Differential population structuring and demographic history of two closely related fish species, Japanese sea bass (Lateolabrax japonicus) and spotted sea bass (Lateolabrax maculatus) in Northwestern Pacific

The Quaternary cold periods in the Northwestern Pacific are thought to have heavily influenced the amount and distribution of intraspecific genetic variation in marine fishes. To estimate the demographic history and genetic structure of Lateolabrax maculatus and L. japonicus in the Northwestern Pacific, 256 individuals were sampled from 19 localities throughout the distribution range of the two species. Mitochondrial DNA variation was analyzed using DNA sequence data from the cytochrome b gene and control region. Nucleotide diversity was much higher in L. japonicus (0.030) than in L. maculatus (0.012). The demographic history of the two species was examined using neutrality tests and mismatch distribution analyses and results indicated Pleistocene population expansion in both species. Estimates of population expansion time suggested earlier population expansion in L. japonicus than in L. maculatus. Molecular variance analyses showed differential genetic structuring for these two closely related species. The results indicated that L. japonicus is panmictic throughout its range. In contrast, populations of L. maculatus showed statistically significant levels of genetic structuring. Pattern of isolation by distance was observed in L. maculatus, suggesting that L. maculatus is in genetic equilibrium. In contrast, L. japonicus did not exhibit isolation by distance.     

Population genetic diversity of Chinese sea bass (Lateolabrax maculatus) from southeast coastal regions of China based on mitochondrial COI gene sequences

Chinese sea bass (Lateolabrax maculatus), an important commercial and recreational fishery species in China, whose natural resources have decreased dramatically for decades due to overfishing and environmental changes. To provide guidelines for the source conservation and management, genetic diversity and population structure of L. maculatus were analyzed based on the mitochondrial cytochrome oxidase subunit I (COI) gene. Totally 192 individuals were sampled from five locations: Qingdao (QD), Chongming (CM), Dongtou (DT), Lieyu (LY) and Fangcheng (FC). After sequencing of a 586 bp fragment of COI gene, 20 haplotypes were defined. H4 (haplotype 4) and H8 were the dominant haplotypes and existed in all populations. Haplotype diversity (Hd) and nucleotide diversity (π) of each population ranged from 0.572 to 0.721 and from 0.00129 to 0.00271, respectively. The highest Hd and π were found in DT and LY populations, while the lowest value of Hd and π were in the QD population. Genetic distance ranged from 0.0031 to 0.0027 within populations and from 0.0018 to 0.0035 between populations. Analysis of molecular variance (AMOVA) revealed that significant genetic divergence was found in QD and FC populations. In addition, neutrality tests and mismatch distribution analysis indicated that this species experienced potential population expansion events.     

Genetic differences between wild and hatchery populations of Korean spotted sea bass (Lateolabrax maculatus) inferred from microsatellite markers

The spotted sea bass, Lateolabrax maculatus, is popular in recreational fishing and aquaculture in Korea. Its natural population has declined during the past two decades; thus, beginning in the early 2000s stock-enhancement programs were introduced throughout western and southern coastal areas. In this study, genetic similarities and differences between wild and hatchery populations were assessed using multiplex assays with 12 highly polymorphic microsatellite loci; 96 alleles were identified. Although many unique alleles were lost in the hatchery samples, no significant reductions were found in heterozygosity or allelic diversity in the hatchery compared to the wild population. High genetic diversity (He = 0.724–0.761 and Ho = 0.723–0.743), low inbreeding coefficient (F _IS = 0.003–0.024) and Hardy–Weinberg equilibrium were observed in both wild and hatchery populations. However, the genetic heterogeneity between the populations was significant. Therefore, genetic drift likely promoted inter-population differentiation, and rapid loss of genetic diversity remains possible. Regarding conservation, genetic variation should be monitored and inbreeding controlled in a commercial breeding program.     

