Abstract:To characterize the structure of Antarctic krill population and to evaluate the fishery resources, we analyzed the stage-specific abundance and distribution of Euphausia superba based on the data collected in the main krill fishing grounds nearby the South Shetland Islands (CCAMLAR subarea 48.1) and the South Orkney Islands (CCAMLAR subarea 48.2) from January 23 to February 13 in 2010. There were two sampling methods used in the survey. One was the vertical haul from 200 m depth to the surface layer using plankton net of which the mesh size was 330 μm. The other was the horizontal haul on surface layer and the mesh size was 500 μm. E. superba was found in 39% of the sampling stations in the surveyed area. The mean density in the water around the South Orkney Islands was 1.92 ind/m3, which was higher than the value of 0.35 ind/m3 in the water around the South Shetland Islands. The developmental stages of the sampled E. superba covered the calyptopsis stages (CI to CIII), the furcilia stages (FI to FIV and FVI), juvenile and adult. Nauplius and furcilia FV were not observed in our samples. Individuals at stages CI to CIII were the most abundant. The length ranges at stages of calyptopsis, furcilia, juvenile and adult were 1.764.96 mm (mean 5.15 mm), 3.9514.80 mm (5.96 mm), 16.8739.85 mm (36.10 mm) and 40.0058.96 mm (43.94 mm) respectively. High growth rates of larval E. superba appeared at stages CIII and FI. According to the portions of individuals from different developmental stages, we deduced that the spawning season of E. superba in the water around the South Orkney Islands started in late September and peaked in early December, which was later than that of krill individuals in the water around the South Shetland Islands. The abundance of E. superba was higher in samples collected with the surface net method than with the vertical haul method, and there was also a remarkable difference in the stage-specific compositions between the two sampling methods. Therefore our study suggested that the surface sampling method could be an effective supplement to the traditional methods in the study of the spatial distribution and the growth of the krill.

Abstract:Tintinnid is one of the major species of microzooplankton, and it has been suggested to be the food source of the first-feeding fish larvae in the marine environment. We studied a large number of large tintinnid in Laizhou Bay during 9 cruises from May to November in 2011 and from March to April in 2012. The study on the horizontal distribution of large tintinnids in Laizhou Bay from March to November enriched the basic data for the development and utilization of fish resources in Laizhou Bay. The total abundance of tintinnid was higher in the inner bay than in the outer bay from March to May. Most tintinnids concentrated in the inner or outer bay from June to November. The temperature and salinity related to the largest tintinnid abundance remained stable in different months, which suggested that tintinnid might be adapted to different temperatures and salinities within a certain range. The distribution of tintinnid in June was consistent with the distribution of fish eggs and larvae, which indicated that tintinnid was the seasonally advantageous for the first-feeding fish larvae as a food source. The richness of tintinnid species ranged from 5 to 20. A total of 17 species were identified as dominant species in the 9 different months. The maximum abundance of Codonellopsis mobilis, a dominant species, was 489×103 ind/m3 in May, but the maximum abundance of another dominant species, Tintinnopsis hemispiralis, was only 4×103 ind/m3 in November. The richness of the dominant species ranged from 2 (in April) to 10 (in July and August). In July and August, the abundances of dominant species were small, and the tintinnids were distributed. In other months, a large proportion of dominant species abundance concentrated in the inner or outer bay, and it dramatically affected the total abundance. C. mobilis, Stenosemella nivalis, Tintinnopsis radix, Tintinnopsis tocantinensis, and T. hemispiralis were the major dominant species that had been dominant for at least 3 months consecutively. Tintinnopsis estuariensis was found in August which appeared nearby the Yellow River estuary, although the salinity was higher than the previous records. It was a newly recorded species.

