Energetics and RNA-DNA ratio in larval red drum Sciaenops Ocellatus

Energetics and RNA-DNA ratio in larval red drum Sciaenops Ocellatus

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Energetics and RNA-DNA ratio in larval red drum Sciaenops Ocellatus
Brightman, Ross Ira
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Tampa, Florida
University of South Florida
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xii, 173 leaves : ill. ; 29 cm


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Channel bass -- Metabolism ( lcsh )
Dissertations, Academic -- Marine Science -- Doctoral -- USF ( FTS )


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Thesis (Ph. D.)--University of South Florida, 1993. Includes bibliographical references (leaves 157-173).

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University of South Florida
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University of South Florida
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F51-00182 ( USFLDC DOI )
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ENERGETICS AND RNA-DNA RATIO IN LARVAL RED DRUM SCIAENOPS OCELLATUS by Ross I. Brightman A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department o f Marine Science in the University of South Florida May, 1993 Major Professor: Joseph J. Torres


Graduate Council University of South Florida Tampa, Florida CERTIFICATE OF APPROVAL Ph.D Dissertation This is to certify that the Ph.D. Dissertation of Ross !.Brightman with a major in Marine Science has been approved by the Examining Committee on March 25, 1992 as satisfactory for the dissertation requirement for the Ph.D. degree. ,.., Examining committee: Mjjorftr

Ross I Brightman 1993 Reserved----


ACKNOWLEDGEMENTS I am most grateful to Dr. J.J. Torres for his continual support and guidance throughout the duration of this project. Thanks also g o to the members of my dissertation committee who provided additional support. I thank J. Donnelly for his technical assistance and patience, J. Harvey for his long hours, G. Tolley for his statistical assistance, J. Chad Edmisten for graphics, and s. Rupe for her editing marvels. Special thanks go to B. Falls, A. Berke and D. Roberts of the Florida Marine Research Institute and the entire crew of the Port Manatee Hatchery. This research was supported by Florida Department of Natural Resources, Marine Resources Grants C-6536 and C-7701. ii.


TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES ABSTRACT CHAPTER 1 : INTRODUCTION General: Red drum Fish energetics CHAPTER 2 : ENERGY GAINED THROUGH INGESTION Introduction Critical period Feeding rate Gut evacuation rate Studies on red drum Materials and methods Maintenance of specimens Critical period Feeding rate Gut clearance rate Daily ration statistics Results Critical period Feeding rate experiments Gut evacuation rate Daily ration Discussion critical period Feeding rate Gut evacuation rate Daily ration Conclusions CHAPTER 3 : ENERGY USED IN GROWTH Introduction Factors that affect growth Methods for measuring growth rate Material and methods Laboratory set-up Pond set-up Growth rate versus ration iii. vi viii X 1 1 4 7 7 7 9 12 13 14 14 16 16 18 18 19 19 19 22 26 27 27 28 30 32 33 35 36 36 37 41 47 47 47 47


Average proximate/elemental composition prey 49 Average proximate/elemental composition larvae 49 Caloric content of prey and larvae 50 RNA-DNA ratio 50 Activities of LDH and cs 51 Results 51 Growth rate versus ration 51 Average proximate/elemental composition prey 65 Average proximate/elemental composition larvae 65 Caloric content of the larvae 74 Indicators of growth 78 Discussion 86 Growth versus ration 86 Proximate and elemental composition 88 Growth versus ration curve; Caloric content 91 Indicators of growth 9 3 Biochemical parameters as predictive tools 97 Conclusions 98 CHAPTER 4 : ENERGY USED IN METABOLISM Introductio n oxygen consumption rate Larval respiration Methods for measuring oxygen consumption Materials and methods O xygen consumption rate Daily respiration costs Results Oxygen consumption rate Daily respiration costs Discussion Oxygen consumption rates Daily respiration costs Conclusions CHAPTER 5 : ENERGY LOST TO EXCRETION Introduction Nitrogen excretion Factors that influence N-excretion O-N ratios Materials and methods Nitrogen excretion Daily excretion O-N ratios Results Nitrogen excretion rates Daily excretion rates O-N ratios Discussion Nitrogen excretion Daily excretion rates O-N ratios Conclusions iv. 1 0 0 100 100 102 104 106 106 108 109 109 116 117 117 119 120 121 121 121 123 124 125 125 126 126 127 127 132 133 134 134 136 137 137


.CHAPTER 6 : ASSEMBLY OF ENERGY BUDGET 138 Introduction 138 Bioenergetics: balancing the budget 138 Assimilation rate and conversion efficiency 140 Materials and methods 141 Energy budget equation 141 assimilation rate and conversion efficiency 142 Results 143 The energy budget 143 Assimilation rate 146 conversion efficiencies 147 Discussion 148 The cost of life 148 Assimilation rate and conversion efficiency 152 Conclusions 154 LIST OF REFERENCES 157 v.


LIST OF TABLES Table 1. Standard length and dry weight of delayed feeding. 2 1 Table 2 Effects of prey concentration on capture success as a function of age. 2 3 Table 3 Evacuatio n rate versus age. Each sample removed represents 10 individuals. 2 6 Table 4. Growth in starved red drum larvae as a function o f standard length and dry weight. 53 Table 5. Standard length versus age in lab-reared and pond-reared red drum. 54 Table 6 Growth in dry weight of red drum as a function o f temperature in laboratory raised larvae. 60 Table 7 Growth in dry weight of red drum as a function of temperature in pond-raised larvae. 63 Table 8. Proximate composition of laboratory-raised red drum larvae at ratio n levels of 0 and 5.0 prey/ml at 20C and 25C and pond-raised larvae at 22C and 32C. 67 Table 9. Elemental composition of laboratory-raised red drum larvae at three prey concentrations and pond-raised red drum at 32C. 73 Table 10. Total caloric content o f red drum larvae at 2soc starved; 25C and fed 5 0 prey/ml; 2ooc and fed 5.0 prey/ml; and 22C and 32C pond-raised. 75 Table 11. Relations between temperature, protein content, RNA/DNA, LDH, cs and growth rate (%BW/d) in red drum larvae. 85 Table 12. Respiration of red drum fed at 5.0 prey/ml. 109 vi.


Table 13. Respiration rates for red drum larvae starved for 24 hours. 111 Table 14. Comparison o f weight-specific respiration rates measured using needle and micro-electrodes. 114 Table 15. Intensity o f feeding to sustain caloric demand o f respiration calculated from total oxygen consumption. 116 Table 16. Mean ammonia and urea excretion in red drum larvae fed t o satiation. 127 Table 17. Mean weight-specific ammonia N excretion rates in red drum larvae fed to satiation, and starved for 24 and 48 hours. 1 29 Table 18. Percent urea production in individuals starved for 24 and 48 hours in 10 ml seawater for 4 hours. Table 19. Daily nitrogen excretion rates of red drum larvae and equivalence in rotifers calculated from mean weight-specific ammonia 131 and urea excretion rates. 1 32 Table 20. Molar oxygen:nitrogen ratios for larvae fed to satiation, and those starved 24 and 48 hours. 134 Table 21. summary of the average daily caloric gains and costs in larval red drum raised in the laboratory at 25C for the first two weeks of life. 144 Table 22 Percentage of required ingested energy allocated to growth, metabolism and excretion. Table 23. Assimilation efficiencies of red drum larvae fed a rotifer diet. Table 24 Gross growth efficiency (K1) and net growth efficiency (K2) for red drum larvae based o n required and predicted numbers of rotifers. Table 25. Percentages of ingested energy allocated to growth, metabolism, and excretion for 145 147 148 species similar in size to red drum. 150 Table 26. Energy allocated to feeding metabolism (SDA) for the first two weeks of life. 151 vii.


LIST OF FIGURES Figure 1 Diagram of rotifer/algae culture unit used in feeding laboratory-reared red drum larvae. 15 Figure 2 Percent survival over the first 14 days of life when first fed a) day 0, b) day 2 c) day 3 d) day 4, and e) day 5. 2 0 Figure 3 Feeding rate of successful capture of larvae of ages of a) 3, b) 6, c) 10 and d) 14 days at ration levels o f 0, 1 .0, 5 0 and 10.0 prey/rnl. 24 Figure 4 Growth in standard length for larvae raised in the laboratory at a) 0 prey/rnl, b) 5 0 prey/ rnl at 20C, and c) 5 0 prey/rnl at 25C. 52 Figure 5 Growth in standard length for larvae raised in ponds at a) 22C and b) 32oc. 55 Figure 6. Growth in dry weight for larvae raised in the laboratory at 25C at a) 0 prey/rnl, b) 0.1 preyj rnl and c) 1 0 prey/rnl. 57 Figure 7 Growth in of dry weight for larvae raised in the laboratory at a) 0 preyjrnl, b) 5.0 prey/ rnl at 20C, and c) 5.0 prey/ rnl at 2soc. 59 Figure 8 Growth in dry weight for larvae raised in ponds at a) 22C and b) 32C. 61 Figure 9 Growth versus ration curve for 2 week-old larvae raised in the laboratory at 2soc fed rations of 0, 1.0, 5.0 and 10. 0 prey/rnl. 64 Figure 10. Growth in protein and lipid for larvae raised in the laboratory at a,d) 0 preyjrnl, b,e) 5 0 prey/rnl at 20C, and c,f) 5 0 prey/rnl at 25C. 69 Figure 11. Growth in protein and lipid for larvae raised in ponds at a) 22C and b) 32C. 71 viii.


.Figure 12. Growth in total calories for larvae raised in the laboratory at a) 0 prey/ml, b) 5.0 prey/ml at 20C, and c) 5.0 prey/ml at 25oc. 76 Figure 13. Growth in total calories for larvae raised in ponds at a) 22C and b) 32C. 77 Figure 14. Growth measured indirectly as RNA/DNA for larvae raised in the laboratory at a) 0 prey/ml, b) 5.0 prey /ml at 2ooc, and c) 5.0 prey/ml at 25C. 79 Figure 15. Growth measured indirectly as RNA/DNA f o r larvae raised in ponds at a) 22C and b) 32C. 81 Figure 16. Growth measured indirectly as LDH and cs activities for larvae raised in the laboratory at a,d) 0 prey/ml, b,e) 5.0 prey/ml at 20C, and c,f) 5.0 prey/ml at 25C. 82 Figure 1 7 Growth measured indirectly as LDH and cs activities for larvae raised in ponds at a) 22C and b) 32C. 84 Figure 18. Absolute oxygen consumption in larvae fed to satiation as a function of a) age and b) weight. 110 Figure 19. Weight-specific oxygen consumption in larvae fed to satiation as a function of a) age and b) weight. 112 Figure 20. Absolute oxygen consumption in larvae starved for 24 hours as a function of a) age and b ) weight. 113 Figure 21. Weight-specific oxygen consumption in larvae starved for 24 hours as a function of a) age and b) weight. 115 Figure 22. Ammonia excretion as a function of age at a) 0 days starvation b) 1 day starvation and c) 2 days starvation. 128 Figure 23. Weight-specific ammonia excretion as a function of age at a) 0 days starvation b) 1 day starvation and c) 2 days starvation. 130 ix.


ENERGETICS AND RNA-DNA RATIO IN LARVAL RED DRUM SCIAENOPS OCELLATUS by Ross I. Brightman An Abstract A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department o f Marine Science in the University of south Florida May 1993 Major Professor: Joseph J. Torres x


Rates of ingestion, growth, oxygen consumption and excretion were measured in larval red drum Sciaenops ocellatus fed at a series of ration levels from 0 to 10.0 prey/ml). The ability to capture prey at low ration levels and the total amount of prey increased with age. Larvae that had not obtained food within 24 hours after yolk-sac absorption were beyond the "point of no return" and died even when offered food. Larvae fed only during daylight hours with gut evacuation rates of 2 17 hours on day 6 and 2.33 hours on day 14. Specific growth rate (%body weight/day) was constant with age for the first two weeks of life, however, percentage of total ingested energy allocated to growth decreased with age. Metamorphosis occurred at a given standard length and dry weight in larvae independent of age and rearing conditions (laboratory and ponds). Growth was successfully predicted using biochemical indices; protein content, nucleic acid ratios, and metabolic enzymes. Each of the indices was affected by temperature and specific growth rate was best predicted by multiple regressions. The percentage of energy lost to respiration increased with age. The difference between starved and fed larvae was determined to be the energy lost to feeding metabolism (SDA), which averaged 50% of total metabolic cost. Absolute xi.


oxygen consumption versus weight had a slope (b coefficient) of 1.02 for fed larvae and 0.70 for starved larvae. Energy lost as ammonia and urea increased with age from 4.3% on day 3 to a constant 11.6% after day 6. Urea production as a percentage of total N-excretion was inversely related to ammonia production in starved individuals and increased with time of starvation. Feces were unable to be measured due to the high assimilation of the rotifers within the gut. ?ills tract xii. Joseph J. Torres Science


1 CHAPTER I. INTRODUCTION General: Red drum In 1766, Linnaeus first described red drum and classified the animal as Sciaenops ocellatus, which translates as "a perch-like marine fish with an eye-like spot". Red drum are one of 22 members of the Family Sciaenidae and can be found along the Atlantic and Gulf Coasts of the United States. Red drum have been reported as far north as the Gulf of Maine but are rarely found north of New Jersey (Smith 1898; Yokel 1966; Lux and Mahoney 1969). The family's common name of "drum" is derived from the fact that many of its members can produce drumming sounds by vibrating their swim bladders with a specialized musculature. Swimbladder morphology, along with otolith and barbel structure, can be used to separate sciaenid genera (Darovec, 1983). Of the 22 members of Sciaenidae the genus Sciaenops is monotypic. Red drum have an advanced swimbladder morphology characterized by a pair of diverticulae that originate from the anterolateral corners and extend backwards, dividing into numerous tubules. Barbels are absent in red drum and, like most sciaenid fishes, they possess a large, thick saccular otolith, with a "tadpole-shaped" sulcus on its inner surface (Chao, 1986).


2 Ramsey and Wakeman (1987) surveyed 22 enzymes and seven structural proteins (40 presumptive gene loci) in red drum to determine genetic variability within the species. They concluded that differentiation into subpopulations was only weakly evident for red drum along the Gulf Coasts of Texas, Louisiana, Mississippi and Florida and the Atlantic Coast of Florida. Cluster analysis of samples of individuals from a population living in Mosquito Lagoon on Florida's easiern coast, however, did indicate a slight genetic difference. This difference was most likely due to the late maturation and breeding in the semi-isolated area of the lagoon. Red drum mature in 3-5 years with the average female producing 500, 000 to 1 mill ion eggs per season (Holt et al. 198lb). Spawning occurs in the ocean along beaches and near inlets and passes (Pearson 1929; Simmons and Breuer, 1966; Yokel, 1966). It was found that red drum in the Gulf of Mexico spawn fro m August to mid-November with a peak in September or October. In southwestern Florida waters spawning occurs in mid-September to mid-February with a peak in October. Atlantic coast red drum spawn earlier in the season-July to December-but also have their peak egg production in October. Spawning can be induced in red drum using hormone injections (Colura and Bass, 1990), for example, or by the conditioning of individuals through regulation of environmental cues. Roberts et al. (1978a) and Roberts (1990), for example, have induced spawning in the laboratory by conditioning adult red drum through


variations in photoperiod and temperature. This enables eggs to be obtained throughout the year for experimentation without sacrificing individuals as is usually the case for the hormone injection method. 3 Holt et al. (1981a) and Holt (1990) described the egg stage and early larval development of red drum in a laboratory environment. They found a mean egg size of 0.95 mm diameter, and that each egg contained an oil globule averaging 0.30 mm diameter. The standard length of larvae upon hatching measured between 1.71 mm and 1.79 mm. Yolk sac larvae were observed to be negatively buoyant and drifted downward (head first) 95% of the time. Roberts et al. (1978a) determined that metamorphosis (post-flexion) of red drum larvae to the post-larvae stage occurred around day 14 at 25C. Larvae at this stage ranged from 5 mm to 7 mm in standard length. Red drum are euryhaline fish with an optimum salinity ranging from 20-40 ppt depending on the life stage of the fish (Simmons and Breuer, 1962). Yan and Thomas (1988) studied osmoregulation in developing larvae and found that changes in prolactin cell size and the number o f chloride cells (site of ion exchange) in larval red drum gill filaments depended on the salinity to which the newly hatched fish were exposed. Individuals exposed to 5 ppt and 45 ppt had no significant changes in chloride cells over the first 7 days of exposure. After 21 days of exposure to their respective salinities, however, a reduction in


4 chloride cells.occurred in the 5 ppt group of larvae and an increase in number was found in the 45 ppt group. Prolactin cell size increased in the fish held at 5 ppt and decreased in fish held at 45 ppt over the 21 days of exposure. Holt and Banks (1989) determined that growth rate was correlated with chloride cell development in larval red drum. They found that changes in salinity during early development affected the rate of larval growth. They exposed the larvae to salinities ranging from 0 ppt to 50 ppt. Growth rates were lower in larvae that had been exposed to salinities higher than 30 ppt and lower than 10 ppt. Reduction in growth at high and low salinities was assumed to be the result of the red drum's poorly developed ability for osmoregulation and the associated metabolic costs of.homeostasis. Holt (1990) found that post-flexion larvae were less susceptible to changes in salinity than were pre-flexion larvae. Once scales had developed on the larvae and reduced the flow of ions across the integument, their tolerance to salinity extremes was greatly increased. It was concluded that some trade-off had occurred between energy used in normal growth and energy needed to maintain homeostasis. Fish energetics The energy utilization of an individual describes how food energy that is ingested is apportioned between the demands made by growth and metabolism and what is lost through excretion. The energy budget is a useful


tool in predicting the energetic boundary conditions needed by an individual to grow and survive. Brett and Groves (1979) described the energy budget by the equation: where I G M E = = = = I = G + M + E ingested energy energy used in growth energy used in metabolism energy lost to excretion 5 Growth (G), or produ6tion, consists of both somatic and reproductive components depending on the age and/or season. Excretion (E) can be broken down into feces, urea and ammonia, and miscellaneous secretions such as mucus. Total metabolism (M) was further subdivided into a number of subcomponents by Calow (1985), who expressed them in the equation: where R is equivalent to the M of the Brett and Groves (1979) equation. These subcomponents included standard metabolism (Rs), or energy expended at rest, routine metabolism (RR), or energy expended in routine activities, active metabolism (RA) and feeding metabolism (RF), an elevation in metabolism that is associated with recently fed animals. Feeding metabolism is sometimes subdivided into energy use due to excitement and that due to the metabolic transformation of foodstuffs, known as specific dynamic action (effect). The difference between the standard metabolic rate and the active metabolic rate is known as the


metabolic scope (Priede, 1985). In order to survive and reproduce, an animal must be able to vary the allocation of incoming energy among subcompartments. For example, spawning salmon focus their incoming energy into reproduction at the expense of other metabolic needs, ultimately leading to their. deaths. 6 Animals will attempt to maximize energetic efficiency by the optimum foraging theory (Priede, 1985) where an animal behaves in such a manner so as to maximize the ratio of incoming energy over energy expenditure. Incoming energy in larvae, like those of the red drum, can be quantified in studies on feeding rates. The first few weeks of life is critical to the recruitment success of larval fish. This critical time is mirrored in the energy utilization pattern of developing larvae: the energy budget.


CHAPTER II. ENERGY GAINED THROUGH INGESTION Introduction Energy gained through ingestion makes up the first component of the energy budget equation. Important subcomponents needed to fully understand the process of ingestion are critical period, feeding rate, and rate of digestion. Laurence (1975) states that acquisition of a minimum ration by larval fish is of prime importance to their survival and successful development. Larvae that do not obtain the proper quantity and quality of food will be adversely affected. The purpose of this chapter is to examine the energetics of feeding and digestion in red drum larvae and t o determine the effects of those processes on survival. Critical period 7 survival of larval fish depends on obtaining food before the effects of starvation result in death. Hjort (1914), Marr (1956), and May (1971, 1974) recognized that fish larvae are particularly vulnerable to starvation after the the yolk-sac is depleted. They postulated that year class strength was determined largely by starvation-induced mortality in the very early life stages of the developing fish larvae. The period of time between yolk-sac absorption


and the time at which a starved fish larva will not feed when presented food has been termed the ''critical" period (Hirano, 1984). 8 The time at which the larvae will not feed has been termed the "point of no return" (PNR) by Yin and Blaxter (1986), who defined it as the period of starvation after which 50% of the larvae, although still alive, are no longer strong enough to feed. The time that the fish reaches the irreversible starvation point is not always evident and may only be realized days later when the fish dies. Ecologically, the PNR is of more significance than the time to death of the larvae (Blaxter and Ehrlich, 1974). Past the PNR the fish is incapable of recovery growth, resulting in the death of the fish even though feeding may occur {Dabrowski and Takashima, 1986). A debate exists on whether the critical period of high susceptibility actually occurs or whether the effects of predation on the larvae are more influential on mortality. Neilson et al. {1986) suggested that poorly fed individuals become more buoyant and are thus more vulnerable to predators in surface waters. Taggart and Leggett (1987) disagreed and stated that even though predation density and larval mortality were positively correlated in larval capelin, the relationship was determined to be statistically insignificant. Those that believe that a critical period exists postulate that the period is determined absolutely by the larv a s energy requirements and can be measured in the


9 laboratory using delayed feeding experiments. Walsh et al. (1981) developed a simulation model that --extrapolated the critical period hypothesis to field studies. The model consisted of interactions of physical and biological submodels for predicting larval fish drift. Larvae that survive are those that have traveled favorable paths through the resources and hazards of their environment. They concluded that the timing of events, both physical and biological, were the key to understanding and predicting the survival success of larval fish. Feeding rate The feeding rate of larval fish is affected by such factors as prey concentration, prey type, type of diet, and prey size. Examination of the stomach contents in larval fish can be used to determine the ef{ects of those factors and how they influence larval survival and growth early in the life of larvae, beginning with first feeding. Prey concentration First feeding in larval fish usually corresponds with the onset of yolk-sac absorption; yolk utilization rates prior to this do not differ significantly when larvae are presented different prey densities (Eldridge, 1981). After yolk-sac absorption the number of larvae that successfully feed within the population, known as the "incidence of feeding'', increases with age. Due to the limited swimming capabilities and, consequently, prey range of younger larvae, prey concentrations have a greater effect early in


10 life. As the larvae become older, increased fin.development allo ws the larvae to range for food more readily in areas with low prey concentrations (Werner, 1981). Houde and Schekter (1981), and Buckley et al. (1987) determined that there was a critical prey concentration (prey/ml) needed for survival in all fish larvae, with the concentration dependent upon the species. They found that of three larval fish species Archosargus, Anchoa, and Achrius Archosargus larvae were best at utilizing intermediate and low prey concentrations. Achrius and Anchoa could only utilize prey effectively at higher prey densities. Bu ckley et al. (1987) obtained similar results with three temperate marine larvae; American smelt, Atlantic mackerel, and haddock. American smelt larvae had the highest surv i val at all feeding levels. In contrast, Atlantic ma ckerel larvae had the highest growth rates but were f ound t o have lower survival than the American smelt at all prey c oncentrations. Larval haddock did not show any trend in growth o r increase in survival with changing prey concentrations. Both feeding studies concluded that species surviving better at high prey concentrations were more likely to be resource limited under low prey conditions. Neither study took diet preferences of the larval species into account. Quality of food: diet and size of prey Th e quality o f the type o f food ingested plays a major r ole i n s u r v i val. Watanabee e t al. ( 1983); Landac et al.


