Abstract:Detailed observations of the embryonic developmental stages of E. possjeticus were obtained by using vivo light microscopy. The embryonic development and its relationship with incubation temperature were studied. The results indicated that there was a positive linear relation between the fecundity and female body length. E. possjeticus displays a direct development, with the eggs developing inside a ventral brood pouch, where they hatch as larvae. The duration of embryonic development was about 220 hours at the constant temperature 21℃. There were two types of observations for embryonic development. One was isolated culture and the other was the fertilized egg carried by the female. The result showed that the fertilized egg of the isolated culture and the fertilized egg carried by the female developed almost simultaneously in the same conditions. In a word, eggs undergo total cleavage for the first few times, and then turn to the superficial cleavage. Early total cleavage appears to be a general feature of amphipod embryogenesis. The hatched larvae had similar morphological characters to those of adults. Temperature showed significant influence on the embryonic development time. Within range of water temperature 15-27℃, the duration of embryonic development decreased with the increase of temperature, while the developmental rate increased. Based on the developmental time at the different temperature, the developmental threshold temperature was calculated to be 6.02℃, and sum of effective temperature was 137.30℃·d.

Abstract:This is the first report of the successful artificial breeding attempts of the giant clam Tridacna squamosa in the South China Sea. Artificial breeding of the fluted giant clam was conducted under the conditions of temperature 27-30℃, salinity 30-33, pH 8.0-8.3 during March to June 2016 at the Tropical Marine Biological Research Station in Hainan. The results showed that the serotonin can effectively promote gamete release; the hatching rate is higher when the ratio of sperm to eggs was maintained at 50-100:1 with zygote density of 15-20/mL; and the initial D-shaped larvae were obtained after 36 h of incubation. It took approximately five days of rearing for D-larvae to develop into the pediveliger larvae, entering metamorphic stage. Introduction of zooxanthellae was carried out after 6 days of rearing, and we found that larval metamorphosis was improved by soaking larvae (30-40 ind./mL) in 3×10⁵-5×10⁵/mL zooxanthellae for no more than 2 h. This ensured that larvae had taken up at least three zooxanthellae cells within their digestive glands. Pediveliger larvae were observed when larvae showed developed gills, secondary shell, and the establishment of zooxanthellae tubular system. After 7-10 days of rearing, spats were observed under the condition of stationary seawater. Spats were cultured using slow-flowing seawater under illumination of 2000-3000 lx, which could have promoted subsequent shell growth and organ development. During this stage, regular cleaning of filamentous algae is required, to prevent entanglement with spats and thus ensure the higher survival rate of spats. After 48 days and 65 days of culture, we obtained about 30000 spats with shell lengths of approximately 1.2 mm and ~5 000 youth with shell lengths of approximately 1.8 mm, respectively. This first study on the artificial breeding for the fluted giant clam can provide theoretical basis and reliable techniques for conducting giant clam aquaculture and transplantation work.

Abstract:In the present study, to examine seasonal changes in the compositions of pond sediment and the subsequent effects on the food sources of cultured sea cucumber Apostichopus japonicus using fatty acid biomarkers, seasonal variations in the fatty acid profiles of sediment samples and A. japonicus were investigated in Jinghai Bay mariculture zone from May 2012 to December 2012. In the present experiment, ratio of 16:1(n-7)/16:0 and EPA were selected as the trophic markers for diatoms, DHA and DHA/EPA for dinoflagellates or protozoa, 20:4(n-6) for brown algae and odd-chain and branched-chain fatty acids (odd & br FAs) along with 18:1(n-7) for heterotrophic bacteria. The results showed that the sediment samples were mainly composed of diatoms, brown algae, dinoflagellates or protozoa and bacteria and their compositions showed significantly seasonal variations as evidenced by the fatty acid trophic markers. The highest ratios of 16:1(n-7)/16:0 and contents of EPA (diatoms) were observed in winter, the highest concentrations of 20:4(n-6) (brown algae) were found in autumn, the highest levels of bacterial markers (odd & br FAs) and 18:1(n-7) were detected in summer and the highest proportions of DHA (dinoflagellates or protozoa) were observed in winter. Correlation analysis showed that the food sources of A. japonicus were significantly and positively correlated with the diatoms, dinoflagellates or protozoa and bacteria. Results of present study suggested that the food sources of A. japonicus were significantly influenced by the seasonal changes in the compositions of sediment.

