Detection of serum and mucosal antibody production and antibody secreting cells (ASCs) in large yellow croaker (Pseudosciaena crocea) following vaccination with Vibrio harveyi via different routes

The kinetics of mucosal and serum antibody response is well as antibody secreting cells (ASCs) production were studied in large yellow croaker following vaccination with inactivated Vibrio harveyi by different routes: oral administration. intraperitoneal (IP) injection and immersion. Indirect ELISA was used to measure the antibody level in serum and cutaneous mucus, and ELISPOT was used to monitor the ASCs derived from gill, blood and head kidney. The data demonstrated that IP injection resulted in the highest antibody levels in the systemic circulation, whereas immersion induced significant antibody levels in mucous. As for the ASCs response, IP injection induced high numbers of ASCs in the head kidney and blood; oral intubation only induced a slight ASCs response in the head kidney: immersion induced a much stronger ASCs response in the gill. These results indicate that mucosal antibodies following immersion immunization are independent of a systemic response and more sensitive, since it could be triggered earlier than serum antibodies. The mucosal antibodies following IP injection immunization may depend oil a systemic immune response. The protective effects of the three vaccination methods were compared by challenging with live V. harveyi. Survival of the three groups of vaccinated fish varied front 40 to 60%. while 100% mortality was found in control fish. Compared with IP and oral vaccination, immersion stimulated higher specific antibody titers in the mucosal system and achieved similar protection, so it is in effective and efficient method for immunizing a large number of fish against V harveyi (C) 2008 Elsevier B.V. All rights reserved.

A genetic map of large yellow croaker Pseudosciaena crocea

Genetic linkage maps were constructed for large yellow croaker Pseudosciaena crocea (Richardson, 1846) using AFLP and microsatellite markers in an F-1 family. Five hundred and twenty-three AFLP markers and 36 microsatellites were genotyped in the parents and 94 F-1 progeny. Among these, 362 AFLP markers and 13 SSR markers followed the 1:1 Mendelian segregation ratio (P > 0.05). The female genetic map contained 181 AFLP and 7 microsatellite markers forming 24 linkage groups spanning 2959.1 cM, while the male map consisted of 153 AFLP and 8 microsatellite markers in 23 linkage groups covering 2205.7 cM. One sex linked marker was mapped to the male map and co-segregated with the AFLP marker agacta355, suggesting an XY-male determination mechanism and this may be useful in the breeding of monosex populations. (c) 2007 Elsevier B.V. All rights reserved.

Isolation and characterization of six microsatellite markers in the large yellow croaker (Pseudosciaena crocea Richardson)

Six polymorphic microsatellite markers were isolated and characterized using an enriched library technique in the large yellow croaker (Pseudosciaena crocea Richardson, 1864), a commercially important marine fish in China. They showed PIC (polymorphism information content) ranging from 0.064 to 0.885 (average of 0.580) and allele numbers ranging from two to 13 (average of 7.5), which were useful for the studies on population genetics and selective breeding of the large yellow croaker.

Induction of triploidy in large yellow crocker Pseudosciaena crocea (Richardson, 1846): effects of pressure shocks and growth performance in the first rearing year

The precociously sexual maturation in large yellow crocker Pseudosciaena crocea has become a serious problem. In an attempt to solve this problem, the production of sterile triploids could be an effective strategy. In this study, triploid P. crocea was obtained by subjecting fertilized eggs to pressure shock. Flow-cytometry analysis was used to assess ploidy level. In terms of triploid rate and hatching rate, the optimal conditions of pressure shock for triploidy induction in P. crocea were 7500 psi for 3 min shock at 3 min after fertilization at 20 degrees C. With the application of these parameters, 100% triploid fish were produced. During the first rearing year, triploid P. crocea had a similar growth performance compared with its diploid counterpart before the age of 8 months and showed a significant advantage at the age of 10 and 12 months in body weight and body length (P < 0.05). At the age of 12 months, the carcass weight of triploids was markedly higher than that of diploid control, and gonadal somatic index was significantly lower than that of their diploid control. During the first rearing year, survival in triploid group was 76.44%, inferior to its diploid control (83.21%).

