Abstract:This study focused on evaluating the effects of feeding frequency on growth performance of Litopenaeus vannamei fed diet with crystalline methionine.The growth experiment was conducted to test three dietary treatments including fish meal(32%)as positive control,fish meal(16%)with methionine deficiency as negative control,and fish meal(16%)with crystalline L-methionine(MET)as the test diet.Feeding frequency trial was conducted in order to explore the difference in absorption of crystalline methionine and amino acid from intact protein(positive control and negative control).Shrimp were fed MET dietary in feeding frequency 2(7:00 and 21:00),4(7:00,12:00,18:00 and 21:00),and 6 times-day(7:00,9:30,12:00,15:00,18:00 and 21:00),the positive control and negative control were fed 4 times per day only.Each treatment was randomly assigned to triplicate groups of 30 shrimps(initial weight 1.27?0.01 g)per aquarium.Shrimp were maintained in aquaria for 5 weeks at water temperature that ranged from 29 to 30 ℃.The results showed that shrimp fed MET diet 6 times per day had no difference in weight gain with positive control(P>0.05).Crude protein contents of shrimp body of shrimp fed MET 6 times per day were significantly higher than that 4 times per day(P<0.05).Total protease activities of shrimp increased with feeding frequency increased,which fed MET 4 times per day and 6 times per day had no difference with positive control(P>0.05).These results indicate that the shrimp fed low fishmeal diet supplemented with methionine had better weight gain,body protein deposition and total protease activities with feeding frequency from 4 to 6 times per day.Increased feeding frequency could improve utilization of free amino acid.

Abstract:In order to understand the muscle nutrient characteristics of the sword prawn Parapenaeopsis hardwickii,the nutritional composition and quality of muscles from the wild and cultured sword prawn were investigated and analyzed by biochemical analysis methods.The results show that the crude fat content of wild group(0.89%)was significantly lower than that of cultured group(1.12%),while the crude ash content of wild group(1.44%)was significantly higher than that of cultured group(1.31%).No significant difference in moisture and crude protein contents of fresh muscles was found between wild(79.42% and 17.94%,respectively)and cultured group(79.28% and 17.95%,respectively).18 kinds of amino acids were found in muscles from wild and cultured groups,and there was no significant difference in the content of 9 kinds of amino acid between wild and cultured groups,while in other 9 kinds of amino acids,the amino acid content of cultured group was significantly higher than that of wild group.Total amino acid(TAA),total essential amino acid(EAA),total non-essential amino acid(NEAA),total delicious amino acid(DAA),and total shrimp flavor amino acid(SAA)content of cultured group(88.08,30.53,47.39,34.47,and 21.72%,respectively)were significantly higher than those of wild group(83.42,29.69,43.59,32.54,and 21.11%,respectively).The essential amino acid index(EAAI)and the ratio of branched chain amino acid to aromatic amino acid(F value)of cultured group(69.10 and 2.12,respectively)were significantly higher than those of wild group(66.41 and 2.07,respectively).The saturated fatty acids(∑SFA)and poly-unsaturated fatty acids(∑PUFA)relative content of cultured group(39.60% and 36.64%,respectively)were significantly lower than those of wild group(41.95% and 39.51%,respectively),while the mono-unsaturated fatty acids(∑MUFA)relative content of cultured group(23.76%)were significantly higher than that of wild group(18.54%).The EPA+DHA relative content of wild group(28.23%)was significantly higher than that of cultured group(25.66%),while no significant difference was observed in the EPA+DHA absolute content of muscle between wild and cultured groups.Therefore,the sword prawn is nutritional,delicious,and artificial culture did not cause abundant accumulation of fat in muscle;on the contrary,it improved protein nutritive value of the muscle.

Abstract:Eighteen half-sib families and fifty-four full-sib families were constructed using methods of nest design and artificial insemination,in which each male mated three females.Sixty 5-month-old shrimp were measured for nine growth traits including the body weight(BW),the total length(TL),the body length(BL),the carapace length(CL),the carapace width(CW),the carapace depth(CD),the first abdominal segment depth(FASD),the third abdominal segment depth(TASD),and first abdominal segment width(FASW).According to quantitative genetics theory,the heritability of each growth trait,and the genetic and phenotypic correlation among the traits were statistically studied utilizing the method of variance and covariance analysis.The results showed that the heritability estimates in the narrow sense from intra-group correlation of paternal half-sib were precise and unbiased,and the heritability values for the above nine traits were 0.460,0.392,0.303,0.234,0.251,0.330,0.282,0.321 and 0.356,respectively.All of them belonged to moderate and high heritability,thus showed a high potential for selective breeding.The estimates of genetic correlation coefficients were 0.750-0.976,where the highest(0.976)was that between the BW and the TL,while the lowest(0.750)was seen between the TL and the TASD.The estimates of phenotypic correlation coefficients were 0.507-0.947,where the highest(0.947)was seen between the BW and the TL,while the lowest(0.507)was that between the FASD and the CD.Very significant difference was detected by t-test(P<0.01)among the nine growth traits for both genetic and phenotypic correlation coefficients,which indicate all the nine traits may respond favorably to direct and indirect selection for growth.