Abstract:This study aimed to investigate the effects of feeding rates on plasma lipid profiles and the mRNA expressions of genes related to lipid metabolism in juvenile blunt snout bream, Megalobrama amblycephala (average initial weight:23.74±0.09 g). A total of 840 fish were randomly distributed into 24 cages and fed with a commercial feed at six feeding rates ranging from 2% to 7% body weight (BW)/day for 60 days. The results indicated that plasma triglyceride and total cholesterol levels increased first, then decreased as feeding rates increased with significant differences observed between treatments (P<0.05). The highest high density lipoprotein and low density lipoprotein concentrations were observed in fish group fed 6% and 5% BW/day respectively. They were significantly higher than those of fish fed 2% BW/day (P<0.05), but showed no statistical difference with those of the other treatments (P>0.05).Very-low-density lipoprotein was the highest in fish offered a feeding rate of 4% BW/day. In addition, feeding rates had a significant effect on the expressions of genes related to lipid metabolism (P<0.05). The mRNA expressions of carnitine palmitoyl transferase-I and II, Acyl-CoA oxidase, apolipoprotein B, fatty acid binding protein and peroxisome proliferators activated receptors all increased significantly (P<0.05) as feeding rates increased from 2% to 4% BW/day, but Acyl-CoA oxidase, fatty acid binding protein and peroxisome proliferator-activated receptor β mRNA expressions are decreased with further increasing ration levels(P>0.05). Lipoprotein lipase expressions of fish fed 3% and 6% BW/day were significantly (P<0.05) higher than that of the other treatments. Liver X receptor and fatty acid transporter protein expressions increased first, then decreased as feeding rates increased with the highest value observed in fish fed 3% and 5% BW/day, respectively. In summary, our results suggest that different feeding rates significantly affect plasma lipid profiles and the mRNA expressions of genes related to lipid metabolism in juvenile blunt snout bream. Low feeding rate (2-3% BW/day) leads to the lower fatty acid transport and oxidation. Higher feeding rate (5-7% BW/day) will result in decreased plasma lipid profiles and the mRNA expressions of genes related to fat deposition. The optimal feeding rate (4-5% BW/day) can maintain fat metabolism balance of juvenile blunt snout bream.

Abstract:In order to decrease dietary fish meal content and culture cost, this experiment was conducted to investigate effects of fish meal replacement by soybean meal on growth performance, body composition, digestive enzyme activities and hepatic biochemical indexes of bullfrog, Rana (Lithobates) catesbeiana. The basal diet contained 300 g/kg fish meal, then fish meal of basal diet was replaced with 0%, 20%, 40%, 60%, 80% and 100% soybean meal to formulate six iso-nitrogenous and iso-energetic diets (D1, D2, D3, D4, D5 and D6, respectively). Bullfrogs were fed with six experimental diets for 60 days. The results showed that no significant differences in feeding rate (FR), survival rate (SR), hepatic somatic index (HSI), viscera somatic index (VSI) and hepatic malondialdehyde (MDA) were found among all treatments. Weight gain rate (WGR), specific growth rate (SGR), protein efficiency ratio (PER) and protein productive value (PPV) of bullfrogs fed with no-fish-meal diet (D6) were significantly lower than those of other groups, while the opposite is true for feed conversion ratio (FCR). Condition factor (CF) and hind leg index (HLI) significantly decreased as the dietary soybean meal increased. No-fish-meal group has the lowest CF among all treatments. Proteases activity in intestine significantly declined as dietary soybean meal level increased, while amylase and lipase activities were just opposite. Fish meal replacement by soybean meal had significant effects on hepatic superoxide dismutase (SOD) and catalases activity (CAT). SOD activity was the lowest in bullfrogs fed D5 diet. While, CAT activity was the lowest in bullfrogs fed D6 diet. Based on these results, we suggest that 60%-80% fish meal could be replaced by soybean meal in practical diets of bullfrog.

