Size hierarchies affecting the social interactions and growth of juvenile Japanese flounder, Paralichthys olivaceus

Japanese flounder Paralichthys olivaceus (T. & S.)juveniles were size-graded and divided into three groups (small, large, and mixture of small and large flounder), and their social interactions (feeding, aggressive attacking and activity) and growth were investigated. The growth of the small flounder was markedly suppressed by the presence of the large flounder. Large flounder did not significantly suppress the overall food intake of the small flounder but exhibited high aggressive attacking on them and consequently inhibited their activity. Size dominance showed little influence on the aggressive behavior, feeding, activity and growth of the large flounder. The large flounder could not effectively defend the food in excess during the experiments ruling out disproportional food acquisition as the primary mechanism responsible for the size hierarchy effect. Aggressive interaction of the large flounder on the small flounder might be an important cause for the growth retardation of the small flounder. In culture, size grading could markedly improve the growth and survival of the early juvenile flounder. (C) 2004 Elsevier B.V. All rights reserved.

Identification of factors affecting the growth and survival of the settling Japanese flounder larvae, Paralichthys olivaceus

A series of experiments were conducted to identify the factors that affected the growth and survival of the settling flounder larvae Paralichthys olivaceus. Settling larvae 24 days after hatching (DAH) were reared in 10-l experimental tanks up to 40 DAH, and two of the following factors were changed as controlled factors in each experiment: light regime (24L:0D or 12L:12D), prey density (1500, 3000, or 5000 Artemia l(-1)), shelter (sand or no sand) and stocking density (5, 10, or 15 fish l(-1)). Early settling larvae (24-35 DAH) experienced little mortality (less than 10% of the overall mortality) that was not significantly affected by above factors. In contrast, late settling larvae (36-40 DAH) suffered high cannibalistic mortality which was significantly influenced by each of the above factors. Larvae experienced significantly lower mortality at 10 fish l(-1) level than at other densities. Larvae at 15 fish l(-1) level had higher mortality than at 5 fish l(-1) when all other factors were identical. Larvae at 3000 and 5000 Artemia l(-1) treatments survived significantly better than at 1500 Artemia l(-1), but no significant differences in larval mortality were found between the two higher densities. Larvae suffered higher mortality at low prey density or at the absence of sand when they were exposed to longer photoperiod. Low stocking density significantly improved the growth of the settling larvae. The average daily instantaneous growth rate (G) at 5 and 15 fish l(-1) treatments were 0.050 and 0.034, with the coefficient of variation (CV) in final length at 16.4 and 23.5, respectively. Daily instantaneous growth rate increased significantly from 0.033 in the 1500 Artemia l(-1) to 0.041 and 0.045 in the 3000 and 5000 Artennia l(-1), respectively, but no significant difference in larval growth existed between the two higher prey densities. These findings suggested that the optimal prey density for growth and survival of the settling flounder larvae at a stocking density of 5 - 15 fish l(-1) was around 3000 Artemia l(-1) . Larvae that were exposed to 24L showed 20% increase in growth ( G = 0.046, CV = 18.7) than those exposed to 12L ( G = 0.037, CV = 20.5). Longer exposure to light significantly improved larval growth, provided sufficient food was available. Sand substrate did not show significant effects on larval growth, possibly because the larvae spent most of the time swimming or feeding in the water column during this stage. (C) 2003 Elsevier Science B.V. All rights reserved.

Cannibalism in Japanese flounder juveniles, Paralichthys olivaceus, reared under controlled conditions

Laboratory experiments were conducted to determine the effects of sand substratum, light, starvation, fish density and size variation on cannibalism among Japanese flounder juveniles, Paralichthys olivaceus. Cannibalistic behavior (biting and swallowing) increased significantly with starvation except in darkness, where juveniles could not find and attack their prey. Cannibalism occurred more frequently in heterogeneous than in the homogeneous size groups. Cannibalism was more frequent in light than in darkness at each starvation level. With sufficient food present, sand had no significant effect on cannibalism among homogeneous size juveniles, but played an important role in discouraging predation among members of the heterogeneous size groups. With sufficient food present, the rate of cannibalism was generally low and there were no significant differences in cannibalism among fish density levels of the homogeneous juveniles, With starvation, the cannibalism rate was significantly higher in high density groups than in the lower ones. Our results indicate that size variation, starvation and Light are the major factors affecting cannibalism in flounder juveniles, while sand and fish density only have a limited effect on cannibalism, depending on the nature of other factors. (C) 2000 Elsevier Science B.V. All rights reserved.