三聚氰胺对花鲈的急性毒性实验研究

本文以花鲈(Lateolabrax japonicus)为实验对象,采用接触、腹腔注射及口服三种致毒方式,进行了非蛋白氮物质-三聚氰胺对花鲈的急性毒性实验研究。口服急性毒性实验中,通过在饲料中添加不同浓度的三聚氰胺（0、500、2000、5000及10000 mg/kg）,进行了21d的花鲈养殖实验。实验结果表明：三聚氰胺溶解度较低,其水溶液没有表现出急性毒性,LC50 > 3500 mg/L;而在腹腔注射致毒方式下,三聚氰胺对花鲈的半致死剂量LD50 = 890.07 mg/kg•w;LD5095%可信限为：778.63－1017.45 mg/kg•w;在口服急性毒性实验中,10000 mg/kg组三聚氰胺降低了花鲈的摄食与生长(p<0.05),饲料系数显著升高 (p<0.05);三聚氰胺对花鲈的存活率、肥满度、肝体比及脏体比均无显著影响 (p>0.05)。饲料中添加三聚氰胺没有显著影响花鲈血清的丙氨酸氨基转移酶、天冬氨酸氨基转移酶、总蛋白、葡萄糖、胆固醇、甘油三酯及尿素氮 (p>0.05),但显著影响了花鲈血清的碱性磷酸酶活性,10000 mg/kg组的碱性磷酸酶活性显著高于其他各处理组 (p<0.05),其他各组间无显著差异(p>0.05)。三聚氰胺对花鲈21d的最大未观察到有害作用剂量（NOAEL, no-observed-adverse-effect-level）为131.99 mg/kg•w•d。     

The effect of tricaine methanesulphonate (MS-222) on the microhaematocrit of fish blood

The effects of the anaesthetic, MS-222, on the microhaematocrit value of freshwater fish have been examined. Blood containing MS-222 showed a higher haematocrit value than blood without the anaesthetic and haemolysis occurred in the former after a variable time depending on the concentration. The results are discussed in relation to previous findings.     

Biochemical characterization of trypsins from the hepatopancreas of Japanese sea bass (Lateolabrax japonicus)

A cationic trypsin (trypsin A) and an anionic trypsin (trypsin B) were highly purified from the hepatopancreas of the Japanese sea bass (Lateolabrax japonicus) by ammonium sulfate precipitation, column chromatographies of DEAE-Sepharose and Sephacryl S-200 HR. Purified trypsins revealed single band on SDS-PAGE and their molecular masses were 21 kDa and 21.5 kDa, respectively. Trypsins A and B exhibited maximal activity at 40°C, and shared the same optimal pH at 9.0 using Boc-Phe-Ser-Arg-MCA as the substrate. The two trypsins were stable up to 45°C and in the pH range from 7.0 to 11.0. Trypsin inhibitors such as Pefabloc SC, PMSF and benzamidine are effective to these two enzymes and their susceptibilities were similar. Apparent K(m)s of trypsins A and B were 1.12 and 0.7 μM and k(cat)s of them were 72.08 and 67.79 S(-1) for Boc-Phe-Ser-Arg-MCA, respectively. The N-terminal amino acid sequences of the two trypsins were determined to the 24th residues, which were highly identical to trypsins from other species of fish while trypsins A and B only shared 45.8% identity. The digestive effect of the two trypsins on native shrimp muscular proteins indicated their effectiveness in the degradation of food proteins.     

Lysine and arginine requirements of juvenile Asian sea bass (Lates calcarifer)

Two separate experiments were conducted to determine the dietary requirements of juvenile Asian sea bass Lates calcarifer Bloch for lysine and arginine. Fish (average initial weight: lysine experiment, 13.12 ± 0.12 g; arginine experiment, 2.56 ± 0.13 g) were given amino acid test diets for 12 weeks containing fish meal, zein, squid meal, and crystalline amino acids. Each set of isonitrogenous and isocaloric test diets contained graded levels of L ‐lysine or L ‐arginine. The feeding rate in the lysine experiment was at 4–2.5% of the body weight day^?1, while in the arginine experiment it was at 10–4% of the body weight day^?1. The fish (20 per tank, lysine experiment; 15 per tank, arginine experiment) were reared in 500‐L fibreglass tanks with continuous flowthrough sea water at 27 °C and salinity of 31 ppt in the lysine experiment and at 29 °C and salinity of 29 ppt in the arginine experiment. The experiments were in a completely randomized design with two replicates per treatment. Survival was high in fish given adequate lysine or arginine. Mean percentage weight gains were significantly different in fish fed varying levels of lysine or arginine. Fish fed high levels of L ‐arginine suffered high mortalities. No significant differences were obtained in the feed efficiency ratios (FER, g gain g^?1 feed) of fish fed graded lysine, although the values tended to increase as the dietary lysine level was increased up to the requirement level. In contrast, in the arginine experiment, significant differences in FER of fish among treatments were obtained; the highest FER was observed in fish fed the diet containing an optimum arginine level. On the basis of the growth response, survival, and FER, the lysine and arginine requirements of juvenile Asian sea bass were estimated to be 20.6 g kg^?1 dry diet (4.5% protein) and 18.2 g kg^?1 dry diet (3.8% protein), respectively. These data will be useful in the further refinement of practical diet formulations for the Asian sea bass.     

