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Hudson, Erica M.
Determining the usefulness of aerobic and anaerobic enzyme assays as proxies for rockfish ecological data
h [electronic resource] /
by Erica M. Hudson.
[Tampa, Fla] :
b University of South Florida,
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Thesis (M.S.M.S.)--University of South Florida, 2008.
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ABSTRACT: Rockfish are commercially and recreationally important, yet due to the in habitat depths at which rockfish inhabit, little is known about their ecology. As a consequence, management of rockfish population as a fishery resource is a work in progress. In particular, changes in physiological condition aver the course of the year is poorly described. This study examined 19 different species of Sebastes from the Southern California Bight over four seasons (late summer, fall, winter, and spring) using metabolic enzyme assays. Enzymes used were lactate dehydrogenase (LDH), malate dehydrogenase (MDH), pyruvate kinase (PK), and citrate synthase (CS). Some muscle composition data (percent water, percent protein, percent lipid, and protein as a percentage of wet mass) were also used to help interpret the enzyme data. Enzyme activity was lowest in the summer when expressed as activity per gram wet weight but when it was expressed per gram protein the trend was reversed. We found that the rockfish tend to have the highest protein as a percentage of wet mass (P%WM) in the spring right before the upwelling period begins and have the lowest P%WM in late summer after the peak of upwelling. Their metabolic poise (represented as CS/LDH) grouped according to locomotory habit (benthic or bentho-pelagic). A mass and oxygen consumption plot also showed that the species group according to locomotory habit. With those known to be benthic grouped together and those species that are known to more actively swimming had higher values. This knowledge could be used to infer whether a rockfish that hasn't been well studied is benthic or bentho-pelagic.
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Advisor: Joeseph J. Torres, Ph.D.
x Marine Science
t USF Electronic Theses and Dissertations.
Determining the Usefulness of Aerobic and Anaerobic Enzyme Assays as Proxies for Rockfish Ecological Data. by Erica M. Hudson A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science College of Marine Science University of South Florida Major professor: Joseph J. Torres, Ph.D. David Mann, Ph.D. Susan Bell, Ph.D. Date of Approval: October 22, 2008 Keywords: Metabolic Poise, Lactate de hydrogenase, Citrate Synthase, proximate composition, Sebastes Copyright 2008, Erica M. Hudson
ACKNOWLEDGEMENTS I am extremly grateful for the support and guidance I have received throughout the course of my degree from Dr. Jose Torre s, my major professor. Funding for this work was provided by NSF grant # 052332 and a NOAA grant #AB133F06SE2766. I would also like to thank the members of my committee for their advice, expertise and guidance throughout this research and especially in the revi sion process of my thesis. Special thanks to Joe Donnelly for all of his help in the lab. Joe started this project and worked most of the kinks out befo re I picked it up. W ithout his knowledge of techniques and the lab in gene ral this process might have ta ken a whole lot longer. Also thanks to Waldo Wakefield from NMFS for hi s insight and coordination of the sampling effort. Also thanks to all my friends and fam ily who have supported me in this effort over the past few years.
i TABLE OF CONTENTS LIST OF TABLES ii LIST OF FIGURES iii ABSTRACT v INTRODUCTION 1 METHODS 7 Sample Collection 7 Enzyme Analysis 9 Protein Analysis 11 Lipid Analysis 11 Dry and Ash Weight Measurement 11 Oxygen Consumption Rates 12 Statistical Analysis 12 RESULTS 13 Enzyme Activities 13 Muscle Proximate Composition 16 Mass and Enzyme Activity 17 Seasonal Trends 17 CS/LDH Ratio 20 Oxygen Consumption 21 DISCUSSION 23 Enzyme Assays 23 Seasonal Trends 26 CONCLUSIONS 27 REFERENCES 28 APPENDICES 33
ii LIST OF TABLES Table 1: Captured size and depth ranges for each species and location and season each species was captured in. 8
iii LIST OF FIGURES Figure 1: Diversity of rockfish species al ong the Pacific coast of North America (Love, Yoklavich, Thorsteinson, Butler, 2002) 1 Figure 2: Sample sites in the southern California Bight 8 Figure 3: This is an ANCOVA representi ng the relationships between all the species averaged over all seasons with the effect of mass removed and the different enzyme activities (expressed per gram wet mass). Note that LDH is the most variable. 14 Figure 4: CS activity versus species ANCOV A with mass effects removed. Note that Bank, Canary, Chilipepper, Vermillion, Widow a nd Yellowtail have significantly higher enzyme activities. 14 Figure 5: Enzyme activity (U/g) VS season 15 Figure 6: Enzyme activity (u/gprotein) VS season 16 Figure 7: Muscle proximate composition am ong all species caught in the upwelling periods (August and September) 17 Figure 8: Nested ANOVA with LDH activity (U/g protein) in each species in each time period 18 Figure 9: Muscle proximate composition with season ANOVA 19 Figure 10: Nested ANOVA with season and musc le percent protein ash free dry weight in each species 19 Figure 11: Lipid concentration in two species in different seasons 20
iv Figure 12: This ANCOVA shows the relatio nship between the CS/LDH ratio (which shows the relative importance of aerobic metabolism vs anerobic metabolism) and species. Some species have a much higher CS/LDH ratio showing that they are more active compared to the other species. 21 Figure 13: The above figure shows mass vs oxygen consumption with each species represented. Some group together more str ongly than others. For example Cowcod is a much larger fish than all the others and therefore groups furthe r to the right. Canary is more active (bentho-pelagic) and is grouped toward the top. Speckled is grouped towards the bottom and tends to be more sedentary, or benthic. 22
v DETERMINING THE USEFULNESS OF AEROBIC AND ANAEROBIC ENZYME ASSAYS AS PROXIES FO R ROCKFISH ECOLOGICAL DATA Erica M. Hudson ABSTRACT Rockfish are commercially and recreationally important, yet due to the in habitat depths at which rockfish inhabit, little is known about their ecology. As a consequence, management of rockfish population as a fish ery resource is a work in progress. In particular, changes in physiological conditi on aver the course of the year is poorly described. This study examin ed 19 different species of Sebastes from the Southern California Bight over four seasons (late summer fall, winter, and spring) using metabolic enzyme assays. Enzymes used were lactate dehydrogenase (LDH), malate dehydrogenase (MDH), pyruvate ki nase (PK), and citrate synt hase (CS). Some muscle composition data (percent water, percent protein, percent lipi d, and protein as a percentage of wet mass) were also used to help interpret the enzyme data. Enzyme activity was lowest in the summer when expr essed as activity per gram wet weight but when it was expressed per gram protein the trend was reversed. We found that the rockfish tend to have the highest protein as a percentage of wet mass (P%WM) in the spring right before the upwelling period begi ns and have the lowest P%WM in late summer after the peak of upwelling. Their metabolic poise (represented as CS/LDH)
vi grouped according to locomotory hab it (benthic or bentho-pelagic). A mass and oxygen consumption plot also showed that the species group according to locomotory habit. With those known to be benthic grouped toge ther and those species that are known to more actively swimming had higher values. This knowledge could be used to infer whether a rockfish that hasnÂ’t been well st udied is benthic or be ntho-pelagic.