Effects of tributyltin on the immune system of Japanese flounder (Paralichthys olivaceus)

Effects of tributyltin (TBT) which has been used for antifouling paint of ship's hulls and fishing nets on the immune system in Japanese flounder (Paralichthys olivaceus) were investigated. After short-term exposure to a high level of TBT, leucocytes in the head kidney from 1-year-old flounder were examined for the proportion of neutrophils in total leucocytes. Also examined were their respiratory burst activities using flow cytometry, the reduction of nitroblue tetrazolium (NBT) and lysozyme activities. Furthermore, long-term exposures to a relatively low level of TBT using young flounder were also carried out. The proportion of neutrophils in total leucocytes prepared from head kidney in each fish exposed to TBT at 20 microg/L for 5 days and the reduction of NBT by leucocytes prepared from the same experimental conditions increase compared to the control group. The contents were 42.0+/-6.8 and 52.5+/-6.3%, respectively. Significant differences of the NBT reduction were observed between 0 and 20 microg/L TBT exposure groups. On the other hand, the respiratory burst activity of cells in the exposure group clearly showed a tendency to decrease compared to the control group. Furthermore, high level of TBT also inhibited lysozyme activity which plays an important role for the bacteriocidal procedures. However, similar results were not obtained in the exposure group with a relatively low level of TBT. To determine the immunotoxic effects of TBT, infection experiments using pathogens which are naturally occurring should be further investigated.

Ecology of aquaculture species and enhancement of stocks: proceedings of the thirtieth U.S. - Japan meeting on aquaculture, Sarasota, FL, Dec. 3-4, 2001

CONTENTS: I. U.S.-Japan Cooperation Open Ocean Aquaculture – A Venue for Cooperative Research Between the United States and Japan.............................................................................. 1 C. Helsley II. Growth, Nutrition and Genetic Diversity Daily Ration of Hatchery-Reared Japanese Flounder Paralichthys olivaceus as an Indicator of Release Place, Time and Fry Quality. In situ Direct Estimation and Possibility of New Methods by Stable Isotope............................ 7 O. Tominaga, T. Seikai, T. Tsusaki, Y. Hondo, N. Murakami, K. Nogami, Y. Tanaka and M. Tanaka Nucleic Acids and Protein Content as a Measure to Evaluate the Nutritional Condition of Japanese Flounder Paralichthys olivaceus Larvae and Juveniles........................................................................................................ 25 W. Gwak Genetic Diversity Within and Between Hatchery Strains of Flounder Paralichthys olivaceus Assessed by Means of Microsatellite and Mitochondrial DNA Sequencing Analysis...................................................................... 43 M. Sekino, M. Hara and N. Taniguchi Tracking Released Japanese Flounder Paralichthys olivaceus by Mitochondrial DNA Sequencing................................................................................ 51 T. Fujii Preliminary Aspects of Genetic Management for Pacific Threadfin Polydactylus sexfilis Stock Enhancement Research in Hawaii........................................ 55 M. Tringali, D. Ziemann and K. Stuck Enhancement of Pacific Threadfin Polydactylus sexfilis in Hawaii: Interactions Between Aquaculture and Fisheries............................................................. 75 D. Ziemann Aquaculture and Genetic Structure in the Japanese Eel Anguilla japonica..................... 87 M. Katoh and M. Kobayashi Comparative Diets and Growth of Two Scombrid Species, Chub Mackerel Scomber japonicus and Japanese Spanish Mackerel Scomberomorus niphonius, in the Central Seto Inland Sea, Japan.................................. 93 J. Shoji, M. Tanaka and Tsutomu Maehara iii Evaluating Stock Enhancement Strategies: A Multi-disciplinary Approach................... 105 T. M. Bert, R.H. McMichael, Jr., R.P. Cody, A. B. Forstchen, W. G. Halstead, K. M. Leber, J. O’Hop, C. L. Neidig, J. M. Ransier, M. D. Tringali, B. L. Winner and F. S. Kennedy III. Physiological and Ecological Applications Predation on Juvenile Chum Salmon Oncorhynchus keta by Fishes and Birds in Rivers and Coastal Oceanic Waters of Japan................................... 127 K. Nagasawa and H. Kawamura Interaction Between Cleaner and Host: The Black Porgy Cleaning Behavior of Juvenile Sharpnose Tigerfish Rhyncopelates Oxyrhynchus in the Seto Inland Sea, Western Japan............................................................................. 139 T. Shigeta, H. Usuki and K. Gushima IV. Case Studies Alaska Salmon Enhancement: A Successful Program for Hatchery and Wild Stocks............................................................................................... 149 W. Heard NMFS Involvement with Stock Enhancement as a Management Tool........................... 171 T. McIlwain Stock Enhancement Research with Anadromous and Marine Fishes in South Carolina...................................................................................... 175 T. I. J. Smith, W. E. Jenkins, M. R. Denson and M. R. Collins Comparison of Some Developmental, Nutritional, Behavioral and Health Factors Relevant to Stocking of Striped Mullet, (Mugilidae), Sheepshead (Sparidae), Common Snook (Centropomidae) and Nassau Groupers (Serranidae)........................... 191 J. W. Tucker Jr. and S. B. Kennedy Participants in the Thirtieth U.S.-Japan Meeting on Aquaculture................. Inside Back Cover iv (PDF has 204 pages.)