Sexual differentiation and juvenile intersexuality in the European sea bass (Dicentrarchus labrax)

Sexual differentiation was studied at the histological level using a mixture of 30 families of sea bass Dicentrarchus labrax . Most of the fish (93%) differentiated into males as usually observed in farmed populations. All testes were differentiated when the males reached 12 cm and no more undifferentiated fish were found from 419 days post-fertilization (p.f.). In 28% of the males, among the biggest, sexual differentiation had already begun at 168 days p.f. (8.3–9.5 cm) and these fish started spermatogenesis in their first year of life. The other males differentiated later and remained immature at the end of their first year of life. Ovaries could be identified at the histological level from the age of 168 days p.f. (7.9–9.0 cm) and the females became significantly longer than the males from the age of 191 days p.f., i.e. during the process of ovarian differentiation. In the studied group, 62% of the males developed intratesticular oocytes. Such intersexuality had no consequence on growth rate. Intratesticular oocytes were also recorded in testes of wild males originating from Atlantic (Britain and Gulf of Gascogne) and West Mediterranean showing that juvenile intersexuality is not restricted to farmed populations but is a widespread phenomenon in sea bass.     

Cloning of Wap65 in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) and expression in sea bass tissues

The complementary DNA encoding WAP65 protein was cloned from the liver of two fish species sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Full-length cDNA sequences were obtained from reverse transcribed total RNA, followed by 5′ and 3′ rapid amplification of cDNA end (RACE) experiments. The full-length cDNA sequence of D. labrax is 1709 bp and the coding sequence is flanked by a 67 bp 5′-UTR and a 358 bp 3′-UTR. The full-length cDNA sequence of S. aurata is 1599 bp, and the coding sequence is flanked by a 48 bp 5′-UTR and a 273 bp 3′-UTR. The deduced amino acid putative primary sequences are composed of 427 and 425 amino acid residues for D. labrax and S. aurata, respectively. They display high homologies with previously described fish WAP65 and other hemopexin-like proteins from rabbit (Oryctolagus cuniculus). Expression of Wap65 has proved to be a natural physiological adaptive answer of teleost fish to warm temperature acclimation. In all fish species studied to date, Wap65 was found expressed mainly by the liver, although other tissues seem able to express Wap65 in response to a warm temperature acclimation, in a specie specific manner. Here, we investigate the tissue specific expression of Wap65 in D. labrax and S. aurata in response to a warm temperature acclimation, by RT-PCR analysis.     

Efficacy of a Listonella anguillarum (syn. Vibrio anguillarum) vaccine for juvenile sea bass Dicentrarchus labrax

The efficacy of a commercial bivalent Listonella anguillarum (serotype 01 and 02) vaccine (MICROViB, Microtek International) was tested on prime- and booster-immersion vaccinated sea bass Dicentrarchus labrax juveniles. We carried out 2 challenge tests on the prime-vaccinated fish, 50 and 90 d after initial vaccination. A second group of fish received a booster vaccination 60 d after the prime vaccination, and were tested with a single challenge 30 d later. Relative percent survival (RPS) was 92 and 84% (both p < 0.01) among the prime-vaccinated fish on the first and second challenges, respectively. The RPS of the booster-vaccinated sea bass was 100% (p < 0.01). Antibody titres were tested only among 10 prime-vaccinated and 10 unvaccinated (control) sea bass, 60 d post-immunisation, and were found to rise to 1/32 in the vaccinated fish. Our results demonstrate that MICROViB immersion vaccine can effectively protect juvenile sea bass from L. anguillarum infection.     