1 INTRODUCTION Rockfishes of the genus Sebastes (family Scorpaenidae) are an abundant and diverse group of fishes occupying the coastal waters of North America. There are over 102 species worldwide, with most of them (~ 96 of those species) found along the Pacific coast of North America and in the Gulf of California (Love, Y oklavich, Thorsteinson, Butler, 2002), with the greatest diversity (~ 56 species) found in the southern California bight. (See Figure 1 to the left) The fish collected during this study occupy a fairly narrow depth range (30m-200m) within an extensive North-South distribution. Rockfishes occupy a variety of habitats ranging from deep benthic and benthopelagic to kelp forests in the nearshore. Not surprising ly, they also exhibit many Figure 1: Diversity of rockfish species along the Pacific coast of N orth America (Love, Yoklavich, Thorsteinson, Butler, 2002)
2 different feeding strategies such as sit and wait, schooling to concentrate prey, and opportunistic feeding. Rockfishes prefer hard bottom areas w ith some habitat complexity such as boulder fields, reefs, oil platforms and kelp fo rests. Behavioral observations suggest that while different species may use the same outcrop and even school together, there is habitat partitioning, with some species bei ng more benthic and others more benthopelagic. Almost all rockfishes are highly valued as food-fishes and are exploited by commercial and recreational fishermen. Rockfish are very long lived (20-100yrs), reaching sexual maturity at 4-10 yrs of age. They have internal fertilizati on and brood their young until their release as hatchlings. Most species reproduce once per year but some species are believed to reproduce year round; in Southern CA most rockfish rele ase their young in early spring to summer after the coasta l upwelling has begun. Their larvae spend up to a year in the pelagic realm and juveniles sp end their first year or more in kelp forests and other shallow habitats. As they grow, they desce nd to greater depths w ith increasing age and size. Rockfish size varies considerably with species but the average adult weight for the group is one kg. Larger species can reach up to 20 kg (Love, Yoklavich, Thorsteinson, Butler, 2002). The southern California bight undergoes s easonal variation in upwelling strength. When averaged bi-monthly, the months of Janua ry and February show the slowest coastal current velocities of the year. Current sp eed picks up in March-April and continues to speed up in May-June. The current velocities peak in July-August and begin to decrease
3 in September-October and November-December. Faster current velocities are correlated with increased upwelling strength. (Winant, Dever, Hendershott, 2003) Proximate composition (protein, lipid, wate r, carbohydrate, and ash content) of nine species of rockfish off the coast of Oregon, showed a slight variation between species but found no differences in com position throughout the year (Thurston, 1960) suggesting that seasonal change in condition may not be evident in these fish. Siebenaller and Yancey (1984) explored the relationship of protein content in white muscle tissue in meso-pelagic fishes from di fferent depths. Their findings suggested that differences in enzyme activity were not due to the general dilution of muscle protein but due to the differences in speciesÂ’ depth of o ccurrence. They attri buted these differences in enzyme activity with depth of occurrenc e to the lower levels of light and food availability at depth which in turn affects metabolism and enzyme activity. In a study on the chemical composition of midwater fishes of the coast of southern California Childress and Nygaard (1973) found that water content increased with depth while protein, lipid, and ash content decreased. Their results sugge sted that fishes occ upying different depths had compositions that scaled with their different needs in locomotion, buoyancy control, and burst swim capability. The enzymes chosen for examination in this present study were L-lactate dehydrogenase (LDH), L-malate dehydrogenase (MDH), Pyruvate kinase (PK), and Citrate synthase (CS). LDH is the termin al enzyme in the anaerobic glycolysis in vertebrate tissues, and ther efore is good indicator of anaerobic capacity and overall condition. MDH plays several roles in ener gy metabolism. The mitochondrial isozyme
4 (m-mdh) is a component of the citric acid cycle and along with the cytoplasmic isozyme (s-mdh), functions in shuttl ing reducing equivalents be tween the mitochondria and cytoplasm but its main role is in aerobic meta bolism. PK is a good indicator of glycolytic capacity and along with LDH is a good indicato r of anaerobic metabolism. CS is found within the mitochondrion and is positioned at the beginning of the citric acid cycle. CS is therefore an important regulat ory site in the citric acid cycle and can be used as a quantitative index of citric acid cycle activity and therefore aerobic activity. The enzymes described above have been used extensively for investigating a variety of metabolic questions in rockfishes and other fish taxa. For example, metabolic activity of deep and shallow living teleosts was compared using enzyme activities in shallow and deep living rockfishes Sebastes and S ebastolobus (Childress, Somero, 1979; Siebenaller, 1983; Siebenaller, Somer o, 1982; Sullivan, Somero, 1980; Vetter, Lynn, 1997; Yang, Lai, Graham, Somero, 1992). Furt her studies examined nutritional state (Sullivan, Somero, 1983) habitat, feeding, and locomotory strategy (Somero, Childress, 1990; Sullivan, Somero, 1980; Yang, Somero, 1993) the relationship of activity with size (Somero, Childress, 1980), changes in enzyme activity with growth rate (Pelletier, Guderley, Dutil, 1993), changes due to temperature effects (Kawall, Torres, Sidell, Somero, 2002; Torres, Somero, 1988a; Wilson, Somero, Prosser, 1974), and adaptations of enzyme activity to living in an oxygen minimum zone (Yang, Lai, Graham, Somero, 1992). Size and depth have been shown to affect enzyme activity in separate studies by multiple researchers. Childress and Somero (1979) studied the scaling effects of the