养殖褐牙鲆细胞色素b 基因序列的初步研究

对养殖褐牙鲆( Paralichthys olivaceus) 的线粒体DNA Cytb 基因的部分序列进行测定,测得的目的DNA 片段的长 度为410 bp ,其A(104 bp) 、T(119 bp) 、C(117 bp) 、G(70 bp) 4 种碱基平均含量分别为25. 4 %、29. 0 %、28. 5 %、17. 1 %。 在28 个褐牙鲆个体中共出现了3 种单倍型。白化褐牙鲆出现的第1 种和第3 种单倍型个体数分别为10 尾(占白 化褐牙鲆样本数的90. 91 %) 和1 尾(9. 09 %) ;6 尾黑化褐牙鲆均出现第1 种单倍型(100 %) ;正常褐牙鲆出现的3 种 单倍型尾数分别为7 尾(占正常褐牙鲆样本数的55. 56 %) 、2 尾(22. 22 %) 和2 尾(22. 22 %) ;测得的序列与既知序列 间在第6 bp 、第19 bp 和第402 bp 碱基处出现差异。由于褐牙鲆Cytb 基因的高度同源性,研究其白化、黑化和正常 状态时出现的序列差异,对于寻找褐牙鲆白化机理研究的分子标记意义重大。

牙鲆一株弹状病毒病原的分离与鉴定

从患病牙鲆中分离鉴定了一株弹状病毒(Paralichthys olivaceus rhabdovirus,PoRV)。用过滤除菌后的患病牙鲆组织匀浆液,接种不同的鱼类细胞,其中有7种鱼类细胞出现明显的病变在对病毒进行挑斑分离后,测定了PoRV的滴度,显示PoRV在敏感鱼类细胞(Grass Carp Ovary,GCO)中的滴度达到106.5TCID50/mL;绘制了PoRV生长曲线;经蔗糖密度梯度离心提纯PoRV,负染及宿主细胞超薄切片的电镜观察,显示PoRV大小约为60nm×200nm。测定了PoRV的

Development and characterization of monoclonal antibodies to spring viraemia of carp virus

Five monoclonal antibodies (mAbs) against spring viraemia of carp (SVCV0504, isolated from common carp in China) were produced from mice immunized with purified virus preparations. The virion of SVCV contains five structural proteins, representing the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (Q. Western blotting analysis revealed that three mAbs (1145, IE10, and 11-17) recognized specifically to a single protein of 47 kDa (N), the mAb 3G4 reacted with, two SVCV0504 proteins of 69 kDa (G) and 47 kDa (N), while the mAb 1A9 reacted with three SVCV0504 proteins of 69 kDa (G), 50 kDa (P), and 47 kDa (N). By indirect ELISA, two mAbs (1H5 and 11-17) showed cross-reactivity with pike fry rhabdovirus (PFRV), but no cross-reactions with the Siniperca chuatsi rhabdovirus (SCRV), Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PoRV) were demonstrated with the five mAbs. Indirect immunofluorescence showed intense fluorescence in the cytoplasm of the SVCV0504-infected epithelioma papulosum cyprini (EPC) cells in areas corresponding to the location of granular structures. The sucrose gradient-purified SVCV0504 particles could be detected successfully by these mAbs using immunodot blotting. mAb 1A9 could completely neutralize 100 TCID50 (50% tissue culture infective dose) of SVCV0504 at a dilution of 1:8. This is the first report of development of the neutralizing mAbs against SVCV. The mAb 1A9 was analyzed further and could be used to successfully detect viral antigens in the infected-EPC cell cultures or in cryosections from experimentally infected crucian carp (Carassius auratus) by immunohistochemistry assay. Furthermore, a flow cytometry procedure for the detection and quantification of cytoplasmic SVCV0504 in cell cultures was developed with mAb 1A9. At 28 h after inoculation with the virus (0.01 PFU/cell), 10.12% of infected cells could be distinguished from the uninfected cells. These mAbs will be useful in diagnostic test development and pathogenesis studies for fish rhabdovirus. (c) 2008 Elsevier B.V. All rights reserved.