Abstract:Macrobenthic organisms are widely distributed in the world and are efficient bio-indicators of environmental change because of their limited mobility. They are also an important part of the basic data in environmental assessment. In this study we evaluated the current macrobenthic diversity and their regional difference in the water around Gulei peninsula, and we also discussed how this distribution pattern was formed. Before the petrochemical production in Gulei national petrochemical base, 27 stations were surveyed in April and May 2011. Twelve stations were located in the east and 15 were in the west of the peninsula. According to the data on totally 145 species, we found there were 69 polychaetes, 20 molluscs and 29 crustaceans, which were the dominant taxa. Macrobenthic species were rich in the water but the number of species was low with an average of 10. The number of species ranged from 2 to 23 at different stations. The occurrence frequency and the dominance index of most species were also quite low. The density and biomass were (111±200) ind/m2 and (14.79±22.03) g/m2 respectively. Higher density and biomass mostly appeared to the west of Gulei peninsula. The average Shannon-Wiener index and Margalef’s species richness index were 2.064 and 2.847 respectively, which were low because the number of species was below 10 at more than half of the stations. The macrobenthic diversity and density in the inner Dongshan Bay were much higher than in the bay mouth and in the east side of Gulei peninsula. Compared to other bays in Fujian, the study area had higher macrobenthic diversity but lower density and biomass. These results suggested that the diversity of macrobenthic organisms in the water around Gulei peninsula was related to the complexity in the sediments. Sandy sediments that were typical in the water were not suitable for the inhabitation of many species at the most stations, which resulted in low species number and density. Furthermore, there was a change in the composition of dominant species compared to the previous data.

Abstract:Half-smooth tongue sole (Cynoglossus semilaevis) is an important aquaculture species in the north coastal area of China. Male and female C. semilaevis exhibit distinct properties, yet their sex determination mechanism is complex and obscure. By genome and transcriptome sequencing of C. semilaevis, we identified a sex-related gene CSFR2, and we then carried out the cloning, expression in E. coli, and 1-step Ni-NTA-based purification to investigate the function of CSFR2. We injected the recombinant CSFR2 protein into C. semilaevis and quantified the amounts of two genes presumably to be affected at the transcriptional level using qPCR. The marker genes were positively affected during the first 72 h following injection. A prokaryotic expression plasmid pET-32a-CSFR2 was constructed transformed into E. coli to produce the fusion protein. HisTrap HP was used for protein purification, and SDS-PAGE electrophoresis was used for fusion protein detection. We injected the fusion protein with liposome into fish, and tested the expression of Cyp19a and Foxl2. The results showed that the expression of Cyp19a and Foxl2 was significantly up-regulated between 6 to 72 h and then returned to the basal level at 72 h after injection. Being a sex-related gene, the activity of the CSFR2 fusion protein could not be directly detected using immunoreaction. We hence examined the activities of other sex-related genes, which could reflect the activity of CSFR2. The results showed that the recombinant CSFR2 protein up-regulated the expression of female-related genes, Foxl2 and Cyp19a, indicating that CSFR2 played a role in sex differentiation by regulating the expression of other sex-related genes. Our study proposed a new strategy in the gene function study, and provided fundamental information for the artificial induction of fish sex reversal.

Abstract:MicroRNAs (miRNAs) are a type of endogenous small single-stranded noncoding RNAs that contain approximately 22 nucleotides (nt). miRNAs are the products of single-stranded pre-mRNAs which have 70-90 nt and a stem-loop structure, and spliced by dicer enzyme. Half-smooth tongue sole (Cynoglossus semilaevis) is an important economic marine fish species. The cloning of C. semilaevis miR-223 precursor was predicted to have a typical hairpin structure, which suggested that the precursor sequence we obtained was reliable. To investigate the role of immune related miRNAs in C. semilaevis, we applied quantitative real-time PCR (qRT-PCR) and studied the expression patterns of miR-223 in different tissues of healthy C. semilaevis. miR-223 was expressed in all tested tissues including head kidney, spleen, gill, intestine, ovary, muscle, stomach, liver, skin, blood, and brain. The expression was the highest in head kidney and weak in brain and blood. We also performed qRT-PCR on three small RNA libraries (CG, NOSG and HOSG) prepared from C. semilaevis immune tissues. The results showed that the expression of miR-223 was very diverse, depending on different tissues and samples. In response to the infection of V. anguillarum at different time-points, there was a great difference in the expression of miR-223 between the control and the challenged group, in tissues including head kidney, intestine, liver and spleen. miR-223 was up-regulated in liver, spleen, and head kidney, and down-regulated in intestine. We infected head-kidney cells with LPS, poly I:C, PGN and RGNNV. It was found that the expression of miR-223 was up-regulated in response to LPS and RGNNV, and was down-regulated in response to PGN and poly I:C. The sequence of miR-223 precursor was obtained through gene cloning and expression profile analysis after infection with bacteria or virus, and it was proved that miR-223 might play a key role in the innate immune response to pathogens. Our study shed lights on the molecular functions of miRNAs in the host-pathogen interaction in C. semilaevis.