(1985), and Hokanson and Lien (1986) looked at the chemical composition of rotifers, copepods, and brine shrimp fed various algae and yeast diets to determine the best food source for larval fish. Rotifers that had been fed a diet of algae were higher in fatty acids than those fed strictly on yeast, resulting in a higher nutritive value. Rotifers of this higher nutritional value resulted in higher growth rates and survival in fish larvae. Brine shrimp, fed to larger larval species, were found to be less nutritional than algae-fed rotifers, resulting in lower growth rates. 11 In terms of a natural diet versus a cultured diet, larvae of spotted sea trout and striped bass fed a diet of copepods were found to have similar growth and survival rates to those fed cultured rotifers (Taniguchi, 1981; Wiles, 1981). Artificial feeds have been given to larval fish in other studies, and in all cases zooplankton food items appear to be superior to dry feeds (Prinsloo and Schoonbe, 1986; Opuszynski et al., 1989). In addition to the species that make Up a diet, the size of a food item is also important. Kendall et al. (1987) found that as larvae grow they need larger food items and select their food according to prey size. Laboratory-raised fish are usually offered a die.t of brine shrimp when larvae become too large for rotifers to provide the necessary nutrition.


12 Gut evacuation rate Stomach contents and their rate of turnover determine ingested energy. Gut evacuation rate, the time for the gut to evacuate a bolus of food from the entire digestive tract, may be very rapid or may take hours. In comparison, the rate o f digestion, or the catabolism of food. particles, may be a fraction of the gut evacuation time. Ellertsen et al. {1981; 1989) found that, in cod larvae, complete digestion of ingested copepod nauplii took just 30 minutes. The remaining non-digestible portion of the nauplii took an additional 3-5 hours to clear the gut. Gut evacuation rate is affected by prey concentration and by the age of the larvae. Werner {1981) and Theilacker {1987) determined that larvae feeding a t high prey densities evacuated their gut more rapidly than those fed at lower concentrations. Faster evacuation resulted in less of the prey items being digested and thus lower assimilation efficiencies. The rate of movement of a food bolus was s l o we r in older larvae. Changes in the digestion and assimilation of nutrients during ontogeny correlate with developmental changes in the alimentary canal {Govoni, 1981). In yolk-sac larvae o f Leiostomus xanthurus {spot) the canal is undifferentiated along its length and does not function. Upon yolk-sac absorption, at the time of first feeding, the alimentary canal becomes functional and forms an s-shaped loop with fore-, mid-and hindgut sections. Baragi and Lovell (1986) found that all digestive enzymes in


13 striped bass larvae are active at first feeding except pepsin, although their activities were much lower (20-60%) than the levels measured at metamorphosis. The surface area of the alimentary canal expands slightly at metamorphosis but is accompanied by no other morphological or histological changes. studies on red drum Feeding ecology o f red drum was described by Peters and McMichael (1987), who examined the stomach contents of wild larval and juvenile red drum. The highest percentage o f empty stomachs at any stage o f development occurred in red drum larvae (84.5%). Copepods were the major prey item o f larval red drum comprising 97% of food items by number and 86% by volume. Cyclopod copepods were the most frequent prey item, followed by calanoids and harpacticoids Diet of the larvae was significantly different from that of other size classes, with less than 30% shared food types. steen and Laroch e (1983) found similar results for red drum larvae and early juveniles (1.8-12.6 mm). Copepods and crustacean nauplii dominated the diets of most fish examined. Crustacean eggs and decapod post-larvae were f ound to be another important food source. Their results showed that, as larvae grew, they fed on increasingly larger prey. Individuals 1.8 to 3.0 mm in standard length fed primarily on copepod nauplii and eggs while larger larvae, 3.0 to 8.5 mm, fed o n copepodites, with copepod eggs remaining an important food item.


Methods and materials Maintenance of specimens 14 Fertilized eggs or yolk-sac larvae were obtained from the Florida Department of Natural Resources' (FDNR) hatchery in Port Manatee, Florida (Mr. Dan Roberts, director). Larvae were transported to the USF Marine Science Lab in St. Petersburg and sorted into 26 liter experimental aquaria at a concentration of 2500-3000 individuals per aquarium. Aquaria were placed in a photoperiod and temperaturecontrolled incubator, and maintained at 25 c and a salinity of 30 ppt. A 13-hour light and 11-hour dark photoperiod was used throughout all experiments. Larvae were first fed rotifers (Brachionus plicatilis) starting at day 3 after hatching until metamorphosis (approximately day 14), when experiments were terminated. Aquaria were aerated and a portion of the saltwater in each was changed daily. Rotifers were obtained from Florida Aqua Farms, Dade City, Florida, and cultured using the procedure of Hoff and Snell (1987) (Figure 1). Rotifers were fed Chlorella once a day to avoid any loss in nutritional value. Seawater for culturing was obtained from offshore in the Gulf of Mexico. The seawater was then treated with bleach (sodium hypochorite, 5 25 % ) to remove any additional plankton species and neutralized with sodium thiosulphate. Seawater salinity was adjusted with distilled water and Tropic Marine Seasalt to achieve the 30 ppt level.


Figure 1. Diagram feeding unit used larvae. of rotifer/algae culture laboratory-reared red drum 15 in


16 Critical period Critical period was determined t o the nearest 24 hours by sequentially delaying the time of first feeding. Three series of experiments were conducted using fertilized eggs from three separate spawns. Eggs were placed in a series of 1 1 finger bowls at a concentration of 100 per bowl. Within eac h experiment 2 replicates were used for each day examined. Food was introduced into the first of the series after a period of 1 day starvation.(day 2 after hatch). Food was added to the second of the series after 2 days of starvation, and so on until the larvae died o f starvation. Dead individuals were removed twice daily and counted to determine percent survival for the first two weeks o f development. When introduced, food concentrations were at a saturating level (5.0 prey/ml) as was determined in the feeding rate experiments. Larvae were examined under a dissecting microscope to determine the time of complete yolk-sac absorption. Feeding rate Feeding rate was estimated using the method of Laurence (1971). Approximately fifty larvae were placed in each of a series of 1 liter finger bowls containing different food concentrations: 0.0, 1 .0, 5.0, and 10.0 rotifers/ml. Larvae were allowed to feed for 6 hours whereupon they were anesthetized with MS-222 to terminate the feeding and prevent regurgitation. Ten t o twenty individuals from each bowl were fixed in a 10% f o rmalin for 2 4 hours and


17 then transferred to isopropyl alcohol for later analysis of stomach contents (Tucker et al. 1984). ---An additional thirty larvae were removed from each bowl, separated into three replicates of ten larvae each and dried at 60C for 24 hours to determine dry weights. The feeding rate experiment was repeated for larvae aged 3, 6, 10, and 14 days. Stomach contents were examined by removing the entire mid-section of each larva up to the notochord. The section was placed in a droplet of glycerin on a glass slide, teased apart with fine forceps and viewed under a compound microscope. Rotifer remains were identified by the presence of two plate-like structures (mastax) surrounded by an outer shell ( lorica) ( H off and Snell, 1987). To determine if n octurnal feeding was occurring, larvae were removed prior to the onset of the light cycle. The relationship b etween intensity of feeding and ration level was fit t o the equation o f Ivlev (1961): where: y = R = a = X = -ax y = R(1-e ) size of a unit ration for a unit time maximum size of the ration at the upper limiting level of food concentration beyond which ration size does not increase coefficient of proportionality prey concentration The relationship was used to generate curves for prey ingested versus prey concentration for days 3, 6, 10, and 14.


18 Feeding incidence was calculated by counting the number of empty guts found within the number of larvae examined at each age and prey concentration. Gut clearance rate The time for gut clearance was determined using the technique of Laurence (1971). Fifty to one hundred larvae were placed in 2 liter finger bowls and fed rotifers dyed with 1.0% neutral red (Crippen and Perrier, 1974). Larvae were allowed to feed for 4 hours at a concentration of 5.0 prey/ml, and then transferred to similar concentrations of non-dyed zooplankton. Ten larvae were removed every halfhour, anesthetized with MS-222, sacrificed, and examined using a dissecting microscope. The stained rotifers could be seen through the larval gut lining. The rate at which the dyed items disappeared from each larva1s gut could then be recorded through time. Digestion rates were determined for larvae of ages 3, 6, 10, 12 and 14 days for 3 separate spawns. Daily ration The average daily ration, or the total number of rotifers ingested, was calculated by combining the data for stomach contents in dry weight at each age (obtained from the feeding rate gut evacuation times. The ingested ration was divided by the initial larval weight to calculate the food consumed as a percent of body weight per day.


19 Statistics All ANOVAs, tests for normality, homogeneity of variance (Bartlett's test) and regression analyses (fitted using the least squares method) were d one using the software package statgraphics, Statistical Graphics Corporation. A probability value of 0.05 was used as the cutoff for statistical significance. Results Critical period Percent mortality per day over the first 14 days of life indicated that the day of initial feeding strongly influences the number of larvae that live to metamorphosis (Figure 2). In all cases yolk-sac absorption was complete by the end of day 3/beginning of day 4, with the rate of absorption unaffected by the timing of initial feeding. Control larvae kept at a ration level of 0 prey/ml were all dead by the end.of day 6. Mortality of the control larvae was not significantly different from the mortality for the treatment groups up t o day 6. Larvae first fed on days 2 and 4 had lower mean survival numbers to day 14 than those first fed on day 3: 2.5% and 3.0% versus 5.5%, respectively. Percent daily mortality versus age between days 6 and 13 for larvae initially fed on days 2 and 4 was significantly different than that in larvae initially fed on day However, by the end of day 14, the three treatment groups were not significantly different. Larvae first fed on day 2 had an average daily mortality rate o f 7.19


100 a! 80 ::> H ::> (/) w u 0::: 0 0 Hl0 a! 80 ::> H ::> (/) w u 0::: 0 0 100 a! 80 ::> H ::> (/) w u 0::: 0 0 3 3 a y = exp <10. 26 + -2.14X r2 = 0.62 6 9 12 15 AGE H ::> (/) w u 0::: 0 0 3 Hl0 a! 80 ::> H ::> (/) w u 0::: 0 0 3 20 b = e x p ( 5 .14 + -0. 337X r 2 = 0.87 6 9 12 15 AGE

9.86%/day over the first two weeks of life. Larvae first fed day 3 and 4 up to day 14 had an average daily mortality rate of 7.12 8 .53%/day and 7 .38 10.0%/day respectively. The large stahdard deviations were the result of the exponential decline in larvae number for the first 6 days. Larvae that were first fed on day 5 (48 hours after yolk sac absorption) only survived to days 8 -9. Mortality of these larvae was not significantly different from the other treatment groups up through day 7, at which point the remainder of the larvae quickly perished within two days. In one experiment 2 individuals first fed on day 5 lived to 21 day 14 when but were markedly underdeveloped and only 2.9 mm in standard length (Table 1). Survival of these individuals may hav e resulted from cannibalism on dead o r dying c ompanions within their b owl. Larvae first fed on day 4 were slightly smaller on day 14 in mean standard length than larvae first fed on day 3 Table 1. Standard length and dry weight of delayed feeding. INITIAL NUMBER STANDARD DRY WEIGHT FEEDING INDIV LENGTH (MM) (UG/INDIV) DAY 2 n = 1 0 3 .43 (0.31) 5 2.5 ( 5.18) DAY 3 n = 11 4 .10 (0.49) 73. 3 (6.60) DAY 4 n = 11 3.50 ( 0. 29) 76.6 (6.13) DAY 5 n = 2 2 .90 ---------( ) = standard deviation; NUMBER INDIV -the number of individuals left of the origina l 500 individuals.


22 Maximum mortality occurred between days 5 and 6 in all treatment groups, corresponding to the maximum survival time of the control larvae. The next highest periods of mortality occurred between days 3 and 4 for larvae first fed on days 2 and 3, and between days 4 and 5 for larvae first fed on day 4, corresponding to the period of yolk-sac reabsorption. Feeding rate experiments Examination of stomach contents showed increased feeding relative to increased larval dry weight (pg/indiv) (Table 2). The percent body weight consumed within the f our hour experiments, at ration levels 5.0 and 10.0 prey/ml, remained nearly constant from day 6 to day 14, ranging between 3 and 6% (1 rotifer = 0 24 pg). In all larvae examined, stomachs were empty following the 11 hours o f the incubators1 night cycle, suggesting that feeding was confined to the duration of the light hours (13 hours) within the incubator. Larval size had a profound influence on the interaction between prey density and the rate (Figure 3) and incidence of feeding. Three-day-old larvae contained 0 rotifers in their stomachs at all experimental ration levels. The presence of oil droplets (remnants of the yolk-sac stage) and an underdeveloped digestive tract indicated that the larvae were still unable to feed. In contrast, twenty-five percent o f the four-day-old larvae (at 5.0 prey/ml) contained r otifers within their guts. Those individuals


23 Table 2. Effects of prey concentration on capture success as a function of age. AGE FEED CONC 3 4 6 8 1 0 14 N 3 N4 N 6 N10 N14 0 1 5 1 0 5 0 1 5 0 5 0 1 5 1 0 0 1 5 5 5 5 5 5 NUMBER FISH 30 30 30 30 20 30 30 20 30 2 0 30 20 20 30 3 0 30 20 20 30 30 30 30 30 STOMACH CONTENTS 0.00 -----0.00 -----0.00 -----0.00 ------2.25 (0.96) 0.00 -----0.00 -----3.5 0 (2.07) 0.00 ------5.38 (2.43) 0.00 ------1.44 (0.73) 11.1 (4.89) 9.11 (4.30) 0.00 ------2.29 (1.92) 13.4 (5.62) 12.0 (5.80) 0.00 -----0.00 -----0.00 -----0.00 -----0.00 -----CAL/M PCT(%) AVG DW (10 ) EMPTY FISH 0.00 0.00 0.00 0.00 1.18 0.00 0.00 1. 84 0.00 2.84 0 .00 0.76 5.84 4.79 0.00 1. 20 7.05 6 .31 0.00 0.00 0.00 a oo 0 .00 100 100 100 100 75 100 100 50 100 25 100 55 10 1 0 100 30 0 0 100 100 100 100 100 17.0 15.3 16.0 16.0 15.3 27.5 28.8 27.0 29. 3 30.5 34.8 44.2 33.0 70.5 74.0 102.2 84.4 ( 1. 0) ( 0. 6) ( 1. 0) ( 1. 0) ( 0 6) ( 1. 0) ( 3. 0) ( 2 0) ( 0. 6 ) (3.5) ( 3 5 ) ( 2 0) ( 8. 7) ( 4. 0) ( 4 5 ) ( 14) ( 5 4) N = Night incubation; ( ) = standard deviation; DW = dry weight in feeding concentration = prey/ml; stomach contents =mean number o f r otifers.


40 ,.... 35 .._, I30 z 25 z B 20 a 15

that had fed averaged 2.25 0.96 rotifers per stomach. Larval red drum begin to feed at the end of day 3/beginning of day 4 in synchrony with yolk-sac absorption. 25 Day 6 larvae with food in their guts at 5.0 prey/ml had an average stomach count of 3.50 2.1 rotifers {range: 1-7 rotifers); 50% of the guts were found to be empty. Day 6 larvae had no rotifers in their stomachs at prey concentrations of 0 and 1.0 prey/ml. Day 10 larvae also had n o r otifers in their guts at 0 prey/ml, but at 1.0 prey/ml 45% of the guts contained prey at an average of 1.44 0 .73 rotifers/stomach {range: 1-3 rotifers). Little difference was observed in the incidence of feeding between 5 0 and 1 0.0 prey/ml; at each ration level 90% of the individuals had fed successfully, with stomachs containing a range of from 3 t o 22 r otifers. The largest {day 14) larvae incubated a t 5.0 prey/ml had a range of 5-27 rotifers in their stomachs, with 0% empty guts. Little difference in these figures wa s observed between ration levels of 5.0 and 10.0 prey/ml; at 10 prey/ml stomach contents ranged from 5-23 with 0 % empty guts. There were no significant differences in feeding success between larvae fed at 5.0 prey/ ml and 10.0 prey/ml. Feeding successes were not significantl y different of larvae agts day 10 and 14 days. Similarly feeding successes of larvae aged 3, 6, and 8 days were not significantly different from each other. However, this younger grouping (3, 6-8) of larvae was significantly different fro m older (10-14) larvae showing a high feeding


26 success, indicating an increase in feeding competence with age. Gut evacuation rate Only those larvae whose stomachs were completely filled with dyed rotifers at the termination of the four-hour incubation period were used to determine gut evacuation times for the larvae of different ages. Dissection of the larvae revealed that the neutral red dye had concentrated in the fluid of the gall bladder and the cells of the lower intestine, making the use of a compound microscope a necessity. Three-day-old larvae did not show dyed intestinal tissue or concentrated dye in the gall bladder. These results reaffirm that day 3 larvae are unable to feed. Evacuation time o f the food b olus changed little with increased age (Table 3 ) Table 3 Evacuation rate versus age. Each sample removed represents 10 individuals. AGE (DAYS) 3 6 10 12 14 RUN 1 0.0 {s=l} 2 0 {s=4} 2.5 {s=5} 2.5 {s=5} 2.5 {s=5} TIME (HOURS) RUN 2 0.0 {s=1} 2.0 {s=4} 2.0 {s=4} 2.0 {s=4} 2.0 {s=4} RUN 3 0.0 {s=1} 2.5 {s=S} 2.5 {s=5} 2.5 {s=5} 2.5 {s=5} MEAN 0.00(----) 2.17(0.29) 2.33(0.29) 2.33(0.29) 2.33(0.29) ( ) -standard deviation, n = the number of individuals sampled each half hour, { s } = number of samples per run.


27 Day 6 larvae had the fastest mean evacuation rate of. 2.17 hours ranging between 2 and 2.5 hours. Days 10 through 14 also ranged between 2.0 and 2.5 hours for gut evacuations with a slightly higher average: 2.33 hours. Daily ration Gut evacuation rates were less than the duration of the feeding rate incubations, indicating gut analyses were based on full guts and a high turnover of food within the digestive system of larval red drum. Calculations for the number of complete gut clearances per 13 hour feeding period were 5.99 for 6 day-old larvae and 5.58 for larvae ages 7 to 14 days. Based on these turnover rates and percent body weight/incubation eaten, day 6 larvae ate an average of 18.3% (20.97 12.4 rotifers) of their body weight in rotifers/day. Older larvae ate slightly more, with a body weight/day with average of 25.4% (32.23 14.56 rotifers) for day 8, 35.0% 27.28) for day 10 and 18.3% (74.77 31.36) for day 14. Discussion Red drum larvae deplete their yolk rapidly after hatching: in 3-4 days at 25C. The feeding strategy of the 25C larvae, consequently, must be to obtain food quickly, within 24 hours after yolk-sac absorption, or face the consequences of irreversible starvation. The crucial 24 hours after yolk absorption appear to be the critical period that determines larval survival.


28 Critical period Red drum larvae have a high mortality rate within the first 6 days of life at 25C in the laboratory when fed a rotifer diet. Larvae that do survive after day 6 are those that have fed during the critical 24 hours after yolk-sac absorption. Larvae that initially fed on day 5 began feeding after the critical period and survived only an additional 24-48 hours. on day 5, therefore, larvae have reached the point of n o return" and cannot overcome the effects o f starvation. There are two major period s of larval mortality in red drum larvae raised in the laboratory at 25C. The highest rate o f mortality occurs between days 5 and 6, and is the cumulativ e result of starvation, even when food is present. This would indicate that a large portion o f the larvae never learn to feed. The second highest period o f mortality occurs between days 3 and 4, within the critical 24 h ours, after which irreversible starvation occurs. Lasker et al. (1970) and Hunter and Thomas (1974) found similar results for Engraulis mordax ( herrin g larvae). They determined that irreversible starvation occurred in E. mordax larvae when they had been denied food for more than 1.5 days after yolk-sac absorption. McG urk (1984) a lso c bserved similar high rates of mortality during this period i n h erring larvae, ranging between 1 8 % and 36% o f the p opulation. Red drum larvae that are fed within 2 4 hours o f day yol k -sac absorption, i.e. those fed initially o n days 2 and


4, have lower survival rates than those fed on day 3. Our data agree with previously reported trends for red drum larvae. Roberts et al. (1978b) found survival percentages --to day 14 of 3 5%, 14.0%, and 4.0% for larvae initially fed on days 2, 3, and 4, respectively. The higher survival rates observed by Roberts et al. (1978) are probably due to the lower stocking densities (2-20 individuals/liter) used in his experiment. 29 Mortality is high during the first weeks of life in all fish larvae that have been reared in the laboratory. Average daily mortality for red drum larvae fed initially on day 3, 7.12%/day, was similar to that found by Laurence (1975) for winter flounder larvae, 5.8-10.1%/d, raised in the laboratory. The l o w percentage of survival to day 14 was also similar t o that f ound by Pedersen et al. ( 1989) for cod larvae raised in enclosures where a 3.0% survival t o metamorphosis was observed. According t o the results achieved in the laboratory t o obtain the highest survival rates, red drum larvae should be fed initially on day 3. The lack of significant difference in mortality between treatment groups by days 13 to 14 may indicate that the larva e were in need of larger prey items when close to metamorphosis. The low survival rates for larvae fed initially on day 2, before first feeding had begun, probably resulted from water quality problems due to uneaten food or the fact that rotifers quickly lose their nutritive value without a food


30 source o f their own. Individuals fed on day 4 were fed towards the termination of the critical 24 hours after yolksac absorption, and many larvae may have already reached the point of no return. Feeding rate Examination of gut contents revealed that only a small portion of the population (25%) ingested rotifers at the time of first feeding. This value was lower than the 50-60% feeding incidence observed in first-feeding plaice larvae (Shelbourne, 1957) o r sand lance larvae (Buckley et al. 1987). According t o these results, three-fourths of the populatio n do not obtain enough energy during the critical 24 hour period to overcome the 11point o f no return11, explaining the high mortality rates associated with yolk-sac absorption and first feeding. The daily mortality rates under laboratory conditions were similar to observed daily m ortality rates of wild larval sea trout; 9%/day (Peebles and T olley, 1988). survival o f the remaining 25% o f the larvae t o day 14 depended on their success in obtaining enough energy to support the demands of growth and respiration. The ability of red drum larvae to capture food successfully was directly related to their size and developmental stage. As red drum larvae increased in age and, consequently, swimming capability, the potential hunting range or search area for prey increased. The increased competenc e of older larvae in seeking food enabled


them to exploit lower prey concentrations better-than younger, less experienced and physically capable larvae. 31 The increase in competence of feeding, or incidence of feeding (Table 2), with age in red drum larvae (25% at day 4 to 100% at day 14) has also been reported for other species of larval fish. Ellertsen et al. (1981}, found for instance, that post-yolk-sac cod larvae exhibited close to 100% feeding success at all prey densities. The l o w feeding incidence in larvae aged from day 4 to day 8 may result from an inability to establish a ''search image" for rotifers as a prey item. Predators that learn to identify a prey item by its appearance and characteristic behavior establish what is referred to as a search image for that prey (Smith, 1976). The higher the degree to. which a search image is established, the better a predator can expl oit a type of prey. In the wild and in hatchery conditions red drum larvae hav e a variety of prey items to c hoose from when to establishing a search image, resulting in higher survival rates (Halstead, personal communication). At all ages examined, the number of prey items captured by each larvae was highly varied. The variability may have reflected the degree to which each larva had established a searc h image for r otifers. The variability in stomach contents observed in red drum larvae has also been reported in laborator y-reared larval anchovy, where the number of items per stomach varies by as muc h as a factor of three ( T l1eilacker, 1987). The mean number o f items in a full gut


32 for anchovy larvae was 15.55 rotifers, slightly higher than that for red drum of equal size: 11.1-13.4 rotifers. Larvae on the l ower end o f the variability for stomach contents range do n o t obtain enough energy for survival and die by the time of metamorphosis. Gut evacuation rate The short gut evacuation times observed in red drum larvae indicate a high demand for energy to supply growth and respiration. Higher turnover of food particles leads to a lower portion o f the ingested energy being catabolized and assimilated. In red drum larvae fed a rotifer diet, most of the rotifers that were ingested were quickly digested (except mastax region) and the remains rapidly e xcreted. The use of neutral red dye as a stain for tracking the rotifer remains t h rough the gut was limited in its success due t o the a lm ost complete digestion o f the r otifers, whic h left l ittle stained material to view. An unexpected result o f interest was the concentration o f the dye within the large gall bladder. Pigments are filtered from the bloo d by the liver and concentrated in the gall bladder for excretion into the intestines. The dye appears in the gall bladder within two hours o f initial feeding indicating a high rate of transport of energy within the larvae along with a rapid digestion and assimilation rate. The rates determined were comparable t o values for similar sized larval large mouth bass, 2-2.8 hours (Laurence 1971) and larval carp ( Cyprinus carpio), 1-8 h ours at


33 temperatures of 1aoc to 29oc (Chiba, 1961). Gut evacuation rates have been found to be longer at lower temperatures. Laurence (1977) found gut evacuation rates averaging 6.6 hours (range: 5.1-8.4 hours) for winter flounder raised at a lower temperature of 8C. Red drum larvae exposed to higher temperatures would be expected to show even higher gut turnover rates, resulting in more energy (daily ration) to allocate to growth and metabolism. Daily ration The feeding strategy of red drum larvae is to maximize the number of prey taken within the daylight hours since feeding does not appear to occur during the night-cycle. The size of the prey, the concentration of prey and the digestibility of the food item determine the handling time for each larvae. The shorter the handling time of the prey item, the more prey can be eaten and, consequently, more energy can be for other metabolic needs such as growth. The large increase with larval age in the number of prey captured (Table 2), and the high rate of turnover of food suggest that red drum larvae require a substantial amount of energy to supply the demands of growth and maintenance. The absence of nocturnal feeding by red drum larvae makes it imperative that all energy needed for growth and be gained during daylight hours. The large energy requirements of growing larvae are satisfied by continuous feeding throughout the diurnal activity period.


