 1992)) occurs between 5 and 15% air saturation, and suggests that metabolic stress is present for these organisms at oxygen concentrations of 5% air sa turation. Such concentrations are present in the lower oxycline, the area of maximum abundance for these species in the water column, and therefore suggests that these two species, like S. subtenuis may undergo slight metabolic stress in their preferred habitat. In addition to oxygen consumption rates, urea excretion rates and % urea-N were the only other parameters that showed signi ficant differences with oxygen level (Figure 2.2). Here, lower temperatures (10C) and decreased environmental oxygen led to an increase in rates for E. inermis and R. rostrifrons while at higher temperatures (17C), E. inermis and S. subtenuis showed the opposite trend. This was particularly interesting, given that within high oxygen treatments, urea ex cretion rates seem to be insensitive to temperature. The formation of urea as an excretory product in crustaceans happens through two different pathways (Claybrook 1983). In the fi rst pathway, the enzyme arginase catalyzes the reaction of the amino acid arginine to or nithine and urea. A second pathway involves the formation of uric acid and subsequently urea from breakdown of purines. Once urea is formed, urease can catalyze the reaction fo r full breakdown into ammonia. Oxygen is only directly involved in one step of the purine catabolism pathway. Changing temperature or oxygen could affect the urea output in two different ways. Firstly, it could signal to the organism to change the substr ates that are being catabolized. A second mechanism would be upor down-regulating the activity or amount of some of the enzymes involved in these pathways, such as arginase or urease. Like most aquatic

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42 invertebrates, marine copepods do not thermo regulate (Willmer et al. 2005), thus cellular temperature largely mirrors environmental te mperature. Enzyme kinetics tells us to expect different rates of reaction at diffe rent temperatures (Weiner 2006), although without testing there is no way to ascertain where maxi mum or minimum rates might occur. While activity of these enzymes is largely unstudied in crustaceans, it has been found that urease is inhibited in denitrifying soil bacteria under high oxygen conditions (Ruan et al. 2009). Additionally, work on rat liver cells has found up -regulation of gene expression in regions coding for arginase I and other enzymes when cellular oxygen levels are increased (Miralle s et al. 2000). This suggests th at upor down-regulation of activity or expression of re levant enzymes in response to different oxygen environments is possible in crustaceans. Studying enzyme kinetics or gene expression levels while varying temperature, oxygen, salinity, etc. would help us better understand how these factors might be controlling urea and ammonia formation pathways. It would also be useful to examine the relative amounts of urea and ammonia produc tion during arginine and purine catabolism, to see what percentage actually utilizes the urease for complete breakdown to ammonia under different treatments. Implications of Oxygen and Temperature Interactions for Nitrogen Cycling in the ETNP Differences in nitrogen excretion products with temperature and environmental oxygen can be important when looking at nitrogen cycling in a study region. For instance, at the ETNP sites, the chlorophyll maximum (usually around 25-50 m depth) often occurred at oxygen levels of 30% air saturation (40-100 M) and temperatures of 15-20C. Community nitrogen excretion rates for E. inermis, S. subtenuis and R. rostrifrons under

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43 conditions of 17C and 20% oxygen satura tion can be estimated using MOCNESS abundance data (data courtesy of K. Wishner and D. Outram). Total nitrogen excretion for these species combined is about 1,266 nmol N m -3 day -1 and, of that, urea-N accounts for about 9%. Had these estimates been made using rates at 100% air saturation, the total N excretion would not change (as there was not a significa nt difference between oxygen levels), but urea-N would account for 35%. As certain types of plankton have different abilities to uptake and/or utilize the various forms of inorganic and organic N (Glibert & Terlizzi 1999, Berg et al. 2003, Soloman et al. 2010), such distinctions are necessary for more accurate modeling and calculations. Thes e results also suggest that estimating N excretion at the base of the upper oxycline (temperatures of 7-12C, oxygen concentrations of 1-10 M (<3% air saturation) is potentially inaccurate using data obtained at 100% air saturation. It cannot be ascertained from this study whether % ureaN would continue to increa se as oxygen levels decrease d, but it seems likely that estimates at full oxygen saturation would not be representative of actual conditions. OMZs are areas of complex nitrogen cyc ling, and it is especially important to understand the sources and cycling of the vari ous forms of nitrogen in these regions. Due to the near anoxic conditions encountered in OMZs, anaerobic nitrogen pathways, including denitrificat ion and anammox (anaerobic amm onium oxidation) are prominent features. Via these pathways, OMZ regions ar e thought to contribute up to 50% of total nitrogen lost from the oceans to the atmosphere (Gruber & Sarmiento 1997, Codispoti et al. 2001). Anammox, a recently discovered path way (Mulder et al. 1995, van de Graaf et al. 1995), combines ammonium and nitrite to produce nitrogen gas (N 2 ) and is believed to be the principal anaerobic pathway in some OMZ systems, including the nearby Peruvian

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44 OMZ (Kuypers et al. 2005, Thamdrup et al. 2006, Hamersley et al. 2007). Anammox bacteria cell abundance is often highes t around the upper boundary of the OMZ core, shortly after oxygen concentrations reach <10 M (Thamdrup et al. 2006, Hamersley et al. 2007, Galn et al. 2009). This also appears to be the case in our study site, as ladderane lipids, which are unique to anammox bacteria (Jetten et al. 2009), were found in the upper OMZ during our 2007 cruise (S. Wakeham, personal communication). Heterotrophic organisms living at depth, like zooplankton, could provide a source of ammonium for this pathway. As OMZs are expanding in our oceans (Stramma et al. 2008, Stramma et al. 2010), it is important to study nitrogen excretion mechanisms in zooplankton to be able to more fully understa nd and predict their role in nitrogen cycling within OMZ systems. Year The interannual variation observed for oxygen consumption rates in shallow-living E. inermis was potentially due to two factorsthe first of which was seasonal differences. As this is a tropical system little seasonal variability was expected. However, wind-driven upwelling in this area influences productiv ity, and wind strength and location can vary throughout the year (Kes sler 2006). While our cruises were close in timing (October-November versus Decembe r-January), some differences in mean annual chlorophyll levels and zooplankton abundances exist between October and January (Fernndez-lamo & Frber-Lorda 2006, Pennington et al. 2006). Seasonal effects on oxygen consumption have been seen in several invertebrates, including ascidians (Hatcher 1991), krill (Torre s et al. 1994), and copepods (Conover 1959,

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45 Conover & Gustavson 1999), although the kri ll and copepod experiments were conducted at high latitudes. A second possibility was the influence of El Nino-Southern Ocean (ENSO) events. Both cruise years coincided with La Nia eventsa strong event was seen in 2007 and a weaker one in 2008. La Nia events have genera lly been associated with higher productivity in the eastern Pacific, rela ted to cooler temperat ures due to a shoaling of the thermocline (Ryan et al. 2006, Kang et al. 2008, Saba et al. 2008). Increased food availability would likely lead to higher rates of oxygen consumption (Gaudy 1974, Bohrer & Lampert 1988), which is the opposit e of what was observed. Differences would more likely be related to temperature, in which the strong La Nia event would produce cooler temperatures and lower meta bolic rates (Barber & Blake 1985, Donnelly & Torres 1988, Ikeda et al. 2001). Depth The major difference observed between E. inermis collected at different depths was lower nitrogen excretion rates in deeper-dwe lling individuals, lead ing to significantly decreased daily % body N turnover. Such ra tes suggested decrease d protein utilization and increased lipid catabolism. This findi ng was supported by highe r O:N ratios in deep individuals during 2008-2009 (median value of 20.0; shallow individuals had a median value of 11.1). Although this difference was not significant (p = 0.06), this result suggested that deeper living adult females ma y utilize a different metabolic strategy at depth. Abundance profiles of ad ult females in this region indicated that a resident population likely existed at depth (L onghurst 1985, Chen 1986, Saltzman & Wishner 1997b). Because particles at depth in the ETN P had little nutritional value (Chapter 4), it

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46 appeared that any feeding occurring in the OMZ may have been supplemented with lipid catabolism. Biomarker studies also suggested that E. inermis females collected in shallow regions and at depth shared a primary food source f ound in the chlorophyll maximum region (Chapter 3). Although feed ing opportunities at depth may be more limited than in near-surface waters, the lo w-oxygen waters within the OMZ afford protection from predators that cannot tolerate the low-oxygen conditions It is unclear whether adult females move between depths on a regular (although not strictly daily) basis, or if there is a certain trigger for ontogenetic migration associated with a life history stage. More work is needed on E. inermis to determine the reasons for their interesting distribution. Conclusions As expected, temperature and species had the greatest overall effect on metabolic rates. In general, increases in te mperature tended to increase metabolic rates. Urea excretion rates were the sole exception, where no trend with temperature observed. S. subtenuis and S. pileatus, which concentrated in the upper 100 m, had the highest metabolic rates, while E. inermis which was found throughout the water column to about 600 m, typically had the lo west metabolic rates. S. subtenuis was an active species, having high protein content, i ndicative of large amounts of mu scle tissue. This species also had decreased oxygen consumption rate s at low oxygen levels and intermediate temperatures (17C), demonstr ating potential oxygen limitati on as shallow as 30 m in the ETNP. This suggested that the vertical distribution of S. subtenuis was likely constrained due to oxygen concentrations in the water column. In contrast, E. inermis had particularly high water and lo w organic content, resultin g in a reduced amount of

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47 respiring tissue for its size, potentially c ontributing to its tolerance for lower oxygen waters. R. rostrifrons which was found predominantly in the upper oxycline, had an intermediate metabolic strategy between S. subtenuis and E. inermis R. rostrifrons had typical water content for a copepod, but appeared to compensate for a limited oxygen supply through decreased activit y. Large lipid reserves cont ributed towards its metabolic needs. When excretion rates were reexam ined as daily body N or P turnover rates, species differences largely disappeared, indicating that there was not necessarily a functional metabolic difference among these species. As S. subtenuis, S. pileatus and R. rostrifrons are circumtropical and subtropical species, it would be interesting to see if the results from this study hold for individuals found in areas without such severe oxygen limita tions. It has been suggested that some characteristics observed in organisms i nhabiting oceanic low oxygen regions are not necessarily adaptations speci fically for life at low oxygen, but rather general taxonomic features that allow them to exploit such a lif estyle (Childress & Se ibel 1998). A regional comparison would help to illuminate whether these copepods respond to low oxygen in this manner due to adaptation, or genetic pre-disposition. One of the most interesting new fi ndings of this study was the relationship between temperature, oxygen leve l, and nitrogen excretion in these species (Figure 2.2). At 10C, low oxygen led to an increase in the amount of urea nitrogen produced relative to ammonium nitrogen in E. inermis and R. rostrifrons. The opposite trend was true at 17C for E. inermis and S. subtenuis. While the general pathwa ys of urea and ammonia production in crustaceans are know n, the mechanisms that regul ate the relative amount of each produced are woefully understudied. Othe r studies have investigated relationships

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48 between urea, ammonium, and amino acid ex cretion with factor s like food source, species, temperature, and life stage (Dagg et al. 1980, Mitamura & Saijo 1980, Conover & Cota 1985, Quarmby 1985, Miller & Roman 2008, Saba et al. 2009). Their findings illustrated that nitrogen excretion is complex and variable among individuals and species. It is particularly important to understand the factors influe ncing nitrogen cycles in OMZ regions. It is thought that such regions contribute up to 50% of total nitrogen lost from the oceans to the atmosphere (Gruber & Sa rmiento 1997, Codispoti et al. 2001), primarily through denitrification and anammox pathwa ys. Our oceans are currently seeing decreases in oxygen concentra tions, increases in temperatures, and expansion of OMZ systems (Keeling & Garcia 2002, Emerson et al. 2004, Stramma et al. 2008, Stramma et al. 2010). Thus, it is particularly important to understand the relationships between temperature, oxygen levels, and zooplankton excr etory products. Future work in this area should include more examination of enzyme levels to better understand how pathways themselves are affected, not just the end products.

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49 CHAPTER THREE Lipid Biomarkers of Eucalanoid Cope pods in the Eastern Tropical North Pacific: Evidence for Differential Use of Wax Ester and Triacylglycerol Storage Lipids INTRODUCTION The presence or relative amount of certain types of lipids, such as specific fatty acids, sterols or hydrocarbons, can be used to determine the source of material in marine ecosystems (Parrish et al. 2000, Dalsgaard et al. 2003, Loh et al. 2008). These biomarker lipids have been used in zooplankton studies to investigate food sources, diet, and trophic position (e.g., Harvey et al. 1987, Graeve et al. 1994a, Graeve et al. 1994b, Hagen et al. 1995, Pond et al. 1995, Albers et al. 1996, Falk-Petersen et al 1999, Falk-Petersen et al. 2002, Peters et al. 2004, Stevens et al. 2004, Br ett et al. 2006, Escrib ano & Prez 2010). Storage lipids are most useful when looking for biomarkers, as those lipid classes generally incorporate dietary fatty acids mo re directly than phospholipids, which are found in cellular membranes and are highly regu lated to maintain ce llular function (Lee et al. 2006). The two major types of storage lipids found in copepods are wax esters (WEs) and triacylglycerols (TAGs). Generally, copepods at high latitude or depth accumulate larger amounts of storage lipids and, of thos e storage lipids, a greater proportion are WEs (Lee et al. 1971a, Lee & Hirota 1973). Add itionally, copepods that undergo diapause

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50 tend to accumulate WEs, which provide energy st ores during diapause (Lee et al. 2006). WEs probably are a better long-term storage lipid, as they usually are mobilized only after TAG depletion during starvation (Lee et al. 1974, Lee & Barnes 1975, Sargent et al. 1977, Hkanson 1984). Two different lipases are likely responsible for mobilization of WEs and TAGs, with TAGs able to be quick ly hydrolyzed for immediate energy needs (Lee et al. 2006). WE catabolism is thought to occur via a hormone-sensitive lipase, perhaps associated with specific life events (preparation for reproduction, diapause, etc.) (Sargent & Henderson 1986). Stor age lipids, particularly WEs, appear to be formed from a combination of direct incor poration of dietary fatty acids incorporation of modified dietary fatty acids, and de novo biosynthesis of fatty acids an d alcohols (Sargent & FalkPetersen 1988, Graeve et al. 1994a, Kattner & Hagen 1995, Gr aeve et al. 2005). As high latitude copepods generally have the highest lipid content as well as the largest proportion of storage lipid, the vast majo rity of copepod biomarker studies have examined high latitude or temperate species. To our knowledge, there are no published papers on copepod biomarkers and lipid profil es in equatorial systems (latitudes lower than 20), and only a few have examined cope pod lipids in detail in latitudes lower than 40 (Lee et al. 1971a, Lee & Hirota 1973, H kanson 1984, Lavaniegos & Lpez-Corts 1997, Saito & Kotani 2000, Sommer et al. 2002, Schnack-Schiel et al. 2008, Escribano & Prez 2010). Members of the copepod family Eucalanidae (genera: Rhincalanus, Eucalanus, Subeucalanus and Pareucalanus) occur throughout the worlds oceans (Grice 1962, Lang 1965, Bradford-Grieve et al. 1999, Goetze 2003). These species, including those present at low latitudes, often have visible storage lipi d sacs (Lee & Hirota 1973, Lee et al.

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51 2006), with total lipid content of 6-69% of dry weight, of which storage lipids usually comprise >40% of total lipids (Lee et al. 1971a, Lee & Hirota 1973, Lee 1974, Morris & Hopkins 1983, Flint et al. 1991, Ohman 1997, Schna ck-Schiel et al. 2008). It is not known why these copepods accumulate such large amounts of li pids, although some eucalanoid species in highly seasonal environm ents have been found to undergo diapause or seasonal dormancy (Kasyi 2006, Schnack-Sch iel et al. 2008). The amount of WEs and TAGs accumulated are variable, and seem to depend on the genus. Rhincalanus spp. consistently show predominantly WE accumu lation (Lee et al. 1971a, Lee & Hirota 1973, Ohman 1988, Graeve et al. 1994a, Kattner et al. 1994, Kattner & Hagen 1995, Sommer et al. 2002, Schnack-Schiel et al. 2008). Eucalanus spp. are more variable, but tend towards TAG accumulation (Lee & Hirota 1973, Lee 1974, Ohman 1988, Saito & Kotani 2000). To our knowledge, no storage lipid pa tterns have been recorded for Subeucalanus or Pareucalanus spp., although some of the unknown Eucalanus spp. identified in Lee and Hirota (1973), may be members of thes e genera, as taxonomic revision of the Eucalanus genus into Eucalanus, Subeucalanus and Pareucalanus genera occurred after that paper (Geletin 1976). Members of all four genera of Eucalan idae occur in the eastern tropical north Pacific (ETNP) (Chen 1986, Sameoto 1986, Vi nogradov et al. 1991, Saltzman & Wishner 1997b). This region is characterized by a severe oxygen minimum zone (OMZ), with dissolved oxygen concentrations of less than 4.5 M often re ported, and values below 0.5 M sometimes observed (Brinton 1979, Same oto 1986, Levin et al. 1991, Vinogradov et al. 1991, Saltzman & Wishner 1997a). The core of the OMZ (the area of lowest oxygen levels) can occur anywhere between appr oximately 300 and 1,000 m with an overall

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52 thickness of the OMZ between 200 to over 1,00 0 m (Fiedler & Talley 2006). Copepods, including the abundant Eucalanidae family, have varied and distinct vertical distributions, likely related to the oxygen environmen t (Chen 1986, Sameoto 1986, Vinogradov et al. 1991, Saltzman & Wishner 1997b). Eucalanus inermis, a species endemic to the ETNP, is found throughout the water column, but has higher abundances at depths associated with the surface chlorophyll maximum and th e upper and lower edges of the OMZ core. Subeucalanus subtenuis, S. subcrassus and Pareucalanus attenuatus are usually concentrated in the shallow euphotic zone. Rhincalanus rostrifrons and R. nasutus on the other hand, have only low to moderate abundances in the upper water column, and instead are concentrated above and below the OMZ core. This suggests that a variety of ecological strategies occur within this famil y, of which different f eeding strategies might be included. Little is known about diet and feeding in eucalanoid copepods. Examination of feeding appendages suggested that many ar e herbivores (Kasyi 2006), although lipid analyses of the high-latitude R. gigas has suggested that this species is an omnivore (Graeve et al. 1994a). Studies of the low-latitude S. pileatus showed that fecal pellet ingestion was comparable to the rate of phytoplankton consumption (Paffenhfer & Knowles 1979). Feeding experiments in the ETNP indicated that adult female E. inermis ingested a wide range of prey, including copepod nauplii, diatoms, heterotrophic dinoflagellates, and ciliates (M. B. Olson and K. Daly, unpublished data ). More work is needed to understand feeding strategies of copepods in th is group. Given the diversity, high abundance, relatively large size (2-6 mm ), variable ecologica l niches, and storage

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53 lipid capacity of eucalanoid copepods of the ETNP, this group re presents a unique opportunity to study lipid biomarkers and f eeding strategies in tropical copepods. The purpose of this study was tw o-fold. Feeding preferences of E. inermis, S. subtenuis, P. at tenuatus, R. nasutus and R. rostrifrons in the ETNP were determined based on a comparison of lipid biomarkers in available food and in copepods. Particulate material was collected at depths of maximum concentration of copepods to identify the available food. Adult E. inermis were investigated in greater detail as it was such a numerically abundant species in this system with a unique vertical distribution. TAGs and WEs were evaluated separately to assess differential incorpora tion of dietary fatty acids between the two storage lipid types. In addition, this study examined differences in lipid classes among and within species. Fatty acid signatures were compared between the two storage lipid classes (TAGs and WEs) as well as phospholipids. Additional information about sterol composition and WE fa tty alcohols is also provided. This is the first comprehensive examination of all lipid classes in tropical copepods. This study investigated the relationships between feeding choices, stor age lipid type, taxonomy, and collection location in determining the com position of the various lipid classes.

