外源铜胁迫下茅尾海南美白对虾营养成分变化分析

通过设置不同的外源铜浓度(0、0. 10、0. 25、0. 50 mg/L Cu2+),分析探讨随外源铜浓度的变化,茅尾海南美白对虾体内铜累积率、氨基酸、灰分、钙、磷、铁等营养成分的变化情况,为茅尾海对虾的安全养殖与重金属污染诊断提供科学的依据。研究表明,当外源Cu的浓度为0. 10 mg/L时,对虾体内Cu的富集累积率为98. 71%;当外源Cu的浓度为0. 25 mg/L时,累积率为99. 21%;当外源Cu的浓度为0. 50 mg/L时,累积率为99. 50%。随着外源Cu浓度的提高,非必需氨基酸、必需氨基酸总体呈下降的趋势,其中丝氨酸、谷氨酸活跃性稍有增加,天门冬氨酸、甘氨酸、丙氨酸、脯氨酸等营养成分的活跃性受到抑制,肌苷酸含量呈下降趋势。外源Cu浓度与灰分含量和Fe、Ca、P含量分别呈显著负相关、高度负相关、显著负相关和微负相关关系。总体来说,外源Cu浓度为0. 10 mg/L时,对虾体内Cu的累积速率最快,氨基酸、Fe的营养成分最易流失;受外源Cu环境影响程度为P>肌苷酸>灰分> Fe> Ca。

几种常用药物对南美白对虾幼体关键发育阶段的毒性影响

研究了四种常用药物对南美白对虾幼体各关键发育阶段生存和变态的毒性影响,通过对试验药物作用机理的探讨以及药物间对南美白对虾幼体各阶段毒性差异的分析,提出了南美白对虾幼体各发育阶段对虾的存活安全浓度和变态安全浓度。

浅谈南美白对虾北方淡水养殖高产技术

详细介绍了南美白对虾北方淡水养殖高产技术,以期指导养殖户提高养殖产量,从而增加经济效益。

Inactivation Kinetics of β-N-Acetyl-D-Glucosaminidase from Prawn (Penaeus vannamei) by Formaldehyde

β-N-Acetyl-D-glucosaminidase （NAGase, EC.3.2.1.52） is chitinolytic enzymes and disintegrate dimmer and trimer a composition of oligomers of N-acetyl-β-D-glucosamine （NAG） into monomer. Prawn （P. vannamei） NAGase is involved in digestion and molting processes. Some pollutants in seawater affect the enzyme activity causing loss of the biological function of the enzyme, which affects the exuviating shell and threatens the survival of the animal. The effect of formaldehyde on prawn （P. vannamei） β-N-acetyl-D-glucosaminidase activity for the hydrolysis of pNP-NAG has been studied. The results show that formaldehyde, at appropriate concentrations, can lead to reversible inactivation of the enzyme, and the IC50 is estimated to be 1.05mol· L^-1. The inactivation mechanism obtained from Lineweaver-Burk plots shows that the inactivation of the enzyme by formaldehyde belongs to the competitive type. The inactivation kinetics of the enzyme by formaldehyde has been studied using the progress-of-substrate-reaction method described by Tsou, and the rate constants have been determined. The results show that k＋0 is much larger than k-0, indicating the free enzyme molecule is fragile in the formaldehyde solution.

Growth and Feed Efficiency of Juvenile Shrimp Litopenaeus vannamei Fed Formulated Diets Containing Different Levels of Poultry by-Product Meal