Effects of ultra-violet irradiation on sperm motility and diploid gynogenesis induction in large yellow croaker (Pseudosciaena crocea) undergoing cold shock

Large yellow croaker, Pseudosciaena crocea, exhibit sexually dimorphic growth, with females growing faster and reaching larger adult sizes than males. Thus, development of techniques for preferentially producing females is necessary to optimize production of these species. We have established a protocol to produce all-female croaker P. crocea through induction of meiotic gynogenesis with homologous sperm. The first set of experiments investigated the ultra-violet (UV) irradiation on sperm motility and duration of sperm activity to determine the optimal UV dosage for genetic inactivation of sperm, yet retaining adequate motility for activation of eggs. Milt from several males was diluted 1: 100 with Ringer's solution and UV irradiated with doses ranging from 0-150 J cm (-2). The results indicated that motility and duration of activity generally decreased with increased UV doses. At UV doses greater than 105 J cm(-2), after fertilization, motility was < 10% and fertilization rates were significantly lower. Highest hatching rate was obtained at 75 J cm -2. A second set of experiments was carried out to determine appropriate conditions of cold shock for retention of the 2nd polar body in P. crocea eggs after fertilization with UV-inactivated sperm by altering the timing, temperature and duration of shock. At 208 degrees C, shock applied at 3 min after fertilization resulted in higher survival rate of larvae at 6 h after hatching. Results of different combinations of three shock temperatures ( 28 degrees C, 38 degrees C or 48 degrees C) and five shock durations ( 4 min, 8 min, 12 min, 16 min or 20 min) at 3 min after fertilization demonstrated that shocks of 12 min gave highest production of diploid gynogens. Statistical analysis revealed that maximum production of diploid gynogens (44.55 +/- 2.99%) were obtained at 38 degrees C. The results of this study indicate that the use of UV-irradiated homologous sperm for activation of P. crocea eggs and cold shock for polar body retention is an effective method for producing gynogenetic offspring.

Cloning and sequencing of the growth hormone gene of large yellow croaker and its phylogenetic significance

Using conserved primers and the PCR reaction, the growth hormone (GH) gene and the 3'-UTR of the large yellow croaker (Pseudosciaena crocea) were amplified and sequenced. The gene structure was analyzed and compared to the GH genes of 5 other percoid fish downloaded from Genbank. Also the GH gene of the large yellow croaker and the genes from 14 Percoidei and 2 Labroidei species were aligned using Clustal X. A matrix of 564 bp was used to construct the phylogenetic tree using maximum parsimony and neighbor-joining methods. Phylogenetic trees by the two methods are identical in most of the clades with high bootstrap support. The results are also identical to those from morphological data. In general, this analysis does not support the monophyly of the families Centropomidae and Carangidae. But our GH gene tree indicates that the representative species of the families Sparidae and Sciaenidae are a monophyletic group.

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。

Studies on frozen storage of ghol (Pseudosciaena diacanthus)

Investigations were carried out on the frozen storage of ghol (Pseudosciaena diacanthus) in the form of fillets. The results indicated that ghol fillets stored at -l8°C remained in a highly acceptable condition up to 20 weeks. However, after this stage, the acceptability steeply declined.

The complete mitochondrial genome of the large yellow croaker, Larimichthys crocea (Perciformes, Sciaenidae): Unusual features of its control region and the phylogenetic position of the Sciaenidae

To understand the systematic status of Larimichthys crocea in the Percoidei, we determined the complete mitochondrial (mt) genome sequence using 454 sequencing-by-synthesis technology. The complete mt genome is 16,466 bp in length including the typical structure of 22 tRNAs, 2 rRNAs, 13 protein-coding genes and the noncoding control region (CR). Further sequencing for the complete CR was performed using the primers Cyt b-F and 12S-R on six L crocea individuals and two L polyactis individuals. Interestingly, all seven CR sequences from L crocea were identical while the three sequences from L polyactis were distinct (including one from GenBank). Although the conserved blocks such as TAS and CSB-1, -2, and -3 are readily identifiable in the control regions of the two species, the typical central conserved blocks CSB-D, -E, and -F could not be detected, while they are found in Cynoscion acoupa of Sciaenidae and other Percoidei species. Phylogenetic analysis shows that L crocea is a relatively recently emerged species in Sciaenidae and this family is closely related to family Pomacanthidae within the Percoidei. L crocea, as the first species of Sciaenidae with complete mitochondrial genome available, will provide important information on the molecular evolution of the group. Moreover, the genus-specific pair of primers designed in this study for amplifying the complete mt control region will be very useful in studies on the population genetics and conservation biology of Larimichthys. (c) 2008 Elsevier B.V. All rights reserved.