Molecular characterisation and inductive expression of a fish protein arginine methyltransferase 1 gene in response to virus infection

Protein arginine methyltransferase 1 (PRMT1) is currently thought as an effector to regulate interferon (IFN) signalling. Here Paralichthys olivaceus PRMT1 (PoPRMT1) gene was identified as a vitally induced gene from UV-inactivated Scophthalmus maximus Rhabdovirus (SMRV)-infected flounder embryonic cells (FEC). PoPMRT1 encodes a 341-amino-acid protein that shares the conserved domains including post-I, motif I, II and III. Homology comparisons show that the putative PoPMRT1 protein is the closest to zebrafish PMRT1 and belongs to type I PRMT family (including PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, PRMT8). Expression analyses revealed an extensive distribution of PoPMRT1 in all tested tissues of flounder. In vitro induction of PoPRMT1 was determined in UV-inactivated SMRV-infected FEC cells, and under the same conditions, flounder Mx wash also transcriptionally up-regulated, indicating that an IFN response might be triggered. Additionally, live SMRV infection of flounders induced an increased expression of PoPRMT1 mRNA and protein significantly in spleen, and to a lesser extent in head kidney and intestine. Immunofluorescence analysis revealed a major cyptoplasmic distribution of PoPRMT1 in normal FEC but an obvious increase occurred in nucleus in response to UV-inactivated SMRV. This is the first report on in vitro and in vivo expression of fish PRMT1 by virus infection, suggesting that PoPRMT1 might be implicated in flounder antiviral immune response. (c) 2006 Elsevier Ltd. All rights reserved.

Characterization and application of monoclonal antibodies against turbot (Scophthalmus maximus) rhabdovirus

Five monoclonal antibodies (mAbs), 1G8, 1H9, 2D2, 2D3, and 2F5, against Scophthalmus maximus rhabdovirus (SMRV) were prepared. Characterization of the mAbs included indirect enzyme-linked immunosorbent assay, isotyping, viral inhibition assay, immunofluorescence staining of virus-infected cell cultures, and Western blot analysis. Isotyping revealed that 1G8 and 1H9 were of the IgG2b subclass and that the other three were IgM. 2D2, 2D3, and 2F5 partially inhibited SMRV infection in epithelioma. papulosum cyprinid (EPC) cell culture. Western blotting showed that all five mAbs could react with two SMRV proteins with molecular masses of approximately 30 kDa (P) and 26 kDa (M). These two proteins were localized within the cytoplasm of SMRV-infected EPC cells by immunofluorescence assay. Also, progressive foci of viral replication in cell cultures were monitored from 6 to 24 h, using mAb 2D3 as the primary antibody. A flow cytometry procedure was used to detect and quantify SMRV-infected (0.01 PFU/cell) EPC cells with mAb 2D3, and 10.8% of cells could be distinguished as infected 36 h postinfection. Moreover, mAb 2D3 was successfully applied for the detection of viral antigen in cryosections from flounder tissues by immunohistochemistry tests.