Abstract:The hypermelanosis on the blind-side of Cynoglossus semilaevis have reduced its market value and affected its availability in the enhancement program, which has adverse effects on the sustainable development of the farming industry. However the underlying mechanisms of hypermelanosis have not been clarified yet. To investigate the cytological mechanism of hypermelanosis on the blind-side of C. semilaevis, we identified three kinds of chromatophores, namely melanophore, xanthophore and iridophore, based on the microscopic observation, and we described their morphology. The melanophore contained black and brown pigment granules and had a relatively larger nucleus. They could be characterized into two types according to the morphology. In contrast, the nuclei of xanthophore and iridophore were relatively small. The distribution and pattern of the three chromatophores in the eye-side and the blind-side skin were examined and compared. We isolated and sequenced the proopiomelanocortin (POMC) cDNA from pituitary using RT-PCR and RACE. The POMC cDNA was 910 bp in length consisting of a 114 bp 5'-UTR, a 154 bp 3'-UTR, and a 642 bp open reading frame that encoded 213 amino acids. The amino acid sequence was composed of the signal peptide, ACTH, α-MSH, βMSH, γ-LPH, and β-endorphin domains, without γ-MSH and most of the joining peptide domains. The amino acid homology analysis revealed that C. semilaevis POMC shared 30%−64% similarity with their fish counterparts. Quantitative PCR results showed that POMC mRNA was mainly expressed in the pituitary and was also expressed in the brain, gonad and the black skin (hypermelanosis) on the blind side. The POMC mRNA expression level in the blind-side black skin was significantly higher than that in the ocular-side and in the blind-side normal skin, which indicated a close correlation between the POMC gene and the hyperpigmentation on the blind-side. Our study provided basic information on the cytological and molecular mechanisms of hyperpigmentation on the blind-side of C. semilaevis.

Abstract:In recent years the aquaculture Takifugu obscurus has been impeded due to the adverse changes in the ecological environments and in the hydrological conditions of the Yangtze River. Here we evaluated the genetic diversity of three cultured populations of T. obscurus and hence provided theoretical data for the potential improvement in T. obscures genetics. We used 21 pairs of microsatellite primers to analyze the genetic variances between the populations with SSR molecular marker technique, and successfully amplified 19 microsatellite markers. In three populations of T. obscurus, the number of alleles (A) was 3−11 with an average of 6.58; the effective number of alleles (Ne) was 1.7−7.8 with an average of 4.5; the value of average observed heterozygosities was 0.156−1.000; the expected heterozygosities (He) was 0.399−0.876 and the mean polymorphic information content (PIC) ranged from 0.353 to 0.858. The mean polymorphic information content (PIC) followed the order of Guangzhou population (0.588) < Shanghai population (0.633) < Jiangsu population (0.655). The genetic diversity coefficients were 0.048, 0.062, and 0.076 respectively with an average of 0.081. There was moderate genetic differentiation among the three populations. The genetic distances between each population and the UPGMA dendrogram showed that Guangzhou population and Shanghai population had the most remote relationship (0.351), and that Guangzhou population and Jiangsu population were the closest (0.204). The three populations were pooled into 2 groups in the cluster analysis—the Guangzhou/Jiangsu group and the Shanghai group. These results suggested that there was a low level of polymorphism information content and moderate genetic diversity in the three populations of T. obscurus. Noticeably the genetic diversity of the farmed Jiangsu population was higher than that of the farmed Guangzhou and Shanghai populations, so there was a high potential in selective breeding. Our study broadened the knowledge on the genetics of T. obscurus and provided necessary information for the artificial breeding.