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54 METHODS Copepod Collection and Measurement Copepods were collected during two cruises to the eastern tropical north P acific (ETNP) during 18 October 17 November, 2007 aboard the R/V Seward Johnson and 8 December 2008 6 January 2009 aboard the R/V Knorr. The cruise transect ran between two major stations: the Costa Rica Dome (9N, 90W) and the Tehantepec Bowl (13N, 105W) (F igure 3.1). Lipid samples were primarily collected at the Costa Rica Dome using bongo tows, Tucker trawls, and MOCNESS (Multiple Opening/Closing Net and Envir onmental Sampling System) (Wiebe et al. 1976) tows in the upper 300 m of the water co lumn. Copepods were collected from their respective depths of maximum abundance. Adult female Subeucalanus subtenuis and Pareucalanus attenuatus were targeted in the upper 50 m, while Rhincalanus rostrifrons and R. nasustus were primarily collected in the 200-300 m range. Eucalanus inermis adult females were collected at both depths (d esignated as shallow a nd deep individuals, respectively) and adult males from near-surface waters. Due to vari ations in abundance and distribution between years, adult female R. nasutus were only coll ected in 2007 and adult female P. attenuatus were only collected in 2008. Immediately after capture, copepods were sorted and individuals of each species were separated into small containers containing 0.2 m filtered seawater at in situ temperature. Copepods were kept at in situ temperatures for approximately 3-12 hours to allow them to empty their guts. All individuals were frozen in cryovials at -80C until measur ements were taken. Prior to lipid extraction, i ndividuals were thawed and quickly measured for total length and prosome length (in mm). Copepods were then grouped into batches of 24-85 individuals and refrozen at -80C until lipid extraction occurred. Wet weights (WWs) (in

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mg) were estimated using length-weight equations derived from measurements on additional individuals collected on both cruises (Chapter 4). Dry weights (DWs) were estimated by conversion from WWs based on the average percent water of each species at each location (Chapter 4). 0 30N 20N 10N 80W 10S 110W 90W 100W 130W 140W 150W 120W Figure 3.1. Map of eastern tropi cal north Pacific sampling site s. Black dots represent the two main sampling regions: the Costa Rica Do me (9N, 90W) and the Tehantepec Bowl (13N, 105W), with the cruise transect drawn between them. Map modified from SWFSC NOAA website (http://swfsc.noaa.gov/). Particulate Matter Collection. Particulates were collected during both cruises at the Costa Rica Dome site. During 2007, water was co llected using Niskin bottles on a CTD Rosette at the chlorophyll maximum (35 m), and layers of high Eucalanus and Rhincalanus abundance (260 and 325 m). 9-30 l wa s pre-filtered from each depth 55

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56 through a 200 m mesh screen to remove large copepods. Subsequently, water was divided evenly between three samples (lipid s, carbon/nitrogen/hydroge n, and protein) and particles were accumulated on a precombuste d GF/F filter for each sample. In 2008, samples for carbon/nitrogen/hydrogen, phosphorus and protein were collected at 29, 275 and 535 m (3-10 l per sample), which corre sponded to the chlorophyll maximum, upper oxycline and lower oxycline, resp ectively. The two deepest de pths also coincided with layers of abundant Eucalanus. Particulates for lipid analyses were collected using a McLane WTS-LV in situ filtration system at 28, 264 a nd 540 m depth. Approximately 2,000 l of water was filtered at each depth, and a 53 m mesh screen was used as a prefilter. Lipid samples were analyzed fr om a subsample of the total lipid extract obtained from particles collected on a double layer GF/F filter array. Particulate Composition. Carbon/nitrogen/hydrogen anal yses on particulate samples were run by the University of California, Sant a Barbara Marine Science Institute Analytic Laboratory. Phosphorus samples were analyzed following the methods of Solorzano and Sharp (1980). Protein conten ts were assessed using the Lowry method (Lowry et al. 1951). Lipid Extraction and Analysis. Lipids were extracted by homogenizing copepods or filters in 2:1 dichloromethane (DCM):methanol (MeOH) using a tissue grinder. Liquid was then transferred to a capped centrifuge tu be containing a few ml of salt water, and shaken. The DCM layer was removed, more DC M was added to the centrifuge tube, and then the process was repeated several times. A total lipid extract was obtained by drying

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57 the extracted DCM with anhydrous sodium su lfate and evaporating the sample using a rotary evaporator setup. The 2008 McLane pump samples were Soxhlet-extracted using 9:1 v/v DCM:MeOH for 8 hours. Extracted lipids were then partitioned into DCM and dried using anhydrous sodium su lfate. Lipid samples proce ssed in this study were a 5% split of total lipids extracted for each filter from the McLane system. Separation of lipid classes was attained with silica columns using 5% deactivated silica gel (Merck silica ge l 60, 70-230 mesh; Wakeham & Volkman 1991). Five of the resulting fractions were utilizedWEs, TAGs, free fatty alcohols and sterols, free fatty acids (FFAs), and phospholipids (PLs) (listed in order of extraction). For this study, the sterol fraction contained both steroid alcoho ls and steroid ketones, which were grouped together. The WE, TAG, FFA and PL fractions were saponifi ed by heating the sample to 100C for two hours with 0.5 N KOH in MeOH. Neutral fractions (fatty alcohols and sterols) were removed first with hexane. The remaining solution was then acidified (pH < 2) and hexane used again to recover the acidic fraction (fatty acids). The fatty acids were converted to fatty acid methyl esters (FAMEs) by addition of diazomethane. The neutral WE fraction and the free fatty alcohol and sterol fraction were converted to trimethylsilyl-ethers (TMS-ethers) using BSTFA (N, O-bis(trimethylsilyl)trifluoroacetamide) and pyridine. Samples were run on a GC (Agilent 6890 gas chromatograph with an FID detector) or GC/MS (Agilent 6890 gas chro matograph coupled to an Agilent 5793 mass spectrometer). FAME fractions were r un on a RTX-WAX column, while TMS ethers were analyzed using a DB-XLB column. Inte rnal standards of me thylnonadecanoate for FAMEs and 5-cholestane for the TMS ethers were added to each sample prior to

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58 injection on the GC. Identif ication of compounds was ac complished using mass spectra and retention times. The mass of each comp ound was converted to moles, and molar percentages were reported unless otherwise not ed. Total lipid mass and percent mass of each lipid class was calculated by summing iden tified lipid compounds in all or relevant fractions. Biomarkers. Lipid biomarkers (summarized in Parrish et al. 2000, Dalsgaard et al. 2003, Loh et al. 2008) were used to investigate copepod diet and pa rticulate material sources. The bacterial contribution to fatty acids was assessed by summing 15 and 17 carbon fatty acids with all branched fatty acids. Diat om input was examined through the fatty acid 20:5(n-3) and the unique diatom marker 16:4( n-1). The ratios 20:5( n-3)/22:6(n-3) having values >1, 16:1/16:0 values >1.6, and C16/C 18 values >2 indicate diatom dominance in the system. Dinoflagellate contribution wa s evaluated using 18:4( n-3), 18:5(n-3) and 22:6(n-3) fatty acids. Ratio values of 20:5(n-3)/22:6(n-3) <1, and high values for C16/C18 and 18:5(n-3)/18:3(n-3) ratios in dicated dinoflagellate dominance. A microzooplankton biomarker 20:4(n-6) also was examined. Sterol markers (names as in Table 3.1) were also used to assess part iculate matter sources. The presence of cholesterol in particulate material wa s indicative of zooplankton input, while 22dehydrocholesterol, bras sicasterol, and 24-methylenecholes terol indicated a contribution by diatoms. Dinoflagellate mark ers included dinosterol and 5 (H)-stanols. Statistics. Cluster analyses were performed using PRIMER 6 to examine relative similarity between samples. Resemblance matr ices for fatty acid or sterol profiles of samples were generated using S17 Bray Curtis similarity test (Bray & Curtis 1957).

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59 Table 3.1. Shorthand, full, and common names of sterol compounds Sterol Shorthand Full Compound Name Common Compound Name C 27 -nor5,22 27-nor-24-methylcholesta-5,22E-dien-3 -ol Occelasterol C 27 5,22 cholesta-5,22E-dien-3 -ol 22-dehydrocholesterol C 27 22 5 (H)-cholest-22E-dien-3 -ol 22-dehydrocholestanol C 27 5 cholest-5-en-3 -ol Cholesterol C 27 0 5 (H)-cholestan-3 -ol Cholestanol C 28 5,22 24-methylcholesta-5,22E-dien-3 -ol Brassicasterol C 28 22 24-methyl-5 (H)-cholest-22E-en-3 -ol Brassicastanol C 28 5,24(28) 24-methylcholesta-5,24(28)-dien-3 -ol 24-methylenecholesterol C 28 24(28) 24-methylcholest-24(28)-en-3 -ol 24-methylenecholestanol 23,24-dimethyl-C 29 5,22 23,24-dimethylcholesta-5,22E-dien-3 -ol dimethyldehydrocholesterol 23,24-dimethyl-C 29 5 23,24-dimethylcholest-5-en-3 -ol dimethylcholesterol C 29 5 24-ethylcholest-5-en-3 -ol Sitosterol C 29 0 24-ethyl-5 (H)-cholestan-3 -ol Sitostanol C 30 22 4,23,24-trimethyl-5 (H)-cholest-22E-en-3 -ol Dinosterol

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60 RESULTS Particulate Matter Co mposition and Lipids. Particulate matter (<200 m) in both years had the highest concentrations in near-surface waters, with lower values seen in deeper waters (Table 3.2). Levels of carbon, nitr ogen, hydrogen, and protein were slightly higher at depth in 2007. At the chlor ophyll maximum (38 and 28 m), overall total material (mg particulates) and hydrogen conten t tended to be higher in 2007 than in 2008, while carbon, nitrogen, and protein concentratio ns were lower. No direct comparisons could be made in overall lipid content, as only the <53 m fraction was examined in 2008, while all particles <200 m were examined in 2007. Table 3.2. Particulate material at the Costa Rica Dome site. Concentrations of different components are in g/l. Lipid concentrations are reported fo r three components: fatty acid (FA), fatty alcohol (FAlc) and sterols. Additionally, percentages of each lipid class (by weight) are described: wax ester (WE), triacylglycerol (TAG) free fatty alcohol (FFAlc), free fatty acid (FFA) and phosph olipid (PL). Besides lipid samples collected in 2008 (<53 m fraction), all concentrations reflect the <200 m particulate fraction 2007 2008 38 m 260 m 325 m 28 m 264 m 540 m Total Material (g/l) 3524.0 1031.3 976.7 2508.8 978.9 974.1 Carbon (g/l) 55.80 13.27 11.85 95.69 10.21 9.47 Nitrogen (g/l) 12.78 2.08 2.16 19.10 1.74 1.25 Hydrogen (g/l) 50.32 15.55 14.53 36.81 12.21 11.37 Phosphorus (g/l) ND ND ND 2.09 0.06 0.12 Protein (g/l) 35.26 11.39 10.79 77.09 5.18 7.88 Lipid (g/l) 1.71 0.52 0.37 0.20 0.02 0.01 Total FA (g/l) 1.44 0.40 0.31 0.15 0.01 0.01 Total FAlc (g/l) 0.03 0.05 0.01 0.00 0.00 0.00 Total Sterol (g/l) 0.24 0.07 0.05 0.04 0.01 0.00 % WE 5.0 11.2 9.0 0.7 4.8 7.2 % TAG 16.0 10.5 8.5 16.0 41.2 20.6 % FFAlc 0.7 0.3 1.6 1.3 0.2 0.7 % Sterol 14.1 12.6 12.2 20.9 32.7 32.5 % FFA 52.8 61.4 63.0 53.6 12.3 22.8 % PL 11.4 3.9 5.8 7.4 8.8 16.2

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61 Lipids in particulate material were dominated by fatty acids (67-85%) (sum of fatty acids in the WE, TAG, FFA and PL fractio ns) in both years and at all depths (Table 3.2). Sterols occupied the next highest pr oportion (12-32%), while fatty alcohols (from the WE and free fatty alcohol fractions) made up the smallest compone nt at 1-10%. 2007 samples had slightly higher fatty acid cont ents and lower sterol content (78-85% and 1214%, respectively) than 2008 (66-78% and 20-33%, respectively) Of the lipid classes, free fatty acids were generally highest (1263%), possibly resulting from the hydrolysis of other fatty acid containing lipids in the particulate ma terial. Other lipid classes included TAGs (10-42%), sterols (12-33%), PLs (4-16%), WEs (1-11%), and free alcohols (0-2%). Cluster analysis revealed close pairing (>75% similarity) between three different groups of particulate total fa tty acid profiles: the two ch lorophyll maxima samples (38 and 28 m), the 2007 deep samples (260 and 325 m) and the 2008 deep samples (264 and 540 m) (Figure 3.2). The 2008 deep samples were more similar to the chlorophyll maxima samples (about 65% similar), while the 2007 deep samples were only 50% similar to the other groups. Major fatty acids for the chlorophyll maxima group included 16:0 (24-32%), 14:0 (14-16%), 16:1 (9-12%), 20:6(n-3) (5-11%), 18:1 (4-9%), 18:0 (38%) and 20:5(n-3) (3-4%) (Table 3.3). Th e 2008 deep samples contained mainly 16:0 (25-28%), 18:0 (9-19%), 18:1 (8-17%), 22:1 (3-9%), 22:6(n-3) (5-7%), 15/17 branched fatty acids (3-5%), and 20:5( n-3) (2-4%). Deep 2007 samp les were dominated by 18:0 (40-52%), 16:0 (29-36%), 18:1 (6-10%) and 16:1 (2-3%). Some fatty acids decreased proportionally with depth in both years, especially the polyunsaturated fatty acids (PUFA), n-3, and 14 carbon fatty acids (Table 3.4). In

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62Samples R. nasutus (2007)-TAG R. nasutus (2007)-WE P. attenuatus (2008)-WE 260 m (2007) 325 m (2007) S. subtenuis (2008)-WE E. inermis M (2007)-WE E. inermis D (2008)-WE E. inermis S (2008)-WE E. inermis S (2007)-WE E. inermis M (2008)-WE R. rostrifrons (2007)-TAG R. rostrifrons (2008)-TAG R. rostrifrons (2007)-WE R. rostrifrons (2008)-WE S. subtenuis (2007)-WE 264 m (2008) 540 m (2008) E. inermis D (2007)-WE 38 m (2007) 28 m (2008) E. inermis S (2007)-TAG E. inermis M (2008)-TAG S. subtenuis (2007)-TAG E. inermis D (2007)-TAG S. subtenuis (2008)-TAG E. inermis M (2007)-TAG P. attenuatus (2008)-TAG E. inermis S (2008)-TAG E. inermis D (2008)-TAG 1 0 09 08 07 06 05 04 03 0 Similarit y Resemblance: S17 Bray Curtis similarity Figure 3.2. Cluster analysis comparing tota l fatty acid profiles for particulate materi al and storage lipi d fatty acid profile s for copepods. Species or depth of sample and ye ar are noted. Storage lipid type is denoted as wax este r (WE) or triacylglycerol ( TAG). For Eucalanus inermis samples are divided into M (adult male), S (adult fe male residing in shallow portion of the water column) and D (adult female residi ng in deeper water).

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63 Table 3.3. Total fatty acid, alcohol, and sterol profiles for particulate samples. Values are in percent molar. Sterol names are in Table 3.1 2007 2008 Fatty Acids: 38 m 260 m 325 m 28 m 264 m 540 m 12:0 0.6 0.3 0.5 0.1 0.5 0.6 13:0 0.1 0.4 0.0 0.4 0.6 0.2 14:0 14.8 0.9 1.7 15.2 6.2 2.4 14:1 1.6 0.3 0.3 0.3 0.3 0.0 15+17 Branched 1.8 1.6 2.0 1.5 3.8 4.3 15:0 1.3 0.8 0.8 0.9 1.6 0.9 16:0 24.0 29.2 35.2 31.9 27.7 25.9 16:1 11.5 2.6 3.1 9.5 9.6 7.4 16:2 4.9 0.0 0.0 0.7 0.4 0.0 16:4(n-3) 0.0 0.0 0.0 0.9 0.0 0.0 17:0 1.1 1.0 1.2 0.3 0.2 0.0 17:1 0.4 0.4 0.4 0.7 2.1 3.3 18:0 7.5 51.6 40.2 3.7 9.1 18.6 18:1 4.8 6.5 9.2 8.9 16.9 8.3 18:2 1.7 0.8 1.1 1.5 1.3 1.6 18:3(n-3) 1.8 0.1 0.1 0.7 0.6 0.0 18:4(n-3) 7.5 0.0 0.1 2.1 0.8 0.2 18:5(n-3) 0.0 0.0 0.0 1.0 0.0 0.0 20:0 2.6 0.5 0.6 2.5 0.8 0.9 20:1 0.0 0.0 0.2 0.2 1.5 1.8 20:4(n-6) 0.0 0.0 0.0 0.4 0.6 0.3 20:5(n-3) 3.5 0.4 0.5 3.6 3.7 2.8 22:0 0.2 0.4 0.1 0.7 0.8 2.3 22:1 1.3 0.3 0.5 0.4 3.9 8.7 22:6(n-3) 5.5 1.4 1.3 10.7 5.1 6.3 24:0 0.0 0.0 0.0 0.0 0.0 0.9 Even C Branched 1.2 0.5 0.5 0.2 1.9 2.0 Other: 0.7 0.1 0.2 0.8 0.2 0.2 Alcohols: 12:0 0.0 0.0 0.0 0.0 32.9 39.7 14:0 41.8 0.0 15.6 0.0 0.0 0.0 15:0 0.0 0.0 0.0 0.0 0.0 9.7 16:0 9.4 0.0 6.4 2.2 40.6 0.0 16:1 0.0 0.0 0.0 0.0 26.5 0.0 17:1 0.0 0.0 0.0 0.0 0.0 0.0 17:2 0.0 0.0 0.0 0.0 0.0 0.0 18:0 31.8 100.0 78.0 29.6 0.0 0.0 18:1 0.0 0.0 0.0 36.5 0.0 0.0 20:0 0.0 0.0 0.0 0.0 0.0 2.6 22:0 16.9 0.0 0.0 0.0 0.0 47.9 22:1 0.0 0.0 0.0 31.7 0.0 0.0 Sterols: C 27 -nor5,22 0.9 0.9 1.1 1.2 2.7 2.7 C 27 5,22 9.9 6.7 7.2 10.1 13.8 10.2 C 27 22 0.7 0.7 0.7 1.1 2.1 1.8 C 27 5 16.5 55.3 39.5 17.1 22.0 15.6 C 27 0 1.2 0.0 1.6 1.8 3.9 3.2 C 28 5,22 17.3 7.5 7.3 14.5 1.5 1.8 C 28 22 1.1 1.9 2.0 1.7 16.2 16.9 C 28 5,24(28) 0.0 0.0 0.0 0.8 3.3 3.7 24-methylcholest-4-en-3-one 31.0 2.8 0.8 29.4 5.4 4.8 C 28 24(28) 2.9 1.9 2.2 3.8 2.9 2.8 23,24-dimethyl-C 29 5,22 2.8 9.9 0.0 2.9 5.3 7.1 23,24-dimethyl-C 29 5 1.1 0.0 14.4 0.6 1.1 1.7 C 29 5 9.3 5.6 15.2 9.6 7.0 16.9 C 29 0 2.3 1.1 1.6 1.3 9.2 3.9 C 30 22 1.0 0.0 0.0 1.8 1.3 2.2 24-ethylcholest-4-en-3-one 2.1 5.7 6.3 2.3 2.3 4.6

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64 Table 3.4. Summary of particul ate lipids and biomarkers in molar percentages. Fatty acids are classified as saturated (SFA), monounsaturated (MUF A) and polyunsaturated (PUFA) forms. Fatty acids al so are described by the total number of carbons in their chai ns (e.g., C14 refers to all fatty acids with 14 carbons). Bacteria l markers refers to the sum of C15, C17 and branched fatty acids. Sterol shorthand notations are described in Tabl e 3.1. Biomarker ratios are defined in the methods section 2007 2008 38 m 260 m 325 m 28 m 264 m 540 m Fatty acids: SFA 49.6 82.8 78.4 54.3 45.2 51.6 MUFA 19.0 9.6 13.3 19.3 32.1 26.3 PUFA 25.4 2.8 3.3 22.1 12.3 11.1 n-3 18.2 1.8 2.1 19.0 10.1 9.2 n-6 0.0 0.0 0.0 0.5 0.6 0.3 C14 16.4 1.3 2.0 15.6 6.7 2.7 C16 41.0 32.0 38.9 43.3 38.8 35.1 C18 24.3 59.2 50.9 18.0 29.1 28.6 C20 6.1 0.9 1.3 6.9 6.6 5.8 C22 6.9 2.0 2.0 11.9 9.7 17.3 Bacterial Markers: 5.7 4.3 4.9 3.8 9.7 10.6 Diatom Markers: 3.7 0.4 0.6 3.8 3.7 2.8 16:4(n-1) 0.2 0.0 0.1 0.2 0.0 0.0 20:5(n-3) 3.5 0.4 0.5 3.6 3.7 2.8 Dinoflagellate Markers: 13.0 1.4 1.4 12.8 5.9 6.5 18:4(n-3) 7.5 0.0 0.1 2.1 0.8 0.2 18:5(n-3) 0.0 0.0 0.0 1.0 0.0 0.0 22:6(n-3) 5.5 1.4 1.3 10.7 5.1 6.3 Microzooplankton: 20:4(n-6) 0.0 0.0 0.0 0.4 0.6 0.3 Ratios: 20:5(n-3)/22:6(n-3) 0.6 0.3 0.4 0.3 0.7 0.4 16:1/16:0 0.5 0.1 0.1 0.3 0.3 0.3 C16/C18 1.7 0.5 0.8 2.4 1.3 1.2 C18/C22 3.5 29.8 26.1 1.5 3.0 1.7 18:5(n-3)/18:3(n-3) 0 0 0 1.3 0 N/A Sterols: Dinoflagellate Markers: 4.5 1.1 3.2 4.9 14.4 9.3 C 27 0 1.2 0.0 1.6 1.8 3.9 3.2 C 29 0 2.3 1.1 1.6 1.3 9.2 3.9 C 30 22 1.0 0.0 0.0 1.8 1.3 2.2 Diatom Markers: 27.2 14.2 14.5 25.4 18.6 15.7 C 27 5,22 9.9 6.7 7.2 10.1 13.8 10.2 C 28 5,22 17.3 7.5 7.3 14.5 1.5 1.8 C 28 5,24(28) 0.0 0.0 0.0 0.8 3.3 3.7 Zooplankton: C 27 5 16.5 55.3 39.5 17.1 22.0 15.6