Gene structure of goose-type lysozyme in the mandarin fish Siniperca chuatsi with analysis on the lytic activity of its recombinant in Escherichia coli

A goose-type lysozyme (g-lysozyme) gene has been cloned from the mandarin fish (Siniperca chuatsi), with its recombinant protein expressed in Escherichia coli. From the first transcription initiation site, the mandarin fish g-lysozyme gene extends 1307 nucleotides to the end of the 3' untranslated region, and it contains 5 exons and 4 introns. The open reading frame of the glysozyme transcript has 582 nucleotides which encode a 194 amino acid peptide. The 5' flanking region of mandarin fish glysozyme gene shows several common transcriptional factor binding sites when compared with that from Japanese flounder (Paralichthys olivaceus). The recombinant mandarin fish g-lysozyme was expressed in E. coli by using pET-32a vector, and the purified recombinant g-lysozyme shows lytic activity against Micrococcus lysodeikticus. (c) 2005 Elsevier B.V All rights reserved.

Complete genome sequence of lymphocystis disease virus isolated from China

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.

Identification of an Edwardsiella tarda surface antigen and analysis of its immunoprotective potential as a purified recombinant subunit vaccine and a surface-anchored subunit vaccine expressed by a fish commensal strain

Edwardsiella tarda is the etiological agent of edwardsiellosis, a systematic disease that affects a wide range of marine and freshwater fish cultured worldwide. In order to identify E. tarda antigens with vaccine potential, we in this study conducted a systematic search for E. tarda proteins with secretion capacity. One of the proteins thus identified was Esa1, which contains 795 amino acid residues and shares extensive overall sequence identities with the D15-like surface antigens of several bacterial species. In silico analyses indicated that Esa1 localizes to outer membrane and possesses domain structures that are conserved among bacterial surface antigens. The vaccine potential of purified recombinant Esa1 was examined in a Japanese flounder (Paralichthys olivaceus) model, which showed that fish vaccinated with Esa1 exhibited a high level of survival and produced specific serum antibodies. Passive immunization of naive fish with antisera raised against Esa1 resulted in significant protection against E. tarda challenge. Taking advantage of the secretion capacity of Esa1 and the natural gut-colonization ability of a fish commensal strain, we constructed an Esa1-expressing recombinant strain, FP3/pJsa1. Western immunoblot and agglutination analyses showed that FP3/pJsa1 produces outer membrane-localized Esa1 and forms aggregates in the presence of anti-Esa1 antibodies. Vaccination analyses showed that FP3/pJsa1 as an intraperitoneal injection vaccine and an oral vaccine embedded in alginate microspheres produced relative percent survival rates of 79% and 52%, respectively, under severe challenging conditions that resulted in 92-96% mortality in control fish. Further analyses showed that following oral vaccination, FP3/pJsa1 was able to colonize in the gut but unable to disseminate into other tissues. Together these results indicate that Esa1 is a protective immunogen and an effective oral vaccine when delivered by FP3/pJsa1 as a surface-anchored antigen. (c) 2010 Elsevier Ltd. All rights reserved.

Isolation and analysis of the vaccine potential of an attenuated Edwardsiella tarda strain

Edwardsiella tarda is an important aquaculture pathogen that can infect a wide range of marine and freshwater fish worldwide. In this study, a modified E. tarda strain, TX5RM, was selected by multiple passages of the pathogenic E. tarda strain TX5 on growth medium containing the antibiotic rifampicin. Compared to the wild type strain, the rifampicin-resistant mutant TX5RM (i) shows drastically increased median lethal dose and reduced capacity to disseminate in and colonize fish tissues and blood; (ii) exhibits slower growth rates when cultured in rich medium or under conditions of iron depletion; and (iii) differs in the production profile of whole-cell proteins. The immunoprotective potential of TX5RM was examined in a Japanese flounder (Paralichthys olivaceus) model as a vaccine delivered via intraperitoneal injection, oral feeding, bath immersion, and oral feeding plus immersion. All the vaccination trials, except those of injection, were performed with a booster at 3-week after the first vaccination. The results showed that TX5RM administered via all four approaches produced significant protection, with the highest protection levels observed with TX5RM administered via oral feeding plus immersion, which were, in terms of relative percent of survival (RPS), 80.6% and 69.4% at 5- and 8-week post-vaccination, respectively. Comparable levels of specific serum antibody production were induced by TX5RM-vaccinated via different routes. Microbiological analyses showed that TX5RM was recovered from the gut, liver, and spleen of the fish at 1-10 days post-oral vaccination and from the spleen, liver, kidney, and blood of the fish at 1-14 days post-immersion vaccination. Taken together, these results indicate that TX5RM is an attenuated E. tarda strain with good vaccine potential and that a combination of oral and immersion vaccinations may be a good choice for the administration of live attenuated vaccines. (C) 2010 Elsevier Ltd. All rights reserved.

