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ICES mar. Sei. Symp., 201: 64-69. 1995 Endogenous nutrient mobilization during egg and larval development in two marine fishes - Atlantic menhaden and spot Hans Jørgen Fyhn and John J. Govoni Fyhn, H. J., and Govoni, J. J. 1995. Endogenous nutrient mobilization during egg and larval development in two marine fishes - Atlantic menhaden and spot. - ICES mar. Sei. Symp., 201: 64-69. Volume measurements of yolk and oil globule, and analyses of protein and free amino acids (FA As) of developing eggs and larvae of Atlantic menhaden (Brevoorlia tyrannus) and spot (Leiostomus xanthurus) were carried out. The ratio of oil globule to yolk is 10 times greater in spot than in menhaden. In spot, the oil globule is resorbed after the yolk, while this occurs simultaneously in menhaden. The total protein content in the spot embryo/larva remains constant from spawning to after first-feeding. The pool of F A A s of spot eggs is about half that of menhaden and their decline correlates with that of the yolk in both species. No sparing of essential FAAs occurs during resorption. T he amount of taurine remains constant during development. It is hypothesized that, in spot, before tissue autolysis begins energy dissipation is based 30% on FA A s and 70% on lipids, while in menhaden the corresponding values are 65% FA A and 35% lipids. H. J. Fyhn: University o f Bergen, Zoological Institute, Allégaten 41, N-5007 Bergen, Norway. [Tel: (+47) 55 213588, fax: (+47) 55 329111], J. J. Govoni: National Marine Fisheries Service, N O A A , Beaufort Laboratory, Beaufort, North Carolina28516-9722, USA. Introduction The energy metabolism of marine fish larvae has attracted much attention, with the focus on the sub strates that are consumed during these early stages when first the yolk and later ingested prey from the plankton represent the sources of nutrients. For the larvae this conversion marks the switch from endogenous to ex ogenous feeding. This transition in larval life is difficult because it corre lates with high mortality in cultured larval populations. It is general consensus that this mortality relates to nutritional inadequacy of the endogenous yolk supply or of the exogenous feed offered the larva at the time of first-feeding. O ur interest in this problem has focused on the devel opment of the alimentary canal (Govoni, 1980; Govoni et al., 1986) and on free amino acids (FAAs) and their importance as a fuel for the marine fish larvae (Fyhn, 1989,1990,1993; Finn et al., 1991,1995a, b; Rønnestad and Fyhn, 1993; Rønnestad et al., 1992a, 1993). The studies on FAA have concerned boreal teleosts such as Atlantic cod (Gadus m orhua), Atlantic halibut (H ippoglossus hippoglossus), lemon sole (Microstomus kitt), and others (Fyhn and Serigstad, 1987; Fyhn, 1989; Finn et al., 1991; Rønnestad et al., 1992a). These fishes typi cally have pelagic eggs with a developmental tem pera ture optimum of about 5°C. The eggs lack an oil globule, so their sole endogenous supply of nutrients is that of the yolk. O ur results for these fishes have shown that FA A s are an important metabolic fuel during the egg and yolk-sac stages. A comparative study on turbot showed that its egg oil globule is an additional fuel for the larva after hatching when increased energy is needed for swimming in connection with the search for prey at first-feeding (Rønnestad et al., 1992b). The present study was initiated to find out whether our findings for FA A are confined to the boreal, coldwater fishes, or whether they apply more generally, and also include warm-water species and fishes with oil glo bules in the egg. Materials and methods Eggs and larvae of the Atlantic m enhaden (Brevoortia tyrannus, Clupeiformes, Clupeidae) and spot (Leiosto- i c e s mar. Endogenous nutrient mobilizations sd. Syrap.. 