5 enzymes due to minimum depth of occurren ce and found that enzyme activity decreased with increasing depth of occurr ence. Somero and Childress (1990) showed that fish with different locomotory strategi es, benthic vs pelagic, had markedly different enzyme activities. They found that in the pelagi c fish there was a higher enzyme activity, presumably due to their greater need for a well developed locomotory ability relative to their benthic counterparts. In a similar study, the effects of nutritional state were studied (fasted vs well-fed) and the enzyme activity in the two fish was significantly affected by the nutritional condition (Yang, Somero, 1993). In fasted fi shes the enzyme activities were much lower than in the well fed fishes and fasted fishes had comparable enzyme activities to field caught fishes Therefore, in order to pr operly interpret the enzyme activities, size, depth and behavior of the individual fish need to be taken into consideration. Many studies have correlated oxygen cons umption rates with enzyme activities (Donnelly, Torres, 1988; Seibel, 2007; Torres, Belman, Childress, 1979; Vetter, Lynn, 1997; Yang, Somero, 1993). CS activity corre lated well with oxygen consumption rates in rockfish off the coast of southern Ca lifornia (Yang, Somero, 1993). The regression equation generated by their work is applicable to other rockfish found in the same region with similar temperature and depth regimes. The aim of the present study was to describe the diversity in enzyme activities and muscle proximate composition within and betwee n closely related species of rockfish, to use those differences to determine metabolic poise and condition and to deduce the
6 underlying causes for differences in enzyme activity and determine if they would be useful in interpreting species seasonal cycles and overall ecology.
7 METHODS Sample Collection Fishes were collected by National Marine Fisheries Service ho ok-and-line surveys off the coast of Southern California during f our separate cruises (see Figure 2 below for sample locations). For each experimental fi sh, a wedge of white muscle from directly behind the head was removed immediately afte r capture and frozen in liquid nitrogen. Muscle specimens were kept in liquid nitrogen or at -80 o C in a cryogenic deep-freeze until used for the enzyme assays described below. Specimen breakdown for the four cruises was as follows. The November cr uise of 2004 collected 52 samples from 4 different species, the April Â’05 cruise collected 66 sample s from 6 species, the AugustSeptember Â‘05 cruise collected 110 sample s from 17 different species and in the September-October Â’05 cruise 35 samples from 5 species were collected. The total sample size was 263 samples from 19 diffe rent species (see Table 1 below). Samples were shipped to Florida in a Dewar contai ning liquid nitrogen which kept samples at approximately -195C. Samples were then stored in a -80 C freezer until analyzed
8 Figure 2: Sample sites in the southern California Bight Table 1: Captured size and depth ranges for each species and location and season each species was captured in. Season location where captured captured Nov '04 Apr '05 Aug '05 Sept '05 samples were caught size range depth range species n n n n (cm) (m) Bank 0 0 3 3 3 35-46 134-238 Blackgill 0 0 2 0 3 52-55 238 Boccaccio 19 14 15 0 1, 2, 3, 6, 8, 9, 10, 13 36-70 83-238 Canary 0 0 1 5 7, 8 35-54 97-109 Chilipepper 0 10 6 1 2, 4, 6 25-43 128-170 Copper 0 0 1 0 8 47 96 Cowcod 0 0 0 16 3, 4, 5, 11, 13 49-79 105-183 Flag 0 0 0 5 4, 5, 7, 11 22-38 40-165 Greenblotched 5 0 6 0 2, 6, 8, 14 24.5-43 78-147 Greenspotted 10 2 5 0 6, 8, 9, 10 23-39 85-128 Greenstriped 0 0 1 0 3 34 187 Mexican 0 0 2 0 6 42-48 143-147 SAN DIEGO LONG BEACH SANTA BARBARACalifornia California 1210'0"W 1210'0"W 1200'0"W 1200'0"W 1190'0"W 1190'0"W 1180'0"W 1180'0"W 1170'0"W 1170'0"W 320'0"N 320'0"N 330'0"N 330'0"N 340'0"N 340'0"N
9 Olive 0 0 6 0 8, 9 33-46 41-97 Speckled 0 0 7 0 9 32-35 86-95 Starry 0 0 20 0 2, 3, 8, 9 18-40 41-138 Swordspine 0 0 1 0 6 22 145 Vermilion 18 11 25 0 1, 2, 3, 8, 9, 10, 12, 13, 14 27-58 46-139 Widow 0 9 5 0 8 36-45 94.7-117 Yellowtail 0 6 4 0 8 36-51 94-115 Enzyme analyses Fish white muscle samples were hom ogenized in 50mM Imadazole/HCl buffer (pH 7.2 @ 20C) using a ground glass homogenizer Samples were kept at ice-bath temperature for the duration of the assays Homogenates were centrifuged at 4500 rpm for 10 minutes at 10C. Samples were placed on ice and the supernat ant was used within three hours to measure enzyme activity. Acti vities were measured at 10C0.2C using a thermostatted CARY 1E UV/Vi sible spectrophotometer with data analysis software. Enzyme activity was expressed in units ( mol substrate converted to product min-1) per gram wet weight of tissue and also in un its (mol substrate converted to product min-1) per gram protein. All enzyme assays follo wed the procedure of Childress and Somero (1979) with the slight m odifications listed below. The activity of LDH was measured by addi ng 10l of the supern atant to 1.0 ml of assay cocktail which consisted of 80mM Im adazole buffer, 5.0mM sodium pyruvate, and 0.15mM of NADH. The reaction was followe d by recording the decrease in absorbance at 340nm resulting from oxidation of NADH. The slope of the initial portion of the tracing was used as th e reaction rate.