This feeding trial was conducted to evaluate the potential of poultry by-product meal (PBM) as a protein source in the culture of Litopenaeus vannamei. Seven isonitrogenous and isoenergetic diets were formulated to near to commercial diet with about 40% protein and 7.5% lipid. Fish meal was replaced by 0, 30%, 40%, 50%, 60%, 70% and 100% of PBM (diets 1-7). The diet with 100% fish meal was used as a control (diet 1). Post-larvae were reared in an indoor semi-closed re-circulating system. Each dietary treatment was tested in 4 replicate tanks (260 L) of 40 shrimp, arranged in a completely randomized design. The shrimps were hand-fed for three times a day to near-satiation (0700, 1200 and 1800) for 60 d. Percentage weight gain, survival, feed conversion ratio (FCR), protein efficiency ratio (PER) and body composition of shrimps were measured. There were no significant differences (P>0.05) in growth performance among shrimps fed diets 1-5 (0-60% fish meal replacement). However, shrimps fed diet 7 (100% fish meal replacement) had significantly lower (P<0.05) growth than those fed diets 1-5 (0-60% fish meal replacement). Shrimp fed diets 2-4 (30%-50% fish meal replacement) showed significantly higher growth than those fed diets 6 and 7 (70% and 100% fish meal replacement, respectively). Survival ranged from 94.7% to 100.0% and did not differ significantly (P>0.05) among different experimental diets. No differences in body composition were found among shrimps fed different diets. These results showed that up to 70% of fish meal protein can be replaced by PBM without adversely affecting the growth, survival, FCR, PER and body composition of Litopenaeus vannamei.

ZResponse to selection,heritability and genetic correlations between body weight and body size in Pacific white shrimp,Litopenaeus vannamei

To quantify the response to selection, heritability and genetic correlations between weight and size ofLitopenaeus vannamei, the body weight （BW）, total length （TL）, body length （BL）, first abdominal segment depth （FASD）, third abdominal segment depth （TASD）, first abdominal segment width （FASW）, and partial carapace length （PCL） of 5-month-old parents and of offspring were measured by calculating seven body measurings of offspring produced by a nested mating design. Seventeen half-sib families and 42 full-sib families ofL. vannamei were produced using artificial fertilization from 2-4 dams by each sire, and measured at around five months post-metamorphosis. The results show that heritabilities among various traits were high： 0.515±0.030 for body weight and 0.394±0.030 for total length. After one generation of selection, the selection response was 10.70% for offspring growth. In the 5th month, the realized heritability for weight was 0.296 for the offspring generation. Genetic correlations between body weight and body size were highly variable. The results indicate that external morphological parameters can be applied during breeder selection for enhancing the growth without sacrificing animals for determining the body size and breed ability; and selective breeding can be improved significantly, simultaneously with increased production.

A genetic linkage map of marine shrimp Penaeus (Fenneropenaeus) chinensis based on AFLP, SSR, and RAPD markers

The Chinese shrimp Penaeus (Fenneropaeneus) chinensis is an important species in marine fishery and aquaculture in China. A female Chinese shrimp Penaeus (Fenneropaeneus) chinensis was captured from west coast of the Korean peninsula and mated with a "Yellow Sea No. 1" male to produce the first filial generation (F1) 100 F2 full-sib progeny from brother-sister crosses between F1 families was used for the mapping study. A genetic linkage map of the Chinese shrimp was constructed, based on 354 markers, including 300 amplified fragment length polymorphism (AFLP) markers, 42 microsatellite (SSR) markers, and 12 randomly amplified polymorphism (RAPD) markers. Forty-seven linkage groups (LGs) were identified. The total map length was 4 580.5 cM, with an average spacing of 11.3 cM, covering 75.8% of the estimated genome size. The construction of this genetic linkage map was part of a genetic breeding program. This linkage map will contribute to the discovery of genes and quantitative trait loci (QTLs) in Chinese shrimp.