Abstract:An 8-week feeding trial was conducted to determine the optimal dietary soy lecithin requirement for juvenile swimming crabs (Portunus trituberculatus) reared in cement pools. Six iso-energetic and iso-lipidic experimental diets were formulated to contain graded levels of soy lecithin, which were supplemented with 0, 10 g/kg, 20 g/kg, 40 g/kg, 60 g/kg, 80 g/kg of soy lecithin respectively. The results indicated that when the dietary soy lecithin was from o to 40 g/kg, WR, MR, PER, SGR and MR were increased significantly, but FCR decreased significantly (P<0.05). The results indicated that optimal soy lecithin in the diet can improve growth performance and feed utilization for juvenile swimming crab. TP, TG and HDL concentration in serum was not affected by the dietary soy lecithin levels, but the contents of CHO and GLU were increased significantly with the soy lecithin increasing from 0 to 40 g/kg, but were not affected by the dietary soy lecithin increasing (P<0.05). The T-Aoc, MDA and LSZ activity in hepatopancreas were not affected by the dietary soy lecithin levels (P>0.05). When the dietary soy lecithin was 60 g/kg, the SOD activity in hepatopancreas was significantly higher than other diets (P<0.05). The content of GPX in hepatopancreas was increased significantly with the soy lecithin increasing from 0 to 40 g/kg, but was not significantly affected by the dietary soy lecithin increasing from 40 g/kg to 80 g/kg (P<0.05). With the soy lecithin supplementation, we also observed that the contents of DHA, EPA, C18:2 and n-3PUFA in muscle were not affected by the dietary soy lecithin (P>0.05), but DHA and EPA in hepatopancreas were significantly increased with the soy lecithin increasing from 0 to 40 g/kg (P<0.05), however they were not significantly affected by the soy lecithin increasing from 40 g/kg to 80 g/kg (P>0.05). With the soy lecithin increacing from 0 to 40 g/kg, the contents of C18:2 in hepatopancreas were very significantly decreased (P<0.01), but the contents of n-3PUFA in hepatopancreas were not significantly affected (P>0.05), while with the soy lecithin increasing from 40 g/kg to 80 g/kg, the contents of C18:2 in hepatopancreas were significantly increased (P<0.05), but the contents of n-3PUFA in hepatopancreas were very significantly decreased (P<0.01). The results showed that the soy lecithin in diet improved the content of n-3PUFA, DHA and EPA in hepatopancreas. Based on two slope broken-line model based on specific growth rate against dietary soy lecithin levels, the optimal dietary soy lecithin requirement was estimated at 41.96 g/kg for juvenile swimming crab.

Abstract:Insulin-like growth factor-I (IGF-I) plays a major role in the control of growth and development in fish. Total RNA was isolated from H. taimen liver tissue. The cDNA encoding insulin-like growth factor I (IGF-I) peptide was amplified by reverse transcription polymerase chain reaction (RT-PCR) strategy using isolated total RNA as template. Recombinant expression plasmid (IGF-I/pS) was constructed by prokaryotic expression vector pS and transformed into E. coli Rosetta. The expression of recombinant IGF-I protein of H. taimen was induced by IPTG. There was a clear target band with expected size between 35~50 kD in SDS-PAGE electrophoresis, and the recombinant protein existed in the form of inclusion body. Purified IGF-I protein was obtained after the inclusion body was denatured/renatured. ELISA results showed that target protein can identify commercial fish IGF-I antibody, indicating that H. salmon IGF-I protein with immune activity was obtained. Cell proliferation assay (MTT) results showed that recombinant IGF-I protein can significantly promote the propagation of CHSE-214, EPC and RTG-2, indicating that the recombinant IGF-I protein had biological activity. The study has laid a foundation for the research of IGF-I function in the growth and development of H. taimen.