201 ( 1995) 65 Table 1. Linear regressions (Y = B0 + B 1X 1) describing yolk and oil globule volume depletion in days post-fertilization for Atlantic m enhaden ( B . tyrannus) and spot (L. xanthurus). Species Cohort Slope (B,) (d -1) Intercept (Bu) (nla) r2 p Yolk Menhaden Spot Spot 1 2 3 -0 .1036 -0.0461 -0 .0383 542 214 210 0.82 0.84 0.84 >0.012 >0.011 >0.011 Oil globule Menhaden Spot Spot 1 2 3 -0.0004 -0 .0019 -0 .0007 3 11 6 0.90 0.87 0.79 >0.004 >0.021 > 0.001- a (nl = 10 9litre). m us xanthurus, Perciformes, Sciaenidae) were studied. These fishes are common along the N orth American eastern coast, and both are commercially im portant for industrial and food fisheries. Both species contain an oil globule in the yolk. Eggs and larvae were reared at 19 ± 1°C and a salinity of 30-35 at the Beaufort Laboratory in accordance with a previously established technique (Hettler, 1984; Pow ell, 1993). Lyophilized samples were shipped to Bergen for biochemical analyses while morphometric measure ments were carried out on live material in Beaufort. A mino acid analyses were carried out using the HPLC technique on tri-chloro acetic acid extracted samples (triplicate samples of 50 pooled eggs or 25 pooled lar vae), and protein by the Lowry technique on 1 M N aO H extracted samples after removal of the FA A fraction as described elsewhere (Fyhn and Serigstad, 1987; Finn et al., 1991). Eggs and larvae from single batch spawnings were used, and the larve were not fed during the experi mentation. 400 In spot, the newly spawned eggs contain a relatively large oil globule of about 7 nl and a yolk sac of about 300 nl (Fig. 1A). The yolk declines to near zero in about 4 days, while the oil globule needs about 7 days to be resorbed. No oil globule resorption seems to occur during the egg stage, while more than 50% of the yolk volume was resorbed at hatching. A delayed resorption of the oil globule compared to the yolk is typical of marine fishes (Javila and Juario, 1987; Rønnestad et al., 1992b, 1994). A t first-feeding, energy for swimming activity may be derived from the remaining oil globule alone, since the yolk reserves are then exhausted. In the newly spawned egg of Atlantic menhaden the oil globule is smaller and the yolk larger than in spot, and both seem to be resorbed before first-feeding (Fig. IB). T he rate of yolk depletion is faster than that of the oil globule during the egg stage. O ther experience with Spot 300: = £ T = 19 ± 1°C 250 - 6 200 3 o>■ 150 ■© 100 - 4 " 2 0 2 6 4 8 10 12 1000 Firstfeeding Hatch _ Atlantic Menhaden 800 - £ a» S J3 600 - C £O > Results and discussion Firstfeeding Hatch 350 400 - 200 - 0 2 4 6 8 10 12 Days post-fertilization Figure 1. Volume of yolk (closed symbols) and oil globule (open symbols) in developing eggs and larvae of spot ( L . xanthurus, upper panel) and Atlantic m enhaden (B. tyrannus, lower panel). The data are mean values of 10 measurements on individual eggs or larvae. Table 2. Volume ratios and dry weight ratios of oil globule to yolk in newly spawned eggs of spot (L. xanthurus) and Atlantic m enhaden (B . tyrannus). Species Spot Menhaden Volume ratio Dry weight ratio“ 1:40 1:400 1:3 1:30 a Estim ated from the data in Figure 1 using a relative water content of 93% for the yolk. H. J. Fyhn and J. J. G ovoni 66 i c e s m ar. s d . s y m P , 201 ( 1995) Table 3. Mensuration estimates of the eggs of Atlantic menhaden (B . tyrannus) and spot (L. xanthurus). Values for the present study are means of 10 measurements. Diameter Species Cohort Menhaden M enhaden M enhaden Spot Spot Spot 1 2 3 Volume Yolk (mm) Oil globule (mm) Yolk (nl) Oil globule (nl) 1.18a 0.95b 1.19“ 0.828 0.816 0.80 0.163 0.22b 860 450c 580 300 280 270c 2 6C 3 _d _e 0.23 0.21 A uthor Present study H ettler, 1984 Powell, 1993 Present study Present study Powell and Gordy, 1980 _e 6 5C a Calculated assuming that the yolk diam eter is 74% of egg diam eter as per H ettler (1984). b Median values. c Calculated with mensuration formulae employed in the present study. d No value given. e The presence of multiple oil globules in the eggs of this cohort precluded measurements. Firstfeeding Hatch 16 C T = 19 ± 