EST-derived SNP discovery and selective pressure analysis in Pacific white shrimp(Litopenaeus vannamei)

Pacific white shrimp has become a major aquaculture and fishery species worldwide.Although a large scale EST resource has been publicly available since 2008,the data have not yet been widely used for SNP discovery or transcriptome-wide assessment of selective pressure.In this study,a set of 155 411 expressed sequence tags(ESTs) from the NCBI database were computationally analyzed and 17 225 single nucleotide polymorphisms(SNPs) were predicted,including 9 546 transitions,5 124 transversions and 2 481 indels.Among the 7 298 SNP substitutions located in functionally annotated contigs,58.4%(4 262) are non-synonymous SNPs capable of introducing amino acid mutations.Two hundred and fifty nonsynonymous SNPs in genes associated with economic traits have been identified as candidates for markers in selective breeding.Diversity estimates among the synonymous nucleotides were on average 3.49 times greater than those in non-synonymous,suggesting negative selection.Distribution of non-synonymous to synonymous substitutions(Ka/Ks) ratio ranges from 0 to 4.01,(average 0.42,median 0.26),suggesting that the majority of the affected genes are under purifying selection.Enrichment analysis identified multiple gene ontology categories under positive or negative selection.Categories involved in innate immune response and male gamete generation are rich in positively selected genes,which is similar to reports in Drosophila and primates.This work is the first transcriptome-wide assessment of selective pressure in a Penaeid shrimp species.The functionally annotated SNPs provide a valuable resource of potential molecular markers for selective breeding.

Predictive ability of genomic selection models for breeding value estimation on growth traits of Pacific white shrimp Litopenaeus vannamei

Genomic selection(GS)can be used to accelerate genetic improvement by shortening the selection interval.The successful application of GS depends largely on the accuracy of the prediction of genomic estimated breeding value(GEBV).This study is a fi rst attempt to understand the practicality of GS in Litopenaeus vannamei and aims to evaluate models for GS on growth traits.The performance of GS models in L.vannamei was evaluated in a population consisting of 205 individuals,which were genotyped for 6 359 single nucleotide polymorphism(SNP)markers by specifi c length amplifi ed fragment sequencing(SLAF-seq)and phenotyped for body length and body weight.Three GS models(RR-BLUP,Bayes A,and Bayesian LASSO)were used to obtain the GEBV,and their predictive ability was assessed by the reliability of the GEBV and the bias of the predicted phenotypes.The mean reliability of the GEBVs for body length and body weight predicted by the dif ferent models was 0.296 and 0.411,respectively.For each trait,the performances of the three models were very similar to each other with respect to predictability.The regression coeffi cients estimated by the three models were close to one,suggesting near to zero bias for the predictions.Therefore,when GS was applied in a L.vannamei population for the studied scenarios,all three models appeared practicable.Further analyses suggested that improved estimation of the genomic prediction could be realized by increasing the size of the training population as well as the density of SNPs.

A Comparative Study on the Nonspecific Immunity of Juvenile Litopenaeus vannamei ever Inhabiting Freshwater and Seawater

A study on the nonspecific immunity of Litopenaeus vannamei ever inhabiting freshwater and seawater was carried out at different molt stages by comparing their total hemocyte count(THC) and respiratory burst(RB) and activity of phenol oxidase(PO), nitric oxide synthase(NOS) and lysozyme(LY). Two-way ANOVA showed that salinity and molt stage independently affected THC and RB and the activity of PO, NOS and LY of juvenile L. vannamei significantly(P < 0.05). The THC and RB and the activity of NOS gradually increased from the post-molt stages(A and B) to the pre-molt stages(D0–D3), which were common in shrimps inhabiting freshwater and seawater. The activity of PO peaked at the inter-molt stage(C), and touched the lowest at the post-molt stage in freshwater and pre-molt stage in seawater. The activity of LY was stable over the molt cycle. The RB and the activity of PO, NOS and LY of juvenile L. vannamei were significantly lower in freshwater than in seawater; whereas THC was significantly higher in freshwater than in seawater(P < 0.05). It was concluded that the post-molt stage(especially stage A) was critical to shrimp culture, which should be intensively attended when L. vannamei was cultured in freshwater.