The diurnal feeding activity and night quiescence observed in red drum larvae has also been reported f o r larval largemouth bass (Laurence, 1971) and winter flounder (Laurence, 1977). Daily ration can be presented as the number of particles eaten in a day (Table 2) or as a weight-specific ration. Houde (1989) determined the number of 0.25 ug particles (equivalent to the average weight of one laboratory reared rotifer) required each day to meet the growth requirements rates of various larval species. A Dicentrarchus labrax DL weighing 90 lJ.g, approximately the weight o f a 14-day-old red drum larva, was found to require an average of 108.9, 0.25 ll9 particles/day, slightly higher than the maximum 106.13 rotifersjday calculated for 14-dayold red drum larvae. In a comparison of this daily ration to that of other species of fish (in terms of percent body weight/day), red drum larvae ingestion rates were similar to those of winter flounder larvae; 10 to 50% BW/d, averaging 28.2% for larvae weighing 10.4 to 667.6 lJ.g/larva (Laurence, 1977). Red drum larvae in our experiment ingested slightly less, averaging 24.3%/day for larvae weighing 17-102 lJ.g/larva. Both rates were well within the range of similar sized larval herring: 20-80% BW/ d (Houde and Schekter, 1981). 34


35 Conclusions The early life history strategy of red drum, as is the case with many marine fishes, is to produced many small eggs that hatch quickly and grow rapidly. Red drum larvae reduce the predation pressure by growing to a large size exponentially. Larvae that do not encounter food 24 hours after yolk-sac absorption on the third day after hatching quickly die from starvation, unable to supply enough e nergy for the demands of the quick growth strategy of the species. The adult red drum maximize survival potential of larvae by releasing eggs in an estuarine environment, thereby exposing larvae to a high probability of satiating food levels during the 24 hour critical period.


36 CHAPTER III: ENERGY USED IN GROWTH Introduction Ricker (1979) describes growth in fish as a series of stages or stanzas. A change from one stanza to another is accompanied by some crisis or discontinuity in development (i.e. hatching, maturation, or a change in habits or habitat). One of the most intriguing stanzas of fish growth is the period between the larval and post-metamorphic stages, where a major reorganization of body structure takes place. A blueprint or bio-algorithm exists in larval fish for growth and development which is slowed or accelerated depending on the environmental conditions (Jobling, 1985). The purpose o f this chapter is to determine the energetics o f growth in red drum larvae from the egg stage to the onset o f metamorphosis using direct measurements (i.e., standard length, weight, and chemical composition determinations) and indirect methods (i.e., RNA-DNA ratios and metabolic enzyme concentrations). L aboratory and pond-raised larvae are compared t o examine differences between the growth of larvae in highly controlled conditions and the maximum growth of larvae raised under the idea l conditions of hatching ponds.


Factors that affect growth rate Environmental factors 37 The two most widely studied environmental factors in field and laboratory experiments have been salinity and temperature. Year-class strength has been shown to correlate to these environmental conditions during the early life stages of the larvae. Studies such as Rothschild (1961) and Alderdice and Hourston (1985} described the effects of temperature and salinity on egg survival and larval growth in the field; they found that highest growth rates were correlated with discrete windows of temperature and salinity. Both studies suggested that there were optimum conditions which resulted in maximum larval survivorship, and that these conditions exist in prime locations f o r spawning. Laboratory studies conducted with controlled levels of temperature and salinity have found that growth correlates positively with increases in temperature at given salinities. Laurence (1978} found that growth rate for larval cod and haddock were positively correlated with a temperature range from 4C to 1ooc. Buckley (1982} similarly determined that for larval winter flounder, optimum growth rates were obtained at 5C and 7oc, with lower rates of growth at 2C and 10C, indicating that growth and temperature were not always positively correlated at extreme temperatures. Positive growth with increases in temperature, therefore, are defined by a lethal lower and


38 upper temperature. Age of the larvae was found to also be a factor in regulation of growth rate through changes in temperatures and salinities. May (1975) found that yolk-sac larvae were more tolerant to changes in temperature and salinity than other embryonic stages. Knowledge of the effects of temperature and salinity on larval growth can aid in rearing larvae out of the wild. Growth rates have been optimized under laboratory and hatchery conditions by controlling temperature, salinity, and water quality conditions. The effects of various conditions on the larvae of red drum have been studied in the laboratory. Holt et al. (1981b) and Arnold et al. (1977) both ran experiments to determine the salinity and temperature conditions for survival and growth of red drum larvae. Larvae were subjected to combinations of four salinities (15, 20, 25, and 30 ppt) and three temperatures (20, 25, and 30C). The best combination of conditions for larval hatching and 24-h survival was determined to be 30 ppt and 25C, conditions that are commonly used in the culturing of red drum. When rearing larvae other environmental factors such as those that affect water quality, must be controlled (Neil, 1990). Water quality conditions that are frequently monitored are: dissolved oxigen; ammonia and nitrite concentrations. Holt and Arnold (1983) found that concentrations of ammonia as low as 0.3 mg NHJ/l significantly reduced survival of newly hatched larvae.


39 Individuals at three weeks of age tolerated twice the concentration of ammonia that they could in the first two weeks. Nitrite concentrations were found to be tolerated up t o 100 mg N02 / l throughout the first two weeks with no significant changes in growth rate. Biological factors Biological factors that affect growth rate include stocking density, competition, prey density and size of prey. Stocking density for optimum survival and growth differs for larvae of different species. High stocking densities may lead to competition between individuals, and energy that is used in competitive interactions between larvae decreases the amount of energy available for growth processes. High stocking densities may also lead to increased problems with water quality resulting in lower growth rates. Klein-MacPhee (1981) found that for southern flounder, 20 individuals/liter was optimum while 80 individuals/liter resulted in the lowest survival and growth rates. In comparison, Roberts et al. (1978b) and Lee et al. (1984) found that red drum larvae had optimum stocking densities of 2 individuals per liter. Larvae raised under conditions of 10 and 20 individuals per liter showed lower growth rates and a higher rate of mortality. However, when substantial amounts of individuals are required for biochemical assays, higher stocking densities are used, ranging between 70 to 100 individuals/liter, as in this study (Lee et al., 1984).


40 The prey density experiments with larval red drum (chapter 2) determined that, at a prey concentration of 5.0 prey/ml, larvae grew optimally. Feeding ration levels with higher prey densities than 5.0 prey/ml did not result in an increase in number of prey eaten (ration), or, consequently, an increase in the larval growth rate. The size of the food particle eaten can also control the rate of growth in larval fish. As larvae get older, the size of available prey becomes important. Pandian and Vivekanandan (1985) observed that even at low densities of prey, a larger prey enhances feeding rate in larvae. Fish were found to be satiated more quickly from fewer larger prey than smaller prey at higher densities. Even after 24 hours of continuous feeding on small prey, larvae may not become satiated. Growth versus ration A growth-ration relationship can be modeled by determining ration acquired at a series of prey densities and combining these data with measurements of growth rate for a specific age. The lowest ration needed to maintain basal metabolism is termed the maintenance ration (Rmaint). The point on the growth-ration curve where efficiency of food utilization is highest defines the optimum ration (R0pt). Ropt may range from a high ration level to a low ration level depending on the species and environmental conditions. Brett (1976) showed that maintenance ration is also affected b y environmental factors (e.g., increasing


41 which lead to increased maintenance costs). Efficiency declines at rations greater than Ropt' up to the maximum ration ( Rmax). Methods for measuring growth rate Histological measurements The compartment within the energy budget of larval fishes that has been given the most attention is the determination of growth rate. Microincrements of otolith growth (daily rings have been used to determine growth rates in a variety of fish larvae [Lough et al. 1982; Boehlert and Yoklavich, 1985; Jones, 1986; Essig and Cole, 1986; Radtke, 1989]). Peters and McMichael (1987), and Comyns et al. (1989) measured growth in wild larval red drum using the daily rings of otoliths. The number of rings formed each day was verified by counting otolith rings in laboratoryreared larvae. The slope o f the linear regression of o tolith number versus age was calculated from the counts and determined to b e not significantly different than one. The results indicated that one ring per day was deposited on the otolith and that the rings were laid down starting on the day of hatch. Thus, wild-caught individuals could be aged in the field. Larval length-frequency distributions in wild-caught individuals were of little utility in determining growth rate due to the larvae's small size range and the two t o three week intervals between sampling time.


42 Other histological methods have examined the cellular structures of various tissues to determine the nutritional status of a larva. O'Connell (1976; 1979) and Theilacker (1978; 1986) used histological criteria for diagnosing the degree of starvation in larval northern anchovy and jack mackerel respectively. Nutritional states based on distinct cellular changes that occurred in tissues of these larvae were believed to be a measure of the expected mortality of the larval populations. Grades were assigned to eleven histo logical characteristic s of the brain, digestive tract, liver, and musculature. Wild-caught larvae were assayed to determine the degree of starvation based upon calibrations using lab-reared specimens O'Connell (1976) determined that an average rating f o r three of the eleven characteristics was sufficient for determining the state of emaciation in a larva. H o wever, determining the degree of starvation-induced emaciation using this technique can be very subjective and morphometric indices, discussed below, are usually included with histological data. Morphometric Measurements The use of b ody measurements such as standard length, head length, eye diameter, body depth at anus, body depth at cleithral symphysis, and body depth at pectoral fin base have been used t o establish morphometric indices of nutritional condition in larval species (Ehrlich et al., 1976; McGurk, 1985; Powell and Chester, 1985). P owell and Chester (1985) found that pre-flexion spot larvae were


43 morphologically similar whether moderately starved or severely starved, indicating that the effects of starvation could not be determined from morphometric mesurements alone. Most studies on larval red drum growth have focused on data easily obtained through the use of morphometric measurements such as standard lengths, width measurements,and dry weights (Roberts et al., 1978a; Lee et al., 1984). Wet weight determination for larval growth can be difficult to obtain due to weight lost from evaporation and handling. A problem with using laboratory-reared larvae to establish both histological and morphometric criteria for general extrapolation is that changes in these measurements may be artificially induced by rearing conditions; thus, other measurements are commonly used. Biochemical Measurements. The proximate and elemental composition of larvae can be used to obtain information on substrate used and conserved during growth. Commonly-measured biochemical parameters, in addition to proximate composition (i.e. proteins, lipids, and carbohydrates) and elemental composition (i.e., carbon, hydrogen, and nitrogen) include nucleic acid concentrations and metabolic enzyme activities. Vetter and Hodson (1983) used data on proximate composition in red drum eggs to locate the source of energy to supply the demands of growth before hatching. They found that lipid reserves in red drum eggs supplied most of the energy for catabolic demands (23. 6 to 16.6 mgjg) up to hatching.


44 Glycogen was the next most highly utilized substance, decreasing from 0 .279 to 0.103 mg/g, while protein was not found to contribute to catabolism. Others (Barron and Adelman, 1984; Fraser et al., 1987; Setzler-Hamilton et al., 1987; Mullin and Brooks, 1988) have used protein content and lipid content to determine growth and nutritional states in larvae that have been caught in the field. Wild-caught individuals were compared to laboratory-raised larvae under varying food concentrations to determine the relative states of proximate composition. Starved individuals were found to be lower in lipids, and protein synthesis was similarly reduced (Setzler-Hamil ton, et al. 19 87) Growth rates estimated for wild-caught larvae using laboratory data correlated well with morphometric data for the wild-caught individuals. Hakanson (1989a; 1989b) found that, in addition to concentrations, the types of lipids present could also be used to determine the condition of arichovy larvae. He found that the concentration of triacylglycerol decreased during starvation, while concentrations of cholesterol remained constant. The elemental composition of larvae can also be used to determine the chemical composition of larvae. The percentages of carbon in a larva can corresponds to the lipid portion of the ciomposition of the larvae. The percent nitrogen, likewise, can be representative of the protein component of proximate composition.


45 In addition to such direct measurements of growth, as in proximate and elemental composition determinations, biochemical assays of nucleic acid ratios and metabolic enzymes are used to measure growth indirectly. A recent type of biochemical analysis that reflects growth rate is the measurement of RNA-DNA ratios (Buckley, Bentle et al., 1981). This technique must be calibrated in the laboratory, but, once generated, it is a useful tool for looking at the nutritional status of field-caught larvae (Haines, 1973), or in monitoring developing larvae in the laboratory. Buckley ( 1984) measured the RNA-DNA ratio in 8 species of laboratory-reared larval fish, and compared the data with growth rates based on protein content. He constructed a model based on the RNA-DNA ratio and temperature that explained 92 % of the variability in protein growth rate. They found that measurements of growth rates for larvae using RNA-DNA ratios were significantly correlated with growth rates determined using measurements of protein content. Barron and Adelman ( 1984), Martin et al. ( 1985), Buckley and Lough (1987), Martin and Wright (1987), Setzler-Hamilton et al. (1987), and Robinson and Ware (1988) have used RNA-DNA ratios in this fashion to estimate the conditions under which species o f larval fish had been growing. These studies demonstrated how wild-caught larvae could be compared with larvae that had been starved


46 and fed under different food concentrations in the laboratory. Through comparison with the laboratory-reared individuals, the RNA-DNA ratios of the field-caught individuals w ere used to determine recent growth rates. Setzler-Hamil ton et al. ( 1987) believed that this information could be used as a predictive tool to determine recruitment of striped bass larvae each year. RNA-DNA ratios that indicated recruitment failure were also correlated with morphometric and histological data that indicated poo r nutritional condition of larvae tested. Westerman and Holt (1988) determined RNA-DNA ratios in red drum larvae under optimum conditions. Faster growing individuals had higher RNA-DNA ratios than the slower-growing cohorts. This was in agreement with the dry weight measurements that were calculated for the individual larvae. Days with large increases in growth were correlated with higher RNA-DNA ratios. Thus they determined that increased RNA-DNA ratios are indicative of recent or current growth in the larvae. Metabolic enzymes such as lactate dehyrogenase (LDH) and citrate synthase (CS) have been used to determine condition of larval fish. Clarke et al. (1992) studied red drum and lane snapper larvae to determine the effects of temperature and nutrition on metabolic enzyme activities. Larvae raised at higher temperatures had higher metabolic enzyme activities in b oth species. Larvae that had been starved o r presented a l ower ration level had lower


47 activities by as much as a four-fold. Dry weight values and protein concentrations in the larvae were also higher at the higher temperatu res, indicating metabolic enzymes can be used as indirect measurements o f growth. Methods and materials L a boratory set-up Larvae were raised at 20C and 25C similar to the methods described in Chapter 2. Prey for lab-reared larvae were provided at four densities, 0, 0.1 1.0, and 5.0 prey items per ml from first feeding (day 3) through the start of metamorphosis (day 14) Prey concentrations were monitored twice daily by removing a 25 ml sample from e ach aquaria, counting the number o f prey in 0.5 ml aliquots and taking the average. Concentrations were adjusted to maintain prey concentrations as necessary. Pond set-up Pond-reared red drum larvae were obtained from the DNR grow out pond s located i n Port Manatee Florida. Two ponds were sampled f o r the first 18 days o f life, one at 32oc and another at 22C Temperature was monitored twice daily, and the average temperature for the two week sampling perio d was used to characterize the ponds. Growth rate versus ration Standard length measurements Standard length of the larvae was measured using a dissection microscope and larvae that had been anesthetized


with MS-222. Measurement was from the snout to the tip of the tail in pre-flexion larvae and to the tip of the notochord in p ost-flexion larvae. Weight measurements 48 Larval growth rates were monitored according to prey concentration. Aquaria with 0, 0.1 and 1.0 prey per ml were checked daily. Aquaria with 5.0 prey per ml was checked every other day, and ponds were sampled an average of every third day sampling occurred each.morning before the larvae began to feed. At each m onitoring interval, 30 individuals were removed for wet, dry, and ash-free dry weight analysis. Ten individual larvae were filtered on to pre-weighed Whatman glass fiber filters and rinsed briefly with distilled water. Standing water was vacuumed off and the samples were placed in pre-weighed micro-centrifuge tubes to prevent evaporation from the larvae and weighed on a Mettler electrobalance f o r wet weight measurements. Growth versus ration curve The percent increase in growth o f the larvae in correlation to the available ration was determined for larvae fed at 0, 1 .0, 5.0, and 10.0 prey/ml for 6 hours. Initial dry weights were determined for 30 14-day-old larvae at the onset of the 6-hour incubations. One hundred larvae were then placed in a series of 1-liter bowls, each containin g one o f the f our ration levels. After the incubations were terminated, stomach contents (as described in Chapter 2) and dry weights were determined. The growth


49 versus ration curve was represented by the regression of the means for growth (%body weight/day) versus ration (%body weight/day) for each o f the four ration levels (Brett, 1979). Average proximate and elemental composition of prey items Prey items were collected in bulk to obtain 50 mg dry weight for proximate and elemental composition measurements. Proximate composition was determined using the methods of Stickney and Torres (1989) and Donnelly et al. (1990). The sample was homogenized with distilled water and analyzed for water, ash, protein and lipid content. Elemental composition was determined using a C :H:N analyzer. Prey samples were taken from the cultured rotifers raised on Chlorella in the laboratory. Average proximate and elemental composition of fish larvae Metho d s used to estimate the proximate and elemental composition of fish larvae were the same as that for prey. Larvae were obtained in bulk (50 mg dry weight) for each day sampled. Pond samples were collected from the hatchery at 0, 2, 6, 10, and 14 days. Laboratory-raised larvae were sampled at prey concentrations of 0, 0.1, 1.0, and 5.0 prey per ml at 0, 2, 6, 10, and 14 days. Protein and lipid values as percent ash-free dry weight ( %AFDW) were multiplied by individual ash-free dry weights to obtain concentrations as


50 caloric content of prey and larvae Caloric content was calculated from compositional data using a value of 0.0048 cal/pg for protein and 0.0095 cal/pg for lipid (Brett and Groves, 1979). RNA-DNA ratio Twenty individuals were removed each sampling period for RNA/DNA content analysis; they were weighed, placed in micro-centrifuge tubes, and frozen at -80C until analysis. RNA/DNA was a nalyzed by first homogenizing the freshly thawed tissue in 1.2 M NaCl and then using the sequential enzymatic method of Bentle et al. (1981). Two 200 pl replicates were taken from the homogenized samples (or standards) and added to a cocktail of Tris buffer adjusted to a pH o f 7.5 (80 mM Tris, 4 mM MgCl2, and 3 2 mM cacl2 ) and ethidium bromide (5 pgjml). Fifteen pl of Proteinase K (10 mg/ml) were added and samples were incubated at 37oc for 9 0 minutes. Sample fluorescenc e was then read on a PerkinElmer spectroflucrometer at 365 nm excitatio n and 590 nm emissio n t o obtain an initial value for the total nucleic acid content of each sample. Twenty-five pl of RNase (5 mg/ml) were added t o digest the RNA and the samples were again incubated at 37C for 30 minutes. After the RNase digestion process was completed. samples were re-read; then, 25 pl of DNase (1 mg/ml) were added and the sample was incubated for an additional 30 minutes before a final reading was taken.