Abstract:Exopalaemon carinicauda is one of most commercially important shrimps in China Sea due to its advantage in reproduction, longer growing seasons, and ecological flexibility. However, a variety of shrimp diseases have become an increasingly serious issue that lead to economic loss. E. carinicauda relies on the innate immunity to fight against invading pathogens. Alpha2-Macrogloblin (α2M) is an inhibitor of diverse proteases ubiquitous in animals. Alpha2M enfolds the target proteases and block their interactions. Therefore α2M plays an important part in avoiding the deleterious effects of its active components. Based on the EST sequence from a cDNA library of E. carinicauda hemocyte，we cloned the cDNA of alpha2-macrogloblin of E. carinicauda (namely Ecα2M) using rapid amplification of cDNA ends (RACE). The Ecα2M cDNA was 4823 bp in length, which contained an open reading frame (ORF) of 4413 bp, a 64 bp 5 untranslated region, and a 346 bp 3 untranslated region. The open reading frame encoded an N-terminal signal sequence of 23 residues, and a mature protein consisting of 1470 amino acids. The molecular mass of this protein was 163.0 kDa and the estimated pI was 5.03. Homology analysis revealed that the amino acid sequence of Ecα2M was highly similar to its homologs in other crustaceans. The similarity of Ecα2M was 80% between E. carinicauda and Macrobrachium rosenbergii. We also analyzed the expression of Ecα2M in different tissues with quantitative real-time PCR. Ecα2M was detected in all tested tissues of E. carinicauda, including hemocytes, gill, hepatopancreas, muscles, ovary, intestine, stomach and eyestalk, and the expression was the highest in hemocytes. After challenged with Vibrio anguillarum and white spot syndrome virus (WSSV), the expression of Ecα2M was up-regulated in hemocytes and hepatopancreas of E. carinicauda. Our results suggested that Ecα2M might play an important role in the prawn immune response.

Abstract:In order to rapidly detect Bacillus cereus and Vibrio alginolyticus from shrimp, we immunized the specific pathogen free (SPF) of New Zealand white rabbits with V. alginolyticus and B. cereus and collected the antisera. The titer of the polyclonal antibody was higher than 1: 2000. The antisera were used as the primary antibody and the goat anti-rabbit IgG-HRP was used as the secondary antibody in indirect enzyme-linked immunosorbent assay (ELISA) for the rapid detection of V. alginolyticus and B. cereus. The optimum dilution of the antiserum was determined to be 1:10000 in indirect ELISA. The optimum coated concentration of the antigen was determined to be 106 CFU/ml. The goat anti-rabbit IgG-HRP was determined to be 1: 1000. We tested the sensitivity of the serum and the lowest detectable concentration of the two strains was 106 CFU/ml. The anti-B. cereus serum had cross reaction only with B. thuringiensis. The cross reaction of the anti-V. alginolyticus serum with other bacterial species in the genus of Vibrio showed negative results. We purified 109 strains of marine bacteria from 9 sample batches including Penaeus monodon, Fenneropenaeus chinensis and Penaeus vanmamei, and these strains were timely identified using the established indirect ELISA method. Six strains of Vibrio were detected and no Bacillus was identified.

Abstract:Frequent outbreaks of viral and bacterial diseases have caused severe economic loss in shrimp farming that has been an important part in aquaculture. The white spot syndrome is one of the most harmful diseases in shrimp culture. It is crucial to develop novel strategies that efficiently protect shrimp against WSSV. In this study, we performed oral administration of diet supplemented with CpG ODNs prepared with large-scale fermentation and Yarrowia lipolytica harboring VP28 (VP28-yl) to farmed Litopenaeus vannamei. After 30-day feeding we challenged the shrimp with WSSV to evaluate the protective effects of CpG ODN and VP28-yl. Shrimp fed with CpG ODN displayed a relative weight gain of (65.8±7.8)% (P<0.05), indicating that CpG ODN might promote the growth of shrimp. After the WSSV challenge, the copies of WSSV in shrimp fed with CpG ODN and VP28-yl were both significantly less compared to the control (P<0.05), and the RPS were 26.7% and 36.7% respectively. The respiratory burst level of shrimp fed with CpG ODN was increased after feeding trial and WSSV challenge. After the WSSV challenge, there was a gradual increase in the apoptosis of haemocytes in the control shrimp. In the contrast, the apoptosis of haemocytes in shrimp fed with VP28-yl was significantly suppressed. Moreover, the expression of STAT mRNA in shrimp fed with CpG ODN and VP28-yl was significantly increased post the WSSV challenge (P<0.05), and there was an opposite trend in the control group (P<0.05). These results suggested that oral administration of CpG ODN and VP28-yl could enhance the antiviral immunity against WSSV, and thus help control viral diseases. These two immunoactive substances could be potentially new therapies in shrimp aquaculture industry.