1”C c 12- sa> G Series 1 Series 2 25- T = 19 ± 1”C ■O Firstfeeding Hatch Series 1 Series 2 o S c 8- 0 2 4 6 8 10 12 Days post-fertilization 0 2 4 6 8 10 12 Days post-fertilization Figure 2. Protein content of eggs and larvae of spot (L. x a n thurus). The values are mean ± s.d. of three samples of 50 pooled eggs or 25 pooled larvae. W here no vertical bars are visible the s.d. is covered by the symbol. Figure 3. Free amino acid (FA A ) content in eggs and larvae of spot (L . xanthurus). The values are mean ± s.d. of three samples of 50 pooled eggs or 25 pooled larvae. W here no vertical bars are visible the s.d. is covered by the symbol. m enhaden (Powell, 1993; Govoni, unpublished results) has established that the oil globule is factually absorbed prior to the yolk, although this has not presently been confirmed. A n early consumption of the oil globule is unusual among marine fishes. The results for menhaden suggest that the nutritional conditions for its larvae at first-feeding are critical for survival, because almost no endogenous reserves are left at this stage. The slopes of the regression lines fitted to the resorbtion data indicate that in both species the yolk is de pleted at a faster rate than the oil is (Table 1). These regressions do not agree well with similar regressions for the Atlantic menhaden of Powell (1993), but this disparity can be explained by the fact that Powell included neither yolk nor oil volumes of the eggs. Neither did he include zero volumes of the day of com plete depletion. His work therefore describes yolk and oil depletion solely for yolk-sac larvae; our data relate to both the egg stage and the yolk-sac stage. The volume ratio between the oil globule and yolk is initially about 1:40 in spot and about 1:400 in Atlantic m enhaden. Since the yolk has a water content of about 93% while the lipids of the oil globule exclude water, the dry weight ratio between the two structures will be about 1:3 for spot and 1:30 for menhaden (Table 2). Mensuration estimates compare reasonably well with values reported in the literature for these species (Table 3). In spot (Fig. 2), we found a constant protein content during the egg and yolk-sac development (regrading the Endogenous nutrient mobilizations IC ES m ar. Sei. Symp.. 201 (1995) 4 80 F irst feeding Hatch 70 60 T3 C 50 O 40 E Hatch —• — Series 1 — O— Series 2 F irst feeding thr val 3 leu T = 19 ± 1”C "O arg C "c E 2 c s 30 s 20 tb 1 10 0 0 0 2 4 6 8 10 10 12 6 Days post-fertilization Hatch Figure 4. Free amino acid (FA A ) content in eggs and larvae of Atlantic m enhaden (B. tyrannus). T he values are mean ± s.d. of three samples of 50 pooled eggs or 25 pooled larvae. Where no vertical bars are visible the s.d. is covered by the symbol. decrease at hatching for series 2, see below). The protein content decreased after first-feeding, probably related to tissue autolysis as found for other fish larvae (Fyhn and Serigstad, 1987; Fyhn, 1989; Rønnestad et al., 1992a, b). The decrease in protein content of series 2 at hatching is due to removal of the chorion. Surprisingly, chorion removal did not show in series 1, although the samples were treated identically in both (i.e. protein was solubi lized by extracting in 1 M N aO H ). The reason for this discrepancy between the two series is not known, although the finding is not unique. Previously, we have obtained similar results between different batches of eggs from the same species (e.g. Atlantic halibut, Fyhn, 1989; Finn etal., 1991). The total FAA pool of spot eggs was about 30 nmol/ egg at spawning, decreasing steeply in a sigmoidal pat tern to a low level after a latency period of about 1 d (Fig. 3). G ood agreement was obtained between the samples from the two series. The decrease correlated with the decrease of the yolk volume, except for the initial delay period, which may not have been revealed in the yolk because of the lack of sampling points. At first-feeding, the FA A pool had attained a low level. Possibly the low