10 The activity of MDH was measured by a dding 30l of the supernatant to 1.0 ml of assay cocktail containing 40mM LesleyÂ’s special buffer (0.2M Imadazole, 0.2M MgCl2), 0.4mM oxaloacetate, and 0.15mM NADH. The reaction was followed by recording the decrease in absorbance at 340nm resulting from oxidation of NADH. The slope of the initial portion of the trac ing was used as the reaction rate. The activity of PK was measured by addi ng 20l of the supernatant to 1.0 ml of assay consisting of PK Â‘coc ktailÂ’ (160mM Imadazole, 200mM KCl, 0.2mM D-Fructose1,6-bisphosphate, 20mM MgSO4), 0.15mM NADH, 1.0mM phospoenolpyruvate, 5.0 mM Adenosine 5Â’-diphosphate, and LDH coup ling enzyme from rabbit muscle solution. The reaction was followed by recording the decrease in absorbance at 340nm resulting from oxidation of NADH. The slope of the in itial portion of the tracing was used as the reaction rate. The activity of CS was measured in an assay medium containing 60l of the supernatant, 50mM Imadazole, 0.4mM 5,5-d ithio-bis(2-nitrobezo ic acid) (DTNB), 0.1mM Acetyl-Coenzyme A. The reaction wa s followed by recording the increase in absorbance at 412nm due to the reaction of the reduced coenzyme-A liberated from the enzymic reaction with DTNB. The rate of ab sorbance increase was first recorded in the absence of oxaloacetate and then after addi tion of oxaloacetate to compute the true CS activity. The blank (no oxaloace tate) was subtracted from the total activity to compute true CS activity.
11 Protein Analysis For protein, homogenate was diluted by a factor of 20 using distilled water. Homogenate was then air-evacuated using ni trogen and placed in the freezer until protein analysis was conducted. Protein composition in white muscle tissue was measured using the method established in Lowry et al. (1951). Absorbance was measured at 750nm. Values were then compared to a standard curve to obtain values for protein content (Lowry, Rosebrough, Farr, Randall, 1951). Lipid Analysis Lipid levels were determined on 200 l of homogenate using the methods of Donnelly et al (1990). Breifl y, lipids were extracted usi ng a mixture of methanol, and chloroform and filtered to removed particulat es. Concentrations were determined using the charring method of Marsh and Weinstein (1966) with stearic acid as a standard. (Bligh, Dyer, 1959; Marsh, Weinstein, 1966; Reisenbichler, Bailey, 1991) Dry and Ash weight measurements One ml aliquots of homogenate were di spensed into pre-combusted, pre-weighed crucibles and dried to a c onstant weight in a 60C oven. Water level (%WM) was estimated from a calculated homogenate dry mass concentration (i.e., DM concentration = total sample DM / total homogenate water volume; where water volume = water added for homogenation + water in tissue; and assuming 1g water 1ml water). Ash content (% DM) was measured following combustion of the dried crucibles at 500C for 3-4 hours.
12 Oxygen Consumption Oxygen consumption ml h-1 (VO2) was calculated using CS activity values (M) in field caught specimens in this regression equa tion generated by Yang and Somero (1993). logVO2 = -2.217+1.042logM (r2 = 0.900) Statistical Analysis Statistics were used to examine the signifi cance of the species specific differences in enzyme activities and composition using ANCO VAÂ’s to account for the effect of mass. Seasonal differences in enzyme activities and composition within species were examined with nested ANOVAÂ’s. All st atistical analyses were conduc ted using Statistica (Statsoft Inc.) with a significance level of p< 0.05.
13 RESULTS Enzyme activities Mean enzyme activities for all species are shown in Appendix A. Activities are expressed as mol substrate converted to product per minute (U or units) per gram wet mass (U g-1 wet mass) and also units per gram protein (U g-1 protein). The overall mean values for each species were calculated, as well as the mean values for each species during each season (Appendix A) Species differences in enzyme activity were examined using only the samples from the August/September cruise to eliminate seasonal effects. ANCOVAs and nested ANOVAs we re calculated with all data values. DuncanÂ’s multiple range test enabled discri mination between homogenous groups. Overall, Widow exhibited the highest LDH activity (expressed in WM) and Bank exhibited the lowest LDH activity both of whic h were significantly different from the rest of the species. Chilipepper had significantly higher MDH values (when expressed in WM) than the rest of the rockfish species. Chilipepper, Bocaccio and Canary exhibited significantly higher PK activity per gram WM. Bank, Canary, Chilipepper, Vermillion, Widow and Yellowtail exhibite d significantly higher CS activity (see Figures 3 and 4 below).
14 Figure 4: CS activity versus species ANCOVA with mass effects removed. Note that Bank, Canary, Chilipepper, Vermillion, Widow and Ye llowtail have significantly higher enzyme activities. Figure 3: This is an ANCOVA representing the relationships between all the species averaged over all seasons with the effect of mass removed and the different enzyme activities (expressed per gram wet mass). Note that LDH is the most variable.