Abstract:White spot syndrome virus (WSSV) is a major fatal pathogen to shrimp. It is known that the b-chain of F0-ATP synthase plays a key role in the synthesis of ATP in all living organisms. Evidence from our previous research indicated that the b-chain of F0-ATP synthase of Litopenaeus vannamei was involved in WSSV infection. However the full-length sequence of the b-chain of F0-ATP synthase in L. vannamei has not been available yet. In this study we cloned the full cDNA using reverse transcription PCR (RT-PCR) and the rapid amplification of cDNA ends (RACE) method. Bioinformatics analysis was performed to predict the amino acid sequence and the secondary and space structure of the b-chain of F0-ATP synthase. We also mapped the homology and phylogenic tree using ClustalX 1.83 and MEGA 4.02. Immuno-histochemical and flow cytometry analysis were carried out to detect the tissue distribution of the b-chain of F0-ATP synthase in L. vannamei. The results showed that the 1129 bp full length cDNA was successfully cloned. Bioinformatics analysis indicated that the full length cDNA had an open reading frame (ORF) of 744 bp that encoded 248 amino acids, and that the predicted molecular weight of the mature peptide was 28.2 kDa. The homology analysis of the b-chain of F0-ATP synthase between species demonstrated that there was a higher similarity between L. vannamei and Caligus clemensi (50%), and Drosophila melanogaster (60%). Immuno-histochemical results showed that the b-chain of F0-ATP synthase was widely distributed in the gill of L. vannamei. Flow cytometry analysis indicated that the b-chain of F0-ATP synthase was distributed on the surface of hemocytes as well as in the cytoplasm of L. vannamei. These results suggested that the b-chain of F0-ATP synthase could be located on the surface as well as in the cytoplasm of hemocytes when mediating the WSSV infection in L. vannamei. Our study shed lights on the further understanding of the role of the b-chain of F0-ATP synthase in WSSV infection.

Abstract:Salinity has significant influences on the growth, metabolism and physiological changes in fish. In this study we explored the growth of juvenile Eleutheronema tetradactylum (7.82±0.43 g) and the Na+/K+-ATP enzyme under five graduated salinities, and found that salinities had impact on the growth and survival of the juvenile fish. We tested the growth performance under salinities of 2, 10, 18, 26 and 34. Along with the increase in the salinity, the final weights, SGR, DWG, GBW and GBL of the juvenile fish all decreased, and there were significant differences (P<0.05) between some salinity groups. Salinity 2 had the highest values of all measurements, and it was significantly different from salinity 18, 26 and 34 (P<0.05), but there was no difference between salinity 2 and salinity 10 (P>0.05). The measurements of salinity 34 were significantly lower than other groups (P<0.05). The feed coefficient of juvenile fish increased as the salinity rose, and some groups showed significant differences (P<0.05). The survival rate of salinity 34 is 72.2%, lower than other groups (P<0.05) of which the survival rates were above 90%. Salinity also affected the Na+/K+-ATP enzyme in the gill of juvenile fish. After 3-day salinity domestication, the activity of Na+/K+-ATP enzyme in some groups was remarkably increased. The enzyme activity at salinity 34 was significantly higher than other groups (P<0.05), and salinity 18 and salinity 26 exhibited lower enzyme activities (P<0.05). After 10 days the enzyme activities at salinity 2, 10 and 34 became slightly lower, and thereafter all salinity groups displayed stable enzyme activities. After a 30-day culture, we found that the optimal salinity for Na+/K+-ATP enzyme was 34 at which the enzyme activity was significantly higher (P<0.05). The enzyme activity at salinity 2 and 10 was insignificantly lower than that at salinity 18 and 26. These results demonstrated that salinity had effects on the growth of juvenile E. tetradactylum and its Na+/K+-ATP enzyme activity.

Abstract:In a high density farming pond, the concentration of nitrite is often much higher than standard due to the excessive release of waste from feeding, breeding, and water pollutions. High nitrite is a prominent harmful factor in aquaculture. It can facilitate the growth of pathogenic bacteria, increase the stress for fishes and shrimp, render them prone to bacterial infection, and consequently lead to disease breakout. Nitrogen-removing has been widely recognized as an effective method to degrade nitrite in the water which comes from food residual and feces. An efficient nitrogen-removing bacterium strain, 201107290102, was isolated from the bio-floc in a shrimp farming pond. It was preliminarily identified as Halomonas venusta because 16S rRNA sequence analysis revealed that it shared 99% similarity in sequence with H. venusta. We next studied factors that determined the nitrogen-removing efficiency of this strain, including C/N ratio, temperature, initial pH and salinity. The optimal conditions were: C/N ratio 15, temperature 37℃, salinity 40, initial pH 10.0. Under the optimal conditions, the nitrogen-removing efficiency could be up to 91.7%. These results suggested that the strain 201107290102 could effectively degrade the nitrite. Our study provided a new tool for improving the bio-floc in shrimp culture.