concentration of some of the F A A is the stimulus for the larvae to feel starved and initiate feeding behaviour by increasing swimming activity in search of exogenous food. In m enhaden, the egg FA A pool at spawning was about twice as large as in spot, and the decrease was almost linear with time (Fig. 4). A t the time of firstfeeding, the pool was much reduced. No sparing of essential compared to non-essential 12 Firstfeedin g tau ser pro 5 •a s 4 'o 3 E ala e 2 1 0 8 10 12 Days post-fertilization Figure 5. Essential (upper panel) and non-essential (lower panel) amino acids in the free pool of eggs and larvae of spot (L. xanthurus). The values are mean ± s.d. of three samples of 50 pooled eggs or 25 pooled larvae. W here no vertical bars are visible the s.d. is covered by the symbol. F A A = free amino acids. amino acids was observed during development as the FAA were consumed. This applied to spot (Fig. 5) as well as to menhaden (Fig. 6). As found typically in other fish species (Fyhn, 1989, 1993; Rønnestad et a i , 1992a, b), the amount of amino acid analogue taurine remained constant during devel opment, becoming the dominant compound in the FAA pool after first-feeding (Figs. 5, 6). Based on our previous experience with eggs and lar vae of the boreal fishes, and these proximate analyses of spot and menhaden larvae, we carried out some calcu lations to estimate the metabolic rate and the pro portional use of amino acids versus lipids as fuels during the early life of these two warm-water fishes. For this we assumed that there is no net synthesis of body proteins, as was actually shown for spot (Fig. 2). Further, we assumed that the FAA s are not plainly lost by diffusive H. J. Fvhn and J. J. Govoni 68 ICES m ar. Sei. Sym p., 201 (1995) 10 H atch First- 8 -O c Hatch -•-th r • val — met — ile —O—leu —o lys -O -a rg feed in g 6 H Lipid c 19 ± 1 C "O c o E ~5 E Firstfeeding £ N 4 £ 0 D ays post-fertilization 10 Hatch Firstfeed in g 12 tau ser pro Figure 8. Estim ated metabolic rate and proportional use of various metabolic fuels in eggs and larvae of Atlantic m enhaden (B . tyrannus). V 0 2 = oxygen uptake rate; F A A = free amino acids. gjy ala TJ' C o E e Days post-fertilization Figure 6. Essential (upper panel) and non-essential (lower panel) amino acids in the free pool of eggs and larvae of Atlantic m enhaden ( B . tyrannus). The values are mean ± s.d. of three samples of 50 pooled eggs or 25 pooled larvae. Where no vertical bars are visible the s.d. is covered by the symbol. F A A = free amino acids. Hatch Firstfeeding ■ FA A ED L ip id P ro te in T = 19 ± 1°C Days post-fertilization Figure 7. Estimated metabolic rate and proportional use of various metabolic fuels in eggs and larvae of spot (L . xanthur us). V 0 2 = oxygen uptake rate; F A A = free amino acids. efflux to the ambient sea water. This is in agreement with recent measurements of larvae of the Atlantic halibut by Rønnestad (1993). Finally, we assumed that the lipid content of the oil globule is consumed in the aerobic energy dissipation of the developing larvae. Although this is not proven, consumption of the oil globule in turbot correlates with its oxygen uptake rate (Fønnestad et a l., 1992a). Given these assumptions, we can calculate the amount of oxygen dem anded to catabolize the actual fuels, and to make a plot for the resulting oxygen uptake during development. For spot, the estimated hourly oxygen uptake rate at 19 ± 1°C follows a curve with a peak of 8-10 nmol in d-1 h -1 around first-feeding (Fig. 7). Probably, the real curve, which is still to be estab lished by measuremënts, will be similar to this calculated curve. Early in development F A A may account for 100% of energy dissipation, while lipids from the oil globule may be the main fuel at first-feeding. Amino acids derived from body proteins seem to become an additional fuel when tissue autolysis starts. The domi nance of lipids at first-feeding leads to speculation as to the actual cellular fuel during this