Effect of Dopamine Injection on the Hemocyte Count and Prophenoloxidase System of the White Shrimp Litopenaeus vannamei

Effects of dopamine injection on the hemocyte count, phenoloxidase activity, serine proteinase activity, proteinase in-hibitor activity and α2-macroglobulin-like activity in L. vannamei were studied. Results showed that dopamine injection resulted in a significant effect on the parameters measured (P < 0.05), while no significant difference was observed in the control group (0.85% NaCl). In the experimental groups,the hemocyte count reached the minimum in 3 h;granular and semi-granular cells became stable after 12 h and hyaline cells and the total hemocyte count became stable after 18 h. Phenoloxidase activity reached the minimum in 6 h, and then became stable after 9 h. Serine protease activity and proteinase inhibitor activity reached the minimum in 3 h, and α2-macro-globulin-like activity reached the maximum in 3 h,and all the three parameters became stable after 12 h. The results suggest that the activating mechanisms of the proPO system triggered by dopamine are different from those triggered by invasive agents or sponta-neously activated under a normal physical condition.

A Comparative Study of Intensive Litopenaeus vannamei Culture on Four Bottom Substrates Without Water Change

The effect of four bottom substrates, oyster shell powder(OP), sugarcane bagasse(SB), a mixture of OP and SB(OS) and fresh soil(FS), on the water quality and bacterial and zooplankton density of intensive shrimp(Litopenaeus vannamei) culture tanks without water change and the growth performance of cultured shrimp were compared in this study. At the end of a 110 days culturing trial, the total ammonium-N(TAN) of the water on SB and the nitrite nitrogen(NO2-N) on OS was significantly lower than that on the other substrates(P<0.05), which coincided with the high density of ammonium- and nitrite-oxidizing bacteria in the water on SB and OS, respectively. The concentration of chlorophyll a(Chl a) increased slowly on OP, SB and OS but remained low on FS. The density of total bacteria on OP, SB and OS was one order of magnitude higher than that on FS, and the density of zooplankton on SB and OS was significantly higher than that on FS or OP(P<0.05). The improved water quality and increased density of bacteria and zooplankton on SB and OS may have had a synergistic effect on shrimp culture, improving its growth performance(high survival rate and yield and low feed conversion rate). SB and OS were more effective for improving the growth performance of intensively cultured L. vannamei without water change than OP and FS. To our knowledge, this study presents the first evidence regarding the effect of different bottom substrates on intensive shrimp culture.

Effect of various Na/K ratios in low-salinity well water on growth performance and physiological response of Pacific white shrimp Litopenaeus vannamei

To investigate the influence of sodium to potassium （Na/IO ratios on the growth performance and physiological response of the Pacific white shrimp （Litopenaeus vananmei）, various concentrations of KC1 were added to low-salinity well water （salinity 4） in an 8-week culture trial. Six treatments with NWK ratios of 60：1, 42：1, 33：1, 23：1, 17：1, and 14：1 were replicated in triplicate. The highest weight-gain rate （3 506±48）% and survival rate （89.38±0.88）% was observed in well water with Na/K ratios of 23：1 and 42：1, respectively, while the feed conversion ratio （1.02～0.01）, oxygen consumption, and ammonia-N excretion rate was the lowest in the medium with a Na/K ratio of 23：1. Gill Na＋-K＋-ATPase activity, as an indicator of osmoregulation, peaked in the treatment where the Na/K ratio was 17：1. The total hemocyte count, respiratory burst, and immune-related enzyme activities （ALP, LSZ, PO, and SOD） ofL. vananmei were affected significantly by Na/K ratios （P〈0.05）. After challenged with Vibrio harveyi, the cumulative mortality of shrimp reared in a Na/K ratio of 23：1 （30±14.14）% was significantly lower than the control （75～7.07）%. In conclusion, the addition of K＋ to low-salinity well water in L. vannamei cultures is feasible. Na/K ratios ranging from 23：1 to 33：1 might improve survival and growth. Immunity and disease resistance are also closely related to the Na/K ratio of the low-salinity well water. The findings may contribute to the development of more efficient K^＋ remediation strategies for L. vananmei culture in low-salinity well water.