Abstract:Selenium-dependent glutathione peroxidase(denoted as Se-GPx)plays an important role in the enzymatic antioxidant defense system. It clears the reactive oxygen species (ROS) to avoid the cellular damage. In order to study the function of Se-GPx in immune system and the ovarian development of Penaeus monodon, the full-length cDNA sequence of Se-GPx from black tiger shrimps (denoted as Pm Se-GPx) was obtained by high-throughput transcriptome sequencing and RACE. The full-length cDNA of Pm Se-GPx is 959 bp, including an open reading frame (ORF) of 639 bp encoding a polypeptide of 212 amino acids, a 5'UTR of 10 bp and a 3'UTR of 310 bp containing a conserved selenocysteine insertion sequence (SECIS) element. The molecular mass of the deduced amino acid (aa) sequence was 23.65 kDa with an estimated pI of 4.61. Blast and phylogenetic analyses suggest that Pm Se-GPx is a new member of the shrimp GPxs family. The tertiary structure of Pm Se-GPx protein presents like a bag and there is a catalysis site, which contains couplet of four conservative residues (cysteine C/selenocysteine U, glutamine Q, tryptophan W and asparagine N). Pm Se-GPx mRNA expression was detected in nine tissues by real-time PCR. Pm Se-GPx mRNA expressed was the highest in the hepatopancreas and moderate in the ovary. The result showed that Pm Se-GPx was expression mainly in hepatopancreas which plays an important role in detoxification. At different stages of ovary development, Pm Se-GPx was expressed highly at StageⅢ and Stage V, which indicated a large number of ROS may be produced during these development stages. Pm Se-GPx may protect the ovary from the oxidization damage. Under the stress of Cu²⁺, the expression of Pm Se-GPx showed a trend of decline, but recovered at 48 h, and then decreased. The result showed that the stress of Cu²⁺ may inhibit the expression of Pm Se-GPx. Under the stress of pH 9, expression trend of Pm Se-GPx first declined, but recovered at 24 h, then rose significantly at the 96 h. The result showed that neutral environment may inhibit the expression of Pm Se-GPx but aqueous alkaline medium may promote it. Presumably, Se-GPx plays an important role during the ovarian development and immune regulation of P. monodon. Our study provides a basis for further research in reproduction and immunology of P. monodon.

Abstract:The early stage of shrimp aquaculture is susceptible to bacterial infection and the bacterial community structure in digestive tract is highly associated with shrimp health. Therefore, it is of great significance to explore the relation between shrimp bacterial community of digestive tract and juvenile shrimp disease. We investigated the intestinal bacterial community composition of healthy and diseased juvenile shrimp (Litopenaeus vannamei) by using Illumina sequencing, and explored Vibrio diverstiy based on 16S rDNA, recA and pyrH gene sequences of Vibrio isolates. The results showed that the average relative abundances of Alphaproteobacteria and Firmicutes are higher in healthy juvenile shrimp digestive tract than those in diseased one, while these of Gammaproteobacteria, Bacteridetes, Actinobacteria and Betaproteobacteria exhibited an opposite pattern and the variation in Actinobacteria is significant. In addition, response ratio analysis at the bacterial family level shows that the relative abundances of Planococcaceae and Bacteriovoracaceae families significantly decreased, while that of Vibrionaceae remarkably increased in diseased juvenile shrimp digestive tract in relation to healthy one. Similarity percentage (SIMPER) analysis revealed that the main contribution to the variation of intestinal bacterial community structure between healthy and diseased shrimp is attributed to 8 OTUs affiliated with Vibrio, unclassified Rhodobacteraceae, Marivita, Cryobacterium, unclassified Microbacteriaceae and Pseudoalteromonas. As to Vibrio assamblege composition, we found that culturable V. sinaloensis strains were the dominant species in healthy juvenile shrimp digestive tract, while V. campbellii is dominant in diseased juvenile shrimp.

Abstract:The cytological changes of nuclear behavior during the process of fertilization and early cleavage of Hemifusus tuba (Gmelin) were continually observed by fluorescent microscope. The numbers of eggs and embryos were also calculated for the whole embryonic development stages. The results showed that mature unfertilized eggs of H. tuba were oval and the average diameter was 0.33 ± 0.03 mm. The nuclear phase was at the metaphase of the first maturation division. At water temperature of 27-28℃, sperm penetrated into cytoplasm of egg 1h after fertilization. The fertilized eggs released the first polar body and the second polar body at 2~3 h. The female pronuclei and male pronuclei formed at 6 h, followed by the first cleavage. The second cleavage happened at 12-24 h, formed 4-cells. The study showed that the zygotes of H. tuba were asynchronous during meiosis and cleavage. In total, the fertilization ratio was about 43.14%, but the zygotes which could develop to 4-cell stage were only 5.70%. In a capsule, many eggs were not fertilized, or stopped developing, and both of them were inferred as the sources of nurse eggs which were consumed by developing embryos. The ratio of eggs unfertilized to eggs that stopped developing was 3 to 2. During the embryonic development, the number of eggs reduced significantly during the 1st to 6th day, only 0.48% of eggs developed to trochophore stage, and this ratio maintained until the juveniles left the capsules for natural environment.