51 Activities of lactate dehydrogenase and citrate synthase Larvae were sampled in bulk every day at a prey concentration of 0 prey/ml. Samples were taken at o, 2, 6, 10, and 14 days for larvae fed 5 prey/ml and those collected in the grow-out ponds. Tissue was introduced frozen into homogenizating medium, ice-cold Tris/HCL (10 mM, pH 7.5 at 10C), and homgenized by hand at 0 to 4oc using conical glass homogenizers with ground-glass contact surfaces (Kontes Glass Co 11Duall11 models). Homogenates were centrifuged at 4500 g for 10 minutes and the supernatants saved for the enzyme analysis. L-Lactate dehydrogenase (LDH, EC 1 .1.1.27; Lactate: NAD+ O xidoreductase) activity was assayed in the pyruvate reductase directio n using methods described in Torres and Somera {1988). Citrate synthase (CS, EC; Citrate: oxa l o a cetate-Lyase CoA-Acetylating) activity was also assayed using the of Torres and Somera (1988). All enzyme activities were a s unitsjgWW, where a unit was 1 of substrate converted to product per minute. Results Growth rate versus ration standard length measurements starved red drum larvae (0 prey/ml) kept at 25C increased in standard length despite the absenc e of prey items (Figure 4 and Table 4). The average size at death on day 6 was approximately 2.89 rnrn, which corresponds to a


5 7 I: I: 4.7 :I: 1-(.!) z w 3.7 ...J 0 Q::

53 daily increase of 0.075 mm/day for days 2-6. surprisingly, these values were similar to larvae fed at 5.0 prey/ml at 25C, which attained an average length of 2.93 by day 6. Table 4. Growth in starved red drum larvae as a function of standard length and dry weight. AGE STANDARD LENGTH DRY WEIGHT (DAYS) (MM) (UG) 0 1. 70 ( --) 29.28 (7.08) 1 2 .69 (0.09) 16.36 (4.52) 2 2.83 (0.06) 16.66 ( 5. 54) 3 2.91 (0.09) 13.48 (4.93) 4 3 .01 (0.15) 10.99 (3.31) 5 2.81 ( 0. 07) 8.13 (3.86) 6 2.89 (0.08) 7.00 ( 1. 80) ( ) = standard deviation Larvae fed ad libitum grew faster at higher temperatures in the laboratory and ponds. Lab-reared individuals at 25C grew to an average of 4 .45 mm by day 14 (Table 5). Flexio n of the notochord within the tail region had begun in the majority of larvae, indicating the onset of metamorphosis. At 20C, day 14 larvae averaged 4 .12 mm, with very few of the larvae demonstrating flexion of their notochord. Pond-reared larvae at 32C had standard length values o f 11.18 mm at day 14 (Figure 5). Notochord flexion occurred on day 8-9 at a length of 4.53 mm, earlier than the laboratory-raised individuals, but similar in size at flexion. Those larvae raised in the ponds at 22C were on avel-9 26 t dav 14 Notocl1or d flexion in these age . mm a .z


54 Table 5. Standard length versus age in lab-reared and pond reared red drum larvae. Day Lab 20 + Lab 25 + Pd 22 + Pd 3 2 + 1 2 .23 0 .00 2 .26 0.00 2 .00 0.00 2.03 0 .00 2 2.34 0.11 2.38 0.12 2.1 9 0.1 9 2.24 0.2 1 3 2.45 0.11 2.51 0 13 2.39 0 .20 2.48 0.24 4 2 .57 0.1 2 2 .64 0.13 2.62 0.23 2.74 0.26 5 2.70 0 .13 2.78 0.14 2.87 0.25 3.03 0 .29 6 2.83 0 .13 2.93 0 .14 3.14 0.27 3.35 0.32 7 2.96 0.13 3.09 0.15 3.43 0 .29 3 .70 0.35 8 3 .11 0 .15 3.25 0.16 3 .76 0.33 4.09 0.39 9 3.26 0 .15 3.43 0 1 8 4.11 0.35 4.53 0 .44 10 3.41 0.15 3.61 0.18 4 .50 0.39 5.00 0.47 11 3.58 0 17 3.80 0 .19 4.93 0.43 5 .53 0.53 12 3.75 0.17 4 .01 0.21 5.39 0.46 6.12 0.59 13 3.93 0.18 4.22 0.21 5 .90 0 .51 6.76 0.64 14 4.12 0.19 4.45 0.23 6.45 0.55 7.48 0.72 15 7 .06 0.61 8.27 0.7 9 16 7 .73 0.67 9.14 0 .87 17 8 .46 0.73 10.12 0.9 8 1 8 9 26 0 .80 11. 1 8 1. 06 Average 0.15 0.17 0 .43 0.54 std dev 0.03 0 04 0.19 0.27 Lab 20 = laboratory raised at 20C; Lab 25 -laboratory raised at 25C; Pd 32 = pond raised at 32C; Pd 22 = pond raised at 22C; + = daily increase in mm/day.


10 CS) v8 :X: 1-(J) 6 z UJ _I Cl 4

56 larvae began on days 9-10, at a size between 4 .11 and 4 .50 mm. Comparison of lab-reared and pond-reared larvae showed that after day 2 standard lengths and developmental stage were vastly different. However, the size at which n otochord flexion (metamorphosis) occurred was similar, no matter what the temperature or feeding c onditions (i.e., pond versus laboratory). Average daily growth rates of lab-reared larvae were 0.17 0.04 mm/day at 2soc and 0 .15 0.03 mmjday at 20C. Pond-raised larvae increased an average of 0 .54 0.27 mm/day at 32C and 0.43 0.19 mm/day at 22C to day 18. Increases in standard length through day 18 were more than three times the rates of lab-reared larvae. The largest increases in larval growth occurre d after metamorphosis, when larval size increased e xponentially. Average daily growth rates up to metamorphosis of pond-raised larvae were about twice those of lab-reared larvae, averaging 0.28 mm/day at 22C and 0.31 mm/day at 32C Weight measurements Growth rates for laboratory-reared larvae at ration levels of o, 0.1 and 1.0 prey/ml at 25C were negative over the test period of 8 days, until death (Figure 6 ) All larvae at ration levels 1.0 prey/ml were dead between 5 and 7 days after hatch. Slopes of the three curves describing the time-dependent decline in dry weight biomass at all three ration levels were not significantly different, however, (p > 0.05, students-t). Dry weight values were


40 (!) 30 :::J ....., I-:I: (!) 20 H w :t >10 a::: Cl 0 0 40 (!) 30 :::J ....., I-:I: (!) 20 H w :t >a::: 10 Cl 0 0 40 (!) 30 :::J ....., I-:I: (!) 20 H w :t >10 a::: Cl 0 0 Figure 6. 2 2 2 AGE AGE a y = exp(3.2 + -0.236X) r 2= 0.60 4 6

58 significantly different from values for larvae raised at a ration level of 5.0 preyjml. Larval wet weights were slightly lower at ration levels of 0, 0.1, and 1 0 prey/ml, but were not significantly different from larvae ages 2-6 fed 5 0 preyjml. Positive growth to metamorphosis was achieved at 5.0 prey/ml, which was determined in Chapter 2 as ad libitum feeding (Figure 7). The relationship between weight and age was best described using an exponential equation. An upward inflection of the growth curve was present between days 2 and 4, the period at which the three lower ration levels had the most decline. Larvae held at 25C and fed 5.0 prey/ ml had higher growth rates than those raised at 20C at the same .ration level inthe laboratory (Table 6}. comparison of variability in the two curves indicated that the growth rates were not significantly different at the two temperatures. Growth averaged 2.86 for the first two weeks of growth in larvae raised at 20C and 10.09 for larvae reared at 25C. Expressed as a percent increase in weight, larvae reared at 20C increased 10.5 %BW/d and those reared at 2soc increased 19.3 %BW/d. Thus, an increase in soc corresponds to a doubling in percent increase and a tripling in average weight in red drum larvae raised in the laboratory. Growth rates for pond-raised red drum larvae (Figure 8) were far higher than those fed rotifers in the laboratory. Larvae raised at 22oc in the ponds increased in size an


150 ,..... (.!) 120 ::J '-" t90 :X: (.!) H w 60 3 >0 30 0 0 150 ,..... (.!) 120 ::J '-" t-90 :X: (.!) H w 60 3 >0 30 0 0 Y = exp(2.02 + 0.176X) r 2= 0. 77 3 6 AGE

60 Table 6. Growth in dry weight of red drum as a function of temperature in laboratory-raised larvae. Day 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 AVG Lab 20 11.54 12.75 14.08 15.56 17.19 19.00 21.00 23.20 25.63 28.32 31. 29 34.57 38. 20 42.21 46.64 51.53 weight increase 0 1. 21 1. 33 1. 48 1. 63 1. 81 2 .00 2.20 2 .43 2.69 2.97 3.28 3.63 4.01 4.43 4 .89 2.86 % 0 10.5 10.4 10.5 10. 5 10. 5 10. 5 10.5 10.5 10.5 10. 5 10. 5 10.5 10.5 10. 5 10.5 Lab 25 12.84 15.32 18.28 21.81 26 .02 31.05 37.04 44.20 52.74 62.94 75.07 89.58 106.88 127.52 152.15 181.54 weight increase 0 2.48 2.96 3.53 4 21 5.03 5.99 7.16 8.54 10.19 12.14 14.51 17.30 2 0 .64 24.63 29.39 10.09 % 0 19.3 19.3 19.3 19.3 19.3 19.3 19.3 19.3 19.3 19.3 19.3 19. 3 19.3 19.3 19.3 Lab 20 = laboratory reared at 20C; lab 25 = laboratory reared at 25C; weight increase = % = percent body weight/day.


16 ""' (S) (S) (S) .-4 12 >< (!) => ....., 8 :I: (!) H w 4 3 >-0::: 0 0 6 8 16 ""' (S) (S) (S) .-4 12 >< (!) => ....., 8 :I: (!) H w 4 3 >-0::: 0 0 6 8 Y = exp(0. 504 + 0.453X ) r 2= 0. 98 10 12 AGE CDAYS) 14 Y = exp(1.06 + 0.471X) r 2= 0.96 10 12 AGE (DAYS) 14 61 a 16 18 b 16 18 Figure 8. Growth in dry weight for larvae raised in ponds at a) 22C and b) 32C.


62 average of 318.27 over 18 days: a 57.3 % BW/d increase (Table 7). Larvae raised at 32C in the ponds increased an average of 799.22 a 60.2 %BW/d increase. An increase of 10C in the pond environment resulted in a twoto three-fold increase in absolute daily weight gain. Comparison with laboratory-raised larvae shows that pondraised larvae grow more than two orders of magnitude faster at the higher temperatures and feeding conditions. N otochord flexion apparently occurs at a prescribed weight point in the development of red drum larvae. Larvae reared in the laboratory at 25C weighed an average of 89.58 at notochord flexion on day 14. Larvae reared at 22oc in the ponds underwent flexion at an average of 97 .40 23.18 on day 9 and at 32C, 102.1 5.10 between days 7 and 8. Growth versus ration curve A growth versus ration curve was constructed for twoweek-old larvae reared at 25C in the laboratory (Figure 9). The ration corresponding to optimum growth (Roptl was estimated to be 23.5 %BW/day, which corresponded to a prey concentration of 5.0 prey/ml and an 18.8% increase in BW/d of the larvae, indicating that most energy was used in growth processes. For larvae to maintain zero growth, or status quo, they must consume 9 % BW/d in rotifers (28.1 rotifers), whic h corresponds to Rmaint= the r ation level needed f o r daily maintenance. Ratio n levels between zero growth and Ropt is left f o r routine metabolism, growth, and


63 Table 7. Growth in dry weight of red drum as a function of temperature in pond-raised larvae. Day Pd 22 (pg) 3 4 10.13 5 15.93 6 25.04 7 39.38 8 61.93 9 97.40 10 153.16 11 240.85 1 2 378. 77 13 595.63 14 936.68 15 1473.00 16 2316.40 17 3642.70 18 5728.80 AVG weight increase 0.00 5.80 9.11 14.34 22.55 35.47 55.76 87.70 137.91 216.86 341.05 536. 32 843.40 1326.30 2085.60 318. 27 % 0.0 57.3 57.3 57.2 57.3 57.3 57.2 57.3 57.3 57.3 57.3 57.3 57.3 57.3 57.3 Pd 32 (pg) 11.9 19.07 30.56 48.97 78.47 125.73 201.46 322.80 517.23 828.76 1327.90 2127.80 3409.40 5462.90 8753.30 14026.00 weight increase 0.00 7.17 11.79 18. 41 29.50 47.26 75.73 121.34 194.43 311.53 499.14 799.90 1281.60 2053.50 3290.40 5272.70 799.22 % 0 0 60. 3 60.3 60.2 60.2 60.2 60.2 60.2 60.2 60. 2 60.2 60.2 60.2 60.2 60.2 60.2 Pd 22 = pond reared at 22C; Pd 32 = pond reared at 32C; weight increase = pg/individual/day; % = percent body weight/day.


40 30 r 20 ,... >

excretion. At ration levels of less than 9% BW/d, the larvae demonstrated negative growth, presumably as 65 the larvae began to combust their own tissues. Larvae fed at 1.0 preyjml declined in weight at a rate of 4 .1% BW/d and consumed a ration of 4 .1% BW/d. At 0 ration level, the larvae lost 6.8% BW/d Rmax could not be determined from the growth-ration curve generated but was assumed to be close t o the value for Ropt Average proximate and elemental composition of prey items Brachionus plicatilis, raised on Chlorella, exhibited a protein level of 32.71% and a lipid level of 9.37% of its ash-free dry weight. carbohydrate level was low, averaging 2 .84% of ash-free dry weight. Using the literature values for the average individual biomass, determined by Hoff and snell (1989) and caloric values recorded by Brett and Groves (1979) as a basis f o r its constituents, our data suggest that each rotifer has an energetic value of 0.000526 calories. Elemental composition of the rotifers showed that the percent carbon was 42.02% AFDW and the percent nitrogen was 10.41% AFDW. The carbon-nitrogen ratio was 3.56. Average proximate and elemental composition of fish larvae Proximate composition Proximate composition can be expressed in three ways: as a percent of wet weight ( %WW), a percent of ash-free dry weight ( % AFDW), or as the total content per larva


66 (pg/individual). Table 8 demonstrates the changes in proximate composition as a function of ration level and age of the larvae. Red drum eggs exhibited a high water content (mean = 94.65 %WW), a high protein content (mean= 42.17 %AFDW) and an intermediate to high lipid content (mean = 19.35 %AFDW). Carbohydrate, generally a vanishingly small fraction of the overall proximate composition of marine species, proved to be so in this case as well (mean =.0.47 %AFDW) and thus was not used as an index of larval growth rates. A marked increase in protein level in the transition from egg to yolk-sac larvae was observed in the larvae raised in the laboratory at 20C and 25C. A 12% to 20% increase in the percentages of protein AFDW occurred, and the increase was manifested as an increase in the total % recovery ( %AFDW) o f organic matter. This suggests the presence o f an undetectable class of compounds (e.g., glycoaminoglycans) in the fertilized egg, but not in day 2 larvae. Alternatively, there is the possibility that these compounds are converted over to protein to provide the musculature for the developing larvae. This trend can be seen in the upswing in protein of day 3 larvae to 59. 68% AFDW, suggesting that the developing musculature commands the major fractio n of the tissue in the early developing fish.


67 Table 8. Proximate composition of laboratory-raised red drum larvae at ration levels of 0 and 5.0 prey/ml at 2ooc and 25C and pond-raised red drum larvae at 22oc and 32oc. Day C Ration 0 25 0.0 1 25 0.0 2 25 0.0 3 25 0.0 4 25 0.0 5 25 0.0 6 25 0.0 Protein % AFDW 43.84(4.47) 57.53(5.38) 55.68(3.97) 59.68(5.41) 58.74(2.48) 62.89(1.20) 62.47(3.28) Lipid % AFDW 9.72(4.16) 18.45(2.08) 22.39(6.02) 21.24(6.06) 16.86(4.35) 12.67(1.72) 13.29(1.28) AFDW AS %DW 91.3(4.5) 94.5(0.2) 90.7(1.8) 90.6(0.9) 90.1(1.2) 94.4(1.4) 92.8(1.2) 90.8(0.7) 92.8(1.0) 91.1(0.8) 89.6(0.7) 91.8(0.6) 93.1(1.6) 91.0(1.0) -------------------------------------------------------0 20 5.0 43.74(4.27) 24.34(1.28) 94.8(7.4) 95.4(0.5) 2 20 5.0 53.08( 4.86) 20.54(3.12) 93.2(2.3) 90.3(1.1) 7 20 5.0 53.19(----) 10.78(----) 91.0(---) 90.3(---) 14 20 5.0 51.73(----) 9.17(----) 87.7(---) 85.7(---) ----------------------------------------------------------0 25 5.0 43.39(0.67) 20.18(2.16) 97.4(2.4) 95.7(0.7) 2 25 5.0 54.64(1.24) 24.54(0.02) 91.8(1.3} 93.5(2.1) 6 25 5.0 52.91(----) 17.23(----) 91.3(---) 88.0(---) 7 25 5.0 54.86(----) 12.46(----) 90.0(--) 86.9(---) 1 2 25 5.0 55.59(----) 11.74(----) 89.4(--) 85.8(---) 14 25 5.0 55.13(2.38) 11.64(0.82) 86.7(0.7) 87.8(0.6) -----------------------------------------------------------0 22 POND 42.70(----) 18.08(----) 92.5(---) 95.0(---) 2 22 POND 56.21(----) 24.82(----) 93.1(---) 91.4(---) 14 22 POND 56.22(----) (----) 87.6(---) 79.3(---) -----------------------------------------------------------0 32 POND 45.62(----) 24.98(----) 97.2(--) 93.9(---) 2 32 POND 54.00(----) 26.36(----) 91.3(---) 92.7(---) 6 .,.., POND 6 5.61(----) 13.05(----) 91.6(---) 88.5(---) 1 0 32 POND 64.16(----) 11.79(----) 87.1(---) 88.0(---) 14 32 POND 64.16(----) 9.87(----) 81.7(---) 89.3(---) ----------------------------------------------------------ROTIFERS 32.71(1.46) 9.37(0.35) 87.4(0.5) 92.2(0.5) ( ) = standard deviation


68 Viewed as a fraction of the total body mass o f each larva, the protein level (%AFDW) shows an increase through time at 0 ration (43.84% to 62.47%) relative to a reduction in lipid (19.72% t o 13.29%), which indicates that lipid was used for energy production during tissue combustion in preference to protein in starving larvae. On a basis, protein actually decreased in starved larvae from 10.5 in newly hatched larvae down t o 4.0 f o r larvae 6 days old. Lipid values declined from 3 pgjindividual on day 3 to 1 pg/individual at day 6. PerceLt water remained high until death at day 6, averaging 91.0% throughout the survival period. The counterpoint to 0 ration data is provided by the data at 5.0 preyjml at 20C and 25C (Figure 10). The upward trend clearly demonstrates accumulation o f energy as protein with little lipid storage. Protein as %AFDW for larvae raised at 2ooc increased from 43.74% as eggs t o 51.73% at day 14. Lipid decreased (%AFDW) from 24.34% as eggs to 9.17% at day 14. Protein concentrations increased from 7.5 pg/individual at day 2 t o 16 pg/individual at day 14. Lipid concentrations increased from 1.6 pg/individual at day 6 to 2.9 pg/individual at day 14. Larvae reared at 25oc increased in protein from 43.39% AFDW as eggs to 55.13% AFDW at day 14. Lipid concentrations as %AFDW decreased from 20.18% as eggs to 11.64% at day 14. Protei n content expressed as pg/individual increased from 7.5 pg/individual at day 2 to 33 pg/individual at day 14. Lipid concentrations decreased with age, with day 6 larvae


a 0 b 0 c 0 Y = exp(2.35 + -0.163X) r2 = 0.44 3 6 9 12 15 AGE 8 H 0 6 '\. (.!) a 4 0 2 H ...J 12 ...J 0 H 8 0 6 '\. (.!) a 4 0 2 H ...J d 0 e 0 f 0 69 Y = exp(1.36 + -0.243X) r2 = 0.42 3 6 9 12 15 AGE

70 averaging 3.5 pg/individual and day 14 larvae averaging 8 pg/individual. The percent water decreased to 88.0% at day 6, less than the value for starved larvae of the same age. The increase in protein %AFDW found in larvae raised at 25C in the laboratory was not significantly different from larvae reared at 20C in the laboratory. Lipid concentrations as %AFDW, however, were found to be significantly different, suggesting that at higher temperatures there is some lipid storage occurring. The proximate composition data set collected on pondraised larvae was smaller than ideal due to problems in obtaining adequate sample sizes through time from the ponds. However, the data on accumulated protein and lipid concentrations give an excellent indication of maximum growth. Pond-raised larvae at 22C and 32C showed faster accumulation of total protein and lipid than larvae raised in the laboratory (Figure 11). Protein concentrations of larvae increased in %AFDW from 42.70% and 45 .62% as eggs to 56.22% and 64 .16% at day 14 f o r 22C and 32C, respectively. Lipid values (%AFDW) decreased from 24.82% to 9.87% at 32C similar to the decrease in lipid percentages in laboratoryraised larvae. Pond-reared larvae at 22C increased in total protein content from 200 pg/individual at day 6 to 480 pg/individual at day 14, while those kept at 32C increased from 200 pg/individual at day 6 to 1200 pg at day 14. Thus, an increase in 1ooc resulted in a three-fold increase in


71 ....... CS) 800 a ....... c >< _J _J H H :> Cl 4 0 0 z H (!) ::J (!) ....., 320 Cl 200 z H a.. H H w _J f-0 a 0 a:: a.. 0 3 6 9 1 2 1 5 1 8 0 3 6 9 12 15 18 A G E < b ....... d _J H H :> Cl z H (!) ::J (!) ....., 320 o200 H z a.. H H w _J f-0 0 a a:: a.. 0 3 6 9 1 2 1 5 18 0 3 6 9 12 1 5 18 AGE < DAYS ) AGE

72 protein pg/individual in 2 week-old larvae raised in the ponds. Lipid values for larvae raised at 22oc and 32oc were similarly much higher than larvae raised in the laboratory with day 14 p6nd larvae averaging lipid contents of 210 pgjindividual and 150 pgjindividual, respectively. Elemental composition Elemental composition was different for larvae fed ad libitum in the lab, those starved in the lab, and those raised in the pond (Table 9). Carbon (%AFDW) declined with age at all ration levels. Eggs obtained from the hatchery were, on average, 48.73 3.98% carbon AFDW. Starved larvae that survived to day 5 were on average 46.06 5.81% carbon AFDW. Larvae raised at a ration level of 5.0 prey/ml (250C) and those raised in the ponds were consistently very similar in % carbon to the other laboratory-reared larvae, ranging from 48.5% at day o to 46% at day 14-18. Nitrogen (%AFDW) remained fairly constant or increased with age at all ration levels. Larvae that were starved had only a slight increase in % nitrogen AFDW from 10.41 0.55% at day 1 to 10.60 0 .23% at day 5. Ration levels of.5.0 prey/ml and pond-raised larvae were again similar in elemental composition with the pond values slightly higher (10.4 to 12.2% for the pond larvae nitrogen AFDW versus 9.9 to 13.1%). carbon-nitrogen (C/N) ratios were higher in larvae kept at a ration level of 0 prey/ml than for larvae raised either at 5.0 preyjml or those from the ponds. Starved larvae had


73 Table 9 Elemental composition of laboratory-raised red drum larvae at three prey concentrations, and pond-raised red drum larvae raised at 32oc. AGE RAT 2 0.1 2 6 0 1 2 3 4 5 1.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 CAR-DW 51.82 51.28 44.69 46.53 ( 9. 16) 46.01 ( 0. 42) 50.38 ( 1. 88) 45.05 ( 4. 99) 44.46 ( 1. 97) 40.93 ( 5. 6 8) NIT-DW 9 .02 9.08 9.59 9.33 ( 1. 08) 9 .18 (0.52) 9 .04 (0.43) 9.21 ( 0. 51) 9.00 ( 0. 16) 9.43 (0.33) CAR-ASH 54.15 56.85 51.37 51.83 (3.98) 48.93 (0.93) 53.99 ( 1.10) 49.96 ( 4. 84) 49.79 (1.92) 46.06 (5.81) NIT-ASH 9.43 10.07 11.02 10.31 (0.55) 10.41 ( 0. 52) 9.70 (0.48) 9.80 (0.50) 10.05 (0.13) 10.60 (0.23) C/N 5.74 5.65 4.65 5.03 (0.19) 5.53 ( 1. 04) 5.73 (0.43) 5.09 (0.26) 4.95 (0.13) 4.34 (0.46) ----------------------------------------------------------0 2 6 7 12 14 5.0 5.0 5.0 5.0 5.0 5.0 45.45 45.79 41.19 41.37 (0.37) 38.31 39.62 (1.16) 10.19 9.45 10.06 10.89 ( 0. 21) 10.65 10.68 ( 0. 4 7 ) 46.66 49.88 45.12 45.77 (0.41) 42.92 45.73 (1.17) 10.46 10.29 11.02 12.03 (0.14) 11.91 12.27 (0.50) 4.46 4.85 4 .10 3.80 (0.03) 3.60 3.73 (0.22) ----------------------------------------------------------0 2 9 15 18 POND POND POND POND POND 38.02 47.15 40.54 41.15 39.49 7.25 9.62 10.84 10.47 11.09 47.70 51.53 44.35 46.66 46.79 9 .10 10.51 11.86 11.87 13.14 5.24 4.90 3.74 3.93 3.56 --------------------------------------------------------ROT 42.02 10.41 3.56 RAT = prey/ml; CAR = carbon; NIT = nitrogen; DW -% dry weight; ASH = % ash weight; ROT = r otifers; ( ) = standard deviation.