The effect of simulated gastrointestinal digestion on shrimp Penaeus vannamei allergenicity

Pen a 1 is a major shrimp allergen that induces an allergy. The aim of this study is to assess the effect of simulated gastrointestinal fluids on the allergenicity of Pen a 1. Purified Pen a 1 from shrimp （Penaeus vannamei） was subjected to digestion in SGF （simulated gastric fluid） and SIF （simulated intestinal fluid） for a set time. The allergenicity of digestive Pen a 1 was analyzed by immunoblotting and Ci-ELISA, using pool sera from patients with shrimp specific IgE. The results showed that Pen a 1 exhibited a decrease in allergenicity with increasing digestion time in the SGF and SIF. However, Pen a 1 exhibited strong resistance to digestive fluids, and all yielded fragments （33 kD, 23 kD, and 14 kD） showed allergic activity. Therefore, anti-digestion may be an important factor for Pen a 1 to induce an allergy.

Comparison of the Respiratory Metabolism of Juvenile Litopenaeus vannamei Cultured in Seawater and Freshwater

Litopenaeus vannamei, a euryhaline species, can be cultured at a wide range of salinities. The emergence of freshwater pond-culture ofL. vannamei is an important prelude to the continued development of shrimp culture in China. In this study, we com- pared the respiratory metabolism of juvenile L. vannamei cultured in freshwater and saltwater by measuring their oxygen consump-tion rate （OCR）, ammonium-type nitrogen excretion rate （AER） and pyruvate kinase （PK） and lactate dehydrogenase （LDH） activi-ties at different molting stages in order to physiecologically characterize juvenile L. vannamei under freshwater conditions. The re- suits showed that OCR was significantly higher in saltwater than in freshwater at all stages of molting cycle. However, variation of OCR among molting stages in saltwater was similar with that in freshwater, and the highest OCR was observed at post-molting stage. At all stages of molting cycle, AER was significantly higher in freshwater than in saltwater, and the highest was observed at post-molting stage. The activity of PK was significantly higher in saltwater than in freshwater. Conversely, the activity of LDH was higher in freshwater than in saltwater in general. Significant variation of PK and LDH activities in molting cycle was observed in saltwater and freshwater. The results indicated that aerobic metabolism of juvenile L. vannamei was more active in saltwater than in freshwater; while its protein metabolism was more active in freshwater than in saltwater.

Responses of Prophenoloxidase System and Related Defence Parameters of Litopenaeus vannamei to Low Salinity

In this study,we investigated the effects of low salinity(26 and 21) on the prophenoloxidase(proPO) system and related defence parameters in the shrimp Litopenaeus vannamei.The results showed that low salinity induced a significant increase of dopamine(DA) concentration in haemolymph at 6 h of the experiment;on the other hand,total haemocyte count(THC),differential haemocyte count(DHC) and PO activity decreased over time to the lowest level at 24 h and remained low thereafter.Serine Protease(SP) and Proteinase Inhibitor(PI) activity in the two lower salinity treatments decreased to the lowest level at 12 and 24 h,respectively,and both recovered to the control level at 72 h.In contrast,α2-macroglobulin(α2M) activity in the two lower salinity treatments peaked at 24 h and then decreased to the control level at 72 h.Therefore,it may be concluded that stress-induced DA plays an important temporary role in neurotransmission and causes immune response in L.vannamei in adapting to salinity changes.