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65 contrast, saturated fatty acids (SFA) and monounsaturated fa tty acids (MUFA) showed no clear trend between depths or years. 18 carbon fatty acids increased between near-surface and deeper samples. 16 carbon compounds were generally constant w ith depth. In 2007, a decrease in 20 and 22 carbon fatty acids was seen with depth, but in 2008 no change was seen in 20 carbon fatty acids and slight in crease was seen for 22 carbon fatty acids at the lower oxycline (540 m). Cluster analysis of steroid profiles (inc luding alcohols and ketones) revealed the same groupings as the fatty acid profiles, although the resemblance within the 2007 deep samples was weaker (only 70%) (See Appendix, Figure A1). Also, similarity between the 2008 deep and chlorophyll maxima sample groups was slightly lower than the fatty acids (only 60% similar). Chlorophyll maxima sterol fraction samples were dominated by 24-methylcholest-4-en-3-one (29-31%), ch olesterol (16-17%), brassicasterol (1418%), 22-dehydrocholeste rol (9-10%) and sitosterol (9-1 0%) (Table 3.3) (see Table 3.1 for sterol name descriptions). 2008 deep samples were dominated by cholesterol (1522%), brassicastanol (16-17%), sitosterol (7-17%), 22-dehydroc holesterol (10-14%), sitostanol (4-10%), and 4-methylcholest-4-en-3-one (4-6%). 2007 deep samples were mostly comprised of cholesterol (39-55%), si tosterol (5-16%), dimethylcholesterol (015%), dimethyldehydrocholesterol (0-10%), brassicasterol (7-8%), 22-dehydrocholesterol (6-8%) and 24-ethylcholest-4-en -3-one (5-7%). Overall, fatty alcohol content was very low and saturated alcohols of 14-22 total ca rbons primarily were observed (Table 3.3). Bacterial markers (C15, C17 and branched fatty acids) showed that contributions from bacteria generally were low (3-6% of total fatty acids), except for 2008 deep samples, where levels increased to around 10%. 16:4(n-1) abundances suggested that

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66 diatoms were most abundant near the surface, bu t largely absent at the deeper depths. In 2007, the diatom marker 20:5(n-3) showed the same trend, however, in 2008, 20:5(n-3) was basically constant with depth. Dinof lagellate markers 18:4(n-3) and 22:6(n-3) showed similar trends in both years, with the highest values near surface and lower values at depth. The dinoflagellate biomar ker 18:5(n-3) was largely absent, except for small amounts (1%) in the 2008 chlorophyll ma ximum sample. The 20:5(n-3)/22:6(n-3) ratios indicated that dinoflagellates were mo re dominant at all depths (values of 0.3-0.7), and there was not a consistent trend with depth. 16:1/16:0 and C16/C18 ratios also indicated relatively low abundances of diat oms overall. The 2008 chlorophyll maximum sample was the only one that indicated possible diatom dominance near surface (C16/18>2). 18:5(n-3)/18: 3(n-3) ratios were all low, due to the absence of 18:5(n-3). Another dinoflagellate indicator the C18/C22 ratio, suggested that there were relatively high dinoflagellate abundances in 2007 deep samples (26-30), but much lower amounts near surface waters and 2 008 deep samples (1.5-3.5). In terms of sterol biomarkers, diatom markers showed an overall decrease with depth, mostly seen in brassicasterol. 22dehydrocholesterol and 24-methylenecholesterol were largely unchanged with depth. Dinofla gellate markers overall showed a slight decrease with depth in 2007 and an opposite trend in 2008. The 2007 decrease primarily was observed in dinosterol, whereas in 2008, 5 (H)-stanols increase d. The 2007 deep samples showed a large increase in choleste rol (the zooplankton marker) (39-55% versus 15-22%), while in 2008, cholesterol was largely unchanged with depth.

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67 Copepod Storage Lipids. Total lipid contents varied among species. E. inermis and P. attenuatus had total lipid contents of 0.06-0.16% of WW or 1.0-2.8% of DW by mass (Table 3.5). Total lipids for S. subtenuis were similar, ranging from 0.24-0.26% of WW or 1.9-2.0% of DW. Rhincalanus spp. had higher total lipid, with R. nasutus lipid content of 1.24% of WW and 8.8% of DW. R. rostrifrons total lipids range d from 2.34-3.10% of WW and 17.6-23.3% of DW. Storage lipid (WE and TAG) accumulation pa tterns varied among different genera (Table 3.5). E. inermis and S. subtenuis accumulated primarily TAGs ( 90% of storage lipid, 12-75% of total lipid). The two Rhincalanus species stored almost exclusively WEs (>90% of storage lipids, 85-97% of total lipids). P. attenuatus similarly tended towards WE accumulation (84% of storage lipid, 41% of total lipid), but also incorporated a considerable pr oportion of TAG (16% of storage lipid, 8% of total lipid). Rhincalanus spp. had only small amounts of non-stor age lipid, with sterols (0.6-4.7%), FFAs (1.5-2.6%), PLs (0.4-1.8%) and free fatty alcohols (0.1-1.0%) each comprising <5% of the total lipid. E. inermis, P. attenuatus and S. subtenuis had lower total storage lipid, so other fractions comprised a larger pr oportion of total lipids. Storage lipids (1376%) usually were still the most abundant fo rm of lipids, followed by FFAs (11-54%), sterols (5-49%), PLs (2-30%) and free fatty alcohols (<1.0%). The amount of each type of lipid class was highly variable within sp ecies and years, and no consistent patterns were seen with interannual variability. Generally, samples with lower percentages of storage lipid had higher percen tages of FFAs, likely due to hydrolysis of PL, TAG and WE fatty acids contributing towards the increased amounts of FFAs (Ohman 1996).

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68 Table 3.5. Lipid classes and total li pids as percent of wet and dry weight of copepods. The percent total lipids are mass per mass. Lipid classes are in mass percent of total lipi d. Lipid class notations are the same as in Table 3.2 Eucalanus inermis Shallow Deep Male Pareucalanus attenuatus Rhincalanus rostrifrons Rhincalanus nasutus Subeucalanus subtenuis 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 %WW 0.14 0.06 0.15 0.11 0.16 0.12 0.15 2.34 3.10 1.24 0.24 0.26 %DW 2.2 1.0 2.5 1.8 2.8 2.1 1.4 17.6 23.3 8.8 1.9 2.0 WE 2.1 0.1 0.5 0.4 1.1 0.2 40.9 85.6 96.5 91.6 2.6 1.0 TAG 36.1 66.8 12.6 72.7 75.2 57.7 7.8 5.2 0.5 2.6 22.3 44.3 FFAlc 0.2 0.2 0.6 0.2 0.1 0.4 0.7 1.0 0.1 0.1 1.0 0.4 Sterol 19.7 8.2 48.8 6.2 7. 5 5.6 8.9 4.7 0.6 2.4 12.2 9.6 FFA 32.0 18.2 28.0 12.8 11.8 26.4 36.8 2.6 1.8 1.5 53.6 15.0 PL 10.0 6.5 9.5 8.1 4.3 9.7 5.0 0.9 0.4 1.8 8.4 29.7

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69 Curtis-Bray similarity matrices revealed that WE and TAG fatty acid profiles within the same copepod sample were usually distinct from each other (Figure 3.2). For E. inermis, S. subtenuis and P. attenuatus there was 60% similarity between the the fatty acids of the two lipid classes. R. nasutus had a slightly higher similarity (~70%). R. rostrifrons showed the most consistency (7884%) between the lipid fractions. Therefore, WE and TAG profiles will be discussed separately. Curtis-Bray similarity matrices indicated that TAG lipid profiles divided into three different groups of samples having 75% similarity: R. nasutus, R. rostrifrons, and a final group containing all E. inermis, P. attenuatus, and S. subtenuis samples (Figure 3.2). R. nasutus was characterized by high 18:1 ( 26%), 16:1(18%), 16:0 (11%), 18:0 (10%), and 20:5(n-3) (8%) (Table 3.6). R. rostrifrons TAGs were primarily composed of 16:0 (33-36%) and 14:0 (25-38%), with smal ler amounts of 18:0 (2-9%), 18:1 (2-6%), 20:5(n-3) (2-5%) and 22:6(n-3) (2-5%). E. inermis, P. attenuatus, and S. subtenuis all showed profiles dominated by 16:0 (12-26%), 14:0 (8-22%), 16:1(1116%), 20:5(n-3) (816%), 18:1 (6-14%), and 22:6(n-3) (2-12%). The fatty acid profiles of the WE frac tion were more diverse (Figure 3.2). R. nasutus and P. attenuatus formed one group with about 80% similarity. Their WEs primarily were composed of 16:1 and 18:1 (54-69% combined), with smaller amounts of 20:5(n-3) (5-9%), 18:2 (2-5%), 22:6( n-3) (1-6%) (Table 3.7). The two R. rostrifrons samples formed a second group, with >90% sim ilarity. This cluste r was characterized by high levels of 14:0 and 16:0 (81-85% combin ed) with some 18:0 (2-5%) and 16:1 (23%). The remaining samples were loosely re lated in a group of 60% similarity. This included E. inermis and S. subtenuis, which accumulated WEs as <10% of storage lipids.

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Table 3.6. Triacylglycerol profiles for copepods. Values are in molar percent Eucalanus inermis Shallow Deep Male Pareucalanus attenuatus Rhincalanus rostrifrons Rhincalanus nasutus Subeucalanus subtenuis 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 12:0 0.0 0.2 0.1 0.1 0.1 0.6 0.3 0.2 0.2 0.4 0.1 0.1 13:0 0.0 0.1 0.1 0.1 0.0 0.1 0.2 0.1 0.2 0.0 0.0 0.1 14:0 21.8 12.3 15.9 10.6 8.3 14.4 11.1 25.2 37.5 3.8 16.3 17.3 14:1 0.1 0.1 0.2 0.2 0.2 0.2 0.2 0.1 0.2 0.2 0.1 0.2 15+17 Branched 1.0 2.4 1.8 2.6 1.6 1.6 1.2 0.8 1.3 0.5 0.7 1.8 15:0 0.5 1.6 1.6 1.7 1.1 1.1 1.5 0.9 1.2 0.2 1.0 1.6 16:0 12.9 20.8 23.0 19.1 14.9 25.4 17.4 35.7 33.2 11.2 19.8 23.0 16:1 14.5 12.3 15.9 11.6 13.2 14.6 12.7 3.0 2.8 18.2 14.3 12.2 16:2 1.6 1.1 1.8 1.4 1.8 1.7 1.5 0.9 0.7 1.1 1.5 1.5 16:3(n-4) 1.4 0.6 0.7 0.9 0. 7 1.1 0.4 1.5 0.1 1.3 0.9 1.3 16:4(n-1) 2.2 0.8 1.3 1.1 0. 8 1.7 1.5 2.1 3.0 1.3 1.8 2.1 Phytanic Acid 2.7 1.3 1.4 1.3 2.0 1.1 0.6 2.2 1.0 6.2 1.5 0.4 17:0 0.0 1.5 0.5 1.5 0.0 0.4 2.0 0.0 0.4 0.0 0.6 1.3 17:1 0.2 0.9 0.7 1.2 0.8 0.5 1.5 0.1 0.8 0.3 0.4 1.1 18:0 1.1 2.4 2.1 2.1 2.2 3.4 3.6 8.2 2.7 10.3 1.5 1.4 18:1 6.4 9.8 7.7 8.3 12.8 10.6 13.7 5.3 2.3 26.0 8.1 9.2 18:2 3.5 2.2 2.8 2.8 3.1 2.2 2.1 1.1 0.4 1.8 3.3 2.4 18:3(n-6) 0.3 0.9 0.0 1.0 1. 4 0.6 0.6 0.6 0.0 0.0 0.5 0.6 18:3(n-3) 0.4 1.1 1.5 1.5 2. 0 1.1 1.0 0.1 0.0 0.3 0.8 0.9 18:4(n-3) 1.1 1.6 1.7 2.6 4. 7 1.1 1.4 0.7 1.3 0.7 1.3 2.4 20:0 0.3 0.6 0.5 0.7 0.9 0.5 0.4 0.7 0.3 0.3 0.2 0.2 20:1 0.2 0.4 0.3 0.4 0.4 0.0 1.0 0.2 0.2 0.9 0.3 0.3 20:3(n-6) 0.6 0.4 0.3 0.5 0. 4 0.0 0.7 0.3 0.4 0.5 0.7 0.0 20:4(n-6) 4.9 1.2 0.7 1.1 1. 2 1.0 1.2 1.2 0.5 1.7 2.9 1.4 20:4(n-3) 0.4 0.4 1.0 0.6 0. 5 0.6 0.6 0.3 0.6 0.5 1.0 0.5 20:5(n-3) 15.8 9.5 9.0 12.0 9. 8 9.4 9.2 4.4 2.8 8.1 12.4 8.5 22:4(n-6) 0.5 0.1 0.0 0.1 0. 0 0.1 0.0 0.0 0.0 0.0 0.1 0.0 22:5(n-6) 0.2 0.5 0.0 0.4 0. 5 0.0 0.7 0.0 0.0 0.0 0.2 0.4 22:5(n-3) 0.9 0.8 0.8 0.9 0. 7 0.4 0.8 0.0 0.8 0.0 1.2 0.5 22:6(n-3) 1.9 8.4 5.0 8.4 11. 3 2.7 6.8 2.8 4.1 3.4 4.7 5.6 24:1 0.3 0.3 0.0 0.4 0.5 0.1 0.0 0.0 0.0 0.0 0.0 0.2 Other: 2.2 3.2 1.6 2.8 2. 3 1.8 4.0 1.4 1.0 0.7 1.6 1.6 70

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71 Table 3.7. Wax ester fatty acid profiles fo r copepods. Values are in molar percent Eucalanus inermis Shallow Deep Male Pareucalanus attenuatus Rhincalanus rostrifrons Rhincalanus nasutus Subeucalanus subtenuis 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 12:0 0.9 7.9 0.0 4.6 0.0 4.7 0.1 0.1 0.1 0.1 0.4 4.7 13:0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.0 0.0 0.0 14:0 7.2 9.2 4.5 10.0 10.5 5.4 1.5 42.1 47.7 2.2 6.3 9.9 14:1 0.0 0.0 0.0 0.0 0.0 0.0 0.4 0.3 0.3 0.4 0.0 0.0 15+17 Branched 1.5 4.9 1.4 0.0 0.0 0.0 0.8 1.0 1.4 0.0 6.8 0.0 15:0 1.4 11.7 2.5 6.8 3.7 6.2 0.2 1.2 1.4 0.0 0.1 3.3 16:0 28.0 31.1 18.8 55.8 54.5 28.1 1.2 39.2 36.5 0.7 24.7 49.5 16:1 3.8 0.0 7.5 0.0 0.0 3.3 23.8 2.5 2.3 35.7 5.3 7.2 16:2 0.0 0.0 0.0 0.0 0.0 0.0 2.5 0.5 0.0 3.6 0.0 0.0 16:3(n-4) 0.0 0.0 0.0 0.0 0. 0 0.0 0.8 0.4 0.0 2.7 0.0 0.0 16:4(n-1) 0.0 0.0 0.0 0.0 0. 0 0.0 0.9 0.7 0.4 1.7 0.0 0.0 Phytanic Acid 0.0 0.0 0.5 0.0 0.0 0.0 1.8 1.8 2.1 3.6 0.0 0.0 17:0 1.5 6.0 1.7 0.0 4.3 4.6 0.0 0.0 0.0 0.0 2.5 6.2 17:1 0.0 0.0 0.0 0.0 0.0 2.2 2.2 0.1 0.2 0.3 0.0 0.0 18:0 21.8 3.0 6.0 14.2 19.4 15.4 0.4 4.8 2.7 0.5 17.4 11.3 18:1 20.8 16.2 13.2 3.5 7.6 23.4 29.8 2.0 1.5 33.2 1.9 0.0 18:2 0.0 0.0 0.0 5.1 0.0 0.0 4.9 0.2 0.2 2.4 2.3 0.0 18:3(n-6) 0.0 0.0 0.0 0.0 0. 0 0.0 1.6 0.2 0.0 0.5 8.2 0.0 18:3(n-3) 0.0 0.0 0.8 0.0 0. 0 0.0 1.3 0.0 0.0 0.4 1.5 0.0 18:4(n-3) 0.0 0.0 1.2 0.0 0. 0 0.0 1.4 0.2 0.2 0.8 0.0 0.0 20:0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.4 0.3 0.0 0.8 0.0 20:1 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.7 0.0 0.0 20:3(n-6) 0.0 0.0 0.0 0.0 0. 0 0.0 1.9 0.0 0.0 0.5 0.0 0.0 20:4(n-6) 0.0 0.0 0.0 0.0 0. 0 0.0 2.4 0.0 0.0 1.6 0.0 0.0 20:4(n-3) 0.0 0.0 0.0 0.0 0. 0 0.0 1.0 0.3 0.0 0.4 0.0 0.0 20:5(n-3) 13.1 8.7 20.4 0.0 0. 0 0.0 8.3 0.8 0.6 5.6 7.0 8.0 22:4(n-6) 0.0 0.0 0.0 0.0 0. 0 0.0 0.3 0.0 0.0 0.0 0.0 0.0 22:5(n-6) 0.0 0.0 0.0 0.0 0. 0 0.0 1.3 0.0 0.0 0.0 0.0 0.0 22:5(n-3) 0.0 0.0 0.0 0.0 0. 0 0.0 0.6 0.0 0.0 0.2 0.0 0.0 22:6(n-3) 0.0 0.0 21.5 0.0 0. 0 0.0 5.3 0.7 1.3 1.6 14.6 0.0 24:1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Other: 0.0 1.4 0.0 0.0 0. 0 0.0 0.0 0.5 0.8 0.5 2.2 6.9

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72 Samples in this group had hi gh levels of 16:0 (18-50%) and varying amounts of 18:0 (322%), 18:1 (0-24%), 20:5(n-3) (0-2 1%), and 22:6(n-3) (0-22%). When the total fatty acid profiles for the particulate matter were compared to the TAG fatty acid profiles of the copepods, the two surface samples (28 and 38 m) clustered closely with the E. inermis / P. attenuatus / S. subtenuis group with >70% similarity (Figure 3.2). Overlaying of copepod and particulate profiles showed that the dominant lipid signatures found in the particulates also were detected in the cope pod lipids (Figure 3.3). A comparison of particulate lipids with WE lipids in copepods indicates that no particulate samples were greater than 60% similar to any of th e copepods, but a loose cluster was formed with E. inermis and S. subtenuis. An overview of the relationships between WE, TAG and particulate lipid pr ofiles can be viewed in Figure 3.2. Copepod Phospholipids. The phospholipids fractions were very similar among copepods (Table 3.8). Major fatty acids included 22: 6(n-3) (20-42%), 16: 0 (18-28%), 18:0 (524%), 20:5(n-3) (5-12%) and 18:1 (4-13%). Cluster analyses indicated that phospholipid fractions among copepods showed >75% similar ity, but still divided into two groups that had >80% similarity (See Appendix, Figure A2). R. rostrifrons formed its own group, which was slightly higher in 18:0 (17-24% versus 5-12%) and lower in 22:6(n-3) (2024% versus 22-42%) than the other species. Copepod Fatty Alcohols and Sterol Fractions Pareucalanus and Rhincalanus spp. were the only groups to accumulate WEs as the primary storage lipid (Table 3.5), and thus the only copepods with significan t amounts of alcohols. The other copepod species that did not accumulate WEs as the primary storage lipid had very small amounts of fatty

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Figure 3.3. Comparison of total fatty acid profil es for particulates from the chlorophyll maxima and copepod triacylglycerol fatty ac id profiles for (A) 2007 and (B) 2008-2009. For each graph, the dark line represents the particulates while triacylglycerols of Eucalanus inermis (males and shallow or deep dwelling females), Subeucalanus subtenuis and Pareucalanus attenuatus are depicted by the patterned lines (see legend) 0 5 10 15 20 25 30 Lipid Profile 12:0 13:0 14:0 14:1 15+17 B 15:0 16:0 16:1 16:2 16:4(n-1) 17:0 17:1 18:0 18:1 18:2 18:3(n-3) 18:4(n-3) 20:0 20:1 20:4(n-6) 20:5(n-3) 22:0 22:1 22:6(n-3) Molar Percentage of Total Fa 73 Particulates (38 m) E. inermis Shallow E. inermis Deep E. inermis Male S. subtenuis tty Acids (%) 0 5 10 15 20 25 30 35 Particulates (28 m) E. inermis Shallow E. inermis Deep E. inermis Male P. attenuatus S. subtenuis A B