Early development, survival and growth rates of the giant clam Tridacna crocea (Bivalvia: Tridacnidae)

Tridacnid clams are conspicuous inhabitants of Indo-Pacific coral reefs and are traded and cultivated for the aquarium and food industries. In the present study, daily growth rates of larvae of the giant clam Tridacna crocea were determined in the laboratory during the first week of life. Adults were induced to spawn via intra-gonadal serotonin injection through the byssal orifice. After spawning oocytes were collected, fertilized and kept in 3 L glass beakers and raceways treated with antibiotics to avoid culture contamination. Larvae were fed twice with the microalga Isochrysis galbana and zooxanthellae were also offered twice during the veliger stage (days 4 and 6). Larval length was measured using a digitizing tablet coupled to a microcomputer. Larval mortality was exponential during the first 48 hours of life declining significantly afterwards. Mean growth rate was 11.3 mu m day-1, increasing after addition of symbionts to 18.0 mu m day-1. Survival increased to ca. 75% after the addition of zooxanthellae. The results describe the growth curve for T. crocea larvae and suggest that the acquisition of symbionts by larvae may be useful for larval growth and survival even before larvae have attained metamorphosis.

Early development, survival and growth rates of the giant clam Tridacna crocea (Bivalvia: Tridacnidae)

Tridacnid clams are conspicuous inhabitants of Indo-Pacific coral reefs and are traded and cultivated for the aquarium and food industries. In the present study, daily growth rates of larvae of the giant clam Tridacna crocea were determined in the laboratory during the first week of life. Adults were induced to spawn via intra-gonadal serotonin injection through the byssal orifice. After spawning oocytes were collected, fertilized and kept in 3 L glass beakers and raceways treated with antibiotics to avoid culture contamination. Larvae were fed twice with the microalga Isochrysis galbana and zooxanthellae were also offered twice during the veliger stage (days 4 and 6). Larval length was measured using a digitizing tablet coupled to a microcomputer. Larval mortality was exponential during the first 48 hours of life declining significantly afterwards. Mean growth rate was 11.3 μm day-1, increasing after addition of symbionts to 18.0 μm day-1. Survival increased to ca. 75% after the addition of zooxanthellae. The results describe the growth curve for T. crocea larvae and suggest that the acquisition of symbionts by larvae may be useful for larval growth and survival even before larvae have attained metamorphosis.

Ensiled product from fish by microbial fermentation

Fish ensilage for animal feed stuff was prepared from Jew fish (Pseudosciaena spp.) and Silver bellies (Leiognathus spp.) by fermentation with pure culture of Lactobacillus piantarum NCIB 6105. The precooked ensiled product gave better product of fish silage (high content of lactic acid, about 5%). Protein Nitrogen content ranged between 1.76 to 1.94%. During storage for one year, the Protein Nitrogen loss was not significant. The material can be used as a supplemental animal ration.

Texturised meat from low cost fish

A comparative study of the suitability of five species of fish, namely, threadfin bream (Nemipterous [sic] japonicus), cat fish (Tachysurus fella), ribbon fish (Trichiurus spp.), barracuda (Sphyraena jello) and jew fish (Pseudosciaena spp.) for the production of texturised meat has indicated that all these species are good source for the purpose. Protein content of the final product from all the species was higher than that prescribed for FPC type A. The product had excellent rehydration capacity and firm and elastic texture. No significant difference was observed in the rehydration capacity of the texturised meat from all the species studied. Salt concentration was found to influence the texture and salt content of 1.5 to 2% was found to result in the desirable firm and elastic texture. Rehydration capacity was not influenced to any significant extent by the salt content.