Effect of fasting on self-feeding activity in juvenile sea bass (Dicentrarchus labrax)

In various experiments under self-feeding conditions, sea bass groups could be divided into three categories regarding feeder actuation: high, low and zero-triggering fish. In all cases few high-triggering fish were responsible for a high percentage of the feed delivery. A question was raised about the role played by feeding motivation in such high-triggering status acquisition. It was approached by applying a 3-week fasting period in order to induce similar negative specific growth rate (SGR) in two groups of fish of similar mean weight but with either a low or a high coefficient of variation for weight (CV_w) (T_low: CV_w ∼ 11%, 3 tanks of 60 fish each; T_high: CV_w ∼ 20%, 3 tanks of 60 fish each). These groups were created to test the consistency of behavioural responses in two different contexts (i.e. two population size-distributions). During the follow-up period of 40 days, the group level feed-demand behaviour was not strongly modified by the fasting period and there were no differences between T_low and T_high groups. Complete growth compensation was the same in all tanks as observed at the end of the experiment. At the individual level, high-triggering fish were exactly the same individuals before and after the fasting period. Up to four high-triggering fish could be observed according to the tank and when several fish were performing high-triggering activity, their rankings were sometimes reversed after the fasting period. High-triggering fish increased their activity levels after the fasting period showing behavioural plasticity. High-triggering status could neither be explained by an initial lower SGR nor a sex effect, nor by any of the measured physiological blood parameters. Thus, individual's triggering activity levels could be related to personality and/or metabolic traits but further research is required to confirm this assumption.     

The immunohistochemical study of nodavirus changes in larval, juvenile and adult sea bass tissue

Owing to its quickness and sensitivity, and the minimal financial effort required, immunohistochemistry is a leading technique for nodavirus diagnosis. The aim of the study was to give detailed results of immunohistochemical changes in the various tissues of larvae, juvenile and adult sea bass (Dicentrarchus labrax L.). Histological sections were treated with polyclonal anti‐noda rabbit IgG, while diaminobenzidine‐peroxidase was used for the enzyme–substrate complex. The development from first changes to vacuolating necrosis was recorded in the brain, spinal cord and retina. For the first time, immunolabelling was noted in the liver endothelium. The other sampled tissue was immunonegative.     

Recruitment in Japanese sea bass,Lateolabrax japonicas (Cuvier, 1828): effects of timing on spawning and larval quality and quantity

This study aimed to elucidate the causes of variability in larval survival and juvenile abundance (recruitment) within and among cohorts of Japanese sea bass (JSB; Lateolabrax japonicus), a winter‐spawning temperate coastal marine fish. Larvae and settled individuals (settlers) belonging to four cohorts were collected from Tango Bay (the Sea of Japan coast) during eight sampling cruises in 2007 and 2008. Larvae were sampled in January and February each year using an ichthyoplankton net, and settlers were collected in February and March each year using a beam trawl. Age of individual larva and settlers was determined and growth history was back‐calculated from otolith microstructure, and the hatch date distribution was computed. Temperature, daily growth rate, size‐at‐age, hatch date, and density data of larvae and settlers allowed elucidating the effects of the timing of spawning and larval quantity and quality (growth rate and body size) on larval survival and recruitment within and among cohorts of JSB. Results showed that cohorts that hatched earlier in the season had higher quantity of larvae, experienced higher mean temperatures and survived better than cohorts hatched later. Recruitment variability among cohorts is determined largely by the initial quantity of larvae, as this explained >97% of the variability in recruitment among cohorts. Within cohorts, larger hatched larvae grew faster than their smaller conspecifics, and the bigger and faster growing larvae survived and settled. Results from this study suggest the following scenarios for recruitment of JSB: (i) earlier spawning in the season promotes larval survival since earlier cohorts are likely to encounter a better temperature and perhaps food conditions, and therefore recruit better than later cohorts; (ii) the initial quantity of larvae appears to be an important determinant of recruitment variability among cohorts; and (iii) the size‐ and growth‐related mechanisms operating during the larval phase appear to start at the time of the hatch.     