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74 Table 3.8. Phospholipid fatty acid profiles fo r copepods. Values are in molar percent Eucalanus inermis Shallow Deep Male Pareucalanus attenuatus Rhincalanus rostrifrons Rhincalanus nasutus Subeucalanus subtenuis 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 12:0 0.1 0.2 0.1 0.9 0.1 0.4 0.5 0.4 1.5 0.2 0.2 0.2 13:0 0.0 0.0 0.0 0.1 0.0 0.1 0.0 0.1 0.0 0.0 0.0 0.0 14:0 3.0 1.3 1.4 2.3 1.3 3.0 3.1 3.0 4.8 1.0 2.1 3.6 14:1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 15+17 Branched 0.3 1.1 0.9 0.8 0.9 0.8 1.0 0.9 0.4 0.2 0.9 0.9 15:0 0.3 0.5 0.5 0.6 0.6 0.4 0.7 0.6 0.7 0.2 0.5 0.6 16:0 24.7 26.0 28.0 24.2 24.8 26.1 26.0 20.8 28.3 18.1 22.3 23.3 16:1 3.7 1.7 2.3 1.8 1.6 2.1 1.9 2.3 1.3 1.7 1.9 1.9 16:2 0.2 0.1 0.1 0.1 0.0 0.1 0.1 0.0 0.0 0.0 0.2 0.0 16:3(n-4) 0.1 0.0 0.0 0.0 0. 0 0.1 0.0 0.0 0.0 0.0 0.0 0.1 16:4(n-1) 0.1 0.0 0.0 0.0 0. 0 0.1 0.0 0.0 0.0 0.0 0.1 0.0 Phytanic Acid 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.3 0.0 0.2 0.0 0.0 17:0 1.6 3.2 2.3 2.9 2.5 2.0 4.1 1.1 1.7 0.7 2.3 3.0 17:1 0.2 0.7 0.3 0.7 0.0 0.4 0.6 0.0 0.0 0.0 0.3 0.6 18:0 8.6 5.4 5.0 5.6 9.8 11.4 10.6 23.6 17.6 11.2 7.5 6.1 18:1 8.2 6.6 9.2 7.3 7.3 5.6 5.6 10.9 6.8 12.6 4.8 7.3 18:2 1.5 0.7 1.1 1.0 0.0 0.6 0.6 0.8 0.6 0.7 1.0 1.1 18:3(n-6) 0.0 0.0 0.0 0.0 0. 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 18:3(n-3) 0.1 0.3 0.3 0.3 0. 0 0.2 0.2 0.2 0.0 0.0 0.3 0.4 18:4(n-3) 0.1 0.1 0.0 0.2 0. 0 0.2 0.0 0.0 0.0 0.0 0.3 0.3 20:0 0.1 0.1 0.0 0.2 0.0 0.2 0.2 0.4 0.4 0.0 0.0 0.0 20:1 0.3 0.2 0.1 0.1 0.0 0.5 0.2 0.4 0.5 0.5 0.1 0.4 20:3(n-6) 0.2 0.0 0.0 0.0 0. 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 20:4(n-6) 5.3 1.4 1.4 1.5 1. 5 1.3 0.8 3.3 2.0 2.5 2.1 2.8 20:4(n-3) 0.0 0.0 0.2 0.0 0. 0 0.1 0.0 0.0 0.0 0.0 0.0 0.3 20:5(n-3) 11.7 7.9 9.9 8.0 9. 9 9.2 5.3 6.6 5.2 9.8 8.4 9.5 22:4(n-6) 1.3 0.0 0.0 0.0 0. 0 0.0 0.0 0.4 0.0 0.0 0.0 0.0 22:5(n-6) 1.8 2.4 1.6 1.8 1. 8 1.6 1.9 1.9 0.0 3.3 1.3 1.7 22:5(n-3) 3.3 0.9 1.1 1.1 0. 0 0.7 0.5 0.0 0.0 0.0 1.2 0.6 22:6(n-3) 22.3 34.0 33.6 29.6 37. 4 29.1 32.2 20.5 23.3 36.9 41.8 34.0 24:1 0.0 4.4 0.0 7.6 0.0 2.6 2.4 1.3 3.2 0.0 0.0 0.8 Other: 0.4 0.8 0.5 1.3 0. 5 0.5 0.3 1.8 0.2 1.5 1.0 0.6

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75 alcohols, and will not be discussed. Each species showed a very different alcohol accumulation pattern (Table 3.9). R. rostrifrons accumulated primarily 18:1 (68-69%), 16:1 (26-27%) and 16:0 (4-5%) fatty alcohols. R. nasutus profiles only contained 16:0 (58%), 14:0 (34%) and 18:0 (6.7%) alcohols, while P. attenuatus had a more general accumulation pattern, with 18:0 (23%), 18:1 (2 1%), 14:0 (18%), 17:0 (14%), 16:1 (14%) and 16:0 (10%) being a lmost equally abundant. Sterol profiles among the copepods were highly conserved, with cholesterol (7596%) and 22-dehydrocholesterol (3-25%) as th e only sterols regularly observed at >1% of total sterols (Table 3.10). Cluster analys es indicated that although all copepods were >85% similar, three different groups of c opepod samples showed >95% similarity (See Appendix, Figure A3). One gr oup was comprised of both R. rostrifrons samples (cholesterol content: 75-76 %), another included both S. subtenuis samples (cholesterol content: 93-96%), and the remaining group included E. inermis, R. nasutus, and P. attenuatus (cholesterol content: 82-89%). Table 3.9. Wax ester fatty alcohol profiles for Rhincalanus and Pareucalanus. Values are in molar percent. Alcohols are only shown for these three species as they are the only ones which accumulated a substantial amount of wax esters R. rostrifrons 2007 2008 R. nasutus P. attenuatus 14:0 0.1 0.0 34.0 18.2 15:0 0.0 0.0 0.9 0.0 16:0 3.9 4.9 58.4 10.1 16:1 26.9 26.4 0.0 13.6 17:0 0.0 0.0 0.0 13.7 18:0 0.3 0.0 6.7 23.0 18:1 68.8 68.7 0.0 21.4

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76 Table 3.10. Sterol profiles for copepods. Values are in molar percent. Sterol notation is described in Table 3.1 Eucalanus inermis Shallow Deep Male Pareucalanus attenuatus Rhincalanus rostrifrons Rhincalanus nasutus Subeucalanus subtenuis 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 C 27 5,22 17.6 14.5 12.4 15.7 12.6 13. 5 13.6 23.4 24.7 9.5 2.8 6.1 C 27 22 0.0 0.0 0.0 0.0 0.0 0. 0 0.0 0.0 0.0 0.0 1.6 0.0 C 27 5 82.4 85.0 86.8 83.9 87.4 86.5 86.4 75.6 75.3 89.2 95.6 93.9 C 27 0 0.0 0.0 0.9 0.0 0.0 0. 0 0.0 1.0 0.0 1.3 0.0 0.0 C 29 5 0.0 0.4 0.0 0.4 0.0 0. 0 0.0 0.0 0.0 0.0 0.0 0.0

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77 DISCUSSION Sampling year accounted for the greates t difference observed between deeper particulate lipid profiles. The 2007 deep samples (260 and 325 m) were very different from the 2008 deep samples (264 and 540 m), although within years differences were small. Surface samples showed little variab ility between years, even though different size fractions were analyzed (<200 m in 2007 and <53 m in 2008). Previous studies have found that specific size fractions do have different lip id signatures, but observed differences can be site specific (Wakeh am & Beier 1991, Harv ey & Johnston 1995, Escribano & Prez 2010). As cr uises were not too far apart in terms of the time of year (October-November versus December-January), this could indicate that little variation occurred between years, and th at most of the lipids occurred in the <53 m particulate size fraction represented in both years. Another possibility is that any interannual or seasonal variability that occurred was compensated for by the difference in lipid profiles of the size fractions. Within years, other parameters, like carbon, hydrogen, nitrogen, protein, and phosphorus content also were si milar among the deep samples. This suggests that little change in particulate material occured between 260 and 540 m, while large changes were seen between the chlorophyll maximum and 260 m. Chlorophyll maxima particulates appeared to be composed of both diatoms and dinoflagellates, with dinoflagellates in hi gher abundance in both ye ars, and both diatoms and dinoflagellates likely pres ent in the <53 m fraction (Harvey & Johnston 1995). Large chain diatoms, which were someti mes observed in this area (personal observations), would probably not have been measured during sampling either year, as they would have been in the >200 m size fraction. Biomarker ratios suggested that

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78 dinoflagellates usually were more abundant th an diatoms, since only the C16/C18 ratio in 2008 surface water particulates indicated diatom dominance (Table 3.4). These results were consistent with microplankton accessory pigment data, which showed that both fucoxanthin and peridinin (i ndicative of diatoms and di noflagellates, respectively) occurred in surface waters in 2007 (J. DiTullio, personal communication), and consistent with microplankton counts from the chlo rophyll maximum in 2007 (M. B. Olson, personal communication), These counts indicat ed that the prey community was largely composed of various types of dinoflagell ates, ciliates, cyanobacteria, autotrophic picoeukaryotes, and heterotrophic nanoflagell ates, with diatoms not appearing in these counts, suggesting low or patchy abundance fo r diatoms. The general agreement between lipids, pigments, and microplankton counts sugg ests that biomarkers can be useful in characterizing basic aspects of the available prey community. Biomarker results suggested that S. subtenuis, P. attenuatus and all E. inermis individuals sampled preferred to feed in the vicini ty of the chlorophyll maximum, an area of maximum particulate concen trations and highest quality particulate material (Table 3.2). These copepods had TAG profiles which were >70% similar to chlorophyll maxima particulates sampled in both years, and ma jor lipid components were similar between particles and copepod storage lipids in both years (Figure 3.3). There were some differences, most notably the much higher va lues of 20:5(n-3) found in the copepods versus particulate material. Th is is not surprising, as 20:5(n-3) is an essential fatty acid, which copepods are unable to manufacture them selves and therefore must acquire from their diet (Brett & Mller-Nav arra 1997). Studies examining assimilation efficiencies of different dietary fatty acids have determined that not all are retained equally, with PUFA

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79 often being preferentially assimilated (Har vey et al. 1987, Hazzard & Kleppel 2003). Additionally, particulate con centrations of 20:5(n-3) w ould be low due to faster breakdown of PUFA in the mari ne environment (Wakeham et al. 1997). The similarities between near-surface particulate matter and co pepod lipids also suggest that they were largely feeding opportunistically, and not discriminating greatly between different particles or prey types. WEs, on the other hand, did not show any strong relationsh ips to particulate material. This was particularly true for R. nasutus, R. rostrifrons and P. attenuatus the copepods that primarily accumulated WEs as th eir storage lipid. Pr evious work with copepods that primarily accumulate WEs have suggested that these lipids are formed through a combination of direct incorporation of dietary fatty acids, modification and incorporation of diet ary fatty acids, and de novo biosynthesis of fatty acids and alcohols (Sargent & Falk-Petersen 1988, Graeve et al 1994a, Kattner & Hagen 1995, Graeve et al. 2005). This is particularly well illustra ted by some high latitude herbivorous copepods, which accumulate many of the fatty acids availabl e in the diet but also have high levels of 20:1 and 22:1 fatty acids and alcohols, which are completely absent in the prey items (Kattner & Hagen 1995, Albers et al. 1996, Pete rs et al. 2004, Stevens et al. 2004, Graeve et al. 2005). As WEs are uncommon in tr opical copepods residing primarily above 500 m (Lee et al. 1971a, Lee & Hirota 1973), most of the conclusions ab out biomarkers in WE lipids have been drawn from high latitude systems. These areas of the ocean are generally characterized by seasonal phytoplankton blooms, where few species make up the majority of prey. In tr opical and subtropical systems, where prey are generally less abundant and more diverse, sp ecific microplankton biomarkers would be expected to be

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80 more scarce in the particulates or copepod lipids. This may lim it the applicability of lipid biomarkers for use in tropical zooplankton that accumulate WEs. Nevertheless, some conclusions can be drawn about the diets of Rhincalanus spp. based on what is known about the other eucala noid copepods collected simultaneously in this region. Total storage lipid profiles suggest ed that the relative amount of the unique diatom marker 16:4(n-1) was similar across species. Percentage s of 16:4(n-1) in E. inermis, S. subtenuis and P. attenuatus ranged from 0.8-2.2%. R. nasutus showed values in this range (1.8%), while R. rostrifrons had slightly lower va lues (0.4-0.8%). The unique dinoflagellate mark er 18:4(n-3) was 1.1-4.7% of total storage lipid in E. inermis, S. subtenuis and P. attenuatus. Both R. rostrifrons and R. nasutus fell below this range, with values of 0.2% and 0.8% respectively. Phytanic ac id, which is derived from chlorophyll breakdown, was 0.4-2.6% of total stor age lipid in E. inermis, S. subtenuis and P. attenuatus R. rostrifrons values also fell within this range (1.8-2.1%), while R. nasutus had the highest amount ( 3.6%). Compared to the E. inermis, S. subtenuis and P. attenuatus, it appeared that R. nasutus may have consumed more chlorophyll-containing phytoplankton and fewer dinoflagellates, whil e consumption levels of diatoms were approximately equal. R. rostrifrons on the other hand, appeared to have consumed the same amount of phytoplankton overall, but fewe r diatoms and dinoflagellates. As adult female Rhincalanus spp. are primarily found at depth at this site (peak abundance at 200400 m, K. Wishner, personal communicatio n), their food source may be largely particulate matter sinking from the surface with a preference for senescent phytoplankton cells.

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81 The difference between lipid profiles in the TAG and WE fractions of P. attenuatus is particularly noteworthy. Storage lipids in this copepod species were primarily WEs, but TAGs made up >15% of st orage lipids, while other copepods in this study had <10% of the non-dominant storage lipi d. Figure 3.4 depicts this difference in storage lipid profiles cl early by comparing the P. attenuatus TAG fraction to E. inermis (which primarily accumulates TAGs) and the P. attenuatus WE fraction to that of R. nasutus (which accumulates primarily WEs). Th ese results bring certain questions to light dealing with the compos ition and type of copepod storag e lipids. The first question is: why would a surface dwelling low-latitude copepod such as P. attenuatus primarily accumulate WEs? Secondly, what do thes e extreme differences between the two fractions indicate about fatty acid origin of the two storage lipids? Accumulating WEs as the primary stor age lipid in copepods has long been thought to occur primarily in deep sea and high latitude herbivorous organisms (Lee et al. 1971a, Lee & Hirota 1973), as prey are often patchy spatially and temporally. Copepods undergoing diapause also generally accumulate WEs (Lee et al. 2006), as those individuals require energy stores for periods of little to no feeding. However, numerous exceptions to these rules have been discovere d. Several marine a nd freshwater copepod species which have dormant stages accumulate primarily TAGs (summarized in Williams & Biesiot 2004). Additionally, some species of high latitude copepods (e.g., Temora longicornis, Acartia clausi, Centropages ham atus, Calanus propinquus, C. simillimus and Euchirella rostromagna ) have been found to accumulate mainly TAGs (Kattner et al. 1981, Hagen et al. 1993, Hagen et al. 1995, Ward et al. 1996). This usually is explained through differing life history strategies, suggest ing that these copepods continue feeding

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2D Graph 14Lipid Profile 12:0 14:0 14:1 15+17 B 15:0 16:0 16:1 16:2 16:3(n-4) 16:4(n-1) Phytanic Acid 17:0 17:1 18:0 18:1 18:2 18:3(n-6) 18:3(n-3) 18:4(n-3) 20:0 20:1 20:3(n-6) 20:4(n-6) 20:4(n-3) 20:5(n-3) 22:4(n-6) 22:5(n-6) 22:5(n-3) 22:6(n-3) 0 10 20 30 40 Molar Percentage of Total Fatty Acids (%) 0 5 10 15 20 25 A B Figure 3.4. A comparison of (A) triacylglycerol and (B) wax ester fa tty acid profiles of Pareucalanus attenuatus. P. attenuatus samples are depicted by the solid lines, while dotted lines in (A) represent 2008 Eucalanus inermis deep female triacylglycerols and (B) 2007 Rhincalanus nasutus wax esters 82

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83 to some degree throughout the winter (u sually through omnivorous or carnivorous feeding) and therefore do not store WEs (Hagen et al. 1993, Graeve et al. 1994a). Omnivorous or carnivorous copepods, however, have been found that accumulate WEs and do not show evidence of seasonal dormancy (Hagen et al. 1995, Albers et al. 1996, Kattner et al. 2003). These species often accumulate shorter chain fatty acids and alcohols, which leads to lower energy WE formation more typical of non-diapausing copepods (Kattner et al. 2003). Given most previous conclusions for WE versus TAG accumulation, there should not be any reason for copepods residing in the upper 300 m of the ETNP to accumulate WEs. WE accumulation does not fit the ge ographical trends, and there was no evidence of diapausing copepods in this area. As chlorophyll levels generally stay between 0.251.0 mg/m 3 in the sampling region (Pennington et al 2006), and similar levels were seen during our cruises (K. Daly, unpublished data), food did not appear to be limiting during any point of the year. The likeliest explanation has to do with taxonomy, which has been already been suggested by several author s (Lee et al. 1972, Hagen et al. 1995, Williams & Biesiot 2004). However, as information a bout lipids has largely been limited to high latitude areas, it is hard to reach firm c onclusions about taxonomic influences, as often only a few species have been examined within a given group due to low diversity within polar regions. Rhincalanus has been the only genus in Eu calanidae to consistently show preferential WE accumulation (Lee et al 1971a, Lee & Hirota 1973, Ohman 1988, Graeve et al. 1994a, Kattner et al. 1994, Kattner & Hagen 1995, Sommer et al. 2002, Schnack-Schiel et al. 2008). Eucalanus spp. from tropical, subtropical, and temperate environments have shown variable WE ve rsus TAG accumulation, although TAG often is

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84 most abundant (Lee & Hirota 1973, Lee 1974, Ohman 1988, Saito & Kotani 2000). No information on lipid classes for Subeucalanus and Pareucalanus spp. have been previously reported for comparison. Data fr om this study suggest that WE accumulation is a dominant strategy in the Rhincalanus spp., with P attenuatus also showing WE preference. E. inermis and S. subtenuis are more similar and primarily store TAG, as suggested by previous Eucalanus findings. These results are consistent with genetic analyses of 16S rRNA and ITS2 gene loci, in which Pareucalanus and Rhincalanus form one monophyletic group and Subeucalanus and Eucalanus form another group (Goetze 2003). Differences between the lipid profiles of WEs and TAGs are largely unexplored. Many studies that examined lipid composition determined the relative amount of each storage lipid class and then looked at total fatty acid an d alcohol profiles, making it difficult to illuminate differences between storage lipids and examine the functional implications. Previous work that reports both TAG and WE lipid profiles for copepods is rare, and has never before included an a ssessment of available food items (Lee et al. 1971a, Lee 1974, Albers et al. 1996, Lee et al 2006). The differences between TAG and WE fractions have been suggested to be due to TAGs being more reflective of recent feeding while WE represented longer-term di et (Lee et al. 2006). Albers et al. (1996) determined that WE profiles can be indicativ e of trophic position, but that TAG did not show any relationship to other ecological parameters. Varia tion between the storage lipid classes often are observed in 14:0, 16:0, 16: 1, 18:1, 20:1, 22:1, 20:5( n-3) and 22:6(n-3) fatty acids, although differences vary by species (Lee et al. 1971a, Lee 1974, Albers et al. 1996, Lee et al. 2006). The one trend observe d consistently was that copepods which

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85 accumulated 20:1 and 22:1 fatty acids often had higher levels in the WE fractions. These fatty acids are typically s ynthesized by high-latitude he rbivorous copepods (Lee 1974, Albers et al. 1996, Lee et al. 2006). One notable exception is Euchaeta antarctica a carnivorous copepod which has hi gher levels of 20:1 in the TAG fraction (Albers et al. 1996, Lee et al. 2006). However, it has been suggested that this copepod likely does not manufacture these MUFA, but attains them through their copepod prey. This is consistent with our suggestion below, that TAGs are more directly refl ective of diet while WEs contain more modified fatty acids. Results from our study suggest that TAGs more directly reflect diet in copepods, while WE contain a greater proportion of modi fied fatty acids. Differences between TAG and WE fractions are illustrated by P. attenuatus lipid composition. TAGs consisted almost entirely of dietary fatty acids, while many of the fatty acids of WEs were likely modified from the diet or synthesized de novo. Examinations of PUFA content of Rhincalanus and Pareucalanus spp. also supported this observation, as the percentages of 20:5(n-3) and 22:6(n-3) were consistently higher in TAG lipids than WEs. As PUFA can only be attained from food sour ces, this suggested a higher proportion of unmodified dietary fatt y acids in TAGs. Such ideas about the origins of WE fatty acids and alcohols have been proposed before (Sargent et al. 1981, Lee et al. 2006). Additionally, it appears that the content of WEs may also be under some phylogenetic control (Graeve & Kattner 1992). For the copepods that primarily accumulated WEs in this study, two well defined groups were seen in cluster analyses based on fatty acid composition (Figure 3.2). R. rostrifrons formed its own group, characterized by 14:0 and 16:0 fatty acids, and R. nasutus and P. attenuatus formed another group, with primarily

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86 16 and 18 carbon MUFA. R. nasutus from other ocean regions also show the same trends, with 16 and 18 carbon MUFA as major c ontributors to WEs or total lipids (Lee et al. 1971a, Lavaniegos & Lpez-Corts 1997, Sommer et al. 2002, Schnack-Schiel et al. 2008). Furthermore, R. gigas from the Southern Ocean also showed dominance of 16:1 and 18:1 fatty acids (Graeve et al. 1994a, Kattner et al. 1994, Ka ttner & Hagen 1995), and a specific pathway for WE fatty acid a nd alcohol biosynthesi s has been proposed (Kattner & Hagen 1995). Total lipid fatty acids for P. sewelli from the Gulf of California showed relatively high levels of 16:1 a nd 18:1 fatty acids (about 12% each), perhaps suggesting similar accumulation patte rns to those observed here in P. attenuatus (Lavaniegos & Lpez-Corts 1997). Similar to R. rostrifrons in this study, R. cornutus from the Gulf of Mexico also shows primar ily 14:0 and 16:0 FAs in storage lipids (Cass et al., in review). Molecular phylogeny usi ng multiple gene loci (16S rRNA, ITS2, 18S rRNA) indicates that R. gigas and R. nasutus are more closely relate d to each other while R. rostrifrons and R. cornutus form their own monophyletic group, supporting the notion that genetic variation among species affects WE fatty ac id composition (Goetze 2003, 2010). One of the major differences in lipid accumulation patterns of Rhincalanus spp. and P. attenuatus was observations of lipid sac size. Lipid sac size was variable in Rhincalanus spp., but it usually took up a large proportion of the main body cavity. Lipid sacs in P. attenuatus, E. inermis and S. subtenuis were more moderate in size, and only occurred in a small part of th e posterior prosome. These visu al observations of lipid sacs were supported by the lipid as % DW and lipid sac mass, where Rhincalanus spp. had much higher lipid values than other coepepods (Table 3.5; Chapter 4). This might help to