15 Enough specimens of Boccacio, Greenspotted, Greenblotched, Chilipepper, Vermillion, Widow, and Yellowtail were caught in each season to compare enzymes across season and species. Most of those species showed a marked seasonality with November showing the highest enzyme activity per gram WM and August and September containing the lowest activity valu es. Activity values expressed as activity per gram protein showed the exact opposite trend with August and September values being the highest and November and April be ing the lowest (see Fi gures 5 and 6 below). Figure 5: Enzyme activity (U/g) VS season
16 Figure 6: Enzyme activity (u/g protein) VS season Muscle Proximate composition Percent protein, lipid, water and ash mean values are listed for each species as well as a mean value for each species during each season in Appendix B. In general, Canary and Widow had the highest percen t water and Speckled and Bocaccio had the lowest. Yellowtail had the hi ghest protein as a percentage of ash-free dry mass (AFDM) and Bocaccio had the lowest. None of these differences were statistically different significant. (See Figure 7 below)
17 Figure 7: Muscle proximate composition among all species caught in the upwelling periods (August and September) Mass and enzyme activity Cowcod outweighed all the other species by 4kg on average with a mean weight of 4.6kg, and Flag was the smallest of all the species with a mean weight of 0.39kg. Overall enzyme activity was more linked with season than with mass (probably due to the fact that mass was also associated with season). Seasonal Trends For seven species enough samples were obtained to compare across different seasons. On average November and April s howed higher enzyme activity values when presented as activity per gram wet weight than the August and September samples. When the data were represented as activ ity per gram protein the opposite trend was
18 observed with August and September values being much higher than those from November and April. Nested ANOVAÂ’s showed that in all species captured in multiple seasons enzyme activity (when expressed per gram protein) was si gnificantly higher in August and September than November a nd April. (See Figure 8 below) Figure 8: Nested ANOVA with LDH activity (U/g prot ein) in each species in each time period Protein expressed either as a percenta ge of wet weight (P%WM) or as a percentage of ash free dry mass (P%AFDM) was significantly higher in the November and April samples than in the August and Se ptember samples. The percent water didnÂ’t change significantly with season. In each sp ecies the same trends were observed with protein concentration, though significance varied directly with sample size.
19 In Bocaccio and Vermilion lipid concentratio ns were determined for November and August samples. The August lipid concentrations were much higher in both species than the November lipid values (p<0.05, ANOVA) (see Figures 9-11 below). Figure 9: Muscle proximate composition with season ANOVA Figure 10: Nested ANOVA with season and muscle percent protein ash free dry weight in each species
F C T S B t h F F igure 11: L i C S/LDH rat i The C T he CS/LD H pecies fell i n B ank, Canar y h eir benthic r igure 12 b el o 0 5 10 15 20 25 30 35 40 Boca c (W i pid concentr a i o C S/LDH rati o H ratio show s n to two gro u y Chilipepp e r elatives: G r o w) c cio ) Bocacci o (S) a tion in two s o was calcu l s the import a u ps dependi n e r and Flag e r eenblotche d o Vermillion (W) V 20 pecies in dif f l ated for all s a nce of aero b n g on locom e xhibited a s d Greenspo t V ermillion (S) f erent season s s pecies and a b ic versus a n otory habit. s ignificantl y t ted, Olive, S Bocac c Bocac c Vermil Vermil s a veraged o v n aerobic me t The benth o y higher CS/ S peckled an d c io (W) c io (S) lion (W) lion (S) v er all seaso n t abolism. o pelagic Co w LDH ratio t h d Starry. (se n s. w cod, h an e
21 Figure 12: This ANCOVA shows the relationship betw een the CS/LDH ratio (which shows the relative importance of aerobic metabolism vs anerobic metabolism) and species. Some species have a much higher CS/LDH ratio showing that they are more active compared to the other species. Oxygen Consumption Oxygen consumption rates were calculated with the regression equations generated by Yang and Somero (1993) on fed a nd fasted scorpenaids. Species with a higher oxygen consumption rate per gram mass were assumed to be more active based on their metabolic demands. Interestingly, in th is analysis as in the CS/LDH ratio, species grouped together according to locomotory strate gy with the more benthic species toward the bottom and the more bentho-pelagic species toward the top of th e plot (see Figure 13 below).
22 Figure 13: The above figure shows mass vs oxygen c onsumption with each species represented. Some group together more strongly than others. For example Cowcod is a much larger fish than all the others and therefore groups further to the right. Canary is more active (bentho-pelagic) and is grouped toward the top. Speckled is grouped towards the bottom and tends to be more sedentary, or benthic. 0.10 1.00 10.00 100.00 1.0010.00100.001000.0010000.00oxygen consumptionmass (in g)Rockfish mass vs oxygen consumption vermillion chilipepper cowcod bank greenblotc hed greenspotte d starry yellowtail widow flag
23 DISCUSSION Enzyme assays Enzyme assays have been used in both fish and invertebrates to determine speciesÂ’ locomotory habits, depth regimes, metabolism, and condition (Bishop, Torres, 2001; Bishop, Torres, Crabtree, 2000; Ca stellini, Somero, 1981; Childress, 1995; Childress, Nygaard, 1973; Childress, Somer o, 1979; 1990; Childress, Taylor, Cailliet, Price, 1980; D'Aoust, 1970; Donnelly, To rres, 1988; Donnelly, Torres, Hopkins, Lancraft, 1990; Drazen, Seibel, 2007; G oolish, 1991; 1995; Goolish, Adelman, 1987; 1988; Ikeda, Torres, Hernandez-Leon, Geiger 2000; Kawall, Torres, Sidell, Somero, 2002; Low, Somero; Pelletier, Guderley, Duti l, 1993; Seibel, 2007; Seibel, Drazen, 2007; Siebenaller, 1983; Siebenaller, 1984; Sieb enaller, Somero, 1978; 1982; Siebenaller, Yancey, 1984; Siebenaller, Somero, 1989; Somero, 1992; Somero, Siebenaller, 1979; Somero, Childress, 1980; 1985; 1990; Sullivan, Somero, 1980; 1983; Torres, Somero, 1988a; b; Torres, Belman, Childress, 1979; To rres, Aarset, Donnelly, Hopkins, Lancraft, Ainley, 1994; Vetter, Lynn, 1997; Webb, 1976 ; Wilson, Somero, Prosser, 1974; Yang, Somero, 1993; Yang, Lai, Graham, Somero, 1992). In the present study, enzyme activities were useful in elucidating the seas onal change in condition of nineteen species of rockfish due either to re productive effects, seasonal food availability due to upwelling or some combination of both.