74 high C/N ratios as eggs, and these values remained high until death at day 5 (4.34 0.46). Pond-raised larvae had values similar to those for 5.0 prey/ml in the laboratory, but were slightly lower at day 14-18 (3.56 versus 3.73). Caloric content of the larvae Larvae that had been starved since hatching declined in total protein and lipid, resulting in a decline in total calories/individual (Table 10 and Figure 12). The data suggest that starved red drum larvae combust their own tissues during starvation at a rate of 0.01135 calories/individual/day after day 2. As a consequence, each individual declines in total caloric value as the protein and lipid in its tissues are used for metabolism and growth. Larvae fed at a ration level of 5.0 preyjml at 20C and 25C increased in total caloric content age (Figure 12). Larvae raised at 20C increased an average of 0.0038 calories/individual/day after day 2 and increased to a total of 0 .0994 calories/individual at day 14. Those larvae reared at 25oc increased at a rate of 0.0160 calories/individual/day, to an average total of 0.2523 calories/individua l at day 14. The limited data on lipid and protein concentrations in pond-reared larvae were used to approximate caloric content at maximum growth. Pond-raised larvae kept at 22C increased in total caloric content from day 2 (0.0782 calories/individual) to day 14 (4.310 calories/individual) (Figure 13). Larvae raised at 32C in the ponds increased


75 Table 10. Total caloric content of red drum larvae at 25C starved; 25C and fed 5.0 prey/ml; 2ooc and fed 5.0 prey/ml; and 22C and 32C pond raised. AGE TEMPERATURE RATION CALORIC CONTENT CAL/D (DAYS (OC) (PREY/ML) (CAL/INDIVIDUAL) 0 25 0 0.0949 ------1 25 0 0.0769 -0.0180 2 25 0 0.0622 -0.0147 3 25 0 0.0504 -0.0118 4 25 0 0.0408 -0.0096 5 25 0 0.0331 -0.0077 6 25 0 0.0268 -0.0063 ------------------------------------------------------------2 6 10 14 20 20 20 20 5.0 5 0 5.0 5.0 0.0535 0.0657 0.0808 0.0994 0.0031 0.0038 0.0047 ------------------------------------------------------------3 6 10 14 25 25 25 25 5.0 5.0 5.0 5.0 0.0961 0.1216 0 .1662 0 .2272 0.0092 0.0148 0.0238 ------------------------------------------------------------2 6 10 14 22 22 22 22 POND POND POND POND 0.0782 0 .2976 1 .1320 4.3100 0.0549 0.2086 0.7945 ------------------------------------------------------------2 32 POND 0.0546 ------6 32 POND 0 .2763 0.0554 10 32 POND 1.3980 0 .2804 14 32 POND 7.0750 1.4190 CAL/D = daily caloric increase.


,... 0.4 :::> H 0 z H 0.3 ...J H 0 z H 0.3 ...J H 0 z H 0.3 ...J r 2= 0.35 6 9 AGE r 2= 0. 85 6 9 AGE

,..... 50 :> 1-t c a z .... ..J .... b c z ....

78 from 0.0546 calories/individual at day 2 to 7.7075 calories/individual at day 14. Average increase in calories/day was 0.3527 calories at 22oc and 0 .5850 at 32oc Indicators o f growth rate Results suggest that the critical 2 4 h our period between the third and fourth day after spawning determines the survival o f early red drum larvae (Chapter 2), and this is reflected in all biochemical indicators examined in this study. Values f o r RNA/DNA and metabolic enzyme concentrations, b oth indirect measurements of growth, reflected the rate o f growth of the larvae and the temperature at which the larvae w ere raised. RNA-DNA ratio RNA-DNA ratio s mirrored the trends observed in larval growth and proximate composition. A decline was observed in RNA/DNA for larvae starved for the first 6 days (0 ration). Downward inflections were observed between day 0 and day 1 due to hatching and between day 3 and day 4 (Figure 14). After day 3 at 0 ration, RNA/DNA reached a plateau (slope not significantly different than 0) at a value of roughly 0.5, indicating that protein synthetic capacity was severely diminished after that time. RNA/DNA in larvae raised at 5.0 preyjml in the laboratory showed n o inflection between days 2 and 4. Rather, the curve exhibited a gradual decline to a plateau of 1.3 at 2ooc and 1.1 at 25C; values at the plateau were not significantly different.


24 20 <1: 16 z c 12 <1: z Q:: 8 4 0 0 24 20 <1: 16 z c 12 <1: z Q:: 8 4 0 0 24 20 <1: 16 z c <1: z 12 Q:: 8 4 0 0 Figure 14. 3 3 3 Y z exp(1.852 + -0.481X) r 2 0. 79 6 9 AGE Y = exp(0.752 + -0.050X) r 2= 0.40 6 9 AGE (DAYS) 79 a 12 15 b 12 15 c 12 15 Growth measured indirectly as RNA/DNA for larvae raised in the laboratory at a) 0 preyjml, b) 5.0 prey /ml at 2ooc, and c) 5.0 preyjml at 2soc.


80 Pond-raised larvae had higher values for RNA/DNA (Figure 15) than the larvae raised in the laboratory. Larvae raised at 22C in the ponds had values between 3 and 4 for 2 week-old larvae. Larvae reared at 32oc had lower RNA/DNA values than larvae reared at 22oc, averaging 1.6 at day 14. The difference between the RNA/DNA values in the ponds was found to be significant. In both the laboratory and the pond-raised larvae, those larvae raised at the higher temperatures demonstrated lower RNA/DNA despite the higher larval growth rates. Metabolic enzymes LDH activities of laboratory-raised larvae increased with age at ration levels of 0 and 5.0 preyjml and temperatures of 20C and 25C (Figure 16). Larvae that were starved continued to produce LDH, though at lower concentrations than fed individuals until death at day 6 Rates of increase were approximately equivalent up to day 6 at both 2ooc and 25C. Larvae reared at 20C had LDH values ranging 20-25 unitsjgWW at day 14. These LDH activities were slightly lower than for larvae raised at 25C, which had LDH values of between 30-35 units/gWW at day 14. Similarly, cs activity increased rapidly to a plateau of approximately 2.0 unitsjgWW by day 3 and remained at that level with increasing age for starved larvae until death at day 6 . Larvae raised at 5.0 prey/ml at 20C also increased rapidly to a plateau of 2 units/gWW which remained relatively constant through day 14. Larvae reared at 2soc


81 4 a 3

""'40 3 3 a C) y = -0.129 + 1. 268X = H 0 .65 z ::::::> '-" >-20 ..... H ::> H t; 10 '-" >-20 ..... H ::> H t; 10 '-" >-20 ..... H ::> H t;10 '-" >-3 ..... H 2 ::> H ..... u 1 '-" >-3 ..... H 2 ::> H ..... u 1 '-" >-3 ..... H ..... CJ) u0 d y = 0 .434 + 0 315X = 0 .59 I 3 6 9 AGE 8 2 12 15 12 15 0 3 6 9 12 15 0 3 6 9 12 15 AGE

83 showed increased cs activity with age, and never reached a plateau. The CS values of 4-6 at day 14 were double the values of larvae reared at 2ooc, but more data needs to be collected on cs to make any definitive conclusions regarding this increase. Activities for both metabolic enzymes were higher in pond-reared larvae than laboratory-reared larvae (Figure 17). Data were again sparse due to the difficulties in collection of the large amount of sample required for both proximate c omposition and enzyme assays. Larvae reared at 22C i n the p onds averaged LDH activities of 45-50 units/gWW, higher than the values for larvae raised at 32C, which a veraged 25-30 units/gWW. The difference between the two ponds w a s determined to be significant and dependent on the temperature at which the larvae were reared. cs activities were higher at 22C, between 4-5 units/gWW, than at 3 2 C between 2-3 units/gWW. Protein c ontent, RNA/DNA, and metabolic enzyme concentration as predictive t ools Specific growth rates could be calculated by the use of multiple regression equations that included a biochemical parameter, and in most cases, a temperature parameter. In all cases, the fit of the observed data to the models was increased by the addition of temperature to the multiple regressions factor, indicating a temperature dependence in eac h case (Table 11}. Predicting specific growth rate from LDH o r cs values alone was not p ossible, since the


84 ...... 60 6 :I a ""' c :::1 :I :I en f-en y = -2.252 + 3.527 X fY 2= 0.586 H 4 ::::> r2 z '-" = 0.98 ::::> r = 0.71 >-30 '-" >3 fH fH 2 ::> fH u f- ::::> '-" '-" >-30 3 f->-fH H ::> 2 H f-fu u

85 partial regression coefficient was not significantly different from zero. Values for protein and RNA/DNA could be used to predict specific growth rates (%BW/d) without a temperature term, but with less fit to the models (lower r 2); half the fit of RNA/DNA with temperature. Percent body weight per day based on protein was least affected by the addition of a temperature term, suggesting a lower effect of temperature on the percentage of protein in the larvae. In all equations, probability levels were highly significant (p S 0.01). The best biochemical parameter for the prediction of %BW/d was achieved using values for cs activity and temperature, followed by RNA/DNA, protein (%AFDW) and LDH activity; all with a temperature term. Table 11. Relations between temperature, protein content, RNA/DNA, LDH, cs, and growth rate (%BW/d) in red drum larvae. y 1 T %BW/d y = 3.41X1 -54.5 0.48* 2 R/D %BW/d y = 17.3X1 0.37* 3 T R/D %BW/d y = 3.88X1 + 21.78X2 -99.7 0.82* 4 T LDH %BW/d y = 3.31X1 + 0.80X2 -72.3 0.66** 5 T cs %BW/d y = 4.25X1 -3.45X2 -69.6 0.98* 6 PRO %BW/d y = 3.56X1 -169.7 0.71* 7 T PRO %BW/d y = 1.58X1 + 2.35x2 -141.7 0.74** T = degrees Celsius; R/D = RNA/DNA; LDH = activity units/g/wet weight; cs = activity/g/wet weight; PRO = protein 9o ash-free-dry weight; = p 0.001; ** = p s 0 .01.

PAGE 100

86 Discussion Red drum larvae appear to have a blueprint for survival. The basic pattern of growth and development was the same for larvae under conditions of varying temperatures and ration levels. Depending on the adversity of. those conditions, the growth and development of red drum larvae were accelerated or retarded. The experiments conducted on the relationship between growth and ration (standard length and weight), and on proximate/elemental composition all indicated that growth was the prime goal for survival. Growth versus ration standard length and weight measurements Standard length data supports the idea of the dependence of developmental stages on a blueprint, the closer correlation of those stages with size than age. Larvae raised in the laboratory and the ponds underwent metamorphosis at roughly the same size, independent of the age of the larvae. In the case of the 32C pond, day 7 larvae were already the size of day 14 larvae reared at 25C laboratory, and were undergoing the same stages of metamorphosis. Analysis of growth rates measured as increases in standard length for each of the laboratory and pond conditions agreed with previously determined rates for red drum at similar temperatures (Holt et al., 19Bla; Holt and Arnold 1983; Lee et al. 1984) and other larval species

PAGE 101

87 (Ehrlich et al., 1976; Fraser et al. 1987 Peebles and ----, Tolley, 1988). None of the above-mentioned studies, however, observed the affects of different ration levels on growth rate. Weight measurements also support the theory of an underlying blueprint for growth. Dry weights at metamorphosis were approximately the same under laboratory and pond conditions, despite the age differences. Red drum larvae followed a pattern of growth, where increases in %BW/d as dry weight were constant with increased age (Table 6 and Table 7 ) The values for dry weight of larvae reared in the laboratory were similar to previously reported values for red drum larvae under similar conditions (Lee et al., 1984; Holt, 1990) although there were some differences. The inflection of weight loss due to hatching and yolk-sac absorption and weight gain occurred earlier than those previously reported. The minimum in weight loss observed by Lee et al. (1984) at day 5 for larvae raised at 24C and fed ad libitum (14.7 occurred on day 3 at 25C (12.8 in this study. Once again the data on the effects of ration on weight were sparing, and this study adds insight into the effects of starvation. The increase in standard length and loss of dry weight in starved red drum larvae conflicted with the idea of a conservation of energy. Dry weight data showed negative growth in larvae fed ration levels of 1.0 prey/ml o r less,

PAGE 102

88 indicating a reduction of organic matter within the larvae. Analysis of wet weight data indicated that as larvae were starved, organic material was replaced by water, resulting in similar wet weights between fed and starved larvae of similar ages. The red drum larva funnels all resources into growth in order to increase the chance of finding food and escaping predators. This tactic appears to be limited in its effectiveness t o 5 days after hatching at low ration levels, when larvae reach the "point of no return", resulting in death. The data collected o n proximate composition of the larvae added to the question of how reductions in organics lead to an increase in length. Proximate a n d elemental c omp osition Prey The proximate composition of the rotifers fed Chlorella in this study was similar to previously reported values for rotifers also fed Chlorella (Watanabe et al. 1983). The rotifers were composed largely of protein, making them an ideal food source for fast growing larvae. Rotifers reared on diatoms have been shown to have higher protein (45.3 4 3 versus 37. 5 1 .67 %DW) and lipid values (20. 1 5.2 versus 10.72 0 .40 %DW) Frolov and Pankov, 1992). Thus, Chlorella was a quick-growing and easily-raised food source for rotifers, b u t lacks some of the nutritional value that a diatom diet provides.

PAGE 103

89 The elemental values of carbon and nitrogen (%AFDW) for the rotifers reflected their high protein (% nitrogen) and low lipid (% carbon) levels. The rapid development and growth rates (Hoff and snell, 1987) of the rotifers resulted in low C/N measurements, and consequently, animals high in protein with small reserves of lipids. Larvae Red drum larvae quickly deplete their lipid reserves to supply the energy demands of growth: The increase in protein { %AFDW) reflects the decline in lipid, and was most evident in the starved red drum larvae. Larvae that have been starved conserve protein up until the time of death in the form of musculature for locomotion. Conservation of muscular proteins allows the animal to swim as long as possible before complete muscle atrophy, and the "point of no return" allowing the larvae to search out prey in other, possibly more productive areas. The loss of dry weight and increase i n water content mentioned earlier in starving larvae, versus the comparable values for larvae fed to satiation, reflect the catabolism of the lipid stores, which results in this increase in water content in fishes {Wallace, 1986). The observed water content values for larvae fed to satiation were similar to those found by Vetter and Hodson (1983) for developing red drum larvae.

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90 The rapid reduction in lipid content found in red drum larvae at all ration levels and conditions has been observed in other species of fish. Fraser et al. (1987) found that larval Atlantic herring had a lipid level of 23% dry weight (176 pg) one day after hatch decreasing to 11% (221 pg) by day 16. These percentages were similar t o those for red drum larvae (20.18% to 11.74%) fed ad libitum to day 14 in the laboratory and in the ponds. Elemental composition of the red drum larvae agreed well with other published values for red drum (Lee et al., 1988) and larval herring of similar size (Ehrlich, 1974a; 1974b; 1975). Elemental composition results also agreed well with our data on proximate composition and yielded some basic information on the character of tissue deposition in developing larvae. Larvae that are growing normally, as in the 5.0 prey/ ml experiments and the ponds show greater increases in protein than in lipid. Assuming that nitrogen content reflects protein and carbon content reflects lipid content, the increase in % nitrogen with age, and the constant or declining % C, mirrored the changes (protein increase, lipid decrease) in proximate composition. This changing elemental composition resulted in a declining C / N in normally growing larvae. Starving individuals had slightly higher C/N than fed individuals as a result of their diminished protein synthesis. Larvae raised in the ponds have the lowest C/N as a result of the high protein levels relative to lipid. Thus, the C / N can be used as an

PAGE 105

91 indicator of physiological status in developing fish. It should be noted, however, that this ratio applies in the opposite fashion to adult fish. A declining C/N in older fish indicates starvation where lipid is laid down as an energy reserve and is combusted before protein. The rapidly accumulating musculature of a healthy, growing fish larva results in a declining C/N, giving the appearance of starvation when, instead, this ratio indicates that protein is accumulating at a faster rate than lipid. Growth versus ration curve; Caloric content Growth versus ration curve The observed growth versus ration curve for 2 week-old red drum was similar in shape to that published by Brett (1979) for larval Cyprinodon macularius. Conversion of equivalent numbers of rotifers to %BW (0.24 Hoff and Snell, 1987) allowed for check and balance with.the caloric content data. Values for maximum growth (Gmaxl of red drum were half those of Taniguchi (1981) who found Gmax for Cynoscion nebulosus at maximum ration (Rmaxl; 46.9% to 56.8% per day for temperatures of 24C and 32C, respectively. However, the growth rate of spotted seatrout has been shown previously to be twice the rate of growth in standard length measurements of red drum (Peebles and Tolley, 1988), so a doubling in Gmax was expected.

PAGE 106

92 Approximately 3/4 (18.8% BW/d) of the total ingested energy (23.9% BW/d) is involved in growth processes. The growth-ration curve enabled the cost of standard metabolism or maintenance at zero growth level, to be calculated. The cost of maintaining zero growth (9% BW/day) was half the rate of Gmax which is found generally in fish species (Brett and Groves, 1979). The remaining energy between zero growth and maximum ration was partitioned into positive net growth (tissue), routine metabolism (discussed in Chapter 4 ) and excretion (discussed in Chapter 5). Caloric content Lubzens (1989) found that the ash-free caloric content for rotifer biomass ranged from 4.8 to 6.7 cal/mg, depending on the size and sex of the rotifers. That range is almost twice the value of r otifers raised in our experiment (2.2 to 4.4 cal/ mg ) Using our data describing the average caloric value of a rotifer, the caloric content of tissue (combined protein and lipid) was converted to the number of equivalent rotifers required to produce the tissue. The decline in caloric content of larvae, starved for the first six days of life is reflected in their average of 0.01121 0.0044 calories/day lost, or the equivalent of 21.3 to 29.6 rotifers/day. This loss in calories resulted from the cost of maintenance and agreed with the 28.1 rotifers required for maintenance metabolism as by the growth-ration curve. Ingestion of a ration less than the rotifers/day required to satisfy maintenance costs resulted in negative growth. To

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obtain sufficient energy for positive net growth many more rotifers than this minimum number must be successfully captured and ingested. For example, larvae raised at 2soc and fed a ration level of 5.0 prey/ml increased by 0.0160 cal/day, or the equivalent of 30 rotifers/day, above the zero growth level. 93 Similarly, in order t o supply enough energy to support the caloric increases in pond larvae, an average of 671 and 1112 rotifers/day would need to be ingested at 22C and 32C, respectively. The energy expenditure in obtaining these quantities o f a single prey would be extensive, however, i t is hypothesized that other food sources such as copepods and their nauplii are required t o maintain pond growth rates. Indicators o f growth Nucleic acid ratios and metabolic enzymes were used as indirect measurements o f growth rate to complement proximate composition and morphometeric data. In both biochemical assays, the values were found to be temperature dependent. RNA-DNA ratio The rapid development of the embryo within the egg between spawn and hatching is reflected in the exponential drop in RNA/DNA. The age of the egg, or proximity to hatch, can be calculated from the nucleic acid ratio. The higher RNA/DNA values observed in red drum eggs contradict findings by McGurk and Kusser (1992) who suggest that the yolk interferes with fluorescence, resulting in lower RNA/DNA

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94 values. After hatching, the rate of growth (%BW/day) for temperature in laboratory (2ooc and 250C) and pond raised (22C and 32C) larvae was approximately the same for each day throughout the experiments. The constant rate of growth resulted in a constant RNA/DNA for each larval age group. Ironically, the conditions of higher temperatures that resulted in higher specific growth rates also resulted in lower nucleic acid ratios. This inverse relationship between RNA/DNA and temperature was also observed by Setzler-Hamilton et al. (1987), who found that in late spring, values for RNA-DNA ratios in striped bass larvae were higher than values measured in hotter, early summer months (spring values were around 3 and summer values were around 2 to 2.5). A high growth rate and high RNA-DNA ratio may be the result of an increase in the efficiency of ribosomes in initiating protein synthesis, or of a slow turnover of ribosomes (Westerman and Holt, 1988). An understanding of what classes of RNA are important in the RNA/DNA and how each class changes in concentration with changes in the growth rate is needed to interpret the relationship between the ratio and growth of larval fishes. For example, an increase in RNA/DNA could result from increases in transcription of messenger RNA coding for metabolic and structural proteins, with little increase in ribosomal RNA. Since 90% of RNA is ribosomal RNA, changes in the rate of ribosomal biosynthesis, rate of turnover of ribosomes, or a

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95 combination of both mechanisms may contribute to the ratio. RNA/DNA can be used to determine the nutritional status despite the lack of definition regarding the functioning of RNA/DNA fluctuations of red drum larvae. Red drum larvae that have been starved show lower RNA/DNA (1 or below) than fed individuals in the laboratory and ponds. Wright and Martin (1985) found similar RNA-DNA ratios of 1 to 2 at 19210C for starved striped bass, while fed striped bass larvae had ratios of 3-3.4 during the first two weeks after hatching. The plateau in RNA/DNA observed in starved red drum larvae between days 2 and 4 was not seen during this period in individuals fed in the laboratory or raised in the ponds. Robinson and Ware (1988) observed a similar trend in RNA-DNA ratios with starvation in the early life of larval Pacific herrings; ratios declined up to yolk-sac absorption, where the ratios plateaued. After 72 hours, the plateau ratios were marked by a downward inflection in RNA/DNA values. The point of no return for these larvae coincided with this inflection point, suggesting that the 72 hrs after yolk-sac absorption was the critical period for Pacific herring. As was true for red drum, the plateau of RNA/DNA values was at a ratio of approximately 2, followed by a decline to just below 1 when the larvae died. Values for RNA/DNA obtained in the laboratory in this study ( 1 to 2 ) were slightly lower than previously reported values for (3-5) red drum larvae (Westerman and Holt, 1988) for red drum larvae and other species of larvae (3-5) (Bulow; 1970; Buckley, 1979; 1980) for similar ages and

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(Bulow; 1970; Buckley, 1979; 1980) for similar ages and size. Westerman and Holt (1988) found values of 4.9 for RNA/DNA in newly hatched red drum, 3.0 for day 4 larvae, and an increase of 60% from days 4-14, closer to those we determined for pond-raised larvae. Metabolic enzymes 96 LDH and cs activities track well with observations on behavior in red drum larvae. Both enzymes show inqreases in activity to day 3 of life, whereupon cs activities level off and LDH activities continue to increase. Burst, or anaerobic swimming, is most typical of larval prey capture which requires sudden darting movements. LDH activity is indicative of this burst swimming potential it can be used as a measure of swimming potential. Production of .LDH increases with age as larvae are forced to expend greater energy in prey capture to fuel their increased growth-energy requirements. Production of LDH continues in starved individuals, indicating the nature of red drum larvae; LDH counts show the larvae's funneling of energy into growth and increased mobility in order to obtain food and escape the pressures of predation. The observed leveling off of cs activity suggests that developing muscle tissues reach a level of metabolic competence at day 3 and remain there at least during this stage in growth up to metamorphosis. Activity of cs has been shown to decline with age in fishes (Siebenaller, 1984), but a decline was not observed at this stage of

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97 development. CS production continued in starved individuals indicating aerobic respiration was vital in the first 3 days o f life. Clarke et al. (1992) found similar values for LDH and cs in red drum larvae raised on wild zooplankton. Values for cs in clarke's study, assuming 87% water content, averaged 3.25 units/gWW for two-week-old larvae. LDH activities averaged 19-26 units/gWW, slightly lower than the values we observed in the larvae raised in the laboratory and ponds. Starved individuals in our experiment increased in the concentrations of LDH and cs where larvae starved by Clarke et al. (1992) did not show this trend. Biochemical parameters as pl-edictive tools The interaction o f temperature with the biochemical indices of growth (RNA/DNA, LDH, and CS) made it necessary t o use multiple regression analyses to describe the. relations. The multiple regressions allowed for specific growth rate (%BW/d ) to be predicted for a larva given a value for a biochemical parameter and a temperature. Buckley (1982) and Buckley et al. (1984) also found the need for multiple regression analyses due to the effects of temperature on RNA/DNA and protein values in several species of larval fish. In each case, the addition of a temperature coefficient increased the correlation coefficient for the specific growth regression equation.