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87 explain why WE and TAG fractions of Rhincalanus were the most similar. In these species, WEs not only dominated the storage lipids they dominated total lipid content. It is possible that Rhincalanus accumulated so many preferred WE fatty acids, that these were also incorporated into the TAG fraction with the dietary FAs, giving a signature not too different from the WE lipids. However, TAG fractions also re flected more direct dietary input (such as higher PUFA), giving a slightly diffe rent signature than the WE fraction. On the other hand, S. subtenuis and E. inermis, which do not have well developed WE biosynthetic pathways, accumulate d dietary fatty acids in the WE fraction along with some modified or newly synthe sized acids and alcohols depending on their genetically-specified lipid pattern. P. attenuatus which had a we ll-developed WE formation mechanism, but only moderate accu mulation, was able to largely retain FAs synthesized for WE in that fraction, and di etary FAs in the TAG fraction. It should be noted that these conclusions may not hold in al l systems, particularly those in which the accumulation of high energy (i.e., long chain) fa tty acids and alcohols are important. For example, the primarily he rbivorous Antarctic copepod, Calanus propinquus primarily accumulates TAGs which have large amounts of 22:1 fatty acids, which are thought to be non-dietary in origin (Kattner et al. 1994, Kattner & Hagen 1995, Albers et al. 1996, Falk-Petersen et al. 1999). The copepods in our study appear to be largely omnivorous and living in food-replete environments, and thus do not require such high energy lipid reserves. This study demonstrates that sterols are not useful as biomarkers for copepods in the ETNP system. For these five different copepod species, profile s were dominated by cholesterol and 22-dehydrocholesterol, sugge sting that sterol composition is highly

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88 regulated. This likely is due to cholestero l having many important functions in cellular membranes, including stabilizing membrane st ructure, affecting membrane permeability and altering the activity of membrane protei ns (Crockett 1998). Such specific sterol compositions are probably attain ed through preferenti al retention of dietary cholesterol and other dietary phytosterols (e.g., brassi casterol (24-methylcholesta-5,22E-dien-3 -ol) and 24-methylenecholesterol (24methylcholesta-5,24(28)-dien-3 -ol)) that can be easily dealkylated to cholesterol, and subsequent conversion of assimilate d sterols to needed forms (Teshima 1971, Goad 1978, Harvey et al. 1989). One major difference between the sterol profiles reported in this study versus previous work is the absence of desmosterol (cholesta-5,24-dien-3 -ol), which is often the second most abundant sterol found in zooplankton (Harvey et al. 1987, Serr azanetti et al. 1992, Serrazanetti et al. 1994, Mhlebach et al. 1999). Desmosterol is usually thought to occur because it is an intermediate in the conversion of dietary phyt osterols to cholesterol (Goad 1978). These studies have all occurred at temperate or polar latitudes, where seasonal phytoplankton blooms contribute to available food. Microplankton counts at our study site indicate that heterotrophic organisms were major components of available prey (M. B. Olson, personal communication). As diet was shown to largel y reflect the available particulate material, heterotrophic prey were likely to be common in the diet, resulting in lower amounts of phytosterols for conversion. It is also possibl e that these copepods have a more efficient or rapid conversion of ingested phytosterols to cholesterol, such that desmosterol was not able to accumulate in the body. The minor interspecific differences observe d in sterol profiles may be related to habitat temperature or temperature range. C holesterol content in copepods can vary with

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89 environmental temperature, such that copepod s at higher temperat ures generally have more cholesterol (Hassett & Crockett 2009). In addition, organisms with higher cholesterol content are less likely to experience heat stress, abrupt phase changes, or high amounts of membrane perturbation with modera te temperature changes (Crockett 1998). In our study region, the thermocline is very shallow, with temperatures varying from about 27C at the surface to 15C at 50 m. The species with the highest cholesterol content, S. subtenuis, is known to primarily inhabit the upper 100 m, which experiences the highest temperatures and greatest temperature range within the water column (Longhurst 1985, Chen 1986, Sameoto 1986). The sp ecies with the lowest cholesterol, R. rostrifrons is usually found below the thermocline and, therefore, experienced the lowest temperatures and smallest variability of these copepods (Longhurst 1985, Chen 1986). However, habitat temperature cannot be the only factor. P. attenuatus which was largely a surface species, and E. inermis which showed peaks in abundance in near-surface and upper oxycline waters, would be predicted to ha ve cholesterol content similar to that of S. subtenuis, when instead intermediate cholesterol levels were observed. There could be differences among copepod species on the cellu lar level, requiring varying amounts of cholesterol for optimal membrane protein function (Crockett 1998). Like sterols, phospholipid profiles are generally highl y regulated, as fatty acid composition is well-accepted to be an important factor in various membrane functions. The copepods in this study illustrated this point well, with phospholipid FA profiles showing >75% similarity among all copepod samples. The major FAs found in these copepods (22:6(n-3), 16:0, 18: 0, 20:5(n-3) and 18:1) have been observed in other

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90 copepod phospholipid profiles as well (e.g., L ee et al. 1971a, Lee 1974, Albers et al. 1996, Scott et al. 2002). Fatty alcohol profiles appeared to be largely species-specific, with R. rostrifrons, R. nasutus and P. attenuatus each having their own distinctive lipid signature. Fatty alcohols of WEs are believed to be synthesized de novo (Sargent et al 1981), indicating that such differences might be expecte d. Fatty alcohol com position has not been previously determined for P. attenuatus or R. rostrifrons Observed profiles for R. nasutus are nearly identical to previous findings (Lee et al. 1971a, Sommer et al. 2002, Schnack-Schiel et al. 2008), and al so similar to those seen in R. gigas (Graeve et al. 1994a, Kattner et al. 1994, Kattner & Hagen 1995). In addition, R. cornutus individuals from the Gulf of Mexico share a fatty alcohol signature with R. rostrifrons reported in this study (Cass et al., in review). This st rongly suggests a geneti c component in fatty alcohol accumulation patterns for thos e copepods that store primarily WE. Bulk lipid contents for eucalanoid cope pods usually are higher (6-69% of DW) than values presented here (1-23% of DW), with Rhincalanus spp. often having the highest lipid contents (>22% of DW) (Lee et al. 1971a, Lee & Hirota 1973, Lee 1974, Morris & Hopkins 1983, Flint et al. 1991, Ohman 1997, Schnack-Schiel et al. 2008). This could be due to several factors. First, Ohman (1997) points out that the method used to determine the lipid mass strongly affects the end result. For example, measuring copepod masses before and after lipid extraction tends to overestimate the amount of lipid mass by 1.5-10 times, compared to values obtained by TLC-FID (thin-layer chromatography-flame ionization detection) analyses (Ohman 1997). This was found to be particularly true for copepods with high water content, like members of the genus

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91 Eucalanus. Such inconsistencies are due to the initial lipid extraction removing more than just lipid components, including carbohydrates, ami no acids, protei ns and salts (Hopkins et al. 1984, Kates 1986). The method employed in this study (summing the lipid peaks found in each fraction) would yield lower estimates of lipids as a percent DW. Reported weighing methods often were not de tailed enough to determine whether lipid weights may be artificially higher in those ot her studies. Another issue encountered was lipid leakage during thawing for length meas urements. This was mostly noted for Rhincalanus spp., as these copepods had the largest amount of lipid stores. Such issues could be avoided by pre-measuring copepods for lipid analyses before initial freezing. A final issue is one inherent in this method. Du e to the large number of steps, it is probable that some lipids were lost by adhering to glassw are or inexact tran sfer throughout the process. If enough sample is available, bul k lipid content should be determined through TLC-FID, with a separate sample used to examine relative amounts of each fatty acid, alcohol, and sterol component. Due to the relatively small si ze of these copepods and the number of individuals available for lipid an alyses, this was not possible in this study. Free fatty acid classes (154% total lipid) for the copepods in this study were also higher on average than would be expected. Free fatty acids are generally a minor component in copepods, usually comprising <3 -4% of the total lipids examined (Lee et al. 1971b, Sargent & Falk-Petersen 1988, Ohma n 1996, Schnack-Schiel et al. 2008). This discrepancy is probably attributable to the length measuring step, where thawing occurred. Ohman (1996) noted that copepods that were not stored properly (temperatures higher than approximately -80C), showed in creased free fatty acid concentrations, as lipases became active and broke down the other lipid components (particularly

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92 phospholipids) into free fatty acids and other molecules. Such breakdown may have occurred during measuring, as copepods were brought to near room temperature and also were subjected to heat from the microscope li ghts. Even though this was only for a short time (usually less than an hour), this wa s the only time period during which copepods experienced temperatures warmer than -80C. This suggests that lipid degradation can quickly occur in zooplankton. As with lipi d leakage, this problem would also be remedied by pre-measuring copepods befo re they were initially frozen. A comparison of particulate and cope pod lipid composition from this study suggests that adult male and female E. inermis, and female P. attenuatus and S. subtenuis likely ingested similar types of food particles from the vicinity of the chlorophyll maximum and had a similar feeding strategy (gen eral particle feeder). This might be expected for P. attenuatus and S. subtenuis which are known to be concentrated in surface waters. E. inermis however, has a broad vertical distribution with a significant proportion of adult females in deeper wate rs and, therefore, having other potential feeding opportunities (Longhurst 1985, Chen 1986, Sameoto 1986). For instance, the lower oxycline may be associated with a peak in particulate orga nic carbon, which could be a food source at depth (Wishner et al. 1995). E. inermis does not appear to have a strong tendency towards diel vertical mi gration (Sameoto 1986, Saltzman & Wishner 1997b); however, our study suggests that deep individuals might occasionally migrate to the surface for feeding, although this might not be on a strictly daily schedule. Feeding preferences for Rhincalanus spp. were more difficult to ascertain via lipid biomarkers, but results suggested that thes e species fed on fewer dinoflagellates, similar levels of diatoms, and similar or higher levels of overall phytoplankton than E. inermis, P.

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93 attenuatus, and S. subtenuis Given that Rhincalanus spp. are largely concentrated below the highly productive surface layer (Longhurst 1985, Chen 1986, Sameoto 1986), feeding was likely on sinking particulate material derived from the chlorophyll maximum. The results of this study also highlighted the differences between TAGs and WEs as storage lipid components. TAGs more di rectly reflected lipid components of food sources and are most useful for lipid biomarker studies, while WEs contained more modified or newly synthesized fatty acids and alcohols. The amount of WEs and TAGs accumulated by copepods in this region appeared to be partially related to taxonomy, and strong suggestion of genetic in fluences were seen in the patterns of fatty acids and alcohols comprising the WE fraction.

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94 CHAPTER FOUR Ecological Characteristics of Eucala noid Copepods of the Eastern Tropical North Pacific Ocean: Adaptations fo r Life Within a Low Oxygen System INTRODUCTION Recent reports indicate that the oceans are decreasing in oxygen in response to global warming, primarily through surface h eating and increased stratification (Keeling & Garcia 2002, Emerson et al. 2004). Addi tionally, regions of the ocean having oxygen minimum zones (OMZs), which are charac terized by oxygen deficient waters at intermediate depths, appear to be expanding (Stramma et al. 2008, Stramma et al. 2010). The suboxic regions in open water OMZs are typically mainta ined as a result of poor ventilation, sluggish circ ulation, oxygen-poor source waters, and decomposition of sinking particles (Wyrtki 1962). Mo st tropical and subtropical regions in the Atlantic and western Pacific oceans have moderate OMZs, with minimum oxygen levels of 60 to 80 M (Paulmier & Ruiz-Pino 2009). One of the largest and most severe open water OMZs is located in the eastern tropical north Paci fic (ETNP) (Paulmier & Ruiz-Pino 2009). The ETNP is characterized by a strong, shal low pycnocline and a pronounced oxycline (Fiedler & Talley 2006), where chlorophy ll, primary production, and copepod maxima occur (Herman 1989). Oxygen concentrations <50 M occur as shallow as 40 m and often reach <4.5 M in the OMZ core (the region of lowest oxygen concentrations) (Brinton 1979, Levin et al. 1991, Vinogradov et al. 1991, Saltzman & Wishner 1997a).

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95 Some oxygen levels have been reported below 0.5 M (Chen 1986, Sameoto 1986, Levin et al. 1991). The vertical oxygen gradients in OMZs structur e biological assemblages and biogeochemical processes. As a result, hab itats of organisms intolerant to low oxygen may be compressed into the shallow, near-surface oxygenated waters (Prince & Goodyear 2006). Members of the family Eucalanidae are dominant copepods in the ETNP, and include all four genera ( Rhincalanus, Eucalanus, Subeucalanus and Pareucalanus) (Longhurst 1985, Chen 1986, Sameoto 1986, Saltz man & Wishner 1997b). Like many other zooplankters in the region, these copepods display a variety of vertical distributions that are likely related to the oxygen e nvironment (Chen 1986, Sameoto 1986, Vinogradov et al. 1991, Saltzman & Wishner 1997b). Eucalanus inermis endemic to the ETNP, is found throughout the upper 1,000 m, often with maximum concentrations in the chlorophyll maximum and the upper a nd lower edges of the OMZ core. Subeucalanus subtenuis, S. subcrassus, S. pileatus and Pareucalanus attenuatus are usually concentrated in the shallow euphotic zone. Rhincalanus rostrifrons [sometimes referred to as R. cornutus rostrifrons (Lang 1965)] and R. nasutus on the other hand, are largely absent from the upper water column, and inst ead are concentrated above and below the OMZ core. This suggests that a variety of eco logical strategies occu r within this family in the ETNP region. Even though eucalanoid copepods are a bundant in the ETNP, little is known about their ecology. As severe OMZ regions appear to be expanding (Stramma et al. 2008, Stramma et al. 2010), understanding the strategies employed by zooplankton in current OMZ systems may help us to predic t the effects of decr easing oxygen on marine

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96 ecosystems in other regions of the ocean. Eu calanidae is a relatively small family of copepods (24 described species) that are distri buted throughout a majo rity of the worlds oceans and occur in coastal and open water systems (Grice 1962, Lang 1965, BradfordGrieve et al. 1999, Goetze 2003). The abundance of eucalanoid copepods in the ETNP system, coupled with their broad distribution in other regions, make them a useful group for comparative studies within and between ecosystems. These copepods also have been the focus of several recent genetic studies, which aimed to further describe separate lineages within species (Goetze 2003, 2005, 2006, 2010, Goetze & Ohman 2010). Consequently their phylogeny is fairly well described, which provides a basis for understanding differences in ecological char acteristics between species and populations within the family. The primary objective of this study was to assess whether eucalanoid copepods employed different ecological strategies in the ETNP OMZ system based on their biochemical, physiological and behavioral ch aracteristics. Based on published vertical distributions in this region (Chen 198 6, Sameoto 1986, Vinogradov et al. 1991, Saltzman & Wishner 1997b), it was hypothesized that P. attenuatus and S. subtenuis would have similar characteristics and strategies, and R. nasutus and R. rostrifrons would be similar to each other, whereas E. inermis would have different strategies relative to the other two groups. The results of this study were compared with findings for eucalanoid copepods in other regions of the world to assess wh ether the observed characteristics were adaptations to the OMZ system, or simply general features of a species or genus. Additional attention was paid to E. inermis, which is one of the most abundant and widely vertically dist ributed copepods in this region (Longhurst 1985, Chen 1986,

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97 Saltzman & Wishner 1997b). Adult females occu r in near-surface wa ters, but a larger resident population also occurs near the OMZ. In temperate environments, E. californicus was observed to undergo a dormant period at depth (Ohman et al. 1998). While a dormant period or stage was not necessa rily expected within a tropical system, it was unclear whether these deep er individuals might repres ent a specific cohort, for instance, a particular stage in the reproductive cycle. Differences in vertical distribution have also been documented between adult ma les and females, with adult males often concentrated at shallower depths than fe males (Longhurst 1985; K. Wishner, personal communication). Therefore, the ecological st rategies of both males and females were examined.

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98 METHODS Collection Site and Methods. Copepods were collected duri ng two cruises to the eastern tropical north Pacific (ETN P) during 18 October 17 N ovember 2007 aboard the R/V Seward Johnson and 8 December 2008 6 January 2009 aboard the R/V Knorr Sampling occurred primarily at the Costa Rica Dome (9N, 90W) and to a lesser extent the Tehuantepec Bowl (13N, 105W) (Figure 4.1). Copepods were collected using bongo tows, Tucker trawls, and MOCNESS (Multiple Opening/ Closing Net and Environmental Sampling System) (Wiebe et al. 1976) tows at depths of high abundances. Adult female Subeucalanus subtenuis and Pareucalanus attenuatus were collected from the upper 50 m, while Rhincalanus rostrifrons and R. nasutus were collected in the 200300 m range. Eucalanus inermis adult females were collected at both shallow and deep depths and adult males collected in the uppe r 50 m. Some CIV or CV stage copepodites also were collected when found in abundance. In addition, adult female R. cornutus were obtained during a cruise to the Gulf of Mexi co (GOM) at a station offshore of Florida (27N, 86W) during June 25-29 2007 aboard the R/V Suncoaster. Additional adult female R. nasutus individuals were collected from the Guaymas Basin, Gulf of California (GOC) (approximate location 27N, 111W) during June 4-12 2007 aboard the R/V New Horizon. These R. cornutus and R. nasutus were both collected using a Tucker trawl. Immediately after capture, adult copepods were sorted and individuals of each species were separated into small containers containing 0.2 m filtered seawater at in situ temperature. Copepods were kept at in situ temperatures for appr oximately 3-12 hours to allow them to empty their guts. Once thei r guts were emptied, copepods collected in the ETNP were divided up for various analyses. Most individuals were frozen at -80C for

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later body content, enzyme, and weight analyses. Some individuals of S. subtenuis, R. rostrifrons and E. inermis were used for end point incubation experiments (Chapter 2). All Rhincalanus spp. collected during the GOM and GOC cruises were frozen at sea, either in liquid nitrogen (GOM) or a -80C freezer (GOC). 0 30N 20N 10N 80W 10S 110W 90W 100W 130W 140W 150W 120W Figure 4.1. Map of eastern tropi cal north Pacific sampling site s. Black dots represent the two main sampling regions, with the cruise tr ansect drawn between them. Map modified from SWFSC NOAA website (http://swfsc.noaa.gov/). Length-Weight Regressions Length to wet weight (WW) equations were generated for the most common species ( S. subtenuis, E. inermis, P. attenuatus, R. rostrifrons and R. nasutus ) so that weights of copepods used in body composition and other analyses could 99

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100 be estimated. Females and immature individu als (when available) were used to obtain a broad size range. Male E. inermis were analyzed separately from immature and adult female E. inermis, as their body shape was different. Th is was the only part of the study which utilized imma ture individuals. In the lab, individuals were thawed and several measurements were made using an ocular micrometer on a dissecting micros cope. Cephalothorax width (CW) was taken from the widest point of the cephalothora x. Cephalothorax length (CL) was measured from the anterior tip of the rostrum to the posterior point of the cephalothorax. Prosome length (PL) was measured from the anterior tip of the rostrum to the posterior of the prosome. Total length (TL) was measured fr om the anterior tip of the rostrum to the posterior tip of the caudal ramus (not includi ng setae). Individuals were then rinsed briefly with deinoized (DI) wa ter to remove salts, blotted dry with a Kimwipe, and transferred to a small aluminum capsule, which was sealed, and wet weight (WW) measured using a Mettler Toledo UMX2 microbalance. Linear regression lines comparing log(CL), log(PL) and log(TL) to log(WW) were generated for each species, and the best fit line between th e three was assessed using the R 2 value. Dry weights (DWs) were calculated using the average per cent water content of each species (methods described below). A subset of 183 individuals (59 E. inermis adult females and late stage copepodites, 18 E. inermis adult males, 10 P. attenuatus 34 R. rostrifrons 11 R. nasutus and 51 S. subtenuis ), were used to calculate WW according to the equation of Kuzmichova (1985). The percent difference be tween this calculated WW and the actual WW was then determined to assess the validi ty of using this gene ral equation. In this

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101 paper, positive percent differences indicate that equations overestimated WW compared to measured values. The same 10 P. attenuatus 51 S. subtenuis, and 34 R. rostrifrons from above, along with 4 R. cornutus from the GOM, were used to assess the validity of species-specific equations published by Gruzov and Alekseyeva (1970) from the Atlantic Ocean to estimate wet weights for these copepod species. Body Composition Subsets of individuals from each species were used for carbon/hydrogen/nitrogen (CHN) content analyses. Copepods were thawed, measured for body size, and then grouped into batches of 5-15 individuals onto pre-combusted (2 hours at 450C) and pre-weighed GF/F filters Copepods on filters were briefly rinsed with DI water using hand-pump filtration and th en dried at 60C for several days prior to weighing on a microbalance. After weighing, filters were placed into pre-combusted 20 ml glass scintillation vials. Copepods and filter blanks were analyzed for carbon (C), hydrogen (H), and nitrogen (N) co ntent at the University of California, Santa Barbara Marine Science Institute Analytic Laborator y. Blank filters a ssessed contamination during handling and sample preparation. Protein content of copepods was determ ined on individuals using the Lowry assay (Lowry et al. 1951). Phosphorus (P) cont ent was assessed on individuals using the method outlined in Anderson and Hessen (1991). For these analyses, copepods were thawed, measured, and then placed in a small amount of reagent in a clean scintillation vial. Copepod integument was disrupted through agitation with a sonication probe. For a subset of individuals, estima tions of storage lipid mass we re made through measurements of storage lipid sacs. Height (h), length (l ), and width (w) were measured and volume

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102 was estimated using the equation for an ellipsoid (V = (4/3)* *(h/2)*(w/2)*(l/2)). Conversions to mass were made based on th e density (reviewed in Sargent 1976) and relative amounts of triacylglycerol or wax es ter known to be accumulated by each species (Chapter 3, Cass et al. in review). Water and ash content was determined us ing batches of 2-10 copepods for each species. Copepods were thawed, measured, dipped briefly in DI water, blotted dry, and sealed into a small aluminum capsule. WW was measured using the microbalance. Capsules were reopened and dried at 60C for several days prior to resealing and reweighing for DW. Capsules were then combusted (500C for 6 hours) and reweighed to obtain ash weight. Enzyme Analyses Levels of lactate dehydrogenase (LDH) were measured on batches of 3-35 individuals using the me thods described in Childress and Somero (1979). Copepods were homogenized using a glass tissue grinde r in a 50 mM imadazole/HCl buffer solution (pH = 7.2 at 20C). Dilution fact ors were either 10:1 or 15:1 l of buffer to mg of wet mass. Activity levels were assessed for all copepods at 10C using a thermostatted Cary 1000 UV/Visible spectrophotometer with data analysis software. Activity levels corresponded to rates of change in abso rbance at 340 nm measured over a 60 second interval due to the oxidation of NADH substrate. Samples were run in triplicate, with the average value reported. Activity level was e xpressed in units (mol substrate converted to product per minute) per gram wet mass or gram protein.