24 Not surprisingly, when looking at the re lative importance of aerobic versus anaerobic metabolism (the CS/LSH ratio) in the rockfishes, they group according to locomotory habit. Those species that have a higher CS/LDH are more aerobically poised suggesting a lifestyle that involves a greater need for aerobic metabolism and therefore more active swimming. Those who have a lower CS/LDH ratio rely more on burst swimming and are more sedentary or benthic and rely more on a sit and wait or ambush prey acquisition strategy. (Love, Yoklavich, Thorsteinson, Butler, 2002) As in previous studies (i.e. Ya ng and Somero (1993)) LDH activity as a standalone value proved to be a good proxy for condition. The high values of LDH/gram protein showed that the fish were in bette r condition (i.e. well fed) during the summer months. The lipid contents in Bocaccio and Vermillion also confirmed this with high lipid contents in the summer and lower contents in the winter. Late r in the year due to growth, reproduction or a less abundant food s upply their lipid reserves became depleted and percent protein increased cau sing them to be leaner. MDH performs two functions in the cell. The first is as an intermediate in the KrebsÂ’ cycle. The second is as a shuttle to allow entry into the mitochondrion of the electrons produced by glycolytic activity during periods when sufficient oxygen is available for aerobic processes. The mitochondr ial membrane is impermeable to cytosolic NADH. Cytosolic MDH regenera tes the oxidized co-factor NAD+ for use in the glycolytic pathway, in turn producing malate from oxalo-acetic acid that can then pass through the mitochondrial membrane and be reoxidized as a KrebÂ’s Cycle intermediate (Lehninger, 1970). Since our assay does not discriminate between the cytosolic and
25 mitochondrial forms of the enzyme, a high activ ity of MDH suggests high activity in both compartments, suggesting in turn a high activity of the glycol ytic pathway. The activity of MDH mirrors that of LDH for mo st species throughout all seasons. CS is an aerobic indicator that also is a good proxy for oxygen consumption. The respiration studies and enzymatic correlati ons by Yang and Somero (1993) on rockfish provided the regression equa tions from which the oxygen consumption data were generated. In Figure 13 Figure 13 you can see that some species, such as Starry and Speckled are much lower than the overall average. Thos e species have been observed to be more benthic in habit, relying on bur st swimming for fight or flight situations. Other species, such as Canary and Chilipepper are higher th an the overall average implying that they are a more active swimming species. Behavioral observations reported in Love et al. (2002) suggest that both Canary and CHilipepper sc hool in groups as benthopelagic species, corroborating the resu lts of the enzyme analyses. The CS/LDH ratio represents the im portance of aerobic versus anaerobic capability of the fish. Organisms that rely mainly on aerobic activity for locomotion tend to be animals which spend most of the tim e actively swimming or otherwise maintaining their position in the water column. Those fish that are more sedentary rely on anaerobic pathways to provide burst swimming in predator and prey interactions. This enzyme data combined with previous behavioral studies shows that there is indeed a noticeable difference between the benthic and th e bentho-pelagic species.
26 Seasonal trends The difference in enzyme activity when expressed as activity per gram wet weight vs. when expressed as activity per gram protei n can be explained by the change in percent protein observed in the samples. The changes in body composition are consistent with what one might expect during those times of year. In the summe r when there is upwelling and increased nutrients which cascade down th e food web, the fish are eati ng and storing excess energy as fat, thus decreasing their muscle water content. Throughout the year as the seasons change and upwelling decreases, primar y production and zooplankton biomass also decrease (Dailey, Reish, Anderson, 1993). This translates down the food web and food becomes scarcer for Sebastes. The fish in turn become le aner and their percent protein increases. Energetic expenditure might also play a role in the seasonal change in body composition. Most rockfish reproduce in the spring and summer months so the changes observed in body composition could be due to reproductive effects.
27 CONCLUSIONS Enzyme activity along with muscle proximate composition can be a very useful tool in evaluating a species physical condition. Increases in protein concentration during the winter months, coupled with decreased li pid and a constant or slightly increasing water level show that the fish are losing we ight and energy stores. The obvious reasons for different physical conditions throughout the year, like upwelli ng, and reproduction are the most likely causes but such a drastic ch ange over the course of a year was not expected. Enzymes can also be very useful in helping a researcher determine what sort of locomotory behaviors an animal is most likel y to rely on due to th e expression of aerobic versus anaerobic enzymes found in their ti ssue. A low expression of aerobic enzymes indicates that the animal most likely relies on anaerobic burst responses whereas a high expression of aerobic enzymes implies that the animal is more active. These findings show that enzymes and muscle proximate composition can be used along with limited observational data on related species to deduce condition and life habits in species that are difficult to obser ve and monitor.