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98 Prediction of growth rates from simple regressions of RNA/DNA versus growth should be restricted to a given temperature range. High temperatures may lead to high growth rates but lower RNA/DNA values, which may be mistaken as poor larval nutrition condition. Thus, collection of temperature data when predicting growth rate from RNA-DNA ratio and/or protein content from larvae in the laboratory and field is a necessity. Conclusion In order to increase their chances for survival, red drum tends larvae have rapid growth rates. The rate of to follow a developmental pattern that growth can be accelerated by increases in temperature and ration level. The trends observed in the data presented here agree well with those reported in the literature for satiated red drum larvae (Mercer, 1984; Holt, 1990; Matlock, 1990). The addition of the effects of varied ration levels on red drum larval growth and biochemical composition adds insight into the survival strategy not evident from previously recorded data on larvae fed to satiation. Many trends in measured growth indices reflected periods of development of the larvae. The initial drops in weight, protein, caloric content and RNA/DNA in larvae reared at zero ration were associated with the hatching process (between day 0 and day 1). A plateau between days 1 and 3 is evident in each of these four variables during the period of development of functional eyes and mouthparts

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99 (Holt et al. 1981b). Between days 3 and 4 a sharp downward inflection was observed in all four variables as the larvae rapidly combusted their own tissues to meet the demands of maintenance metabolism. The drops in weight, protein content, and caloric value coupled with the severely diminished ability for protein synthesis strongly suggest that red rum larvae have less than 24 h after yolk-sac absorption on day 3 to find nutrition before irreversible starvation occurs. These findings were in agreement with data collected in the critical period experiments. The larvae raised in the ponds represented maximum ration and growth rate for red drum larvae, and the range of this optimum figures of growth rate can be used to predict survival. Pepin (1989) used growth histories to estimate larval fish mortality. Larval populations with the highest mean growth rates had the least rate of mortality. Based on this theory larvae raised in ponds should have a higher percent survival overall. Biochemical assays can be used as indicators of growth rate, but in most cases the values are ultimately determined by the temperatures at which the larvae are growing. The multiple regressions obtained from the biochemical and temperature interactions can be used as predictive tools in determining growth rates for red drum larvae.

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100 CHAPTER IV: ENERGY USED IN METABOLISM Introduction Brett and Groves (1979) state that the primary function of food, before any energy storage or somatic growth can be achieved, is to meet maintenance requirements. The strategy of red drum larvae is to funnel most of the ingested energy into growth processes. To maximize energy used in growth, maintenance costs must therefore be minimized. The purpose of this chapter is tc determine the maintenance costs of red drum larvae in their first 2 weeks of life. Oxygen Consumption Rate Oxygen consumption rate is an indirect calorimetric measure of the rate at which food is combusted. It is equivalent to the sum total of the costs of heart rate and circulatio n cellular maintenance, and movement. Depending on its stage in life, a fish may use 20% to 50% of its total ingested energy for oxygen consumption, with growth being the other major sink (Brett and Groves, 1979). oxygen consumption can be expressed in absolute terms: o 2;individual/hr, or as a weight-specific rate: o 2;weight/hr. Absolute oxygen consumption increases with body mass (Schmidt-Nielson, 1988), and may be expressed as the following allometry (Imabayashi and Takahashi, 1987):

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where y = oxygen consumption x = body size a, b = coefficients Values for the slope (b coefficient) for a wide variety of 101 organisms range between 0.67 (scaling directly with surface area) and 1.0 (scaling directly with mass) (Schmidt-Nielson, 1988) Both p oikilotherms and homeotherms usually exhibit slopes of intermediate value, 0.75, although some exceptions such as some insects and pulmonate snails have b values closer to 1.0. A b value of 1.0 indicates that the rate of oxygen consumption scales directly with body mass. Most physiological processes involving the transport o f oxygen are surface-related, however, resulting in b values of less than 1.0 As a consequence, weight-specific oxyge n consumption decreases with increasing b ody mass (Schmidt-Nielson, 1988) In additio n t o biotic factors such as body size, respiratory surface area, and degree of activity (swimming speed), oxygen consumption in fish is influenced by abiotic factors such as ambient temperature, season, and the hypoxic and hypercapnic conditions of the respiratory media (Prasad, 1986). Cost of maintenance increases with increasing temperatures, leading to higher oxygen consumption rates. In regions of lower oxygen tension fish must increase ventilatio n similarly resulting in higher maintenanc e costs for respiration a s well. Each abiotic factor influences

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oxygen consumption differently during different stages in the life history of fishes (Fry, 1971). Larval respiration 102 Respiration in eggs and yolk-sac larva is believed to occur through cutaneous diffusion (de Silva, 1974). At hatching, most species of fish lack respiratory pigments and are completely transparent. The blood of these larvae becomes pink weeks or months later upon metamorphosis, which marks the time of blood pigment development and advanced gill filament formation (Weihs, 1981; Blaxter, 1986). Fyhn and Serigstad (1987) measured oxygen consumption in cod (Gadus morhua) eggs and larvae. They observed that oxygen uptake gradually increased during the egg stage (16 days) and the first 4 days after hatching. Subsequently, oxygen consumption leveled off, coinciding with the time of yolk absorption. Larvae that were fed exhibited a pattern of continual increase in absolute oxygen consumption with size (Prasad, 1986). The behavior of larvae affects their respiration. Larval anchovy, Engraulis mordax, exhibit periods of continuous swimming interspersed with periods of rest during the first 3 days after hatching. Hunter (1972) believed this behavior served a respiratory function for larval anchovies, moving the larvae from regions depleted in oxygen as a result of cutaneous respiration. Weihs (1981), however, suggested that the behavior might be a means to counter sinking due to the negative buoyancy of the larvae,

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rather than as an aid to respiration. Larval fish show various physiological adaptations which aid them in respiration. During ontogeny, larval 103 hemoglobin is replaced with adult hemoglobin which is characteristically different (Dabrowski, 1986a; 1986b). Larval hemoglobins.have higher oxygen affinities than adult hemoglobins, reducing the problems of HC03 accumulation and lactate 1ons after vigorous exercise. The activity of enzymes involved in aerobic and anaerobic respiration changes as the larva develops. Siebenaller (1984) f ound that the activities of kinase, malate dehydrogenase and citrate synthase (CS) were nearly one order of magnitude greater in larval Sebastolobus alascanus during pelagic life than in juveniles or adults. He believed that the initial high concentrations reflected the high energy demands o f aerobic metabolism associated with the high levels o f locomotion and protein synthesis required during larval development. I n contrast, larval coregonids initially maintained the three glycolytic enzymes, phosphofrucokinase, pyruvate kinase, and lactate dehydrogenase (LDH) at a constant low level until glycolysis became important in energy supply (Forstner et al., 1983). Wieser et al. (1985) showed that larval salmonids which remain in the hypoxic bottom substrate, maintai n high levels o f anaerobi c enzymes earlier than pelagic larvae. Clearly, the activ ity o f metabolic. reflecte the life h istory o f a given species of larvae.

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104 Methods of measuring oxygen consumption Oxygen consumption rates can be measured directly by chemical titrations and oxygen electrodes. The standard chemical titration method used is the Winkler method, which has been used to determine oxygen consumption in larval fish (Klyashtorin and Musayeva, 1977; Theilacker, 1987). However, the Winkler method is not suitable for the continuous monitoring of oxygen consumption that allows the investigator to look at short-term variability in rates. For these reasons experiments involving gas permeable electrodes now dominate oxygen consumption research. Clark et al. (1956) first used a membrane-covered polarographic electrode to determine oxygen concentrations in blood plasma. A problem associated with the oxygen electrode was that a significant amount of oxygen was consumed by the the electrode itself. T o eliminate the problem of a low oxygen boundary layer on the membrane caused by the electrode's oxygen consumption, the medium that was being monitored wa s stirred. Stirring the medium, however, restricted measurements to large volume chambers and animals that could cope with stirring. Giere (1973) tried another approach and measured dissolved gases in marine sediments with a naked platinum electrode. The data collected were highly variable, and this system was determined to be unreliable. Revsbech et al. ( 1979; 1980 ) used membrane-covered platinum electrodes smaller than those of Clark t o measure disso lved oxygen in

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marine sediments. These electrodes were designed with tip diameters of 2-8 urn, and were referred to as needle electrodes. These smaller electrodes provided a better spatial resolution and less distortion of the physical environment than the Clark "macro" electrodes had. The 105 needle electrodes did not consume oxygen and did not have to be placed in a stirred medium. Revsbech and Ward (1983) found that the smaller needle electrodes could function under a variety of environmental conditio n s and in a variety o f media. Another advantage they f ound was that the 90% res p onse time needed to detect changes in oxygen concentrations was less than 0 2 seconds. Pui et al. (1978) found that these smaller electrodes could also be used t o determine chemical concentrations of ammonia, carbon d i o xide and urea, suggesting that these electrodes could be u sefu l in a variety of biological experiments. Before the present study, measurements of larval fish respiration had been conducted only on large individual larvae, or on groups of smaller larvae, that provided sufficient biomass to produce a change in the partial pressure o f o2 (P02) detectable by conventional Clark electrodes. To measure the rates on small, individual larvae, the small needle electrodes (Revsbech and Ward, 1983) used previously for e xamining P02 in pore waters are ideal. The needle electrodes are very small and have a much fin e r p oint than the C lark electrodes, thus allowing for

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106 smaller volumes of water and smaller numbers of animals to be used in determining oxygen consumption. Multiple animals usually have to be used to produce a measurable signal for fish larvae 2 to 3 days old, which may result in the "group effect" that Imabayashi and Tallahashi {1987) found in postlarval and juvenile red sea bream. The present study is the first time needle electrodes have been used to measure individual larval fish oxygen consumption rates. Methods and materials Oxygen consumption rate oxygen consumption was measured in red drum ranging in age fro m 3-18 days. Individuals that were used in respiratory determinations were o f two types: those fed ad libitum and transferred immediately to respiration chambers, and those that had been starved for 24 hours. Oxygen partial pressure was monitored in the respiratory chambers using both unstirred "micro" and "needle" polarographic oxygen electrodes (respectively, Clark et al., 1956; Revsbach and Ward, 1983) as individuals or groups of individuals reduced the oxygen levels to low (0-40 mm Hg) partial pressures. Starved individuals were monitored using the needle electrodes and fed larvae were monitored by both micro and needle electrodes. Electrodes were calibrated before and after each experimental run using air-and nitrogen-saturated seawater.

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Respiratory chambers were manufactured from plastic 1 ml and 10 ml syringes. Each chamber was filled with seawater filtered through a 0.45 millipore filter, and capped with the electrode fitted with an a-ring. The respiratory chamber within the syringe barrel was thus defined at one end by the syringe plunger and at the other end by the oxygen electrode. Single individuals were run using a 1 ml syringe and the needle electrode set-up. 107 Groups of 2-4 individuals were run in 10 ml syringes using a micro electrode. Chambers were kept at 24oc ( 1 .ooc). Data were recorded using a digital computer controlled data logging system for the micro electrodes and a linear chart recorder system for the needle electrodes (Donnelly and Torres, 1988). For data acquired with the chart recorder, the oxygen consumption rates were computed directly from the slope of the recorded data. Rates were calculated from the data-logger as means of P02 values for 10 minute periods. A mean rate for the entire rate was determined from the means for all 10 minute periods within the run. The lowest and highest 10 minute mean rate for each run represented the minimum and maximum rate, respectively. After each run, individuals were rinsed with distilled water and dried at Gooc for 24 hours, then weighed using a cahn Electrobalance for dry weight measurements. It should be noted that the number of replicate experiments for larvae starved 24 hours was lower than that for fed individuals

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108 since many of the larvae that survived the period of starvation were not hardy enough to be good experimental subjects. Daily respiration costs A daily oxygen consumption rate was calculated using average mean and minimum respiration rates for each day sampled. Average mean rates were used for the 13-hour light period; minimum values were used to represent oxygen consumption during the 11 hour inactive period in the dark when the larvae were not feeding (Chapter 2). Maximum rates reflected the highest measured oxygen consumption, and occurred most often within the first thirty to sixty minutes of the experiment. Holliday et al. (1964) found that respiration of newly hatched herring larvae dropped during inactivity at night to 1/2 the daytime rate. Rates dropped off one hour after darkness and increased to daytime rates 3 hours prior to the light cycle. This pattern of activity was attributed to the circadian rhythms of the larvae. Brett and Groves (1979), and Houde (1989) also found that night-time respiration rates in a variety of larval fish species declined to 1/2 the value of daytime rates. To be consistent with these prior investigations minimum respiration rates were used to represent night-time energy costs. Daily oxygen consumption rate was converted to calories expended using an oxycalorific equivalent of 0.00463 02 (Brett and Groves, 1979). Calories were converted to

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109 "rotifer equivalents" using the relationship o f 0.000526 calories/rotifer determined from the proximate com position data in Chapter 3. Results Oxygen consumptio n rate Starved versus fed larvae Absolute oxygen consumption rates for individuals fed a ration o f 5.0 prey/ml increased with increasing dry w eight and as a consequence, with the age o f the larvae as well ( Table 12 and Figure 18). Rates ranged between 0 .04 and 0.14 pl/individual/hr for the first week o f growth. During Table 1 2 Respiration of red drum larvae fed at 5.0 prey/ml. DAY N MDW UL/INDIV/HR MEAN Q02 3 4 7 8 9 10 1 2 14 16 17 1 8 6 17.0 4 24.1 2 31. 5 4 34. 1 8 46.1 5 52.0 6 68.6 6 109.0 1 9 1.7 2 155.0 1 172.0 0 .0473 0.1128 0 .1402 0.1238 0.2466 0 .2844 0.2593 0.3412 0.5383 0 .7316 0 .7688 2 .78 (0.93) 4.68 (1.02) 4.45 (2.59) 3 63 (1.73) 5.35 (1.47) 5.47 (0.83) 3 .78 (1.94) 3.13 (1.56) 5.87 ( --) 4.72 (2.63) 4.47 ( --) 1.15 (0.43) 4 .56(0.65) 2.54 (1.38) 7 .48(1.71) 1.53 (1.35) 11.7(3.30) 1.35 (1.17) 8 .28(2.79) 2.01 ( 1 13) 11.1(6.27) 3.22 (1.46) 8.91(3.85) 1.24 (0.33) 10.8(2.79) 1.20 (0.54) 8.08(3.18) 1.46 (0.05) 12.4(7.61) ( ) = standard deviation; M D W = mean dry weight in pg; Q02 = weight-specific respiration in pl 02/mg D W /hr; N = number of sample runs. the second week of growth, values increased sharply from 0.14 to 0 .34 pl/individual/hr reaching 0 77 pl/individual/hr

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1.0 Q: :I: 0.8 ::> H c z 0 6 H ...J :;) 0 4 ....., z UJ (!) 0.2 >>< 0 0 1.0 Q: :I: 0.8 ::> H c z 0.6 H ...J :;) 0 4 ....., z UJ (!) 0.2 >>< 0 0 Figure 0 0 a Y = 0.014X 1.28 r 2= 0 .69 3 b Y = 4. 35X1. 02 r 2 = 0 78 0.04 6 110 9 12 15 18 AGE
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111 at 18 days after hatching. The slope of the regression line (b) for oxygen consumption versus age was 1.28 0 .12; it was 1 .02 0.08 for oxygen consumption versus weight. Weight-specific oxygen consumption (Q02l varied between 2 and 6 02/mg DW/hr with a mean of 4.23 1.09. 02/mg DW/hr. The slope of the regression of weight-specific oxygen consumption versus age was not significantly different from zero, indicating a constant rate per unit body mass f o r the first two weeks of life (Figure 19). Absolute oxygen consumption rates of individuals starved for 24 hours also increased with weight and age (Table 13 and Figure 20). Oxygen consumption versus dry weight had a higher coefficient of determination than that of oxygen 2 consumption versus age (r of 0.50 versus 0.39). The slope for the regression lines (b) of oxygen consumption versus age were 0.98 0 .40 and 0.70 0.23 for that of oxygen consumption versus weight. Table 13. Respiration rates for red drum larvae for 24 hours. DAY RUNS MDW UL/INDIV/HR Q02 3 6 17.0 0.0473 2 .78 (0.93) 5 2 20. 7 0.0786 3.80 ( 1. 36) 6 3 16.2 0.0271 1. 67 (0.93) 10 3 36.1 0 .1147 3.18 ( 1. 98) 11 2 91.7 0.0963 1. 05 (0.22) 15 "") ...) 77.9 0.2516 3.23 (1.61) 17 2 180.0 0.8622 4.79 (1.43) ( ) = standard deviation; MDW = mean dry weight in Q02 = weight-specific respiration in

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10 a 8 :z: (!) 6 J: ..J :::::l ......, 4 N 0 0 2 0 0 3 10 b 8 :z: (!) 6 J: ..J :::::l ......, 4 N 0 .. 0 .. 2 0 0 0.04 6 9 AGE 112 18 .. 0 2 Figure 19. Weight-specific oxygen consumption in larvae fed to satiation as a function of a) age and b) weight.

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1.0 ,.., a 0::: :I: 0.8 ::> Y = 0.112X 0 9 7 5 H 0 r 2 = 0.39 z 0 6 H _J ::::> 0.4 '-" z w (.!) 0 2 >>< 0 0 0 3 6 9 12 15 18 AGE r 2 = 0. 5 0 H 0 z 0.6 H _J ::::> 0.4 '-" z w (.!) 0 2 >>< 0 0 0 0.04 0.08 0 1 2 0 .16 0 2 I.JEIGHT < M G ) Figure 20. Absolute oxygen con sumption in larvae starved for 24 hours as a function o f a) age and b ) weight 113

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Absolute and weight-specific oxygen consumption of starved larvae was one-half or less that of fed larvae at all ages except day 17 (Figure 21). The slopes of the 114 regressions_ of weight-specific oxygen consumption versus age and weight were not significantly different than zero. As was true for fed larvae, starved larvae maintained the same oxygen consumption rate per unit body weight over the first two weeks of life. However, average weight-specific rates for starved individual were equivalent to the minimum oxygen consumption rates of fed larvae. Comparison of electrode measurements Oxygen consumption rates obtained with the needle electrodes were not significantly different from those obtained with micro-electr6des (Table 14). The needle electrodes, however, allowed measurements to be made on individual larvae in smaller volumes of water. Table 14. Comparison o f weight-specific respiration rates measured using needle and micro-electrodes. AGE MEAN Q02 NEEDLE N MICRO N DAY 6 4.12 (0.56) 2 2.43 (1.76) 2 DAY 7 3.63 (1.73) .... 4.45 ( 2. 59) 2 G DAY 9 5.35 (1.48) 4 5.53 (1.22) 2 DAY 14 3.13 (1.56) 3 3.56 (1.48) 6 DAY 18 4.47 ( --) 1 4.72 (2.63) 2 Q02 = pl/mg/hr; ( ) -standard deviation; N = number of runs

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115 10 a y = -0.437x0290 8 r 2= 0.13 ,... :X: J: 6 ...J -....; 4 (\J 0 0 2 0 0 3 6 9 12 15 18 AGE CDAYS) 10 b y = -0.303x322 8 r 2= 0.16 ,... :X: 6 J: ...J -....; 4 (\J 0 0 2 0 0 0.04 0.08 0.12 0.16 0.2 WEIGHT CMG) Figure 21. Weight-specific oxygen consumption in larvae starved for 2 4 hours as a function of a) age and b) weight.

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Daily respiration costs Respiratory costs increased from 0.00385 calories/day (7.32 rotifers) at day 3 to 0.02765 calories/day (52.55 rotifers) at day 14 (Table 15). Table 15. Intensity of feeding to sustain daily caloric demand of respiration calculated from total oxygen consumption. AGE (DAYS) 3 4 6 7 8 9 10 12 14 16 17 18 DAILY RESPIRATION COST (CALORIES/DAY) 0.00385 0.00914 0.00429 0 .01136 0 .01003 0.01998 0.02304 0 .02101 0.02765 0.04362 0.05928 0.06229 DAILY NUMBER PREY ITEMS (ROTIFERS/DAY) 7.32 17.38 8.15 21.60 19.07 37.99 43.81 39.94 52.55 82.93 112.70 118.43 The metabolic demand of 52.55 rotifers for day 14 larvae represents the combined costs of standard, routine and feeding metabolism and costs of growth. Based on the the figure of the maximum rotifers ingested (106) 2), red drum larvae contribute a significant amount of energy used in metabolic processes. 116

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Oxygen consumption rates Starved versus fed larvae Discussion 117 Absolute oxygen consumption of red drum larvae was similar to that reported for other larval species. Fyhn and Serigstag (1987) found absolute oxygen consumption rates of 0.05-0.100 02/individual/hour in cod larvae for the first 5 days of life at 5C, which was identical to the measured values for red drum. Houde and Schekter (1983) reported rates of 0.080 .14 02/individual/hr for Archosargus rhomboidalis reared at 26C, which increased to 0.44-0.62 pl 02/individual/hr in 41-66 pg individuals. Red drum larvae values were slightly lower at 0 .05-0.14 pl/individual/hr for 21-31 pg individuals, and 0 .26-0.28 pl/individual/hr for 52-68 pg larvae. The regression o f absolute oxygen consumption versus weight in red drum had a greater slope than either the average 0.75 found for other animal species (SchmidtNie lson 1988), or the slope of 0 .88 for fish in general (Brett and Groves, 1979; Houde and Schekter, 1983; Prasad, 1986). When standard error o f the slopes and 95% confidence intervals were compared from the reported values for other species and red drum, h owever, all slopes were not significantly different from the 0.88 o f Brett and Groves. The b coefficient for red drum was well within the range of 0.60 -1.24 found by H oude and Schekter (1983) for similar sized Archosargus rhomboidalis larvae and 0.8865 for Esomus

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118 danricus larvae (Prasad, 1986}. The slope of 1.02 0.8 exhibited by red drum larvae indicates that oxygen consumption was a direct function of body mass (1.0) rather than surface area (0.67). Weight-specific metabolism was similar to that reported in larval fish of equal size at similar temperatures and used to determine total energetics (Houde, 1989). Weight-specific and absolute oxygen consumptiqn rates for two-week-cld red drum that had been starved for 24 hours were similar to values reported for other larval species of equal weight that had also been starved. Davenport and Lanning (1980) found Q02 values of 1.1-1. 6 pl/mg DW/hr for starved larval cod and a mean rate of 1.9 pl/mg DW/hr for starved larval winter flounder reared at 5C. (1989) found total oxygen consumption rates of 0.08-0.12 pl/individual/hr for larval bay anchovy (20-40 pg), reared at 24oc. The b coefficient for starved larvae (0.697 in red drum) has also been reported for starved larval herring, Clupea harengus (0.68). Both values are within 0.02 units of the classical weight exponent of 0.67. The high mortality of starved red drum larvae has been documented in other species of sciaenids. Powell and Chester (1985) found that spot larvae were highly vulnerable to starvation in the preflexion stage versus the flexion and post-flexion stages, underscoring the critical relation between food and survival in the early life history of larval fishes.