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103 Survivorship Studies Metabolic end-point experiments were run on various species of eucalanoid copepods (see Chapter 2). Expe riments were run at 6, 15, and 100% air oxygen saturation. Only experiments where all individuals survived were used for rate measurements. Here, survivorship duri ng all metabolic experiments at 10C was investigated. Experiments at 6 and 15% sa turation levels were approximately 12-15 hours in duration, while 100% saturati on experiments were 24-36 hours. Statistical Analyses Statistical analyses were run in SigmaPlot 11.0. One and two way ANOVAs and t-tests were used to compare the central trend of data. When raw data did not fit the assumptions of equal variance or normality, ranked data were substituted or a comparable non-parametric test was util ized. Significance was assessed at = 0.05. Unless otherwise noted, values listed in the text are means and standard deviations.

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104 RESULTS Environmental Parameters and Copepod Distributions. Temperature, oxygen, and fluorescence profiles for the Tehuantepec Bowl and Costa Rica Dome stations during the 2007 and 2008/2009 ETNP cruises are depicted in Figure 4.2. Temperature profiles were generally similar between stations and year s. Fluorescence prof iles showed two major peaks in the Tehuantepec Bowl region, while only one peak was observed at the Costa Rica Dome. In both locations, oxygen rapidl y declined between the surface and about 50-60 m. In the Costa Rica Dome, however, oxygen intrusions were present between 100 and 300 m, resulting in higher oxygen concentra tions at these mid-depths than at the Tehuantepec Bowl site. Eucalanoid copepod vertical distributions at the Cost a Rica Dome during 2007 were similar to those reported previously (K. Wishner, personal communication). Daytime abundances of adult female Eucalanus inermis were highest between 200-400 m (1-15 M O 2 ) and 30-60 m (20-50 M O 2 ) with a small peak at 500-600 m (1-2 M O 2 ) (Figure 2.1). Adult males were found between 30-110 m (15-50 M O 2 ). Subeucalanus subtenuis adult females had peak abundan ces in the upper 60 m (>20 M O 2 ). Adult female Rhincalanus rostrifrons were found primarily at 200-350 m (2-15 M O 2 ) during the day, with a smaller number pres ent at 40-60 m depth (20-30 M O 2 ). R. nasutus adult females were only found in lo w numbers at 250-300 m (4-7 M O 2 ) and 550-600 m (1.5-3 M O 2 ).

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Oxygen Concentration (M) 0 50100150200250Depth (m) 0 100 200 300 400 500 Temperature (C) 051015202530 Fluorescence (mg/m 3 ) 0.00.20.40.60.81.01.21.41.6 Oxygen (M) Temperature (C) Fluorescence (mg/m 3 ) Oxygen Concentration (M) 0 50100150200250Depth (m) 0 100 200 300 400 500 Temperature (C) 051015202530 Fluorescence (mg/m 3 ) 0.00.20.40.60.81.01.2 Oxygen (M) Temperature (C) Fluorescence (mg/m 3 ) Oxygen Concentration (M) 0 50100150200250Depth (m) 0 100 200 300 400 500 Temperature (C) 051015202530 Fluorescence (mg/m 3 ) 0.00.20.40.60.81.01.21.41.6 Oxygen (M) Temperature (C) Fluorescence (mg/m 3 ) Oxygen Concentration (M) 0 50100150200250Depth (m) 0 100 200 300 400 500 Temperature (C) 051015202530 Fluorescence (mg/m 3 ) 0.00.20.40.60.81.01.2 Oxygen (M) Temperature (C) Fluorescence (mg/m 3 ) A B C D Figure 4.2. Water column oxygen, temperatur e, and fluorescence profiles in the upper 500 m at two stations during the eastern tropi cal north Pacific cruises. 2007 profiles are depicted for the (A) Tehuantepec Bowl and (B) Costa Rica Dome. 2008-2009 cruise profiles are shown for the (C) Tehuant epec Bowl and (D) Costa Rica Dome. Length-Weight Equations Length to WW equations are listed in Table 4.1. For all species, TL or PL had the strongest linear asso ciation with WW. With the exception of E. inermis males (R 2 = 0.83) and S. subtenuis females and juveniles (R 2 = 0.79), all R 2 were 0.92. The lower R 2 values were due to scatter of data over the entire size range evaluated in E. inermis males and in the larger size classes of S. subtenuis individuals 105

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106 collected in 2008. The R. rostrifrons wet weight equation proved adequate for estimating R. cornutus WW (average difference 1.0% with a standard deviation of 7.4%); hence R. cornutus and R. rostrifrons were pooled to generate WW equations. Few length or weight measurements have been reported for eucalanoid copepods from the ETNP. Instead, weights were ofte n estimated using general copepod weight equations or equations produced using spec ies in different loca tions (e.g., Gruzov & Alekseyeva 1970, Kuzmichova 1985). A compar ison indicated that the general equation published by Kuzmichova (1985) did not ade quately estimate WW fo r these species of copepods. Only 14.8% of the calculated values fell within % of the measured WWs. Even with more lenient standards, only 22.4% and 40% of values fell within % and 20% of the measured WW, respectively. Specifically, Kuzmichovas equation underestimated WW on average by 11, 25, 34, 30 and 24% for E. inermis, S. subtenuis, R. cornutus, R. rostrifron s and Pareucalanus attenuatus, respectively. Gruzov and Alekseyevas (1970) species-s pecific equations ha d a better overall fit. For S. subtenuis, the percent difference was -5.3%, with a standard deviation of 5.5% and overall range of -20.5 to 5.9%. No one size class was better predicted by this equation. For P. attenuatus, WW tended to be overestimated (13.5 16.8%), by the equation, although adult females (WW > 1.60 mg) had differences of <5%. R. cornutus usually was overestimated as well (11.0 5.1%). R. rostrifrons which appeared to have a similar length-WW relationship as R. cornutus in this study, was also compared with Gruzov and Alekseyevas R. cornutus equation, and WW was f ound to be consistently overestimated (29.0 10.5%). This indicates that species-specific equations generated using copepods from the specific colle ction site are preferable for WW

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107 Table 4.1. Length to weight rela tionships for eucalanoid copepods. Equations for estimating wet weight (WW) from prosome lengt h (PL) or total length (TL) are shown, along with the R 2 value of the regression line, and N is sa mple size. The size range of PL and WW of individuals used to generate the equation are also noted. The average percent water content of each species the standa rd deviation and N, number of measurements, are listed. Finally, equations for conversion of WW to dry weight (DW) is given for each species or group. Note that the R. cornutus and R. rostrifrons were combined to generate the WW equation, but DWs are calculated separately Species Equation for WW (mg) R 2 N Range of PL (mm) Range of WW (mg) % Water SD (N) Equation for DW (mg) E. inermis Female + Juvenile Log(WW) = 3.5 150*Log(PL) 1.8065 0.98 59 3.30 6.04 0.94 7.93 93.90 0.49% (105) DW = WW*0.0610 E. inermis Male Log(WW) = 3.5067*Log(PL) 1.7489 0.83 17 3.54 4.20 1.93 2.89 94.18 0.31% (3) DW = WW*0.0582 P. attenuatus Log(WW) = 5.5694*Log(TL) 3.3186 0.92 10 3.20 3. 85 0.85 1.78 89.30 2.43% (3) DW = WW*0.1070 R. rostrifrons Log(WW) = 3.1550*Log(PL) 1.6602 0.95 48 2.23 3. 28 0.25 0.98 80.57 0.61% (3) DW = WW*0.1943 R. cornutus Log(WW) = 3.1550*Log(PL) 1.6602 0.95 48 2.23 3. 28 0.25 0.98 86.73 2.26% (20) DW = WW*0.1327 Log(WW) = 3.2178*Log(TL) 1.9708 0.97 30 2.70 3. 95 0.93 1.53 85.91 2.83% (12) DW = WW*0.1409 87.18 1.43% (52) 0.65 1.18 2.67 3. 22 51 0.79 Log(WW) = 2.8628*Log(TL) 1.6010 R. nasutus S. subtenuis DW = WW*0.1282

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108 estimation. Additionally, eucalanoid c opepod WWs should not be estimated using generalized copepod equations. Body CompositionSpecies Differences. Body composition was variable among adult females of E. inermis, S. subtenuis, P. attenuatus, R. nasutus and R. rostrifrons in the ETNP (Table 4.2). Percent water conten t (% of WW) was signi ficantly higher in E. inermis (93.9%) than R. rostrifrons, R. nasutus, and S. subtenuis (mean values of 86.687.2% of WW). P. attenuatus had intermediate water content (89.3%) and high variability. Ash content followed the same trends, with E. inermis having the highest values (41.8% of DW), while S. subtenuis, R. nasutus and R. rostrifrons showed significantly lower values (13.9-18.0% of DW). P. attenuatus again had intermediate ash content (22.7% of DW). C content was lowest for E. inermis based on %WW and DW, and highest for R. rostrifrons and R. nasutus (Table 4.2). E. inermis C content was signifi cantly lower than R. nasutus R. rostrifrons and S. subtenuis, based on WW and DW. However, when converted to % of ash-free dry weight (AFDW), E. inermis was more similar to P. attenuatus and S. subtenuis and all three were lower than Rhincalanus spp. N content (%WW) was significantly different between S. subtenuis and E. inermis, which showed the highest and lowest values, respectively (Table 4.2). When converted to % of DW, P. attenuatus and S. subtenuis grouped together with high values, while E. inermis and Rhincalanus spp. were lower and simila r. When examining % of AFDW, Rhincalanus spp. showed the lowest values while S. subtenuis and P. attenuatus had the highest, about twice as much. E. inermis fell between the two.

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109 Table 4.2. Body composition for eastern tropical nor th Pacific adult female copepods. Parameters are listed in terms of percent dry weight (DW), wet weight (WW), and ashfree dry weight (AFDW) when applicable. Unless otherwise noted, per centages or weights are reported as the mean standard deviation (number of replicates). C:N, C:P, and N:P denote molar ratios involving carbon (C), nitrogen (N), and phosphorus (P) content E. inermis P. attenuatus R. rostrifrons R. nasutus S. subtenuis WW (mg) 5.36 0.92 (140) 1.45 0.37 (8) 0.68 0.06 (34) 1.25 0.22 (4) 0.92 0.11 (102) DW (mg) 0.34 0.07 (140) 0.18 0.08 (6) 0.10 0.02 (25) 0.23 0.01 (3) 0.12 0.02 (64) Water (% WW) 93.9 0.5 (105) 89.3 2.4 (3) 86.7 2.3 (20) 86.6 (1) 87.2 1.4 (52) Ash (% DW) 41.8 4.1 (105) 22.7 8.1 (2) 13.9 7.6 (20) 16.2 (1) 18.0 3.8 (52) Carbon % WW % DW % AFDW 1.48 0.28 (34) 25.2 3.0 (35) 44.5 5.3 (35) 3.29 0.26 (3) 38.4 0.7 (3) 51.8 0.8 (3) 7.82 1.61 (5) 52.2 3.6 (5) 61.6 5.8 (5) 8.95 0.44 (2) 55.2 0.3 (2) 67.7 0.1 (2) 5.44 0.55 (12) 38.8 2.6 (12) 49.3 3.5 (12) Nitrogen % WW % DW % AFDW 0.32 0.04 (34) 5.5 0.5 (35) 9.7 0.9 (35) 0.80 0.07 (3) 9.3 0.2 (3) 12.6 0.2 (3) 0.81 0.07 (5) 5.5 0.8 (5) 6.5 1.2 (5) 0.88 0.03 (2) 5.4 0.4 (2) 6.7 0.6 (2) 1.34 0.13 (12) 9.5 0.5 (12) 12.1 0.7 (12) Hydrogen % WW % DW % AFDW 0.23 0.05 (34) 4.0 0.9 (35) 6.8 1.2 (35) 0.46 0.09 (3) 5.4 1.1 (3) 7.3 1.6 (3) 1.23 0.33 (5) 8.2 1.3 (5) 9.6 1.0 (5) 1.19 0.09 (2) 7.3 0.3 (2) 9.0 0.3 (2) 0.72 0.13 (12) 5.1 0.8 (12) 6.5 1.0 (12) Phosphorus % WW % DW % AFDW 0.027 0.007 (59) 0.45 0.13 (59) 0.78 0.22 (59) 0.085 0.035 (3) 1.06 0.43 (3) 1.38 0.56 (3) 0.048 0.013 (18) 0.36 0.10 (18) 0.42 0.12 (18) 0.049 0.014 (9) 0.37 0.10 (9) 0.44 0.12 (9) 0.125 0.019 (33) 0.98 0.15 (33) 1.19 0.18 (33) Protein % WW % DW % AFDW 1.63 0.27 (51) 26.9 4.2 (51) 46.2 7.2 (51) 2.47 1.01 (6) 23.0 9.5 (6) 29.8 12.2 (6) 3.23 0.57 (14) 24.3 4.3 (14) 28.3 5.0 (14) 3.93 0.55 (9) 27.9 3.9 (9) 33.3 4.7 (9) 7.04 1.48 (32) 54.9 11.6 (32) 66.9 14.1 (32) Lipid Sac Mass 1 % WW % DW % AFDW 0.28 (0.05-0.54) 145 4.5 (0.9-.8.5) 145 7.7 (1.5-14.6) 145 0.08 (0.03-0.20) 30 0.7 (0. 3-1.8) 30 0.9 (0.4-2.4) 30 7.91 (6.27-11.12) 76 63.8 (50.5-92.4) 76 74.1 (58.6-107.4) 76 4.16 (2.94-6.41) 33 31.0 (21.9-47.8) 33 37.0 (26.2-57.1) 33 0.00 (0.00-0.06) 165 0.0 (0.0-0.4) 165 0.0 (0.0-0.5) 165 C:N 1 5.4 (4.9-5.7) 35 4.8 (4.8-4.9) 3 12.1 (9.1-13.1) 5 11.9 (11.2-12.6) 2 4.8 (4.7-4.9) 12 C:P 2 141.6 100.0 418.5 472.0 112.4 39.9 23.7 N:P 2 26.4 20.8 37.4 1 Due to non-normality or use of ratios, these va lues are listed as median (25th-75th quartile) number of replicates 2 These ratios are only estimates which were calculated usin g the average values for C, N, and P for each species

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110 H content per unit WW was significantly lower in E. inermis than S. subtenuis and Rhincalanus spp. (Table 4.2). Per unit DW, th ree distinct groups were formed, which were statistically distinct from one another. E. inermis had the lowest H content, P attenuatus and S. subtenuis showed intermediate levels, and Rhincalanus spp. had the highest values, which we re twice as high as E. inermis Conversion to AFDW grouped E. inermis with S. subtenuis and P. attenuatus, which were all lower than Rhincalanus spp. P content was highest in S. subtenuis and lowest in E. inermis based on WW, a significant difference (Table 4.2). Per unit DW, E. inermis was intermediate between two statistically distinct groups: S. subtenuis/P. attenuatus and Rhincalanus spp. These trends continued when converted to % of AFDW. Protein content per uni t WW was lowest in E. inermis and highest in S. subtenuis E. inermis showed significantly lower protein levels than S. subtenuis and Rhincalanus spp. (Table 4.2). S. subtenuis also had significantly high er protein contents than P. attenuatus. On a per unit DW basis, S. subtenuis was significantly higher than all other species. Comparisons using AFDW indicated that P. attenuatus and Rhincalanus spp. had the lowest protein contents, while S. subtenuis had levels about twice as high. E. inermis had intermediate protein levels. C:N ratios were lowest in S. subtenuis and highest in Rhincalanus spp. (C:N of about 4 versus 10) (Table 4.2). There were significant differences observed between S. subtenuis and R. rostrifrons, R. nasutus and E. inermis C:P and N:P ratios (estimated from average C, N and P values for each species) showed two distinct groups. For both ratios, Rhincalanus spp. had higher values than the other three species.

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111 Storage lipid mass (estimated through measurement of lipid sac dimensions) was lowest in S. subtenuis and highest in Rhincalanus spp., particularly R. rostrifrons (as percent WW, DW and AFDW) (T able 4.2). Statistically, Rhincalanus spp. grouped together, and had significantly higher storage lipid mass than the other three species. E. inermis also had significantly more storage lipid than S. subtenuis Body Composition Eucalanus inermis Comparisons between male and female E. inermis, as well as between shallow and deep-dwelling E. inermis females showed few differences in body composition (Table 4.3). Males had significantly higher C, N, protein content, and C:N ratios than females. Interannual variabil ity was observed in P content for all E. inermis with 2008 yielding significantly lower concentrations of P. Shallow dwelling females collected in 2007 had significantly highe r water content and lower C and N than other E. inermis female groups. 2008 shallow females showed significantly lower protein cont ent than other female groups. For CHN content analyses, shallow E. inermis females collected in 2007 were grouped based on collection site and analyzed separately. Collection sites included the Tehuantepec Bowl (13N, 105W), Costa Rica Dome (9N, 90W), and an intermediate station (1041.41N, 96.60W). One-way ANOVAs comparing CHN content at these stations found significant differences for all three parameters, particularly for comparisons based on WW.