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34 Appendix A: Mean enzyme activities plus or minus the standard deviation for all species Table 2a: The average of all enzyme values ex pressed (U/g wet mass) for all species Species LDH MDH PK CS Bocaccio 91.874.00 32.381.35 50.553.21 0.530.024 Cowcod 50.105.13 21.271.96 56.674.37 0.410.021 Bank 52.4412.68 15.882.17 44.1915.33 0.650.05 Canary 93.2717.63 45.3010.42 77.1015.41 1.360.14 Chilipepper 107.9711.07 45.622.58 79.616.76 0.990.055 Flag 29.234.94 27.424.74 19.552.38 0.570.095 Greenblotched 82.866.82 24.032.89 30.385.87 0.400.064 Greenspotted 80.737.93 24.462.09 34.374.8 0.550.069 Olive 59.259.17 20.281.91 15.251.25 0.390.022 Speckled 77.608.75 24.121.41 11.111.07 0.360.064 Starry 53.304.27 16.741.12 9.930.84 0.310.013 Vermillion 85.762.62 28.510.97 33.092.55 0.750.034 Widow 108.186.94 28.321.44 36.074.24 0.740.033 Yellowtail 71.297.34 27.291.80 27.314.96 0.790.074 Table 2b: The average of all enzyme values expressed (U/g protein) for all species Species LDH/Protein MDH/Protein PK/Protein CS/Protein Bocaccio 810.9946.87 299.1722.55 409.2518.62 4.770.31 Cowcod 796.3987.24 318.5325.54 865.1365.72 6.490.42 Bank 871.43196.00 269.5541.18 721.30232.32 11.041.13 Canary 1434.27265.66 686.73145.59 1176.00243.28 21.062.30 Chilipepper 897.5892.65 428.1952.24 675.0944.26 9.731.41 Flag 465.5076.89 427.4655.16 325.4859.37 8.951.11 Greenblotched 915.9989.99 251.2020.79 291.9026.79 3.930.16 Greenspotted 678.0370.97 198.9612.63 263.2624.81 4.140.35 Olive 970.98191.20 330.8348.77 244.5529.00 6.410.61 Speckled 1357.18212.99 425.1157.14 185.7615.17 5.780.51 Starry 1073.76157.25 324.0642.23 209.7342.23 6.000.64 Vermillion 1209.74153.56 363.8838.51 313.7527.37 9.080.75 Widow 1167.57217.86 293.0845.19 305.1419.56 7.42.94 Yellowtail 676.82148.68 243.4332.69 202.6518.90 6.950.90
35 Appendix A (Continued) Table 2c: The average of all enzyme values expressed (U /g wet mass) for all species in November Â‘04 Species LDH MDH PK CS Bocaccio 110.3226.40 37.0010.43 62.9318.57 0.610.17 Greenblotched 101.0717.21 32.236.65 47.1716.95 0.63.01 Greenspotted 95.8131.9 28.727.29 41.5720.29 0.700.21 Vermillion 84.7117.95 30.786.49 43.9413.82 0.860.26 Table 2d: The average of all enzyme values expressed (U /g protein) for all species in November Â‘04 Species LDH/Protien MDH/Prot ein PK/Protein CS/Protein Bocaccio 699.78141.04 234.4355.15 400.83118.51 3.951.24 Greenblotched 671.0995.70 213.5935.58 313.23107.39 4.190.23 Greenspotted 653.20254.03 194.3954.86 275.80126.89 4.741.47 Vermillion 562.95104.86 204.0835.62 289.8784.25 5.751.75 Table 2e: The average of all enzyme values expressed (U/g wet mass) for all species in April Â‘05 Species LDH MDH PK CS Bocaccio 93.5121.44 31.286.71 58.4117.94 0.580.13 Chilipepper 137.5826.42 46.984.79 97.1323.07 0.980.18 Greenspotted 68.146.87 27.332.70 44.816.19 0.680.01 Vermillion 82.5219.59 31.666.45 44.9513.47 0.890.23 Widow 97.7919.45 27.026.28 45.2711.10 0.770.15 Yellowtail 70.9414.68 29.634.51 38.239.13 0.910.17 Table 2f: The average of all enzyme values expressed (U/g protein) for all species in April Â‘05 Species LDH/Protien MDH/Prot ein PK/Protein CS/Protein Bocaccio 561.55115.48 187.8735.63 350.49102.88 3.510.76 Chilipepper 797.46130.69 273.8731.44 564.38126.72 5.660.93 Greenspotted 412.2741.29 165.3916.43 271.1437.28 4.110.07 Vermillion 504.09115.41 192.7831.65 273.5475.39 5.461.31 Widow 654.64106.87 180.8535.87 304.2774.48 5.150.83 Yellowtail 449.7384.21 187.5622.43 240.8043.15 5.791.13
36 Appendix A (Continued) Table 2g: The average of all enzyme valu es expressed (U/g wet mass) fo r all species in August Â‘05 Species LDH MDH PK CS Bocaccio 66.9711.37 27.577.55 27.568.96 0.400.13 Bank 26.467.55 16.126.09 17.271.87 0.650.19 Canary* 64.14 24.55 25.62 1.22 Chilipepper 72.1628.40 37.925.66 53.475.41 1.010.32 Greenblotched 67.6913.39 17.214.98 16.403.24 0.230.04 Greenspotted 55.6223.30 14.813.15 15.824.19 0.200.07 Olive 59.259.17 20.281.91 15.251.25 0.390.022 Speckled 77.608.75 24.121.41 11.111.07 0.360.064 Starry 53.304.27 16.741.12 9.930.84 0.310.013 Vermillion 87.2520.25 25.326.70 14.045.82 0.620.17 Widow 126.9127.48 30.662.28 19.536.41 0.710.08 Yellowtail 71.8435.42 23.806.04 10.941.84 0.630.23 *only one specimen caught in this season Table 2h: The average of all enzyme values expressed (U /g protein) for all sp ecies