Abstract:The cultivation of ornamental fish has been gaining more and more popularity and commercial importance. Consequently ornamental fish diseases have also caught attentions. Aequidens rivulatus is one of the most important ornamental fish in commercial fisheries in Tianjin of China. In recent years mass death of A. rivulatus has caused heavy economic losses. In this study we aimed to identify the biological characteristics of the pathogens which caused diseases in A. rivulatus. A dominant bacterium 071901 was isolated from the kidney of diseased A. rivulatus and was confirmed to be pathogenic to A. rivulatus in artificial challenge test. We examined the morphological, physiological and biochemical characteristics of this bacterium, and analyzed 16S rRNA and cfb gene (GBS-specific gene cfb, CAMP factor) for further identification. The 16S rRNA gene and cfb gene were partially sequenced and compared with data from the GenBank database. BLAST showed that 16S rRNA gene and cfb gene of strain 071901 were highly similar to their counterparts registered in GenBank. Based on the morphological observation and physiological and biochemical characterization, strain 071901 was identified as Streptococcus agalactiae. Phylogenetic trees of S. agalactiae based on 16S rRNA gene indicated that strain 071901 was branched into the same cluster as S. agalactiae and showed the highest similarity to S. agalactiae. The results of molecular analysis of cfb gene were consistent with the previous results from the biochemical assays. We then performed antimicrobial susceptibility assay for 30 antimicrobial agents and found that strain 071901 was susceptible to 19 agents including erythromycin, azithromycin and florfenicol, and was highly resistant to 11 agents including enrofloxacin, bacitracin and furazolidone. In conclusion our results demonstrated that S. agalactiae was the pathogen that caused high mortality in A. rivulatus.

Abstract:To better understand the use of sulfa drugs in aquaculture, especially in the culture of Fenneropenaeus chinensis, we studied the pharmacokinetics of three sulfa drugs in F chinensis using the high performance liquid chromatography (HPLC). The three selected sulfa drugs were sulfadimidine (SM2), sulfamethoxydiazine (SMD) and sulfadiazine (SD). The water temperature for the experiment was (24.6±2.4)℃. Healthy shrimp received oral administration of the drugs at a dose of 100 mg/kg. The results showed that the hemolymph concentration-time course of SM2, SMD and SD conformed to a two-compartment open model with the first order absorption after oral administration. Major pharmacokinetic parameters such as T1/2β, AUC, Vd, CL, Tmax and Cmax were 25.812 h, 34.066 mg/L•h, 94.553 L/kg, 2.608 L/h•kg, 2 h, 1.07 mg/L respectively for SM2, and 46.446 h, 45.39 mg/L•h, 97.207 L/kg, 1.504 L/h•kg, 1 h, 1.17 mg/L for SD, and 66.296 h, 65.917 mg/L•h, 40.015 L/kg, 0.763 L/h•kg, 2 h, 2.00 mg/L for SMD. These suggested that SMD was more widely distributed and absorbed than SM2 and SD in F. chinensis. The elimination half-life showed the order of SM2SD>SM2. As for the drug clearance rate, SMD had the fastest rate and SM2 had the slowest rate. Therefore in 72 hours after the oral administration, SMD had the most residues and the SM2 had the least residues in F. chinensis. In another word, SMD had the longest-lasting pesticide effect. Taking no account of other factors such as the cost and side effects, SMD could be the most suitable sulfa drug according to its pharmacokinetic characteristics. The comparison of the elimination half-life showed that SMD had the most enduring efficacy.