Microsatellite DNA polymorphism of Japanese sea bass (Laterolabrax japonicus) inhabiting Chinese and Japanese coasts

Twenty‐two microsatellite DNA markers were developed for Japanese sea bass (Laterolabrax japonicus), of which 19 were independent from each other and at Hardy–Weinberg equilibrium across the three populations of Japanese sea bass inhabiting Chinese coasts (defined as China group) and the five populations inhabiting Japanese coasts (defined as Japan group). These 19 markers were used to determine the number of alleles and the expected heterozygosity across the eight populations. The majority of individuals (93.8–98.8%) of the three populations of the pre‐defined China group were assigned to an inferred cluster, and 90.9–94.6% of the individuals of the five populations of the pre‐defined Japan group were assigned to the other. The average number of alleles across the 19 loci was significantly lower in the China group than in the Japan group (10.3 vs 15.4), however, the average expected heterozygosity across the 19 loci of the China group was similar to that of the Japan group (0.743 vs 0.750). An effective population size reduction (i.e. bottleneck effect) was detected in the China group (P = 0.00357), which may have resulted from either over‐catching or glaciations or both. The pairwise F_ST among populations of the China group (0.019–0.029) and among populations of the Japan group (0.003–0.021) were lower than those between the populations of the China group and the populations of the Japan group (0.076–0.101). The average pair‐wise F_ST between the populations of the China group and those of the Japan group reached 0.075, and the variation between the China group and the Japan group accounted for 7.16% of the total. Nei’s original measures of genetic distances among the populations of the China group and the Japan group ranged from 0.123 to 0.145 and from 0.055 to 0.123, respectively, while that between the populations of the China group and the populations of the Japan group ranged from 0.326 to 0.450. Japanese sea bass is able to disperse over a long distance; however, our observations demonstrated that it cannot migrate across a possible barrier existing between Chinese and Japanese coasts. Most individuals of the Zhoushan population of the China group were assigned to two inferred clusters, and most individuals of Ariake Sea, Tokyo Bay and Ishikawa populations of the Japan group were assigned to three inferred clusters, indicating that these locations were the gathering grounds of Japanese sea bass.     

Population genetic structure of the sea bass (Lateolabrax japonicus) in Korea based on multiplex PCR assays with 12 polymorphic microsatellite markers

Population genetics has been recognized as a key component of policy development for fisheries and conservation management. In this study, natural sea bass (Lateolabrax japonicus) populations in three ocean basins in Korea were assessed using multiplex assays with 12 highly polymorphic microsatellite loci; 203 alleles and similarly high levels of genetic diversity [mean number of alleles (N_A) = 14.43, mean expected heterozygosity (He) = 0.84] were detected. All populations showed significant heterozygote deficiency at four loci, which could be explained by the presence of null alleles. The genetic population subdivision was low and was significantly different according to F-statistics (overall F _ST = 0.003, R _ST = 0.005). However, this substructure was not supported by an analysis of molecular variance test, analyses of isolation by distance or Bayesian analysis. The passive dispersal of eggs/larvae via the main currents appears to facilitate gene flow. The possibility of a recent genetic bottleneck was observed in all three populations of L. japonicus, indicating that overfishing and degradation of the environment in recent years has led to a decline in the sea bass populations in Korea. Our study demonstrates that sea bass in Korea do not appear to be genetically partitioned and should be managed as a single unit; however, the potential for a rapid loss of genetic diversity remains. Information regarding the genetic characteristics of Korean sea bass populations has important implications for fishery management and conservation efforts and will aid in the sustainable exploitation of fishing resources and the preservation of biodiversity.     