in August Â‘05 Species LDH/Protien MDH/Prot ein PK/Protein CS/Protein Bocaccio 1184.68295.62 485.09147.56 474.78140.91 7.022.28 Bank 453.40158.54 276.26118.91 293.1154.30 11.153.91 Canary* 1229.91 470.81 491.30 23.36 Chilipepper 1158.10521.14 593.4373.29 844.24130.80 15.815.04 Greenblotched 1120.08246.69 282.5476.83 274.1475.69 3.730.64 Greenspotted 834.03360.07 221.5452.53 235.0562.08 2.970.99 Olive 970.98191.20 330.8348.77 244.5529.00 6.410.61 Speckled 1357.18212.99 425.1157.14 185.7615.17 5.780.51 Starry 1073.76157.25 324.0642.23 209.7342.23 6.000.64 Vermillion 2018.271250.65 562.15318.36 350.11281.91 13.245.55 Widow 2090.87692.19 495.09104.83 306.7379.39 11.512.52 Yellowtail 1017.47627.28 327.24124.96 145.4318.43 8.713.93 *only one specimen caught in this season
37 Appendix A (Continued) Table 2i: The average of all enzyme values expressed (U /g wet mass) for all species in September Â‘05 Species LDH MDH PK CS Cowcod 50.105.13 21.271.96 56.674.37 0.410.021 Bank 78.4318.17 15.655.75 71.1136.71 0.650.06 Canary 99.1045.64 49.4526.16 87.4131.39 1.390.38 Chilipepper* 26.84 78.36 61.36 1.12 Flag 29.244.94 27.434.74 19.562.38 0.580.095 *only one specimen caught in this season Table 2j: The average of all enzyme values expressed (U /g protein) for all species in September Â‘05 Species LDH/Protien MDH/Prot ein PK/Protein CS/Protein Cowcod 796.3987.24 318.5325.54 865.1365.72 6.490.42 Bank 1289.48163.91 262.85105.67 1149.50506.52 10.931.97 Canary 1475.14718.87 729.91380.78 1312.94550.68 20.616.16 Chilipepper* 335.62 979.96 767.30 13.96 Flag 465.5076.89 427.4655.16 325.4859.37 8.951.11 *only one specimen caught in this season
38 Appendix B: Mean proximate composition values for all species in all seasons Table 3a: Mean proximate composition values for a ll species averaged over all seasons Species P%AFDM %water P%WM Ash%DM Bocaccio 62.023.57 75.850.33 13.830.71 12.820.41 Cowcod 30.411.13 76.480.40 7.170.31 6.660.64 Bank 29.591.64 77.870.81 7.640.21 5.991.41 Canary 32.801.56 78.560.47 7.20.36 6.521.13 Chilipepper 61.416.84 76.020.43 10.521.31 12.831.03 Flag 31.191.78 77.920.61 8.890.37 6.271.11 Greenblotched 48.497.26 76.910.46 8.441.42 10.150.55 Greenspotted 61.595.06 76.740.31 11.270.99 12.670.83 Olive 30.411.83 77.270.56 7.230.42 6.400.54 Speckled 29.085.72 74.891.5 7.770.89 6.280.42 Starry 28.172.78 76.200.60 9.830.55 5.940.34 Vermillion 55.883.74 77.650.35 10.480.73 10.950.39 Widow 64.336.97 78.410.74 11.831.16 11.711.13 Yellowtail 65.797.92 77.740.94 10.421.37 12.480.52 Table 3b: Mean proximate composition values for all species in November Â‘04 Species P%AFDM %water P%WM Ash%DM Bocaccio 74.806.58 75.142.72 17.071.33 15.674.99 Greenblotched 73.226.67 77.621.14 9.240.68 15.032.12 Greenspotted 72.646.38 76.611.30 13.121.08 14.883.24 Vermillion 74.355.21 76.981.37 12.951.02 15.022.96 Table 3c: Mean proximate composition values for all species in April Â‘05 Species P%AFDM %water P%WM Ash%DM Bocaccio 82.453.39 77.321.35 11.560.97 16.512.47 Chilipepper 84.005.52 76.52.04 12.430.82 17.194.61 Greenspotted 81.596.11 77.51.56 9.850.02 16.520.42 Vermillion 85.123.71 78.561.21 10.360.87 16.341.56 Widow 82.255.95 79.012.18 13.290.83 14.874.57 Yellowtail 84.156.13 77.972.34 11.360.91 15.771.27
39 Appendix B (continued) Table 3d: Mean proximate composition values for all species in August Â‘05 Species P%AFDM %water P%WM Ash%DM Bocaccio 26.783.54 75.391.73 11.860.71 5.774.85 Bank 30.694.78 78.202.84 9.850.52 5.963.81 Canary* 26.7225978 78.7754938 8.05 5.215164381 Chilipepper 28.122.21 75.421.19 7.50.54 6.390.85 Greenblotched 27.891.53 76.321.67 7.780.53 6.091.34 Greenspotted 31.521.82 76.731.30 8.140.34 6.731.16 Olive 30.411.83 77.270.56 7.230.42 6.400.54 Speckled 29.085.72 74.891.5 7.770.89 6.280.42 Starry 28.172.78 76.200.60 9.830.55 5.940.34 Vermillion 28.6314.23 77.743.41 8.692.10 5.431.65 Widow 32.0810.94 77.333.63 8.871.18 6.370.55 Yellowtail 38.2511.47 77.424.17 9.031.14 7.561.08 *only one specimen caught in this season Table 3e: Mean proximate composition values for all species in September Â‘05 Species P%AFDM %water P%WM Ash%DM Cowcod 30.411.13 76.480.40 7.170.31 6.660.64 Bank 28.503.70 77.551.25 5.430.61 6.030.89 Canary 34.022.68 78.521.29 7.030.66 6.793.05 Chilipepper* 35.21659652 74.8537437 9.7 7.996656999 Flag 31.191.78 77.920.61 8.890.37 6.271.11 *only one specimen caught in this season