Abstract:The method of transmission electron microscope was used to study the tissue distribution and formal characteristics of the microsporidian, Ameson sp. in the host Portunus trituberculatus. The results showed that the microsporidians were distributed mainly in the skeletal muscle, hemolymph, stomach and gut, no found in the heart, hepatopancreas, gonad and nervous tissues. Some meronts were found in the host skeletal muscle, connective tissue of the stomach and gut, indicating that the microsporidian was able to multiply in these tissues, especially in the skeletal muscle cells.A large number of microsporidians of different periods implied the tissue tropism of this microsporidian. The meronts and other multiplicative cells of the microsporidian were observed only in host cells, and the mature spores could exist within host cells and extracelluar matrix. A total of six existing forms of mature spores were found. The three within host cells:① The free spore without a membrane surrounding directly parasitized within the host cytoplasm. ② The spore was surrounded by a single membrane with the microvilli-like projections partly dissolved. This form was generally observed within an agranular cell (i.e. the professional phagocyte). ③ The spore was surrounded by lamellar membranous whorls and the microvilli-like projections was clearly visible. This form was found in nonprofessional phagocytes. The three within the host extracelluar matrix:① The spore was free without a membrane surrounding. The microvilli-like projections were clearly visible. ② Several spores were surrounded by a humoral encapsulation. The microvilli-like projections were clearly visible. ③ The spore was free without a membrane surrounding. The exospore and microvilli-like projections both disappeared. The present study sheds light on the tissue distribution of microsporidian and existing form of the spore, and provides important data for the further study on the migration of microsporidia in the crab.

Abstract:Previous studies showed that the petroleum ether extracts from the submerged macrophytes Potamogeton crispus significantly inhibited the growth of Karenia mikimitoi. Further, four antialgal activity compounds (1-4) were successfully isolated through a combination of silica gel column chromatography and repeated preparative thin-layer chromatography in this paper. These four antialgal compounds showed antialgal activities against K. mikimitoi. Furthermore, their structures were identified on the basis of spectrum data. There are pentacosyl alcohol, 1, 2-benzenedicarboxylic acid, 1, 2-bis(2-methylhexyl) ester, palmitic acid, and 1, 2-benzenedicarboxylic acid, 1, 2-bis(5,5-methylhexyl) ester. These four compounds were isolated from P. crispus for the first time, and compounds 1, 2, and 4 were isolated from the submerged macrophytes for the first time. This paper provided solid experiment foundation for further study. Furthermore, at the concentration of 50 μg/mL, these four antialgal compounds significantly inhibited the growth of Heterosigma akashiwo; and pentacosyl alcohol and 1, 2-benzenedicarboxylic acid, 1, 2-bis(5,5-methylhexyl) ester exhibited more significant antialgal activity against Prorocentrum donghaiense and Phaeocystis globsa.