period of develop ment. Totally, FA A s seem to account for about 25% of the energy production of the developing spot larva, while lipids derived from the oil globule account for 60% , and body proteins for an additional 15%. Restricting the period of calculation to that before tissue autolysis sets in , the proportional use of F A A and lipids as fuels would be about 30% and 70% , respectively. Although no data on protein are currently available for embryonic and larval m enhaden, the same calcu lation procedure and assumptions as for spot reveal a more rounded oxygen uptake curve with a peak value of about 5 nmol i n d -1 h -1 at first-feeding (Fig. 8). FAA s ICES mar. Sei. Symp., 201 (1995) seem to be the dominating fuel, and account for about 65% of the total dissipated energy. Lipids from the oil globule add to the FAA fuel, especially around firstfeeding, as in the spot. It is likely that the use of lipids for swimming activity associated with first-feeding is a general phenomenon in fish larvae. Conclusions Spot embryos contain a larger oil globule than menha den, and seem to rely more on lipids than on FA A in its early life energy dissipation. Lipids seem especially im portant around the time of first-feeding, possibly as a fuel for swimming activity. Using proximate analyses as a basis for an hypothesis, one can estimate oxygen uptake rate of larval spot and menhaden reaching levels of about 5-10 n m o l ind“ 1I T 1 at 19 ± 1°C. These are not unrealistic values for fish larvae at this temperature. During development and before tissue autolysis begins, energy dissipation seems based 30% on FA A and 70% on lipids in spot, while in menhaden the corresponding values are 65% FAA and 35% lipids. These hypothetical values need verification by measurements. O ur results indicate that FA A s are likely to be an im portant metabolic fuel during the early life stages also for warm-water fishes, although lipids become the domi nant fuel after hatching in species with a high ratio of oil globule to yolk volume. A recent study on the warmwater gilthead sea bream, Sparus aurata (Rønnestad et al., 1994), confirms this suggestion. Acknow ledgem ents H JF acknowledges the extensive cooperative relations enjoyed with the research institutions of Pivers Island, Beaufort, USA, inspiring discussions with Ivar R ønnes tad and Roderick Nigel Finn, as well as excellent techni cal assistance provided by Maria Sula Evjen. William H ettler provided laboratory spawned eggs. The study was partially supported by the Norwegian Fisheries Research Council and the Nansen Foundation. References Finn, R. N ., Fyhn, H. J., and Evjen, M. S. 1991. Respiration and nitrogen metabolism of Atlantic halibut eggs (H ippog lossus hippoglossus h .). Mar. Biol., 108: 11-19. Finn, R. N., Rønnestad, I., and Fyhn, H. J. 1995a. Respir ation, nitrogen and energy metabolism of developing yolk- Endogenous nutrient mobilizations 69 sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.). Comp. Biochem. Physiol, 111A: 647-671. Finn, R. N., Widdows, J., and Fyhn, H. J. 1995b. Calorespirometry of developing embryos and yolk-sac larvae of turbot (Scophthalmus maxim us). Mar. Biol, 122: 157-163. Fyhn, H. J. 1989. First feeding of marine fish larvae: Are free amino acids the source of energy? Aquaculture, 80: 111-120. Fyhn, H. J. 1990. Energy production in marine fish larvae with emphasis on free amino acids as a potential fuel. In Nutrition in wild and domesticated animals, pp. 176-192. Ed. by J. Mellinger. Karger, Basel. 289 pp. Fyhn, H. J. 1993. Multiple functions of free amino acids during embryogenesis in marine fishes. In Physiological and bio chemical aspects of fish development, pp. 299-308. Ed. by B. T. W alther and H. J. Fyhn. University of Bergen, Bergen. 355 pp. Fyhn, H. J. and Serigstad, B. 1987. Free amino acids as energy substrate in developing eggs and larvae of the cod ( Gadus morhua). Mar. Biol., 96: 335-341. Govoni, J. J. 1980. 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