Abstract:Spotted halibut, Verasper variegates has been a target model in the studies of reproductive biology and molecular biology. However, litter is known about the dietary requirements of this species. In this study, we determined the optimal ratio of protein to lipid for this fish and analyzed the combined effects on the growth，digestive enzyme activities and blood biochemical parameters in spotted halibut. Fish meal and casein were used as protein sources, and soybean oil and fish oil served as lipid sources. Nine experimental diets were formulated as combinations of three levels of crude proteins (40%, 45%, and 50%) and three levels of crude lipid (8%, 12%, and 16%), namely P40L8, P40L12, P40L16, P45L8, P45L12, P45L16, P50L8, P50L12 and P50L16. They were randomly distributed into 27 cement ponds (2 m×1 m×1 m) with 20 fish/pond for 83 d in an in-door flow-through aquarium system. The results showed that the survival rate of fish was not significantly affected by the levels of protein and lipid (P>0.05). The weight gain, specific growth rate and feed intake were significantly reduced along with the increase in the dietary lipid (P<0.05). Compared to P40L16, the weight gain in P50L8 was 33.63% higher and the specific growth rate was 31% higher. The feed efficiency was significantly boosted along with the increase in the dietary protein (P<0.05). The feed efficiency in P50L8 was 24.09% higher than P40L8, and the weight gain, specific growth rate, feed intake and feed efficiency in P50L8 were significantly higher than those in other groups. The activity of stomach protease was first increased and then decreased along with the increase in dietary protein. The protease activity in the hepatopancreas in P50L8 group was significantly higher than that in P50L12 group (P<0.05), but showed no significant differences with other groups (P>0.05). The activity of the stomach lipase was first decreased and then increased along with the increase in the dietary protein. The activity of intestine lipase in P50L8 group was higher than other groups, but there was no significant difference between all groups (P>0.05). The increase in dietary protein resulted in significantly higher level of total proteins (P<0.05). Glutamic pyruvic transaminase and Glutamic oxalacetic transaminase in P50L8 group were higher than other groups, but there were no significant differences between all groups (P>0.05). Triglyceride and total cholesterol in P50L8 group were significantly lower than P45L12 and P45L16 groups (P<0.05), but showed no significant differences with other groups (P>0.05). These results indicated that increased dietary lipid level might not cause the protein-sparing effect. Therefore the recommended formula for the best growth performance was 50% protein, 8% lipid, 104.31 mg/kcal P/E and 6.63 P/L.

Abstract:In this study we investigated the effects of vitamin E on the reproduction and the egg quality of tongue sole Cynoglossus semilaevis. Broodstock were fed for 10 weeks with diet groups Ec, El and Eh that were supplemented with 0, 200 and 1200 mg/kg vitamin E (DL-α-tocopherol acetate) respectively. The results showed that subjects in the Eh group (1200 mg/kg Vitamin E) displayed advantageous development of gonad, and improvement in the relative fecundity, buoyant egg ratio, fertilization rate, hatching rate ratio and larvae length, as well as higher quality of larvae. The larvae survival rate, larvae length at 12 days post hatching and survival activity index of larvae (SAI) of the Eh group were higher than those in the Ec group (P<0.05). The development of gonad and the larvae length in the El group (200 mg/kg vitamin E) were promoted, however the fecundity, buoyant egg ratio, fertilization rate and hatching rate ratio were not changed compared to those in the Ec group (P>0.05). Along with the increase in dietary vitamin E, the activity of superoxide ismutase (SOD) in serum was significantly increased and the level of malonaldehyde (MDA) was reduced (P<0.05). The levels of dry matter and vitamin C in eggs were significantly higher in the El and Eh groups (vitamin E supplementation groups) than in the Ec group (P<0.05), but there was no difference between the El and Eh groups. Furthermore, the addition of vitamin E in the diet also increased the levels of PUFA, n-3PUFA, n-3HUFA and n-6PUFA in eggs and thus improved the egg quality. These results demonstrated that vitamin E supplemented in the diet could promote the development of gonad and improve the fecundity, the quality of eggs and larvae of tongue sole, and that 1200 mg Vitamin E per kg was optimal for the reproductive performance of tongue sole.