Abstract:Maternal transfer of immune factors from mother to eggs has been reported in many aquatic species,and these factors played important immune roles in developing embryos and larvae.However,the study on maternal immunity of shellfish remains lacking.In this study,maternal transfer and bacteriostasis of a C-type lectin in scallop(Patinopecten yessoensis)were analyzed.We detected the mRNA expression pattern of the C-type lectin gene in ovary and early developing larvae using the real-time quantitative PCR method,and tested the antibacterial activity of maternal C-type lectin in eggs by colony-forming unit(CFU)assay.The results of qRT-PCR showed that the expression of C-type lectin gene in ovary after bacterial challenge was time-dependent,and it was up-regulated gradually from 2 h after injection of bacteria and reached the peak at 8 h which was 6.2 times compared to the control group,and then dropped progressively to the original level.On the other hand,the fact that mRNA was found both in the eggs and larvae indicated that it could be transferred from mother to offspring.The mRNA expression in bacterial challenge group was significantly higher than those in normal group except for the 36 h point.The CFU assay revealed that the cytoplasm of eggs with protein concentration of 200 μg/mL and 400 μg/mL had lethal effect on the bacteria Vibrio anguillarum,and the function weakened significantly once the antibody of C-type lectin was added,which indicated the C-type lectin from mother had antibacterial activity.It is concluded that the maternal C-type lectin can transfer from mother to the offspring and has antibacterial activity.

Abstract:In order to study genes related to pearl formation in freshwater pearl mussel and their regulation mechanism,a 1 437 bp cDNA sequence of calreticulin gene fromHyriopsis cumingii(HcCRT)was obtained by rapid amplification of cDNA ends(RACE).It consisted of a 231 bp 5′-untranslation region(UTR),a 615 bp 3′UTR and a 591 bp open reading frame(ORF).The inferred amino acids sequence was composed of 196 amino acids,including a signal peptide of 21 amino acids and a mature peptide of 175 amino acids.The molecular weight of the peptide was predicted to be 22.4 ku,with a theoretical isoelectric point of 5.01.Amino acid sequence analysis showed that there was no obvious amino acid sequence of membrane domain.Results of the ProtScale online analysis showed that the protein was hydrophilic protein.Homology analysis indicated that HcCRT amino acid had a conservative sequence of calreticulin family and was highly conserved with Danio rerio(77%),Crassostrea gigas(70%),Pinctada fucata(70%).The sequence analysis showed that HcCRT shared two potential calreticulin family signature motifs with CRT from other species,KHEQNIDCGGGYLKVF and IMFGPDICG.The prediction results of H.cumingiiHcCRT protein secondary structure and tertiary structure indicated that the protein contains the alpha helix and beta folding.Real-time quantitative PCR(RT-PCR)showed that HcCRT is expressed in a wide range of tissues including the mantle,blood,gill,foot,liver,kidney,intestine and muscle,with the highest level of transcripts in mantle,followed by blood,while there is rare expression in other tissues.These data suggested that HcCRT might be involved in pearl formation of H.cumingii.This study which was first time to obtain HcCRT gene fromH.cumingii played an important role in exploring pearl formation of H.cumingii.

Abstract:In order to control Fusarium isolated from shrimp culture environment producing T-2 toxin,the inhibitory effect and antibacterial mechanism of lipopeptides from Bacillus natto on Fusarium was studied in the article.The inhibitory effect was studied through the influence of lipopeptides on the germination and growth of Fusarium spores; and the observation of the ultrastructure of the spores,the mycelium morphology and the mycelium permeability was performed through electron microscope,ordinary microscope and fluorescence microscope respectively to study the antibacterial mechanism.When the concentration of lipopeptides was 1 mg/mL,the inhibition rate on the germination of Fusarium spores reached 78.8%,and the cultivation biomass of Fusarium was 24.5% of the control group without lipopeptides on the third day.Under the condition of the same concentration,the microscopic observation under scanning electron microscope revealed that the spores of Fusarium presented moniliform state under the influence of lipopeptides; ordinary microscope revealed that the mycelia became nonseptate after the treatment of lipopeptides; and fluorescence microscope revealed that lipopeptides could increase the membrane permeability of the Fusarium mycelium.