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112 Table 4.3. Body composition for eastern tropical north Pacific Eucalanus inermis copepods. Parameters are listed in terms of percent dry weight (DW) or wet weight (WW). Samples are separated by sex, collection depth, and year. Un less otherwise noted, percentages or weights ar e reported as the mean standard deviation (number of replicates). C:N, C:P, and N:P denote molar ratios involving carbon (C), nitrogen (N ), and phosphorus (P) content Eucalanus inermis Female Shallow Deep Male 2007 2008 2007 2008 2007 2008 WW (mg) 5.05 1.16 (46) 5.22 0.87 (34) 5.67 0.41 (8) 5.80 0.43 (52) 2.26 0.29 (12) 2.55 0.20 (8) DW (mg) 0.28 0.05 (46) 0.35 0.07 (34) 0.38 0.04 (8) 0.38 0.04 (52) 0.16 0.01 (6) 0.16 0.04 (3) Water (% WW) 94.3 0.4 (31) 93.8 0.4 (26) 93.6 0.5 (4) 93.6 0.4 (44) 94.2 0.3 (3) ND Ash (% DW) 43.7 4.1 (31) 42.5 3.7 (24) 35.4 4.8 (4) 40.4 3.2 (41) 38.9 6.4 (3) ND Carbon % WW % DW 1.35 0.28 (15) 23.5 3.0 (15) 1.58 0.33 (8) 25.1 2.8 (8) 1.66 0.15 (3) 27.8 1.4 (4) 1.57 0.16 (8) 27.1 1.4 (8) 1.72 0.12 (3) 30.8 2.8 (3) 1.82 0.14 (3) 31.7 6.7 (3) Nitrogen % WW % DW 0.30 0.04 (15) 5.2 0.5 (15) 0.34 0.04 (8) 5.5 0.3 (8) 0.33 0.04 (3) 5.6 0.3 (4) 0.34 0.02 (8) 5.9 0.4 (8) 0.33 0.03 (3) 6.0 0.3 (3) 0.36 0.01 (3) 6.2 1.0 (3) Hydrogen % WW % DW 0.23 0.07 (15) 3.9 1.0 (15) 0.23 0.03 (8) 3.7 0.3 (8) 0.19 0.01 (3) 4.9 1.5 (4) 0.24 0.03 (8) 4.1 0.5 (8) 0.25 0.03 (3) 4.5 0.6 (3) 0.26 0.01 (3) 4.5 1.0 (3) Phosphorus % WW % DW 0.033 0.007 (16) 0.56 0.12 (16) 0.022 0.007 (13) 0.37 0.11 (13) 0.028 0.004 (15) 0.48 0.07 (15) 0.025 0.007 (15) 0.39 0.10 (15) 0.030 0.009 (13) 0.51 0.16 (13) 0.020 0.004 (13) 0.35 0.08 (13) Protein % WW % DW 1.72 0.17 (14) 29.6 2.9 (14) 1.33 0.28 (15) 22.8 4.8 (15) 1.76 0.14 (10) 27.7 2.1 (10) 1.79 0.12 (12) 28.1 1.8 (12) 1.65 0.42 (14) 28.4 7.2 (14) 1.81 0.41 (14) 31.1 7.0 (14) Lipid Sac Mass 1 % WW % DW 0.57 (0.46-0.84) 37 9.2 (7.5-13.6) 37 0.09 (0.03-0.38) 36 1.6 (0.5-6.7) 36 0.11 (0.04-0.32) 38 1.7 (0.7-5.1) 38 0.34 (0.06-0.53) 34 5.3 (1.0-8.3) 34 0.26 (0.01-0.48) 41 4.4 (0.2-8.2) 41 0.31 (0.03-0.67) 41 5.3 (0.4-11.6) 41 C:N 1 5.1 (4.9-5.5) 15 5.3 (5.1-5.5) 8 5.7 (5.7-5.8) 4 5.3 (5.0-5.7) 8 6.1 (5.8-6.2) 3 6.2 (5.8-6.2) 3 C:P 2 105.7 185.5 153.2 162.2 148.0 235.1 N:P 2 20.2 39.9 34.3 26.1 30.1 24.4 1 Due to non-normality or use of ratios, these va lues are listed as median (25th-75th quartile) number of replicates 2 These ratios are only estimates which were calculated using the average values for carbon, nitrogen and phosphorus for each spe cies

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113 Body Composition Rhincalanus spp. The body composition of R. nasutus females was similar among individuals collected from the ETNP and the GOC. Due to a similarity in % water, trends observed were similar in terms of WW and DW (Table 4.4). R. nasutus individuals from the ETNP had significantly higher N and pr otein content than females from the GOC. On the other hand, indivi duals from the GOC had significantly higher storage lipid mass. Comparisons of the closely related R. rostrifrons from the ETNP and R. cornutus from the GOM showed more distinct differe nces. Percent water was significantly higher in R. rostrifrons (86.7 versus 80.6% WW), leadin g to major differences in body components in terms of WW, but fewer diffe rences in terms of DW. Per unit WW, R. cornutus showed significantly higher C, H, N, P, protein, and lipid sa c mass. However, per unit DW, significant differences were on ly observed for H and protein content, for which R. cornutus still had higher values than R. rostrifrons Enzyme Activity. Lactate dehydrogenase (LDH) activity was detected in all E. inermis adult males and females, as well as in adult female R. rostrifrons and R. nasutus (Table 4.5). LDH activity levels in S. subtenuis and P. attenuatus were below detection limits for this assay. In E. inermis, shallow dwelling males showed the highest activity levels, while deep dwelling females had the lowest, a statistically significant difference. Results were similar using both units per gr am WW and per gram protein. Statistical comparisons were not able to be made among species, owing to small sample sizes. LDH activity per gram WW appeared to be similar among adult females of E. inermis, R.

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114 rostrifrons and R. nasutus However, in units of activity per gram protein, E. inermis showed 2-3 times higher activity, on average, than Rhincalanus spp. Table 4.4. Body composition for Rhincalanus cornutus from the Gulf of Mexico and R. nasutus from the Gulf of California. Parameters are listed in terms of percent dry weight (DW) or wet weight (WW). Unless otherwise noted, percentages or weights are reported as the mean standard deviation (number of replicates) R. cornutus R. nasutus WW (mg) 0.80 0.12 (10) 1.11 0.13 (11) DW (mg) 0.12 0.03 (3) 0.15 0.04 (14) Water (% WW) 80.6 0.6 (3) 85.8 2.5 (11) Ash (% DW) ND 15.3 3.8 (11) Carbon % WW % DW 10.53 0.48 (3) 54.2 2.4 (3) 7.39 0.91 (3) 52.5 2.5 (3) Nitrogen % WW % DW 1.14 0.02 (3) 5.9 0.1 (3) 0.74 0.03 (3) 5.3 0.3 (3) Hydrogen % WW % DW 2.02 0.06 (3) 10.4 0.4 (3) 1.44 0.21 (3) 10.2 0.7 (3) Phosphorus % WW % DW 0.078 0.027 (7) 0.40 0.20 (7) 0.043 0.011 (14) 0.32 0.08 (14) Protein % WW % DW 6.90 0.64 (5) 35.5 3.3 (5) 3.30 0.70 (16) 23.4 5.0 (16) Lipid Sac Mass 1 % WW % DW 13.3 (8.8-17.5) 24 68.7 (45.4-90.1) 24 10.3 (8.3-14.8) 28 72.8 (58.5-104.5) 28 C:N 1 9.2 (9.0-9.4) 3 10.2 (9.6-10.4) 3 C:P 2 135.0 171.9 N:P 2 14.6 17.2 1 Due to non-normality or use of ratios, these values are listed as median (25th-75th quartile) number of replicates 2 These ratios are only estimates which were calculated using the average values for carbon, nitrogen and phosphorus for each species

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115 Table 4.5. LDH activity for eas tern tropical north Pacific copepods. LDH activity was measured in units ( mol substrate utilized per minute) per gram wet weight (WW) or protein. Activity levels are re ported as the mean standard deviation (number of samples). BD denotes levels of LDH that were below detection. All assays were determined on homogenized copepods with 1:10 dilutions except P. attenuatus, which was a 1:15 dilution. Species LDH Activity (Units per g WW) LDH Activity (Units per g Protein) E. inermis Female Shallow Deep Male 2.29 1.31 (8) 1.73 1.00 (6) 2.34 1.02 (3) 1.12 0.38 (3) 3.96 0.29 (2) 139.0 79.4 (8) 108.9 66.6 (6) 154.3 66.9 (3) 63.5 21.3 (3) 229.1 16.9 (2) P. attenuatus BD (1) BD (1) R. rostrifrons 1.59 (1) 49.3 (1) R. nasutus 1.43 (1) 36.4 (1) S. subtenuis BD (2) BD (2) Survivorship Studies. Survivorship was high (96-100%) for S. subtenuis, E. inermis and R. rostrifrons in the 15 and 100% oxygen saturation le vel metabolic experiments at 10C (Figure 4.3). In contrast, at 6% oxygen saturation ( 20 M O 2 ), only E. inermis, R. rostrifrons and R. nasutus still had high survivorship (96, 93, and 87%, respectively), while S. subtenuis had only 22% survivorship after 12-15 hours.

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0 20 40 60 80 100 120 6% 15% 100%Percent Oxygen SaturationSurvivorship (% of Total) E. inermis S. subtenuis R. rostrifrons R. nasutus 23 23 14 64 209 32 139 357 0 49 0 109 Figure 4.3. Percent survivorsh ip of eucalanoid copepods du ring metabolic incubations. Bars represent the percentage of individu als surviving at the end of an incubation experiment at 10C and either 6, 15, or 100% oxygen saturation. Numbers above bars indicate total number of individuals used in experiments. Results are shown for Eucalanus inermis (white columns), Subeucalanus subtenuis (striped columns), Rhincalanus rostrifrons (checkered columns), and R. nasutus data (dotted columns). 116

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DISCUSSION Results of this study indicated that adult females of Eucalanus inermis, Pareucalanus attenuatus, Subeucalanus subtenuis, Rhincalanus rostrifrons and R. nasutus could be separated into four groups base d on similar ecological attributes within genera. Eucalanus inermis E. inermis an endemic species from the eastern tropical Pacific, was found throughout the upper 1,000 m of the water column, with peaks in abundance near the chlorophyll maximum and above and below the OMZ core (Chen 1986, Sameoto 1986, Vinogradov et al. 1991, Sa ltzman & Wishner 1997b). E. inermis body composition was characterized by high water and low organic matter content per unit WW. This low level of body tissue fo r its size allowed E. inermis to have a low metabolic rate (Figure 4.4) compared to S. subtenuis and R. rostrifrons (Chapter 2). E. inermis also had moderate LDH activity, indicating an ability to utilize the glycolyt ic pathway for energy production under hypoxic conditions (Hocha chka & Somero 2002). LDH activity, coupled with low metabolic rates, likely allows E. inermis to have a high tolerance for low oxygen environments. Although E. inermis body contents were low ba sed on WW relative to other eucalanoid species, comparing com ponents on the basis of %AFDW put E. inermis within the range of the other species. Co mparisons based on AFDW were useful because they looked only at the tissue components, and di d not include salt or water content. One interesting trend that emerged with AFDW comparisons was that E. inermis actually had intermediate protein levels (Table 4.2). Thus, E. inermis had more muscle mass per unit 117

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0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 10 17 23Temperature (C)O2 Consumption (nmol (mg WW) -1 hr -1) E. inermis R. rostrifrons S. subtenuis 23 4 1 6 1 0 5 5 12 Figure 4.4. Oxygen consumption rates for Eucalanus inermis, Rhincalanus rostrifrons, and Subeucalanus subtenuis at different temperatures and under conditions of oxygen saturation. Rates are medians for specifi ed temperatures, with bars noting the 25 th and 75 th quartiles. Numbers are the number of replicate experiments. Rates are for E. inermis (white columns), R. rostrifrons (checkered columns), and S. subtenuis (striped columns). AFDW than Rhincalanus spp. or P. attenuatus although not as much as S. subtenuis suggesting an intermediate activity level. As E. inermis had a wide vertical range, muscle might (i.e. protein) might be needed for making long migra tions (Donnelly et al. 1993). Fatty acid profiles of storage lipids suggested that adult E. inermis largely fed omnivorously on surface material (Chapter 3) regardless of their collection depth, indicating that all individuals came to the surf ace periodically to feed. Compared to the other copepods in this study, E. inermis had a moderate amount of storage lipid, primarily composed of triacylglycerols (Chapter 3) Storage lipid sac mass was higher per unit WW and DW than those of S. subtenuis and P. attenuatus but not nearly as high as 118

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Rhincalanus spp. These moderate lipid stores would supply them with energy during times of reduced feeding at depth. Relativ ely low metabolic demand and ability to use alternative metabolic pathways is another strategy that would allow E. inermis females to reside in low oxygen waters for periods of time. Few differences were seen among the three groups of adult E. inermis examined in this study. It appeared that males had hi gher C, N and protein levels than females, possibly indicating greater muscle mass and organic matter. These males might have been more active than females, as they o ccurred almost exclusively in the upper 120 m of the water column (Longhurst 1985; K. Wishne r, personal communication). This would leave them less exposed to low oxygen than the females, which showed peak abundance around 300-350 m during this study (K. Wi shner, personal communication). LDH levels also appeared to be variable, with males having the highest activities. As males had about half the mass of females, these activity differences could be due to a difference in size, which has been found to a ffect enzyme activity rates (Thuesen et al. 1998). It was also possible that males, ha ving higher protein leve ls, might have higher metabolic rates which require supplementati on of anaerobic pathways in relatively low oxygen conditions encountered in the upper 100 m. Unfortunately, no metabolic data were available for males. Additionally, LD H assays were primarily carried out to establish presence or absence of LDH, so replicate numbers were low. Further samples should be run to ascertain firm differences among the groups. This study also hoped to illuminate differences between deeper and shallower dwelling adult females, as ther e appeared to be a resident population at depth. In more temperate and seasonal environments, E. elongatus and E. californicus were seen to 119

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undergo ontogenetic migration and have a dorma nt period at depth (Ohman et al. 1998, Kasyi 2006). While dormancy was not expected within the ETNP sy stem, it was unclear whether these deeper individuals might repr esent a specific cohort, for instance, a particular stage in the reproductive cycle. Comparison of shallow and deep females showed differences, although th ese were primarily based on either 2007 or 2008 shallow samples which had significantly higher or lower proportions of a body component compared to all other shallow and deep gr oups. Additionally, substantial variation was seen in the shallow females in terms of C, H, and N content based on collection site. This suggested that differences observed in this study were likely the result of natural variability captured during sampling. Previous work had shown that this species likely reproduces continuously, due to the co-occurr ence of all naupliar and copepodite stages during sampling (Longhurst 1985, Hidalgo et al. 2005a, b). This might make it difficult to observe cohort variability through body composition studies, as cohorts might be overlapping, obscuring potential di fferences. It has been sugge sted that changes in mean body size might be useful for distinguishing cohor ts in this species (H idalgo et al. 2005a), and deep females did appear to be larger than shallow ones during both sampling years (Table 4.2). However, this was likely an artif act of the copepod sorting process, as size comparisons using full MOCNESS data showed no change in average size of adult females with depth (K. Wishner, persona l communication). A dditional comparisons using oxygen consumption rates indicated si milar metabolic rates between the two groups, although metabolic ratios suggested in creased lipid utilization by the deeper individuals (Chapter 2). This was consistent with lipid biomarker data, which suggested that all adult E. inermis fed primarily near the chlo rophyll maximum (Chapter 2). 120

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Feeding at depth was rare, and thus lipid st ores could be used as an energy supplement when individuals resided there. Therefore, data from this study were inconclusive to ascertain if deep and shallow females repr esented different cohorts. Further work comparing egg and gonad development in shallow and deep individuals would help to determine if there is vert ical separation based on timing in the reproductive cycle. Subeucalanus subtenuis S. subtenuis employed a different strategy than E. inermis. S. subtenuis is reported to primarily occur in the upper 100 m of the wate r column, in more oxygenated waters (Chen 1986, Sameoto 1986, Saltzman & Wishner 1997b; Figure 1). Their vertical distribution a ppeared to be limited due to oxygen levels rather than a preference for surface waters. S. subtenuis did not have detectab le levels of LDH, indicating little anaerobic ability to help cope with oxygen-limited conditions. Survivorship studies showed that only 22% of adult females were able to survive 12-15 hours at <20 M oxygen, while survivorship was 100% in more highly oxygenated water (Figure 4.3). Thus, vertical distributions would be lim ited, as oxygen levels of <20 M occurred as shallow as 50 meters in the Tehuantepec Bowl and somewhat deeper in the Costa Rica Dome (Figure 4.1). Indeed, S. subtenuis abundance decreased to almost zero at depths where oxygen levels were < 20 M during 2007 (Figure 2.1). Of the five species, S. subtenuis had the highest protein co ntent. This suggested that they were the most active, and therefore required a greater muscle mass. The higher activity also was supported by metabolic data (Figure 4.4), which showed that S. subtenuis had the highest metabolic rate compared to R. rostrifrons and E. inermis across a large temperature range. The large amount of muscle wa s likely due to higher activity levels. Observations of live S. subtenuis indicated that they were more active than other 121

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eucalanoid species observed in this study. As S. subtenuis was found within a relatively narrow depth range, vertical migration probably did not occur to any great extent. Instead, activity was within the surface layer, and possibly included predator avoidance and prey capture. Their food sour ce appeared to be the same as E. inermis shallow particulate material (Chapter 3). However, they did not accumulate as much storage lipid as E. inermis, although these two species shared tr iacylglycerols as the major storage lipid (Chapter 3). This sugge sted one of several possibilitie s. First, their metabolism may be sufficiently high that little exces s lipid was available for storage. Another scenario is that storage lipids were not necessary for this su rface-dwelling, active copepod, and therefore they did not prioritize their accumula tion. As this species may not experience food limitation or undergo di apause, storage lipids would not be necessary. S. subtenuis had 2-3 times higher P content than Rhincalanus spp. and E. inermis. As was observed with C:N ratios, copepods we re separated into tw o groups based on C:P and N:P ratios. The first gr oup, with higher ratios, was Rhincalanus spp. The second group was comprised of S. subtenuis, P. attenuatus and E. inermis This suggested a similar overall biochemical strategy with regards to elemental composition for S. subtenuis, P. attenuatus, and E. inermis, even though individual elemental levels varied based on weight percent. Rhincalanus spp. showed a separate strategy, seemingly characterized by lower P and higher C content than the other group. While N:P ratios were high, this was likely due to lower P rather than higher N, given the low protein levels of this species (Sterner & Elser 2002). Higher C may be attributed to high levels of storage lipid, which was a major contributor to C and H content (Sterner & Elser 2002, 122

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Ventura 2006), both of which had the highest concentrations in Rhincalanus spp. The majority of body P content is primarily attribut ed to nucleic acids a nd nucleotides, as well as some from phospholipids (Sterner & Elser 2002, Ventura 2006). The lower P levels in Rhincalanus species may also be due to their larg e levels of storage lipids, leaving phospholipids to occupy a smaller proportion of lipid material. Lipid composition data (Chapter 3) indicated that the proportion of phospholipids was often 5-10 times higher in E. inermis, P. attenuatus, and S. subtenuis than in Rhincalanus spp. Furthermore, differences in P content based on nucleotid e levels is a promising explanation, as RNA/DNA levels may vary am ong species depending on condition or timing within the reproductive cycle (Bmstedt 1986). Thus, elemental ratios emphasized a basic difference in biochemical strategies between Rhincalanus spp. and the three other eucalanoid species. Rhincalanus rostrifrons and R. nasutus. R. rostrifrons and R. nasutus together comprised the third group. R. rostrifrons was much more abundant than R. nasutus in the ETNP however, they both had a similar vertical distribution (Chen 1986, Sameoto 1986, Saltzman & Wishner 1997b). Abundances were hi ghest right above the OMZ core, in the upper oxycline. In addition, lower abundances of individuals also occurred in the surface waters and below the OMZ core in the lower ox ycline. Some reports specifically placed these species at the edge of the OMZ core, but not within it, and suggested that these species were limited by oxygen concentrations below 0.5 M O 2 (Sameoto 1986, Vinogradov et al. 1991, Saltzman & Wishner 1997b). During this study, Rhincalanus spp. were only observed in waters with 1.5 M O 2 while E. inermis was found in 123

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waters of <1.5 M O 2 (K. Wishner, personal communication). Like E. inermis, LDH activity was observed in Rhincalanus spp. However, based on activity per gram protein, LDH activity levels were only about one-h alf to one-third th e levels found in E. inermis This may point towards a lower anaerobic cap acity, explaining their absence from the lowest oxygen regions. However, within taxa trends have been found with size (Thuesen et al. 1998). The WW of E. inermis was approximately 5-10 times higher than WWs of Rhincalanus spp., indicating a large size gap. Differe nces in LDH activity might be due to such a relationship, and not necessarily represent differential adaptation to OMZs. Even though the metabolic rate of R. rostrifrons was similar to that of S. subtenuis at 10C, its tolerance for low oxygen was superior. Survivorship at <20 M was more than 85% for Rhincalanus spp., compared to only 22% for S. subtenuis. Their swimming activity level also was much lower generally than S. subtenuis, and often little to no motion was observed in Rhincalanus spp. during sorting. Protei n levels indicated that S. subtenuis had approximately twice as much muscle mass as Rhincalanus spp., providing further support for observations of la rge differences in activity levels. One of the most dis tinctive features of Rhincalanus spp. was the large storage lipid sac that occupied a major portion of its body cavity. Th e percent of wet mass of storage lipids was more than 10 times that of E. inermis, P. attenuatus or S. subtenuis. Rhincalanus spp. also had higher C and H content (the major components of lipids), as well as C:N ratios near 10, due to high lipid an d low protein levels. The need for such large amounts of storage lipids in this envi ronment was not clear, and may be due to a genetic predisposition. Rhincalanus spp. globally are known for their extremely high total and storage lipid levels, with total lipid levels between 16-69% of dry weight (Lee & 124

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Hirota 1973, Morris & Hopkins 1983, Kattner et al. 1994, Sommer et al. 2002, SchnackSchiel et al. 2008). Storage lipids, often present in a large oil sac (Lee et al. 2006), commonly comprised 61-92% of total body li pid in adults (Lee et al. 1971a, Lee & Hirota 1973, Graeve et al. 1994a, Kattner et al. 1994, Kattner & Hagen 1995, Sommer et al. 2002, Schnack-Schiel et al. 2008). Additionally, the composition of storage lipids for this group was primarily wax esters, not triacylgly cerols as found in E. inermis and S. subtenuis Predominance of one type of storage lipid over an other is thought to occur due to a variety of factors. For instance, accumulating wax esters as the prim ary storage lipid in copepods has long been thought to occur primarily in deep sea (>600 m) and high latitude herbivorous organisms (Lee et al. 1971a, Lee & Hirota 1973), as well as those undergoing diapause (Lee et al. 2006), as those individuals require surplus energy stores for periods of little to no feeding. Rhincalanus spp. in the ETNP, however, did not meet those crite ria, as there was no evidence or necessity for diapause, and they occurred primarily above 400 m. This is only one of many exceptions to the ru les that have been noted in the literature (e.g., Kattner et al. 1981, Hagen et al. 1993, Hagen et al. 1995, Ward et al. 1996, Williams & Biesiot 2004). In the case of the Eucalanidae family, there is likely a large genetic component controlling storage lipid ac cumulation patterns (Chapter 3). Genetic analyses of 16S rRNA and ITS2 gene loci indicated that Pareucalanus and Rhincalanus form their own monophyletic group and Subeucalanus and Eucalanus form another, consistent with the observation of was ester versus triacylglycerol dominance (Chapter 3; Goetze 2003). Lipid biomarker analyses suggested that the feeding strategy of R. nasutus and R. rostrifrons was different than that observed in the other three species. While E. 125