Stage-specific gene expression during fish spermatogenesis as determined by laser-capture microdissection and quantitative-PCR in sea bass (Dicentrarchus labrax) gonads

The role of genes implicated in the regulation of spermatogenesis and their patterns of expression is still poorly understood. In this study, we took advantage of the cystic arrangement of the teleost testis to set up a laser capture microdissection procedure to isolate cells from cysts containing spermatogonia, spermatocytes, spermatids, or spermatozoa. We then used quantitative PCR to determine the stage-specific expression patterns of the germ cell marker vasa; gonadal aromatase (cyp19a); estrogen receptors (ers) alpha, beta1, and beta2 (era, erb1, and erb2, respectively); 11beta-hydroxylase (cyp11b1); androgen receptor beta (arb); insulinlike growth factor 1 (igf1); and sox17. vasa had the highest mRNA levels, followed by genes involved in androgen metabolism (cyp11b1 and arb). Most genes associated with estrogen metabolism (cyp19a, era, and erb1) had a lower expression, whereas igf1 and sox17 exhibited the lowest mRNA levels. Comparison of changes in mRNA levels revealed five patterns of gene expression, in general with progressively lower expression seen as spermatogenesis advanced. igf1 and sox17 were exclusively expressed in spermatogonia-containing cysts, suggesting effects during the proliferative stage. Genes involved in androgen synthesis (cyp11b1) and action (arb) peaked during the early stages of spermatogenesis and then sharply decreased. In contrast, genes associated with estrogen action, particularly erb2 and era, showed a more gradual decrease. Together, these results demonstrate the usefulness of fish models and suggest that whereas androgens are required at high levels and may exert their major actions at the initial stages of spermatogenesis, estrogens are also essential, albeit required at lower levels, and with a more generalized influence.     

饥饿对于鲈肌肉、肝脏和血清主要生化组成的影响

于 2 2 93± 2 15℃条件下 ,在室外水泥池 (3m× 2m× 1m)中对正常鲈 (2 85 2 6± 6 5 4 g)和患脂肪肝病鲈 (4 6 4 71± 5 4 2 2g)进行为期 9周的饥饿处理。分别在实验开始后第 0周、 3周、 5周、 7周和 9周取样 ,以观察饥饿对于鲈内脏相对重量、肌肉肝脏和血清主要生化指标的影响。研究表明 ,鲈对饥饿耐受能力较强 ,在饥饿时首先快速动用肠系膜脂肪和肌肉脂肪作为能量供应 ,而在整个饥饿阶段则主要以肌肉蛋白质作为能量来源 ,肝脏中能源物质在饥饿中并无明显减少 ,故不是鲈饥饿时的主要供能物质。饥饿时 ,肌肉和肝脏中的水分和脂肪含量呈现负相关 ,尤其在肝脏中表现明显。鲈血清中脂肪酶、甘油三酯、胆固醇、低密度脂蛋白和高密度脂蛋白在饥饿中表现出周期性和阶段性的变化 ,其中正常鲈表现出有规律的波浪状图形 ,而脂肪肝病鲈则表现出山峰状图形 ,说明脂肪肝病鲈代谢机制不如正常鲈灵敏 ,9周的饥饿并不能减轻或消除鲈的脂肪肝病.     

Preliminary study of the effects of commercial lactobacilli preparations on digestive metabolism of juvenile sea bass (Dicentrarchus labrax)

Two forms of the same commercial product (SORBIAL, Allonnes, France), one with live bacteria (PSA) and the other with heat-inactivated bacteria (PSI), containing a mixture of 2 strains of lactobacilli and their growth medium were tested as a diet complement for juvenile sea bass (Dicentrarchus labrax) during a 103-day experiment. In addition to zootechnical parameters (survival, growth, conformation), some effects on digestive metabolism were studied, including enzymatic, ultrastructural and microbial aspects. Microbial preparations improved survival rate. The ventral, dorsal and operculum malformations which usually occur in juveniles did not appear in those receiving PSA and PSI. Furthermore, they stimulated, but not constantly, trypsin and acid phosphatase activities. Intestinal ultrastructure showed an increase in the number of endocytosis vesicles at the apical pole of enterocytes in fishes receiving enrichments. Bacterial flora was not modified in terms of quantity, especially the lactic acid bacteria counts, which were not changed in fishes receiving live lactobacilli (PSA). The mode of action of these multiple beneficial effects appears complex and could be caused by different molecules inside the bacterial cell or excreted into their medium.