Abstract:Porphyra haitanensis is one of the most important economic marine crops of China.For any cultivar,the correct identification of species or forma of the cultivated strains is necessary to ensure a well-bred cultivation and good production quality.However,because the gametophytic blade of Porphyra is morphologically simple and marked variations can occur as environmental conditions change,it is very difficult to precisely identify the species or forma of cultivated strains based only on their morphological characteristics.With new advances in molecular biology,molecular markers and DNA fingerprinting techniques have become routine for the identification and classification of many crops,including seaweeds.The strain of “Z-26” of P.haitanensiswas selected by the laboratory of germplasm improvement and the application of P.haitanensis in Jimei University which has the characters of high-temperature tolerance and high yield,and it has been widely cultivated in south China.In order to construct the technology of variety identification for “Z-26”,firstly,300 primers of RAPD were used to scan the specific markers of 6 new strains of P.haitanensis and 9 specific RAPD markers of “Z-26” were selected.After cloning and sequencing,two specific RAPD markers of “Z-26” were transformed into the SCAR markers successfully,the length of the 2 SCAR markers was 540 and 242 bp,respectively.Secondly,after verification by 4 different experiments,we can affirm that the 2 SCAR markers were the specific and stable markers of “Z-26”.At last,based on the 2 SCAR markers,after optimization of experimental conditions,the technology of multiplex PCR which was used to identify the variety of “Z-26” was constructed.The result supplied a simple,fast and reliable technigue for variety identification of “Z-26”.

Abstract:Vibrio vulnificus is a marine seafood-borne pathogen that will cause death in susceptible individuals after consumption of raw or uncooked contaminated seafood around the world.So,early detection and identification of V.vulnificus strains in food and clinical samples is essential for diagnosis and reducing the incidence of food-borne disease.PCR assay has been one of the most important and extensive method to detect pathogenic bacteria.Previous study depended on vvhA as the target gene to detect this bacterium.In this study,we constructed a local BLAST database and identified 34 candidates as V.vulnificus-specific target genes distributed in 3 isolates(CMCP6,MO6-24/O,and YJ016)with published completed genome.Among these candidate-specific targets,VV1_2692,VV2_0075,and VV2_0939 genes are known for their functions,while the rests encode hypothetical protein of unknown function.To evaluate the specificity of above 3 genes,PCR amplification of genomic DNA from a V.vulnificus strain resulted in a product with predicted length,whereas no products were detected from 14 non-V.vulnificus bacterial strains.The minimum detectable limits of VV1_2692,VV2_0075,and VV2_0939 genes were 10³,10¹,and 10² cfu/mL,respectively.A total of 137 seafood samples(e.g.,fish,prawn,crab,shell)from Hangzhou city were detected by both PCR assay and biochemical method.Among them,39 V.vulnificus isolates were detected using biochemical method,while 38,39,39,and 39 V.vulnificus isolates were detected using PCR assay of VV1_2692,VV2_0075,VV2_0939,and vvhA genes,respectively.The negative result of VV1_2692 probably resulted from its relative high detectable limit,and both VV2_0075 andVV2_0939 genes might be suitable species-specific targets to detect V.vulnificus in seafood.Moreover,28.5% of 137 seafood samples contained V.vulnificus,and oyster had the highest ratio(68%),suggesting status of V.vulnificus pollution was extremely serious in coastal seafood of Hangzhou city,especially oyster.

Abstract:The fish spotted scat Scatophagus argus,as a new resource of marine aquaculture,is popularized in South China in recent years.Studies on the gonadal development can provide the theoretical knowledge for the artificial propagation of Scatophagus argus.Generally,after two years of growth,some individuals can reach sexual maturation in the south of China.In this study,histological observation of gonads was performed based on paraffin section technique and HE staining method.The linear alignments of tubular structures are observed in the testis,which indicates that the testis of the Scatophagus argus belongs to the tubular type.The development of testis may be divided into 5 stages,i.e. multiplication of spermatogonial cells,growth of spermatocytes,maturation of spermatocytes,emergence of spermatoblasts,maturation of spermatozoa,etc.The average gonado somatic index of the testis was between 0.2% and 1.5% .The gonado somatic index(GSI)reached the peak at the stageⅤ,and the the peak of hepato somatic index(HSI)was observed at the stage Ⅳ.The oocyte deveolpment was divided into 5 phases and ovary development consisted of 5 stages accordingly.The average gonado somatic index of the testis was between 1.2% and 14.5%.The gonado somatic index(GSI)reached the peak value at the stage Ⅴ and the peak of hepato somatic index(HSI)was found in stage Ⅳ.The yolk nucleus and follicle membrane are observed at phase Ⅱ of oocyte.The number of yolk granules and oil droplets increased rapidly at the phaseⅢ,and subsequently yolk granules fused into pieces at the phase Ⅳ Apart from a large number of oocytes at the phase Ⅴ,there are still many oocytes of phase Ⅱ,Ⅲ and Ⅳ in ovaries at the stage Ⅴ.The similar condition was also found in ovaries after spawning.It indicates that oocyte development in Scatophagus argus is asynchronous.