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119 Daily respiration costs Daily mean respiration rates represent routine activity costs (Fry, 1971) for diurnal activity in day 14 larvae. Minimum rates were about one-half those rates and were more representative of standard metabolic costs during periods of inactivity (i.e., nocturnal activity). Brett and Groves (1979) also found that routine rates were about twice standard rates. The cost of standard metabolism of 0 .0137 calories (26 rotifers) was slightly lower than the cost of maintenance metabolism of 0.0148 calories (28.1 rotifers) as determined by the growth-ration curve (Chapter 3). This relationship has also been found for fish in general (Brett and Groves, 1979). The reduction of oxygen consumption in starved versus fed individuals reflects the decrease in energy expenditure associated with feeding metabolism, otherwise known as specific dynamic action, or SDA (Kiorboe et al., 1985; 1987; Kiorboe, 1989). Recent work (Jobling, 1981b; 1983) suggest that SDA may represent an energy expenditure associated with growth. The traditional view of SDA suggests that growth and SDA are in direct competition for ingested energy. Diets that produce a high SDA would consequently result in lower amounts of food energy available for growth. The alternative view would be that diets that promote high growth will also induce high energy costs as SDA. The 40% to 60% reduction in metabolic rates in starved individuals indicates that the majority of metabolic

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120 above maintenance in f e d larvae appear to be composed of the energy involved in the assimilation of food, feeding, digestion, excretion, and biochemical transformations (=SDA), with little energy being used for swimming activity. Observation of larval activity during daytime hours agree with the notion of l o w energy expenditures. Most larvae remained posed towards the substrate (sides of tank) waiting for rotifers to pass in front of them. Comparison with the data from ingested energy indicates that metabolic cost s are substantial compared to overall energy allocation. The majority o f energy expended on metabolic processes is related to SDA, which would be expected from an individual trying to funnel all energy into growth and survival. Jobling (1985) states that SDA is specifically related to growth and that individuals growing slowly demonstrate little c hange in metabolic rate f ollowing feeding. Based on SDA values, red drum larvae s h o w a survival strategy based on a high growth rate. conclusions Red drum larvae have a high energy demand to meet the costs o f metabolism. Th e strategy for rapid growth in the red drum larvae results in an increase of feeding metabolism, which is 50% o f the total metabolic costs. These results agree with the findings observed in Chapter 3, which suggest red drum larvae are geared for rapid growth. The amount of ingested energy remaining after cost of respiration and growth are covered shoul d be lost as excretion.

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121 CHAPTER V: ENERGY LOST TO EXCRETION Introduction J obling {1981a) states that measurements of total nitrogenous excretion show the effects of environmental and nutritional factors o n protein metabolism. Ingested energy not used in growth and respiration is lost in the form of feces and non-fecal nit rogen {ammonia and urea). The purpose of this chapter i s to determine the amount o f energy lost as nitrogenous waste in the form of ammonia and urea and the effects o f starvation o n nitrogen excretion. Nitrogen excretion A portion of the food energy ingested by an individual is indigestible and lost as feces {up to 20% in older fish; Brett and Groves, 1979). In larval fishes, feces are small and very difficult to collect and are rarely measured. Their contribution t o t otal excretion in fish larvae varies with the fraction of refractory material in the diet. Of the remaining food energy that i s digestible, a portio n is lost as non-fecal nitrogen, mainly as ammonia and urea. Brett and Groves {1979) state that energy lost through nonfecal excretion ranges from 3-10% o f total ingested calories.

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122 Non-fecal nitrogen originates from two sources. Nitrogen produced from the breakdown of food, mainly protein, is termed the exogenous portion. Nitrogen produced from the catabolism of tissue proteins (Brett and Groves, 1979) is termed the endogenous fraction of nitrogen excretion. It is difficult to distinguish between the two sources of nitrogen but approximations can be made. Endogenous nitrogen excretion can be estimated by measuring nitrogen excretion after an empirically defined time interval for larvae on a complete, but low-nitrogen, diet. However, larval fish that feed mainly on live foods make timed control of dietary nitrogen measurements difficult. Instead, data on nitrogen excretion of starved animals can provide an approximation of the endogenous fraction {Jobling, 1981a) for all larvae. Teleosts are primarily ammonotelic, excreting most of their nitrogen in the form of ammonia, mainly through their gill epithelium. Ashley {1975) and Pandian {1975) determined that urine was a minor route for nitrogen excretion in most fish species and that 60% to 80% of fish nitrogenous waste as ammonia was lost through the gills. Most species of sharks and some species of lungfish produce urea in large quantities. Sharks use urea in osmoregulation and lungfish produce this less-toxic form of nitrogen during periods of estivation when water is unavailable for ammonia excretion. Urea is primarily excreted in the urine. In adult teleosts urea usually constitutes a minor portion of

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123 nitrogen excretion. Urea requires energy for its production from ammonia precursors, and researchers debate the existence of a mechanism for urea production in teleosts. Rice and Stokes (1974) examined the metabolism of nitrogenous wastes in eggs and alevins of rainbow trout, and determined that one source of urea production is the dietary degradation of arginine by arginase in freshwater teleosts. cvancara (1969) suggested that another source for urea production was purine metabolism. Brown and Cohen (1960) found that most adult fishes lack the enzymes of the ornithine-urea (O-U) cycle needed to produce urea, suggesting that these species have lost their ability to produce these enzymes due to gene deletions. Factors that influence N-excretion Buckley and Dillman (1982), and Klummp and von Westernhagen (1986) measured nitrogen excretion in larvae of several fish. species and compared the larval rates to those of older individuals. They found that the rate of nitrogen turnover varied with age, but that it was more rapid in larvae compared to adults. The rapid turnover was attributed to the faster growth rates of larvae compared to those in later stages of development. Along with the effects of age, daily fluctuations in ammonia and urea excretion resulting from different stocking densities, handling stress or diet (Burrows, 1964) have been measured in hatchery-reared fish. Dabrowski et al. ( 1987)

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found that the type of ration fed to larval fish affected the rate of ammonia excretion; an increase occurred when fish were fed live food and good quality dry food. 124 Jobling (1981a) found that rates of ammonia excretion increased with increasing temperature in plaice larvae and were scaled with body weight to the power 0.67, suggesting a surface-area dependence (Schmidt-Nielsen, 1988). Similarities in ammonia excretion and oxygen that parallel growth rates have been observed in larval fishes. Davenport et al. (1983) found low rates of ammonia excretion and oxygen consumption in larvae (Gadus morhua) with slow growth and developmental rates. Developing cod larvae showed a twelve-fold increase in ammonia excretion from spawning to hatching (25 days), accompanied by a tenfold increase in oxygen consumption. As a result ammonia excretion is a good indicator of protein synthesis and thus growth in larva l fishes O-N ratios Data collected simultaneously on ammonia excretion and oxygen consumption can be used to determine the biological substrate being combusted as fuel by the larvae by using the ratio of oxygen consumed to the nitrogen produced (O:N ratio) (Davenport et al., 1983). O:N ratios of 8 or below indicate that pure protein is being combusted. Values greater than 20 suggest that lipids are the primary source of energy (Bayne, 1973). Larvae depleting their yolk-sac lipid reserves should thus e xhibit high O:N values, while

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125 fast-growing larvae actively synthesizing protein with little or no lipid deposition or combustion should have low O:N values. Methods and materials Nitrogen excretion Nitrogen excretion rates were determined for larvae of the same ages and feeding states as those used in the oxygen consumption experiments (Chapter 4). Larvae aged 3-14 days were split into 3 feeding groups: those fed to satiation, those starved for 24 hours and those starved for 48 hours. Five sets of ten larvae were removed from each feeding group and placed into a series of 10 ml screw-top centrifuge tubes. Each tube was filled with charcoal-filtered seawater and sealed after the addition of the larvae. Two control tubes were also filled with charcoalfiltered seawater. An equal amount of water from each feeding bowl was added to one control tube to measure any ammonia addition caused by transfer of the larvae. The other control tube was filled with 10 ml of the charcoalfiltered seawater to serve as a blank. Larvae were incubated for 4 hours at 25C. Larvae were subsequently filtered from the tubes using 50 mesh netting, dried at 60C for 24 hours, and then weighed. The remaining sample water was split into aliquots and immediately capped and frozen for later analysis. Concentrations of ammonia and urea for each sample were determined with an Alpkem autoanalyzer (Price and Harrison, 1987).

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126 Daily excretion Total daily energy loss in the form of nitrogen excretion was calculated by summing individual hourly ammonia and urea excretion rates, based on the 4 hour incubations. Mean daily rates for larvae fed to satiation were used as representative excretion rates for the 13-hour diurnal activity period. Rates for larvae starved for 24 hours (1/2 the satiated values) were used as an estimate of the 11-hour dark period when larvae become inactive and metabolic rate dropped to one-half the diurnal rate (discussed in Chapter 4). Ammonia concentrations were converted to calories using a value of 0.00489 NH3 {Elliot and Davidson, 1975); urea concentrations were converted to caloric equivalents using a value of 9.096 urea, the heat of combustion of urea (Kleiber, 1961). Calories expended as ammonia and urea were summed for the 24 hour period. Calories were converted to "rotifer equivalents" by using the value of 0.000526 calories/rotifer that was determined determined in proximate composition experiments (Chapter 3). O:N ratios O : N ratios were calculated using oxygen consumption data {Chapter 4), and data for both total nitrogen excretion (ammonia and urea) and ammonia excretion only. Total nitrogenous waste was determined by doubling the concentration of urea-N (two ammonia molecules within each

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127 urea molecule) and adding the concentration of ammania-N. Weight-specific oxygen consumption in 02/mg/ h was converted t o 02/mg/h by using the volume to molar equivalence in gases of 22.4 liters/m o l The of 02 were than multiplied by 2.0 to get the atomic oxygen concentration O). These values were divided by the rates of weight-specific ammonia excretion and weight-specific total nitrogenous excretion to obtain O:NH3 ratios and O:N ratios, respectively. Results Nitrogen excretion rate Absolute nitrogen excretion rates increased with age in terms of both ammaniaN and urea-N (Figure 22). Concentrations of ammaniaN, in larvae fed to satiation, increased from an average of 0.00659 0.001 NH3/individual/h o n day 2 t o 0.06659 0.0133 NH3/individual/h on day 14 (Table 16) Table 16. Mean ammonia and urea excretion in red drum larvae fed to satiation. AGE N AMMONIA UREA %EXCRETION 2 6 1 0 14 ( ) fish 5 0.007 (0.001) 10 0.01 9 (0.007) 10 0.040 ( 0 0 1 2 ) 10 0.067 ( 0. 013) /H (X 10-.... ) 0.325 (0.20) 1.333 (0.38) 2.215 (0.40) 3.918 ( 0 7 8 ) --------URE A (X 10 ) 0.7925 ( 0 7 5) 32.8 2.0575 (0.25) 27. 4 3 .0375 (0.30) 22.3 4.2050 (0.43) 17.8 = standard deviation; N = the number of samples ( 10 per sample)

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0.10 a :I: 0.08 '\. :> H 0 0.06 z H '\. M 0.04 :I: z (.!) :::J 0.02 0 0 0.10 b :I: 0.08 '\. :> H 0 0.06 z H '\. M 0.04 :I: z (.!) :::J 0.02 0 0 0 .10 :I: 0.08 '\. :> H 0 0.06 z H '\. M 0 .04 :I: z (.!) :::J 0.02 0 0 y = -0.01 + 0.005X r 2 = 0. 72 c 3 6 AGE CDAYS) y = 0 .004 + 0 001X r 2= 0.27 3 6 AGE
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129 Weight-specific ammonia excretion for larvae fed to satiation remained constant over the ages tested. It decreased slightly after day 6, but the slope was not significantly different from zero (Table 17 and Figure 23). Values ranged from 0.394 x 10-3 at day 2 to 3 0.753 x 10 at day 14. Weight-specific ammonia excretion was significantly different only in 2 day-old larvae. Absolute urea-N production increased with age of the larvae but at a lower rate than ammania-N (Table 16): Table 17. Mean weight-specific ammonia-N excretion rates in red drum larvae fed to satiation, and starved for 24 and 48 hours. AGE FED STARVED 21 3HOURS STARVED HOURS (X 10-3 ) (x 10 ) (X 10 ) 2 0.394 (0.091) -------------------------6 1.103 (0.563) 1.855 ( 0. 918) 2.153 (1.822) 10 0.841 ( 0. 318) 0.756 (0.095) 1.107 (0.844) 14 0.753 (0.177) 0.404 (0.209) 1. 040 (-----) ) = standard deviation from 0 .7925 0.75 x 10-4 urea/individual/h on day 2 to 4.2050 0.43 x 10-4 urea/individual/h on day 14. Urea-N values as a percentage of total nitrogen excretion decreased from 32.8% on day 2 to 17.8% on day 14. Ammonia excretion increased with age in red drum larvae starved for 24 hours. Larvae starved for 48 hours showed no change in nitrogen excretion with age; the rate

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4 .-i CS) CS) 3 CS) >< 'V :J: 2 (.!) M 1 :J: z (.!) 0 0 4 .-i CS) CS) 3 CS) >< 'V :J: 2 (.!) M :J: 1 z (.!) 0 0 4 .-i CS) CS) CS) 3 >< 'V :J: 2 (.!) M 1 :J: z (.!) 0 0 Figure 23. 3 3 3 Y = 0.001 + -0.000X r 2= 0.01 6 9 12 AGE
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131 remained at approximately 0.01 NH3/individual/h. Thus, the absolute rate of ammaniaN excretion decreased with increasing period o f starvation (Table 18). Very few pre-flexion larvae survived the 2 days of starvation. Those that did were sufficiently weak that transfer to the sample tubes resulted in a high mortality rate and thus a low number of data points. Larvae that were starved for 1 day excreted at a rate approximately 38% less than fed larvae, dropping t o 0.013 57 NH3 /individual/ h at day 6 and to 0.02567 NH3/individual/h at day 14. Table 18. Percent urea production in individuals starved for 24 and 48 hours in 10 ml of seawater for 4 hours. AGE N AMMONIA UREA %EXCRETION UREA (X 10-..;) (X 10 ) 6 S1 1 0 0.014 (0.008) 0 .660 (0.20) 0.4648 ( 0 1 1 ) 63.9 6 S2 10 0.011 (0.006) 0.643 ( 0. 13) 0.5238 (0.70) 60.8 10 S1 5 0.015 ( 0 00.4) 0 .883 (0.23) 0 .6678 {0.85) 60.5 10 S2 5 0.011 (0.009) 1.003 ( 0. 53) 0.7025 ( 0. 28) 58.4 14 S1 5 0.025 (0.001) 2.265 (0.07) 0 .4160 (0.25) 32.7 14 S2 1 0.019 (-----) 2.210 (----) 1.1055 ( ----) 45.8 S1 = 24 hours starved; S2 = 48 hours starved; ( ) = standard deviation; N = the number of samples (10 fish per sample) Mean weight-specific ammonia excretion rates increased slightly with duration of starvation in larvae starved for 24 and 48 hours, and were highly variable. Mean weight-specific ammonia excretion rates among fed individuals, and those sta rved for 24 and 48 hours, were not significantly different on days 6 and 1 0 Thus, the

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132 reduction in absolute ammonia excretion was the result of smaller individuals producing at the s ame weight-specific rate as fed individuals. Absolute urea excretion increased with age in larvae that were starved for 24 and 48 hours. Urea was a higher percentage of the total nitrogen excretion in starved than fed individuals by a facto r of 2:1. Daily excretion rates Daily rates of nitrogen excretion (calories/ individual/d) increased with age (Table 19). Expressed as caloric equivalents, nitrogen excretion increased from 0.68 x 10-3 calories at day 2 tc 6.81 x 10-3 calories at day 14. The amount of energy lost t o excretion expressed in rotifer equivalents (0.000526 calories/rotifer) increased from 1.30 to 12.9 rotifers from day 2 to day 14, respectively. Tabl e 19. Daily nitrogen excretion rates o f red drum larvae and equivalence in rotifers calculated from mean weightspecific ammonia and urea excretion rates. AGE UREA/DAy3 TOTAL 3 (DAYS) (CA L X 10-_,) (CAL X 10 ) (CAL X 10 ) 2 0.47[0.90] 0.21[0.40] 0 .68[1.30] 6 1.94[3.68] 0 .55[1.04] 2.49[4.72] 6 S 1 0 .58[1.10] 1.23[2.34] 1.81[3.44] 6 S2 0.94[1.79] 1.39[2.64] 2.33[4.43] 1 0 3.23[6.13] 0.83[1.58] 4 .06[7.71] 1 0 S1 1.28[2.441 1.77[3.37] 3.05[5.81] 10 S2 1.46[2.77] 1.86[3.54] 3.32[6.31] 1 4 5.70[10.8] 1.11[2.12] 6 .81[12.9] 14 Sl 3 .28f6.12] 1.10 [ 2.10] 4.38[8.33] 14 S2 3.22[6.11] 2.93[5.58] 6.15[11.7] Sl starved 24 S2 -starved for 4 8 hours; [ ] = the caloric equivalence i n r otifers.

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133 The decline in the rate of ammania-N excretion with increased period of starvation was offset by the increase in the rate of urea-N excretion in starved larvae. The total amount of nitrogen excreted remained the same with starvation, the percent contribution of ammonia and urea to total excretion changed, with urea dominating in starved individuals. Caloric values for rates of excretion represent the chemical bond energy in NH3 and urea that wa s either not assimilated or was lost to the system after the deamination accompanying pr6tein turnover. Excretion rates together with the amounts of energy used in growth (Chapter 3}, and respiration (Chapter 4), allow calculation o f assimilation efficiencies (Chapter 6}. O-N ratios The l ow values for O:N ratios based on both ammania-N (O:NH3 ) and total nitrogen (ammania-N and urea-N) excretion rates indicated that protein wa s the combusted fuel source (Table 20). Values for O:NH3 ratios ranged between 4 and 10 f o r all ages. O:N ratios values based on combined ammania-N and ureaN excretion were lower, ranging between 1 and 8. Larvae starved for 24 and 48 hours had lower O:N ratios than fed larvae. starved larvae had O:N ratios ranging between 1 .75 and 3.75 and O:NH 3 ratios between 4.3 and 8.08.

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134 Table 20. Molar oxygen:nitrogen ratios for larvae fed to satiation, and those starved for 24 and 48 hours. AGE (DAYS) 2 6 6 S1 6 S2 10 10 S1 10 S2 14 14 S1 OXYGEN (J.lM/MG/H) 0.244(0.021) 0.367(0.183) 0.401(0.087) 0.401(0.087) 0.598(0.183) 0.189(0.062) 0.189(0.062) 0.321(0.091) 0.288(0.005) AMMONIA (].lM/MG/H) 0.024(0.005) 0.084(0.039) 0.089(0.039) 0.110(0.026) 0.050(0.018) 0.044(0.006) 0.065(0.050) 0.049(0.008) 0 .024(0.012) O:NH3 10.94(2.44) 6.97(3.12) 4.89(1.74) 8.08(5.17) 10.82(2.07) 4.31(0.58) 6 .30( --) 6.81(1.64) 5.58(0.18) O:N 7.56(1.49) 6.01(3.02) 1.76(0.63) 2.90(1.86) 8.87(1.70) 1.75(0.26) 2.63( --) 5.94(1.37) 3.75(0.12) ( ) = Standard deviation; S1 = starved 24 hours; S2 = starved for 48 hours. Discussion Nitrogen excretion These data on nitrogen excretion are the first reported for red drum larvae and add to the limited data set for larvae in general. Absolute ammonia excretion rates of red drum larvae were similar to those reported by Klumpp and von Westernhagen (1986) for similar size Pleuronectes platessa (0.025-0.050 NH3 /individual/h at 100 j.lg and 11 days old) and Blennius pave (0.05-0.10 ].lg NH3/individual/h at 100 11g and 5 days old). Absolute ammonia production in fed larvae reflected both protein turnover of food and body tissues and turnover of protein of b o d y tissue f o r starved individuals. Weight-specific rates were the same for fed and starved individuals, ineicating that protein turnover continues even in the absence o f food. Thus the apparent decrease i n abso lute amn,ci1:..3. Excretion with starvation was caused by the

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135 decrease in weight due t o starvation and not a reduction in p r otein turnover. Pre-flexion red drum larvae did not show the decline in weight-specific ammonia excretion with age that has been observed in other larval fishes (Klummp and von Westernhagen, 1986) This decline may appear at a later stage in the development of red drum larvae, perhaps after metamorphosis. The constant weight-specific excretion rates with age found throughout the first two weeks of mirrored weight-specific oxygen c onsumption rates (Chapter 4 ) Thus, ammonia excretion rates appea r t o f ollow a weight-specific pattern similar t o those observed in oxygen consumption and percent growth in the preceding chapters. Values obtained in the present study for the percentage o f nitrogenous waste in the f o rm of urea agree well with previou sly. reported values. Jobling (198la) found that 15% t o 25% o f nitrogenous waste in plaice larvae occurred in the form o f urea which agrees w ell with values o f 18% to 33% for red drum larvae. Ammonia increased as a percent o f the total nitrogen excretion with age i n red drum larvae fed to satiation. Production o f urea may play a role in survival during the larva's early life. Urea costs m ore energy to produce than ammonia, but it is less toxic and is used in osmoregulation and as an excretory produc t during estivation by some species, as n oted earlier. Yan and Thomas (1988) found t hat gill chloride cells, the major teleost osmoregulatory site, develop later in the ontogeny of red

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136 drum, closer to the juvenile stage. Production and retention of urea to increase blood osmolarity, in a similar manner to the elasmobrachs, may serve as a form of osmoregulation in the early stages of life. The percent increase in urea production with starvation has been observed in other fishes under hatchery conditions (Brett and Zala, 1975). The constant weight-specific nitrogen excretion in starved individuals, despite the changes in relative.contributions of ammonia and urea, is indicative of continued protein catabolism. However, the protein catabolised in starved individuals was the protein contained within the larvae1s own tissues. Urea normally contributes less than 50% of the total nitrogen excreted and is most often overlooked, but the results here suggest urea is an excellent indicator for starvation in larval fishes. The decrease in the percentage of urea excreted in larger red drum larvae may be an indicator of the increased ability to survive starvation closer t o metamorphosis. Daily excretion rates The absence of measurable fecal production indicated that the majority of energy lost to excretion in red drum larvae was in a non-fecal nitrogen form. Brett and Groves (1979) determined that 7% of ingested energy was lost to excretion as n on-fecal nitrogen and that 20% was lost in the form of feces. Estimates of larval red drum non-fecal nitrogen excretion indicate a slightly higher percentage

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137 (1013%) o f ingested energy in day 14 larvae, with little if any energy lost as feces. Daily nitrogen excretion rates for red drum larvae were comparable to those for similarlysized Paralichthys dentatus larvae (Buckley and Dillman, 1982). O:N ratios The nitrogen excretion data in larval red drum suggest that protein was the major catabolic substrate, with little, if any, energy being stored in o r produced from lipid reserves. Low O:N ratios in well-fed red drum larvae as well as those starved for 24 and 48 hours indicated a protein fuel source in all cases. Ingested rotifers were protein-rich (Chapter 3) which almost certainly contributed to the l ow O:N ratios of fed individuals. The slightly lower O:N ratio values found in starved larvae when compared to fed individuals suggests an even stronger dependence on protein for metabolic. fuel, combusted at the expense of tissue protein. Conclusions The absence of measurable fecal production resulted in non-fecal excretion accounting for all the energy lost as excretion. The low percentage of energy lost as nitrogen excretion (maximum 12 rotifers at day 14) indicated a high assimilation o f the energy obtained fro m the r otifer diet. The energetics and strategies involved in urea produc tion in early red drum larvae need t o be investigated further, since these a ppear t o be important factors i n larval survival.