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inermis, P. attenuatus and S. subtenuis all seemed to feed in the vicinity of the chlorophyll maximum, Rhincalanus spp. likely fed on sinking particulate material derived from the surface (Chapter 3). Pareucalanus attenuatus P. attenuatus comprised a final group. This was surprising, as the vertical distributi on of this copepod was similar to that of S. subtenuis P. attenuatus was found primarily in the upper 100-150 m, w ith peak abundances from about 30-100 m (Chen 1986, Sameoto 1986, Saltzman & Wishner 1997b). P. attenuatus also shared several other characteristics with S. subtenuis Both species had high levels of P, and C:P and N:P ratios were similar to each other and to those in E. inermis. Also, both P. attenuatus and S. subtenuis had no detectable LDH activ ity, indicating little anaerobic capacity. These results suggested that P. attenuatus also resided in ne ar-surface waters due to an inability to tolerate prolonged exposure to low oxygen. Storage lipid mass, C and H content, and C:N ratios also were similar between P. attenuatus and S. subtenuis However, P. attenuatus primarily accumulated wax ester storage lipids while S. subtenuis accumulated mostly triacylglyerols. As men tioned above, this difference might be due to genetic factors. Biomarker analyses of these storage lipids indicated that S. subtenuis and P. attenuatus likely shared a common food source at the chlorophyll maximum (Chapter 3). Another major difference between S. subtenuis and P. attenuatus was protein content. P. attenuatus had protein levels and presumably muscle mass that were simlar to those in Rhincalanus spp., the least active c opepod group. They also had slightly higher water and ash content than Rhincalanus spp. and S. subtenuis, indicating that less tissue 126

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material was present in the body. Thus, this species appeared to occupy an intermediate position between the other three groups in terms of an ecological strategy. Like S. subtenuis, P. attenuatus remained in near-surface waters to minimize exposure to extremely low oxygen levels. Thus, substantia l lipid storage was not necessary, as food levels are relatively consiste nt throughout the year. Reduc ed activity levels and lower respiring tissue per WW are strategies that would help this species tolerate lower oxygen levels observed in the upper 50 to 100 m. Unfortunately, P. attenuatus was only present in low abundances in our sampling area, limiting the number of measurements. Additional studies of this species would be useful in further elucid ating the extent of differences between P. attenuatus and the other eucalanoid species of this region. Comparison with Other Eucalanoid Copepods While it was apparent that these four groups of copepods utilized different strategi es within the ETNP region, it was difficult to ascertain if such findings were specific to this region or based on more general differences among taxa. In a review on adap tations to low oxygen zones, Childress and Seibel (1998) pointed out that many features of organisms inhabiting low oxygen regions of the water column are adaptive, but not n ecessarily adaptations specifically for life in such areas. In other words, some orga nisms are able to e xploit low oxygen zones because of previously evolved traits, and not due to adaptations that have evolved specifically in response to oxygen limitation. To determine if pa tterns observed here were similar to other systems, ETNP copepod traits were compared with traits in conspecifics and congeners from other collection sites. 127

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R. nasutus collected from the Guaymas Basin region of the Gulf of California (GOC) had a similar vert ical distribution to R. nasutus in the ETNP, with peak abundances between 250-500 m (Vinogradov et al. 2004). Like the ETNP, the Guaymas basin had a pronounced oxygen minimum zone (see Figure 1 in Rosa & Seibel 2010), where oxygen levels were as low as 2 M during our collection period (ETNP values reached approximately 0.9 M). Globally, R. nasutus is known as a subsurface species (Grice 1962, Lang 1965) with reported maxi mum abundances around 300-500 m (Ohman et al. 1998, Schnack-Schiel et al. 2008). I ndividuals from the GOC had higher storage lipid content, on average, than those in th e ETNP. However, the fatty acid composition of the two were extremely similar (Cass et al ., in review), suggesting that there was not a difference in lipid accumulation strategies. Di fferences in overall storage lipid could be due to a variety of factors, including co llection time (June versus October-November), timing in reproductive cycle, or differences in food abundances at the two locations. Individuals from the ETNP al so had significantly higher protein content, 20% higher in the ETNP samples compared to the GOC samples. This could also be due to differences in collection season or location variability, or could represent a real trend of higher protein content in ETNP individuals. Furt her sampling throughout the year would be helpful in determining the underlying processe s influencing these trends. Overall, it appeared that differences between the two sample sites were minimal, and these copepods were similar in most aspects of body composition. Body composition data reported in the literature for R. nasutus have been somewhat variable. R. nasutus from the California Current regi on appeared to be similar to those of the ETNP and GOC, with protein content around 25% of DW. Water content 128

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was slightly lower (82% of WW), likely related to the higher lipid content (55% of DW) (Ohman et al. 1998). The California Curre nt region also has low oxygen (<20 M in some areas), although not as low as the ETNP and GOC (Paulmier & Ruiz-Pino 2009). R. nasutus from the northern Pacific Ocean ha d similar amounts of carbon and hydrogen (52.2 and 8.3% of DW, respectiv ely) and water content (86.5% of WW) as individuals in this study (Omori 1969). However, N cont ent was almost twice as high (9.9%), suggesting higher protein and muscle mass in th ese individuals. Si nce the north Pacific has higher oxygen concentrations than the ETNP, GOC and California Current regions (Paulmier & Ruiz-Pino 2009), this suggest s that individuals in higher oxygen environments may have higher protein cont ents and greater muscle mass, indicating higher activity levels. R. nasutus off the coast of western Africa (minimum oxygen levels >40 M; Paulmier & Ruiz-Pino 2009) were repo rted to have protein contents of 11.3% of WW (Flint et al. 1991). However, these va lues were reported based on estimated WWs using the equations of Kuzmichova ( 1985), which we found to underestimate R. nasutus WW by approximately 30%. Taking that in to account, protein levels could be around 8% of WW, about twice as high as values reported in this study. Flint et al. (1991) reported that R. nasutus collected in the east ern boundary currents of the Pacific Ocean also showed similar protein levels to those collec ted in the Atlantic. Such collection sites would be similar in oxygen levels to the California Current site of Ohma n et al. (1998), in which similar levels of protein were found between R. nasutus individuals in that region and the ETNP. This contradicts the idea th at low oxygen may be associated with lower protein levels. Further research is ne eded to explore this relationship in R. nasutus 129

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Flint et al. (1991) repor ted no LDH activity in R. nasutus It was unclear whether individuals for this assay were from the Atlantic or Pacific collection sites, allowing no speculation concerning the relationship betw een LDH activity and environmental oxygen conditions. However, anot her enzyme study examining Calanus pacificus determined that LDH activity was present, when Flint et al. reported no activity (Thuesen et al. 1998). In our assays, many individuals were co mbined to ensure that activity would be detected. Thus, it is possible th at LDH activity can be found in R. nasutus outside of our ETNP study site, but that an insufficient amount of material or a less sensitive assay was used in the Flint et al. (1991) study. R. cornutus individuals were collected from th e eastern Gulf of Mexico (GOM). Genetic work using 16S rRNA and ITS2 loci grouped them closest to R. rostrifrons out of the four Rhincalanus spp. (Goetze 2003, 2010). Historically, R. cornutus and R. rostrifrons have been considered se parate subspecies of R. cornutus (Lang 1965), however Goetzes recent work supports separation into two species. Lipid profiles suggested high biochemical similarity between th ese two species (Cass et al., in review). The GOM is different from the ETNP in that oxygen levels are always above 100 M (Morrison & Nowlin 1977, Jochens & DiMarco 2008, Paulmier & Ruiz-Pino 2009). Per WW, R. cornutus showed significantly lower water content (80.6 versus 86.7% WW) and higher C, H, N, P, protein, and lip id sac mass compared to that in R. rostrifrons. On a DW basis, R. cornutus had a 50% higher protein content than R. rostrifrons Water, protein, and lipid content in our specimens were similar to those reported for R. cornutus collected in the same area several decades earlier (Mo rris & Hopkins 1983), indicating that these values were typical for this spec ies in this location. The depth of maximum 130

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abundance noted in Morris and Hopkins (1983) of 350 m was consistent with distributions of R. rostrifrons in the ETNP, ruling out vertic al distribution as a factor influencing protein levels. Protein levels reported for R. cornutus in the western Atlantic (corrected for WW estimates) also were higher (4.7% of WW) than in R. rostrifrons (Flint et al. 1991). This area has interm ediate oxygen levels between the GOM and ETNP. Unfortunately, it was hard to tell if these differences in protein were due to species-level differences or reflective of th e oxygen environment. Further measurements of R. rostrifrons from additional sites in the Pacific Ocean are needed to interpret trends between protein content and oxygen environment. Only one other paper (Flint et al. 1991) investigated body composition (C, P, protein, and lipid) of E. inermis and values were comparable to the study here. LDH activity levels report ed by Flint et al. (1991), however were much higher than those observed in this study. Instead, values fr om this study were similar to many deepdwelling calanoid copepods in the California Current system (Thuesen et al. 1998). Even though Eucalanus spp. from Flint et al. (1991) were not collected within the ETNP, they were collected from low oxygen areas. The congener E. hyalinus also was reported to have a similar body composition per WW as observed for E. inermis. Additional work on E. hyalinus in the GOM noted high water conten t (92.2% of WW) (M orris & Hopkins 1983). E. californicus from the California Current had similar values for protein (20% DW), and water content (93% of WW) as E. inermis, but slightly higher values for lipid contents (11% of DW) (Ohman 1997, Ohman et al. 1998). This variation in lipid was not surprising, as evidence for dormancy in E. californicus was found, which would require more extensive lipid stores It is thought that the Eucalanus genus comprises a distinct 131

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physiological group, characterized by high wate r and low organic matter content, leading to less actively respiring tissue and allowing a lifestyle more similar to gelatinous plankton (Flint et al. 1991, Ohman et al. 1998). Such char acteristics would allow them to exploit some of the benefits of the lethargic lifestyle employed by typical gelatinous organisms, including decreased metabolic ra te and only slight ne gative buoyancy. This would be advantageous in low-oxygen conditions but does not necessarily seem to be an adaptation specifically for such an environment. E. hyalinus was found in the highoxygen GOM region, and still had high water content and low organic matter (Morris & Hopkins 1983). In the GOM location, this species did have slightly higher protein content (2.7% of WW) than that reported for E. inermis, and 3 times higher protein content than E. hyalinus values reported by Flint et al. (1991) in low oxygen regions. This further supports the idea that high protein contents might be associated with life in higher-oxygen environments for this c opepod group. The only member of the Eucalanus genus which did not show high water content was E. bungii collected in the northern Pacific, an area of higher oxygen content than the ETNP (Omori 1969, Paulmier & RuizPino 2009). This species had only 88% water, possibly due to the hi gher C (50% of DW) and N (7.6% of DW) content than E. inermis. Its C levels were comparable to Rhincalanus spp., suggesting that large lipid accumulation accounted for this difference. N content was about 40-50% higher than E. inermis which suggests slightly higher protein content in E. bungii However, direct comparisons between these two species have limited applicability, as the collection areas differ greatly in terms of seasonality and other environmental factors. 132

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No previous research was available for body composition on S. subtenuis and P. attenuatus. However, Morris and Hopkins (1983) reported on water, protein, lipid and ash contents of S. monachus and P. sewelli in the GOM, which are closely related to S. subtenuis and P. attenuatus, respectively (Goetze 2003). S. monachus was slightly smaller than S. subtenuis (0.07-0.08 mg DW), but had simila r protein contents (5.3% of WW). Water content was lower (83.5% of WW), possibly due to the large amount of lipid material (9.3% of WW). These levels were similar to R. cornutus in the same region, indicating substantial lipid accumulation for S. monachus S. monachus in the GOM had a peak of maximum abundance at 550 m, substantially deeper than S. subtenuis in the ETNP. Generally, S. monachus is classified as a subsurface epiplanktonic species, while S. subtenuis is epiplanktonic (Fleminger 1973). This deeper distribution might explain some of the lipid accumulation (Lee et al. 1971a), as food is not as regularly abundant at depth. This also suggests that S. monachus was not a good comparison species, as many differences might already exis t that are unrelated to oxygen levels. P. sewelli and P. attenuatus are both described as epip lanktonic species, with P. sewelli showing a circumgloba l distribution, while P. attenuatus is restricted to the Pacific Ocean (Lang 1965, Fleminger 1973). Despite this, P. sewelli had a peak in maximum abundance at 600 m in the GOM (M orris & Hopkins 1983). Water content was similar between P. sewelli and P. attenuatus and P. sewelli lipid content was low relative to all the eucalanoids examined conc urrently. The main difference between the two species was that P. sewelli showed higher protein content (43% of DW), similar to that of Subeucalanus spp. It was unclear whether this indicates that P. attenuatus in general had low protein content, or P. attenuatus specifically in the ETNP had low 133

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protein content. Regardless, we can c onclude that low protein content is not characteristic of the entire Pareucalanus genus. Low lipid conten ts might be conserved across species, as P. sewelli still showed low lipid levels despite their depth in the GOM. Conclusions In conclusion, this study determined that E. inermis, S. subtenuis, P. attenuatus, R. rostrifrons and R. nasutus formed four groups based on genera that differed in biochemical, physiological, and ecological characteristics in the ETNP. R. rostrifrons and R. nasutus were the only two species simila r enough to classify together. E. inermis was able to tole rate exposure to low oxygen through adopting a physiological strategy more similar to gelatinous plankton than typical copepods. Adult females had high water content and low organic matter, re sulting in a decreased metabolic demand. This species also had an alternative anaerob ic pathway, supporting its tolerance to low oxygen conditions. Rhincalanus spp. were characterized by particularly high lipid content and low protein levels. LDH was present in moderate levels, leading to tolerance for low oxygen conditions when coup led with low general activity. S. subtenuis appeared to avoid the OMZ, as survivorship for this species was much lower than for E. inermis or Rhincalanus spp. at <20 M O 2 S. subtenuis also had the highest protein and lowest lipid levels of the group. P. attenuatus showed a similar vert ical distribution to S. subtenuis, but had a different lipid accumulation st rategy and one of the lowest protein levels, indicating low muscle mass and activity. These factors appeared to separate the two species in this region. It was difficult to tell whether some of the observed features of these species were adaptations to conditions in the ETNP OMZ system, or if they were simply 134

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characteristics of the species or genera. P. attenuatus, E. inermis and Rhincalanus spp. in the ETNP generally had lower protein content th an congeners or conspe cifics residing in higher oxygen environments. Such differences in protein levels (i.e., muscle mass) might be adaptive for low oxygen environments, as lo wer activity levels (a potential behavioral adaptation) would lead to d ecreased metabolic demand. Ho wever, too few comparisons exist in the literature to draw firm conclusi ons. Metabolic studies thus far have not recorded large differences in metabolic rates between populations of sp ecies that reside in high versus low oxygen environments (Child ress & Seibel 1998). Nevertheless, these studies often measured routine or resting metabolic rates (RMR), in which activity was minimal (Withers 1992). RMR can differ base d on maximal activit y rates, as an energetic lifestyle may lead to increased m itochondrial density or size of energetically active organs (Reinhold 1999). However, activ e rates can be 5-20 tim es higher than the standard metabolic rate (measured or extrapolated as the rate for zero activity) (Hochachka & Somero 2002), so it is still likely that majo r differences would be most pronounced during activity. The possibili ty of different pr otein levels and in situ active metabolic rates poses an interesting question for future research dealing with adaptations to low oxygen zones. 135

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CHAPTER FIVE Summary of Major Conclusi ons and Future Research The research presented here assessed meta bolic processes, diet, storage lipids, body composition, and behavioral strategies of a dominant zooplankton group, the eucalanoid copepods in relation to environmental conditions within the eastern tropical north Pacific (ETNP) oxygen minimum zone (OMZ). One major finding was that the interaction between temperature and oxygen c oncentration strongly influe nced urea excretion rates for Eucalanus inermis, Rhincalanus rostrifrons and Subeucalanus subtenuis Knowledge of such interactions is necessary for accura te modeling of nitrogen cycles in OMZ and other oceanic regions. Nitrogen cycling in OM Zs is particularly complicated due to the inclusion of significant amount s of denitrification and ana mmox (a recently discovered pathway which utilizes ammonium as a substr ate). Further studies comparing urea and ammonium excretion rates of zooplankton in OMZ and non-O MZ regions would provide a better understanding of the role of zooplan kton as a source of urea and ammonium, as well as improve our ability to predict the effects of oxygen and temperature on metabolic rates. Lipid analyses revealed previously unknown information on feeding strategies and lipid storage in eucalanoid copepods Lipid biomarkers indicated that E. inermis, S. subtenuis, and Pareucalanus attenuatus all fed on a similar food source near the 136

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chlorophyll maximum, and did not discriminate greatly among partic les during feeding. R. nasutus and R. rostrifrons appeared to be feeding deeper in the water column, perhaps on sinking particulates recently derived from su rface material. In addition, an unexpected result was the differing roles of wax esters and triacylglycerols as storage lipids for this copepod group. Triacylglycerols were more re flective of diet, while wax esters were primarily based on modification of dietary fa tty acids and de novo synthesis. These results suggested that genetic influences play a greater role in determining lipid contents than previously believed. As few eucalanoid species have been examined in detail for lipid components, future work should expand upon existing knowledge to determine the extent of genetic influences on lipid accumulation in this family. Overall, eucalanoid copepods utilized many di fferent ecological strategies in the ETNP OMZ system. E. inermis was found at lower oxygen concentrations than any other species examined and showed significantly lower metabolic rates due to its high water content. This species also had moderate levels of activity for lactate dehydrogenase (LDH), which catalyzes the anaerobic pathway in copepods, and would aid in survival under low oxygen conditions. S. subtenuis on the other hand, wa s not well-adapted for living at low oxygen, as it had relatively high metabolic rate s, no detectable levels of LDH activity, high activity levels and protein co ntent, and low survivorship at <20 M.. This was unsurprising given that S. subtenuis was generally found w ithin the upper 80 m of the water column. P. attenuatus had a similar vertic al distribution as S. subtenuis yet was distinct from S. subtenuis. P. attenuatus had lower protein content and a different lipid accumulation strategy, indicating a separate ecological grouping. R. nasutus and R. rostrifrons were similar in most respects and appeared to have the same ecological 137

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strategy. Both primarily resided in the upper oxycline and were characterized by high storage lipid contents, relatively high routine metabolic rates but low activity levels and protein content, moderate levels of LDH activity, and overall high survivorship at <20 M. It was unclear whether the observed char acteristics were adaptations specific to OMZ regions. It is possible that some of these character istics are unique to the ETNP populations or species, but they may also repr esent more general ta xonomic features. For example, high water content in Eucalanus spp. is common to this genus throughout the oceans. Nevertheless, information about su ch characteristics is important to better understand how zooplankton in oxygenated ecosy stems might respond to the expansion of OMZs or to increased stratification and decreased oxygen concentrations due to climate warming. Improved knowledge of current OMZ ecosystems will enhance our ability to model and predict changes in e nvironmental processes and the response of marine food webs. 138

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APPENDICES 152

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Appendix A: Extra Figures 260 m (2007) 325 m (2007) 38 m (2007) 28 m (2008) 264 m (2008) 540 m (2008)Samples 100 90 80 70 60 50 Similarit y Resemblance: S17 Bray Curtis similarity Figure A1. Cluster analysis for the sterol fraction of partic ulate samples. Samples are designated by depth and year. 153

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R. rostrifrons (2007) R. rostrifrons (2008) E. inermis S (2007) R. nasutus (2007) S. subtenuis (2007) E. inermis M (2008) P. attenuatus (2008) E. inermis S (2008) E. inermis D (2008) E. inermis M (2007) E. inermis D (2007) S. subtenuis (2008)Samples 1 0 09 59 08 58 0 Similarit 7 5 y Resemblance: S17 Bray Curtis similarity Figure A2. Cluster analysis for copepod phos pholipid fatty acid profiles. Samples are designated by species and collection year. For Eucalanus inermis, samples are divided into M (adult male), S (adult female residing in shallow portion of the water column) and D (adult female residing in deeper water). R. rostrifrons (2007) R. rostrifrons (2008) S. subtenuis (2008) S. subtenuis (2007) R. nasutus (2007) E. inermis M (2008) P. attenuatus (2008) E. inermis D (2007) E. inermis M (2007) E. inermis S (2007) E. inermis S (2008) E. inermis D (2008)Samples 100 95 90 85 Similarit y Resemblance: S17 Bray Curtis similarity Figure A3. Cluster analysis for copepod sterol profiles. Samples are designated by species and collection year. For Eucalanus inermis, samples are divided into M (adult male), S (adult female residing in shallo w portion of the water column) and D (adult female residing in deeper water). 154