Abstract:Sablefish (Anoplopoma fimbria) has been studied for years but was only recently acknowledged as a new marine aquaculture species. To better understand the nutritive composition of its muscles, we measured and analyzed the general nutrients, amino acids, fatty acids, major and trace elements as well as vitamins, using biochemical methods. The results showed that the percentages of the moisture, crude proteins, crude fat, crude ash and total carbohydrate in the fish muscles were 69.92%, 16.48%, 10.64%, 1.69% and 1.27% respectively. In dry samples, the total amino acids (TAA) accounted for 43.81%, the ratio of total essential amino acids to total amino acids (WEAA/WTAA) was 40.68%, and the ratio of total essential amino acids to total nonessential amino acids (WEAA/WNEAA) was 81.74%. The constitutional rate of the essential amino acids met the standards of Food and Agriculture Organization of the United Nations /World Health Organization (FAO/WHO). The ratio of branched-chain amino acid amount to aromatic amino acid amount (BCAA/AAA) was 2.51, which was close to the normal value in humans. The level of flavor amino acids (FAA) was 16.94% and accounted for 38.67% of the total amino acids, which was higher than that in the main mariculture species in the north of China. According to the amino acid score (AAS) and chemical score (CS), the first limiting amino acid in the muscles was Cystine plus Methionine with an essential amino acid index (EAAI) of 76.35, implying that A. fimbria had relatively balanced amino acid composition. The ratio of unsaturated fatty acids to saturated fatty acids was 2.81, indicating a high fat quality of the muscles. Our results also suggested that the muscles of A. fimbria were rich in major and trace elements such as P and Zn, and vitamins such as VA, VB6 and VB3. In conclusion, A. fimbria may be enriched with flavor amino acids, unsaturated fatty acids, major and trace elements and vitamins, therefore it could be a valuable aquaculture species due to the nutritional advantage.

Abstract:Enriched in proteins and with similar amino acid compositions to the terrestrial vegetables, Porphyra has become an important economic marine algae with high nutritional and medical values in China. In order to obtain peptides holding antibacterial activity, Porphyra yezoensis was utilized as raw material and water, NaCl, HCl and NaOH were used as the solvents. Water-dissolved, salt-dissolved, acid-dissolved and alkaline-dissolved proteins were prepared by the extraction, ammonium sulfate salting-out, dialysis and freeze-drying, respectively. The four types of proteins were hydrolyzed respectively by pepsin, papain, neutral protease, trypsin, acid protease and compound protease under the optimum conditions for each enzyme. Twenty-four hydrolysates were selected to evaluate the antibacterial activity to Staphylococcus aureus, Micrococcus tetragenus, Bacillus subtilis and Escherichia coli that were used as the indicator bacteria. Moreover, the proteins were divided into 7 portions by fractional precipitation with different saturation degree of ammonium sulfate; and their hydrolysates were fractioned to peptide fragments with different molecular weights by ultrafiltration. The best antibacterial peptides were evaluated by comparing the inhibitory effects of hydrolysates to the indicator bacteria. The result showed that the zymolyte of water-soluble proteins hydrolyzed by pepsin presented the strongest antibacterial activity. Thus, antimicrobial peptides were achieved via fraction precipitation by ammonium sulfate for the proteins, ultrafiltration for their zymolyte, and the ammonium sulfate saturation at 40%−50%, and the range of molecular-weight was less than 5 kDa. In addition, factors (such as temperature, medium pH and antimicrobial peptides concentration) that affect the antibacterial activities were also studied. The results indicated that the antimicrobial peptides from Porphyra yezoensis possessed good thermal and acid-base stability, which may be candidate for a safe food preservative.

Abstract:Aeromonas veronii is a highly mortal pathogenic microorganism in the loach aquaculture. To screen the antagonistic bacteria and to develop microbial preparation against the pathogens, the samples were collected from loach breeding pond and the bacteria strains were isolated and screened for the ability to inhibit the size of the zone of the strains. The active substances were separated and purified with the ammonium sulfate precipitation and ion exchange chromatography, and the molecular weights were revealed with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A strain named 4-1-3 was isolated and screened from the samples collected from the bottom sediments of a loach breeding pond in the Lianyungang city, and it identified as Pseudomonas fluorescens based on its morphology and the analysis of 16S rRNA gene sequence, designated as P. fluorescens 4-1-3. The strain strongly inhibited the growth of A. veronii, with 15.2 mm inhibition zone diameter. The antibacterial substance produced by the strain P. fluorescens 4-1-3 is an extracellular protein which can be precipitated using 60% ammonium sulfate and eluted by 600 mmol/L NaCl through Sepharose DEAE Fast Flow. The molecular weight of P. fluorescens 4-1-3 is around 100 kDa determined by SDS-PAGE. In this study, we obtained a strain that can inhibit the growth of A. veronii and we found a new bacteria antagonistic substance produced by P. fluorescens. The present study provides evidence to the improve disease control in loach breeding and other aquaculture.