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138 CHAPTER VI: ASSEMBLY OF THE ENERGY BUDGET Introduction Kleiber (1961) writes in The Fire of Life: Life involves much more than chemical potential, work and heat. Nutrition, especially modern nutrition, is concerned with more than the supply of energy, yet energy transfer remains an important aspect of physiology in general and of nutrition in particular. Familiarity with the basic concepts of animal energetics should therefore be an essential preparation for students of physiology, biochemistry, and nutrition. Kiorboe (1989) compared energy budgets of adults and larvae in several fish species. He determined that larval fishes were growing at nearly the maximum possible efficiency, limited only by the biochemical costs of biomass transformation that are reflected in assimilation and conversion efficiencies. This chapter will assemble the major components of the energy budget (Chapters 2 to 5) during the first 2 weeks of life for lab-reared red drum larvae to determine those assimilation and conversion efficiencies. Bioenergetics: balancing the budget Major insights into the early life history strategy of a species can be obtained by balancing the major energy compartments (i.e., growth, metabolism, and excretion) into a bioenergetic model or energy budget. For example, larvae

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139 like red drum that start small and must allocate a large portion of their ingested energy into growth, attempt to maximize the possibility of survival by using a strategy of early, rapid increase in size . Individuals with an early life history strategy of this type are in turn the result of a reproductive strategy where fecundity is maximized by the production of large numbers of small eggs, resulting in larvae that start small and develop rapidly in the hospitable waters of an estuarine environment. In contrast, slower growing individuals are generally expected to have larger egg slower rates of development, and less energy allocated to growth, as shown in the studies of Pepin and Myers (1991). Few studies on larval fishes have collected data for all the components of the energy budget. Most studies have relied on values for closely related species to fill in missing measurements in one or more compartments and balance energy budgets. Once the four major components of the energy budget have been determined and balanced through laboratory work, however, subcompartments such as assimilation and growth conversion efficiencies can be calculated with much greater precision and reliability than those found in studies relying on alternate-species approximations.

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140 Assimilation rate and conversion efficiency Assimilation rate is the percentage of ingested food energy utilized by an individual for maintenance and growth (Brett and Groves, 1979), and comprises the difference in energy content between the food ingested and the feces produced. Feces represent the nondigestible fraction of the diet and may also include sloughed intestinal epithelial cells, mucus, catabolised digestive enzymes and bacteria. Assimilation rates in fast-growing +arval fish are believed to be quite high. Theilacker (1987) found rates as high as 92% for northern anchovy larvae fed strictly on a rotifer diet. She estimated assimilation efficiency by combining the energy of metabolism and growth and dividing it by the energy consumed. Assimilated energy can be calculated in this way when energy lost as feces cannot be accounted for, a common problem in studies of larval fish where fecal material is both vanishingly small and diffuse (Theilacker, 1987). Assimilation efficiencies show the ability of larval fish to extract energy from food items, and are computed as gross and net conversion efficiencies. Gross conversion efficiency (K1) is the percentage of total ingested energy allocated to growth. Net conversion efficiency (K2) is the quantity of ration in excess of maintenance level that is converted into flesh. Houde and Schekter (1983) found that gross growth efficiencies in the larvae of three species (Archosargus rhomboidalis, Achirus lineatus, and Anchoa

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141 mitchilli) ranged from 11% to 41%, and that net growth efficiencies ranged from 39% to 57%. They estimated that 32% to 83% of ingested energy could be excreted in feces and urine. Conversion efficiency rates change during the life history of an individual. Older, slower-growing adult fish usually have lower food conversion efficiencies; since energy once devoted to growth is funneled into resulting in a declining efficiency of somatic growth even with an increase in body mass. At the other end of the life cycle, Marr (1966) found that the highest growth efficiencies in larvae were associated with the conversion of yolk in early development. Salmonids showed gross conversion efficiencies of 65% to 75% as yolk-sac larvae, but only 35% as post-larvae. Materials and methods Energy budget equation Energy allocation between the major compartments was described by the equation of Brett and Groves (1979): where I = G + M + E I G M E = = = = ingested energy energy used in growth energy used in metabolism energy lost to excretion Values were obtained from the regression of total calories versus age (Figure 12) for growth, and weight-specific oxygen consumption versus age ( Figure 19) for metabolism;

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142 predictived values. Values derived from the regressions .were compared to mean values for growth (Table 10) and for metabolism (Table 15); required values. Assimilation rate and conversion efficiency The percentage of energy assimilated was calculated using the equation (Houde, 1989): where A G M I = = = = A = (G + M)/I assimilated energy energy used in growth energy used in metabolism energy consumed Gross conversion efficiency was calculated using the equation: K1 = (G/I) X 100 where G = growth rate, and I = ration of food ingested. Gross conversion efficiency may be expressed in terms of wet weight, dry weight or caloric content. Ne t conversion efficiency was calculated by subtracting the ration needed for basal metabolism (Rmaint) from the total ingested energy (Brett and Groves, 1979): K2 = G/(I -Rmaint) X 100 Net conversion efficiency is a measure of the capacity to convert that fraction of ration that is in excess of maintenance level into flesh. Net conversion efficiency can also be calculated using the energy used in growth divided by the energy assimilated: G/A (Houde, 1989; Kiorboe, 1989).

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143 Results The energy budget Ingested energy was calculated in three ways. The first was based on the average number of rotifers counted in individual stomachs representing each age group of larvae: observed energy intake. The second was calculated by summing the mean calories used in growth (Table 10), respiration (Table 15), and excretion (Table 19) for each age group: the required energy intake. Lastly, ingested energy was calculated using values from regression equations of growth (Figure 12) and respiration (Figure 19) and mean excretion rates (Table 19) for each age group: the predicted energy intake. Table 21 compares the calories and number of rotifer equivalents for observed, required, and predicted energy budgets. The caloric value of rotifers ingested by red drum larvae as determined by direct counts was lower than the summed energy requirements of growth, respiration, and excretion in both required and predicted values. The difference between the observed and summed values was greater in younger larvae, probably due to an underestimation of the rate of gut evacuation and thus the number of rotifers ingested by younger individuals. Another possibility for the difference is the variability in the caloric values of the rotifers (Chapter 3). Ingestion rates based on required and predicted values were close, within 1 rotifer for days 3, 6, and 14, and within 4 and 10 rotifers

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144 Table 21. summary of the average daily caloric gains and costs in larval red drum raised in the laboratory at 25C for the first 2 weeks o f life. AGE 3 0 R p 4 0 R p 6 0 R p 10 0 R p 14 0 R p INGESTED ROTIFERS 0 31.1 30.4 13 36.8 32.3 21 39.30 40.60 61 79.60 66.00 75 111.00 112.00 GROWTH ENERGY -0.0118[22.43] 0.0088[16.70] 0.0096[18.25] 0 .0088[16.70] 0.0092[17.49] 0.0085(16.10] 0.0148(28.13] 0.0112(21.30] 0.0238(45.20] 0.0153[29.00] RESPIRATION ENERGY 0.00385[7.32] 0.0065(12.30] 0.00914[17.4] 0.00760[14.4] 0.00900[17.1] 0.01040(20.0] 0.02304(43.8] 0.01950[37.0] 0 .02765[52.6] 0.03660(69.6] EXCRETION ENERGY 0.00068[1.30] 0.00068[1.30] 0.00060[1.14] 0.00060[1.14] 0.00249[4.72] 0.00249[4.72] 0.00406[7.71] 0.00406[7.71] 0.00681[12.9] 0.00681(12.9] ( ] = number of rotifer equivalents (1 rotifer = 0.000526 calories; R = summation of growth; metabolism and excretion; P = predicted from regression equations; = standard deviation.

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145 on days 6 and 10, respectively. Total calories used for growth, respiration, and excretion increased with age, with the largest single increase observed i n metab o lic costs. Total energy devoted t o respiration and excretion based on required and p redicted values increased with age at a faster rate tha n t h a t for growth, resulting in a decline with age in the fraction o f the total energy devoted t o grow t h ( T a ble 22). Growth commanded the h i ghest fraction of total energy used by day 3 to 6 larvae, with the percentages dropping from 72.2% on day 3 to 40.7% on day 14 based on required values. Percentage of energy dedicated to growth dropped 55.0% on day 3 to 26.0% on day 1 4 based on values o btained from caloric regression equations, on day 14. The overal l percentage drop was equivalent in the required and regression-generated data. Table 22. Percentage of required ingested energy allocated to growth, metabolism and excretion. AGE INGE STED GROWTH RESP IRATION EXCRETION 3 72.2 (55. 0 ) 23 6 (40.7) 4 2 ( 4 3 ) 4 100 49.6 (51.7) 47.3 (44.8) 3.1 ( 3 5 ) 6 100 44 5 ( 39.7) 43.5 (48.7) 12.0 (11.6) 1 0 100 35.3 (32.3) 55.0 (56. 1) 9.7 ( 11.6) 1 4 100 40.7 {26.0) 47.4 ( 6 2 4) 11.9 (11.6) ( ) = values based on d a t a determined from regression equations

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146 Energy allocated for metabolism was lower than that used for growth based on required values in larvae between day 3 and 6, and between day 3 and 4, based on predicted values. Respiration costs increased from 23.6% on day 3 to 47.4% on day 14 (required) and 40.7% at day 3 to 62.4% (predicted) on day 14. The percent increase in energy allocated to metabolism with age was thus similar in required and predicted respiration data. The smallest fraction of the energy budget at all ages was the energy lost to excretion, ranging from 3% to 12% in both required and predicted values. Assimilation rate The absence of measurable fecal production in red drum larvae fed on a strictly rotifer diet made it necessary to calculate assimilation rate using the of Theilacker (1987) described earlier. Rotifer remains were completely digested in the stomachs examined indicating a high assimilation efficiency, which was borne out in the calculated assimilation efficiencies (Table 23). Assimilation efficiencies decreased with age in required, predicted and observed data. Percentage of energy decreased from 96.9% at day 3 to 88.1% at day 14 for required values and from 96.5% at day 3 to 88.4% at day 14 for predicted values. Assimilation efficiencies based on the observed maximum number of rotifers eaten increased from 81.8% at day 10 to 92.0% at day 14. The large variability of observed. data made assimilation and conversion efficiencies based on

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147 required and predicted data more reliable. Table 23. Assimilation efficiencies of red drum larvae fed a rotifer diet. AGE I (G + M)/I REQ PRED OBSER REQ PRED OBSER G+M+E G+M+E I(*) G+M+E G+M+E I(*) 3 0.00000 0.00000 0.00000 ----4 0 .01934 0.01700 0 .00684.0032 96.9 96.5 6 0 .02069 0 .02139 0.01105.0063 88.0 88.4 10 0.04190 0.03476 0.03209.0142 90.3 88.3 81.8 14 0 .05826 0.05871 0.05576.0163 88.1 88.4 92.0 REQ = required values; PRED = predicted values; OBSER: I(*) = % values based on observed data; I = calories, (G + M)/I = assimilation efficiency percentage. Conversion efficiencies Gross conversion efficiencies (K1) we r e variable but tended to decrease with increasing age (Table 22 and Table 24). Net growth efficiencies (K2) were higher than gross growth efficiencies but also declined with increasing age (Table 24). K2 values based on G/(I-Rmaintl decreased from 58.5% on day 6 to 43.9% on day 14 for required data and from 50.4% at day 6 to 29.0% at day 14 for predicted data. Based on G/A, K2 values decreased from 58.5% on day 4 to 29.5% at day 14.

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148 Table 24. Gross growth efficiency {K1 ) and net growth efficiency {K2) for red drum larvae based on required and predicted numbers of ingested rotifers. AGE K1 R maint K2 K2 *G/I G/{I-Rmaint) G/A -----------------------------REQ PRE D REQ PRED REQ PRED 3 0 .00532 4 49. 6 51.7 0.01176 51.2 58.5 6 44.5 39. 7 0.00384 58. 5 50.4 56.7 48.7 10 35.3 32.3 0 .01491 57.7 41.5 41.4 48. 2 14 40. 7 26.0 0.00556 43.9 29.0 45.1 29.5 *G/I-values from Table 22; Rmaint =calories {Table 15); G / I -Rmaint = percent; REQ = values determined from regression equations. Discussion The cost of life Red drum larvae compensate for their small egg size, small yolk supply and short incubation time by using a rapid growth rate in their very early life history to quickly attain a large size. The strategy for survival is one of using ingested resources as efficiently as possible to maximize growth. The danger in this type of early life history strategy is that there is little room for error in an environment filled with constant challenges, potentially resulting in high mortality rates. The window of survival for red drum larvae reared at in the laboratory is the 24 hours after day 3, {the completion of yolk-sac absorption) during which the larvae must locate a food source and learn to capture prey successfully.

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149 The energy budget for red drum larvae is based on the premise that biological systems conform to the laws of thermodynamics (Kleiber, 1961; Brett and Groves, 1979). The estimated energetic requirements of growing red drum larvae are 0.00894 to 0.01946 calories (17 to 37 rotifers/day) for day 4 larvae and 0.05576 to 0.058912 calories (106-112 rotifers/day) for day 14 larvae. These numbers agree well with estimates for Chysophrys major (red sea bream) and Lamanda yokahama (flatfish) larvae (Hoff and Snell, 1987). Estimates of larval feeding rate based on required and predicted ingested energy (G+M+E) were closest to observed ingested energy in older larvae. Day 14 larvae exhibited the smallest difference in ingestion rates for observed, required and predicted values; 106 to 111 and 112 rotifers/day, respectively. The reduced disparity between observed and summed energy intakes as larvae aged may result from the selection of less successful larvae out of the population by day 14. The resulting average number of food items captured by older larvae was more representative of a healthy, successful population. The relative contributions of growth, metabolism, and excretion to the energy budget of red drum larvae were similar to those for other species of fish larvae (Table 25). The percentage of ingested energy that was allocated to growth in red drum larvae based on predicted values, agreed well with predicted percentages found in a composite of subtropical species (Houde, 1989); 26.0% versus 28.7%,

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Table 25. Percentages of ingested energy allocated to growth, metabolism, and excretion for species similar in size to red drum. 150 SPECIES G M E SOURCE Engraulis mordax 0.020-0.089 15.5 (33.3-46.8) 0.010-0.036 (16.6-18.9) Pseudopleuronectes -------------------arnericanus 8.0 (2.0-14.0) (55.0-65.0) Archosargus --------------------rhomboidalis 26.0 (21.0-41.0) (6.0-31.0) Achirus lineatus --------0 .064-0.089 28.0 (16.0-33.0) (41.1-56.1) BOREAL 10.0 COMPOSITE TEMPERATE 20.0 COMPOSITE SUBTROPICAL 30.0 COMPOSITE 0 .00045* ( 28.7) 0.00092* (28.7) 0.00139* (28.7) 0 .00076* (48.4) 0.00113* (35.3) 0.00150* (31.1) 0.00036* ( 22.9) 0.00115* (36.0) 0.00194* (40.2) G, M, and E in calories/day; ( ) = percentage; 1 = Theilacker (1987); 2 =Laurence (1977); 3 =Houde and Schekter (1983); 4 =Houde (1989); = 1 2 3 3 4 4 4

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151 respectively. However, percentages based on required values (40.7 to 44.5) for red drum larvae were more similar to the amount of energy allocated to growth of northern anchovies aged days 5 to 14 days (33% to 47%) deteimined by Theilacker (1987). Metabolic costs were higher for red drum larvae (40.7% to 62. 4 % ) than the values for other species at the same temperature; 31. 1 % (Houde and Schekter, 1983; Houde, 1989). Percentages of ingested energy allocated to metabolism were similar to metabolic rates of colder species such as winter flounder (55% tb 65%) (Laurence, 1977), and the boreal composite (48. 4 % ) (Houde, 1989). The higher metabolic costs of red drum larvae reflect a high percentage of energy allocated to feeding metabolism (SDA), which increases in older larvae (Table 26). Table 26. Energy allocated to feeding metabolism (SDA) for the first two weeks o f life. AGE 4 6 10 14 REQ = FED RESP (CAL /INDIV/D) 0 .0076 0.0104 0.0195 0.0366 required values; regression equations. STARVED RESP (CAL/INDIV/D) 0.0044 0.0056 0 .0091 0 .0148 PRED -values % REDUCTION % INGESTED (SDA) ENERGY REQ PRED 42.1 19. 9 18. 9 43.2 18.8 21.0 53.3 29. 3 29. 9 59.6 28. 3 37.2 determined from

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152 The difference in metabolic rates between fed and starved red drum larvae is the portion of energy contributing to the SDA (Chapter 4). SDA ranges from 47.8% of total metabolic costs at day 3 to 59.6% at day 14. Based on required energy values, 11 .3% and 28 3 % of total energy ingested was used in SDA at day 3 and day 14, respectively. A similar analysis with predicted values yields a figure of 19.5% attributable to SDA at day 3 and 37.2% at day 14. Thus, a substantial amount of ingested energy was allocated to SDA in red drum larvae, reflecting the high energy requirements of growth processes. Assimilation rate and conversion efficiency In order to meet maintenance requirements and continue to add tissue at a high rate, growing larvae fed constantly during daylight hours. Increased swimming ability with age enabled larvae to increase the number of prey captured per unit time, but a higher portion of food energy was lost to respiration. The very complete and rapid digestion of rotifers observed in our feeding experiments resulted in high assimilation efficiencies (88% to 96%) This contrasts with other species' assimilation efficiencies, which have been reported as closer to the 70% used by Laurence (1977) for fish in general and the 65% to 75% of Ware (1975) for fish in general. Theilacker (1987) determined that assimilation efficiencies were SO% to 60% in larval northern anchovies. Similarly, Houde and Schekter (1983) found values for subtropical species (Anchoa mitchilli,

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153 Archosargus rhomboidalis, and Achirus lineatus) ranging from 24% to 74%. The latter two studies reported lower assimilation efficiencies than those of red drum larvae. The high assimilation of prey items by red drum larvae in the laboratory is most likely a result of their diet. Hoff and Snell (1987) reported that the dominant component of rotifers, protein, was highly (89% to 94%) digestible. Klumpp and von Westernhagen (1986) found that fish species in general exhibited high protein absorption efficiencies, usually exceeding 90%. Consequently, the high digestibility of rotifers may contribute to the already high assimilation efficiencies of larval red drum. Conversion efficiencies for red drum larvae were similar to those reported for other species. Klumpp and von Westernhagen (1986) found that conversion efficiencies for 9 to 15 day-old Pleuronectes platessa were 38% to 55% (K1), and 45% to 63% (K2), well within the range for similarly aged red drum larvae. Both the present study and that of Klumpp and von Westernhagen (1986) indicate that the gross efficiency of 15% to 35% determined as average for aquatic consumers by Welch (1968) is a low estimate, especially for young larval fish which exceed it by a factor of two. The decrease in conversion efficiency with age noted in this study has also been observed in other fish species (Ivlev, 1945; Eldridge et al., 1982}. Parker and Larkin (1959) attributed such decreases to the channeling of a greater portion of food energy to maintenance costs

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154 associated with increased swimming and feeding metabolism. The high initial values of 3-day-old larvae reflect the high conversion efficiencies of yolk combustion which has been observed in salmonids and sardines (Brett and Groves, 1979). Net growth efficiencies for red drum larvae indicated a high capacity to convert the ration in excess of maintenance into tissue. Brett and Groves (1979) found an average value of 36% net efficiency for larval fish, lower than the values observed for red drum. Red drum larvae ages 10 and 14 days had net efficiencies (29% to 48%) similar to the values of 16% to 56% reported for young perch (Mills and Forney, 1981) and the 38% to 57% reported for subtropical species (Houde and Schekter, 1983). Conclusions The early life history strategy of red drum is similar to other subtropical fish species, where few larval.cohorts find the local conditions required for maximum growth and survival (Houde, 1989). The stages of development were controlled by size and weight and were independent of the age of the larvae. Ration level and temperature control the rate of this development, with higher temperatures and increased ration having the highest growth and shortest stage durations; as shown by the values obtained from the 32oc pond larvae. Larger ingestion rates were required to support the high growth rates and associated lower mortality. Protracted spawning, serial spawning and batch spawning are strategies used by low latitude fish like

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155 the red drum to insure that some larvae hatch during periods when conditions produce high growth rates conducive for survival. The high demand for growth was reflected in the energy budget equation, where most ingested energy was channeled to growth and respiration processes. The high rates for energy expenditure of respiration reflect the cost of feeding metabolism (SDA} or the cost of growth processes. Red drum larvae that were starved decreased in calories equivalent to the calories gained in body tissue in larvae that were fed. The constant weight-specific rates for daily growth, nitrogen excretion and oxygen consumption in larvae fed and starved reflect the need for continued growth in size in accordance with the species survival blueprint, even at the expense o f the body tissues of the larvae. The high assimilation efficiencies of larvae fed a rotifer diet reflected the high digestibility of the food item, resulting in l o w amounts of energy lost as excretion. Excretion as urea, though comprising only a small fraction of total ingested energy, may be useful as a predictive tool in identifying starvation in larval fishes. The use of technology such as the needle electrodes allows for metabolic rates to be measured on a single larva and avoids the possible group effects that may occur. In addition, multiple regressions based on biochemical indicators which include a temperature coefficient can be useful predictive tool s for growth rates. The combination

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156 of these techniques allows for an increased resolution and power for prediction of the bioenergetics of hatchery and field-caught red drum larvae.

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