Abstract:In order to select the most outstanding groups of Pacific oyster (Crassostrea gigas) with golden shell color, a total of 25 full-sib families were constructed successfully by using the method of nested design in June 2014. These parental breeders were individuals of C. gigas with golden shell cultured in 2013, which had been selected for four generations by families breeding and populations breeding based on golden shell coloration. In the meanwhile, we produced the control families by using unselected individuals as parents. We measured and compared the phenotypic growth traits and the average survival rates among these families during the larval and juvenile stages. The results indicated that the average values of the growth performances and the survival rates of these golden shell families were greater than those of the control, at the larval stages, the shell height and the average survival rates of all the golden shell C. gigas families were greater than the average values of the control by 2.27%~16.67% and 1.72%~9.40%, respectively; at the juvenile stages, the shell height, shell length and total weight and the average survival rates of all the golden shell C. gigas families were greater than the average values of the control by 10.04%~19.79%, 6.56%~17.78%, 10.44%~32.92% and 0.20%~4.26%, respectively. The differences in growth and survival rates were significant among these full-sib families, based on the great performance of fast growth and high survival, the G19 and G28 were the most excellent families of all the golden shell families of C. gigas in early and medium terms. At the age of 11 months, the shell height, shell length and total weight of G19 and G28 were greater than the average values of the total families by 4.78%, 8.22%, 12.38% and 7.61%, 4.02%, 9.04%, respectively, and were greater than the average values of the control by 15.31%, 15.31%, 24.11% and 18.42%, 10.85%, 20.42%, respectively. The average survival rates of these two families were greater than the average values of the total golden shell families and the control by 11.70%, 12.71% and 11.92%, 12.94%, respectively. Our results suggest that the growth performance superiority of the C. gigas with golden shell coloration is significant and the potential for the selection of the survival rate needs to be studied. The families G19 and G28 can be used as valuable genetic materials for the following breeding and relative research. Furthermore, these results may also provide theoretical supports for genetics and breeding programs of golden shell Pacific oysters.

Abstract:Actual position often deviates from the original design position in the installation of artificial reefs for the construction of sea ranching program. How to estimate the deviation error at unit artificial reef and artificial reef groups was the precondition of evaluation of artificial reef building quality. In this paper, three different types of artificial reefs were chosen to do the research, which were harbor artificial reef area, island-reef area, open artificial reef water area. Five indexes were used to calculate and analyse the deviation error between actual artificial reef and designed artificial reef at unit artificial reef level, which were gravity center, peripheral area, overlapping area, monocase number, and average spacing between monocases. The results showed that the error of each index was mainly normal distribution, however, error of index in one artificial reef area may follow different types of reasonable fitting distribution. The same error index of different artificial reef areas may have the same fitting distribution, and the fitting distribution of different error indexes may be the same. Gravity center, overlapping areas and monocase number have the same limit error in different areas, the standardizing distribution scope were[0, 1],[0, 1],[0, 0.451] respectively. Limit error was different from other indexes, generally, distribution scope of peripheral area was[0, 1], and distribution was[0, 1] and[0, 0.890] separately of the average spacing between monocased and total error. Based on the analysis of distribution pattern and distribution scope of artificial reef, the deviation errors between actual artificial reef and designed artificial reef can be divided and can be used to evaluate the quality of artificial reef building.

Abstract:In this study, the toxic mechanism of Procambarus clarkii responding to high pH stress was investigated through analyzing the effect of high pH on enzymes activity and histological structure of gill and hepatopancreas. Based on the 96 hours median lethal concentration, Procambarus clarkii were exposed to two pH levels:pH 7.6 (control group) and pH 10.2 (experimental group). Each group was respectively sampled for measuring activity of Na⁺-K⁺-ATPase, lactate dehydrogenase (LDH), fumarate reductase (FRD) in gill, measuring activity of superoxide dismutase (SOD), catalase(CAT) and content of malondialdehyde (MDA) in hepatopancreas, and observing the histological structure of gill and hepatopancreas at 0, 2, 8, 24, 96 h. The results showed that the value of pH LC₅₀ was 10.194 at 96h. Activity of gill Na⁺-K⁺-ATPase, LDH, FRD in experimental group presented the trend of rising and were significantly higher than those in control group(P<0.05) after 8 h exposure. With pH stress time prolonging, the variation trend of malondialdehyde content in experimental group was similar to LDH and FRD. Activity of hepatopancreas CAT in experimental group presented the trend of rising firstly and declining afterward. Activity of hepatopancreas SOD was decreased significantly(P<0.05) after 24 h exposure. Procambarus clarkii exposed to high pH displayed histological alterations in the gill and hepatopancreas. Gill tissues showed respiratory epithelium cells were exfoliated gradually, and cuticle rupture. The hepatopancreas indicated basement membranes of hepatopancreas tubule were ruptured, the enlarged size and increased number of vacuoles in hepatopancreas tubule, and the number of hepatocytes was decreased distinctly. The results indicated that high pH stress will affect the metabolism of Procambarus clarkii and will arouse oxidative stress, The histological structure of gill and hepatopancreas will also be affected by high pH stress, as well as their biological functions.