牙鲆与大黄鱼卵黄囊期发育阶段生化与能量代谢研究

于1999年5－6月间在实验室内对暖温性底层鱼类褐牙鲆(Paralichthys olivaceus)和暖水性近底层鱼类大黄鱼(Pseudosciaena crocea)进行了自受精卵开始到早期仔鱼的发育生态学实验。褐牙鲆和大黄鱼的培育温度分别为17.4±0.3 ℃和23.2±0.3 ℃，盐度均为31±0.2‰，遮光静养。通过测定其受精卵细胞的干重、湿重、卵黄和油球体积以及细胞发育过程中的耗氧率、氨排泄率、铵离子含量、游离氨基酸、蛋白质、总脂、脂肪酸等参数的变化（大黄鱼仅限于卵黄和油球体积、耗氧率、氨排泄率、铵离子含量、游离氨基酸和蛋白质），分析不同发育阶段的生化代谢和能量代谢特征；结合已有的研究成果，比较鱼类发育能量学的基本规律。结果表明：1．在受精卵分裂和胚体发育中，褐牙鲆和大黄鱼均呈现明显的铵离子累积现象，其间产生的氨一部分排到环境中，另一部分在卵中积累，孵化时随着卵膜的破裂而排出，导致氨排泄率出现一个峰值。2．游离氨基酸是早期发育阶段的一个重要能源物质，分别构成了褐牙鲆能量代谢的35％和大黄鱼能量代谢的28％。此外，在受精卵发育阶段，游离氨基酸还被用来合成蛋白质，所以在受精卵发育阶段，蛋白质非但不减少，还会有明显的增加。3．在孵化前后，伴随着卵膜的破裂和卵周液的流失，干重和蛋白质含量均有明显的减少。4．卵黄囊期仔鱼早期阶段，蛋白质含量略有增加，这说明还有一部分的游离氨基酸被合成了蛋白质。其后，蛋白质含量呈现下降趋势，表明蛋白质也已被用做能源物质。5．褐牙鲆受精卵中最主要的多不饱和脂肪酸是DHA和EPA，二者合计占多不饱和脂肪酸总量的86％。比较油球体和总脂含量的变化特征，不难发现脂肪酸主要是位于油球中。这说明来自油球的脂类是褐牙鲆仔鱼在这个阶段的主要能源物质，来自油球的中性脂肪NL的脂肪酸FA是从孵化到开口这段时间主要的能源物质。磷脂PL的脂肪酸FA只是被很少的利用（为中性脂肪NL的30％）。6．游离氨基酸FAA是褐牙鲆与大黄鱼在受精发育阶段最重要的能源物质，而在孵化后来自油球的中性脂肪NL的脂肪酸NLFA被用做主要能源物质，蛋白质则是在开口之前就用做能源物质，但其作用还不是很大。主要能源物质的转变可以从氮熵NQ的变化看出来，氮熵呈现一种先降低后升高的态势。但就整个早期发育阶段来说，无氮的脂类所起的作用更大一些。分解的主要中性脂肪酸是那些含量最丰富的种类，而与他们的饱和度无关。7．褐牙鲆从卵受精后8小时到仔鱼开口，能够将卵黄消耗的焓的59.7％转化为生长（P），有43.6％因代谢而散失，还有极少部分0.4％因排汇而丢失。有一定的实验误差在里面。8．褐牙鲆是适温较低的暖温性底层鱼类，卵子较小，发育时水温较低，发育较慢；大黄鱼是适温较高的暖水性近底层鱼类，卵子较大，发育时水温较高，发育较快，所以褐牙鲆与大黄鱼在早期发育阶段所采取的能量对策是不一样的。褐牙鲆游离氨基酸占总的能量代谢的35％，而大黄鱼只占28％，相对而言，暖水性鱼类比暖温性鱼类更为依赖脂类，氨基酸所起的作用相对较小一些。9．依据已有研究成果，不难得出：鱼类早期发育过程中主要能源物质变化和能量代谢牲征有物种特异性。对于含一个油球的鱼类来说，基于脂肪的能量代谢是重要的；而对于不含油球的鱼类来说，氨基酸的作用更为重要一些。这些研究对于仔鱼在开口时营养成分的确定及满足、死亡率以及生活史特征具有十分重要的科学价值。