Identification of a novel C-type lectin from the shrimp Litopenaeus vannamei and its role in defense against pathogens infection

Acting as one of the pattern recognition receptors （PRRs）, C-type lectin is believed to mediate pathogen recognition and plays an important role in the clearance of pathogens as part of the innate immune system. In this work, a novel C-type lectin gene （named LvLecl） was cloned from the shrimp Litopenaeus vannamei, The ORF of LvLecl is 510 bp, encoding 169 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids at the N-terminal and a carbohydrate recognition domain （CRD） at the C-terminal. LvLecl was mainly expressed in the hepatopancreas. Real-time PCR analysis indicated that the level of LvLecl transcripts significantly changed in the hepatopancreas after the shrimp were artificially challenged with LPS, Micrococcus lysodeikticus and white spot syndrome virus （WSSV）. RNAi-based silencing of LvLecl resulted in increases in mortality when the shrimp were challenged with WSSV, and the median lethal time was reduced compared with controls. Although there was no characteristic ＂EPN＂ （Glu-Pro-Ser） or ＂QPD＂ （Gin-Pro-Asp） motif, the recombinant LvLecl, expressed in Escherichia coli BL21 （DE3）, could also agglutinate M. lysodeikticus and Vibrio anguillarum. The agglutinating activities were calcium-dependent and could be inhibited by D-mannose, D-glucose, D-galactose and N-Acetyl-D-mannose. These results suggest that LvLecl might be involved in the immune response against WSSV and bacterial infections and contribute to non-self recognition as a pattern recognition receptor in the innate immune system of the shrimp L. vannamei.

BAC end sequencing of Pacific white shrimp Litopenaeus vannamei: a glimpse into the genome of Penaeid shrimp

Little is known about the genome of Pacific white shrimp (Litopenaeus vannamei). To address this, we conducted BAC (bacterial artificial chromosome) end sequencing of L. vannamei. We selected and sequenced 7 812 BAC clones from the BAC library LvHE from the two ends of the inserts by Sanger sequencing. After trimming and quality filtering, 11 279 BAC end sequences (BESs) including 4 609 paired- ends BESs were obtained. The total length of the BESs was 4 340 753 bp, representing 0.18% of the L. vannamei haploid genome. The lengths of the BESs ranged from 100 bp to 660 bp with an average length of 385 bp. Analysis of the BESs indicated that the L. vannamei genome is AT-rich and that the primary repeats patterns were simple sequence repeats (SSRs) and low complexity sequences. Dinucleotide and hexanucleotide repeats were the most common SSR types in the BESs. The most abundant transposable element was gypsy, which may contribute to the generation of the large genome size of L. vannamei. We successfully annotated 4 519 BESs by BLAST searching, including genes involved in immunity and sex determination. Our results provide an important resource for functional gene studies, map construction and integration, and complete genome assembly for this species.

TAT Improves in vitro Transportation of Fortilin Through Midgut and into Hemocytes of White Shrimp Litopenaeus vannamei

Fortilin is a multifunctional protein implicated in many important cellular processes. Since injection of Pm-fortilin reduces shrimp mortality caused by white spot syndrome virus (WSSV), there is potential application of fortilin in shrimp culture. In the present study, in order to improve trans-membrane transportation efficiency, the protein transduction domain of the transactivator of transcription (TAT) peptide was fused to fortilin. The Pichia pastoris yeast expression system, which is widely accepted in animal feeds, was used for production of recombinant fusion protein. Green fluorescence protein (GFP) was selected as a reporter because of its intrinsic visible fluorescence. The fortilin, TAT and GFP fusion protein were constructed. Their trans-membrane transportation efficiency and effects on immune response of shrimp were analyzed in vitro. Results showed that TAT peptide improved in vitro uptake of fortilin into the hemocytes and midgut of Litopenaeus vannamei. The phenoloxidase (PO) activity of hemocytes incubated with GFP-Fortilin or GFP-Fortilin-TAT was significantly increased compared with that in the control without expressed fortilin. The PO activity of hemocytes incubated with 200 μg mL-1 GFP-Fortilin-TAT was significantly higher than that in the group with the same concentration of GFP-Fortilin. Hemocytes incubated with GFP-Fortilin-TAT at all concentrations showed significantly higher nitric oxide synthase (NOS) activity than those in the control or in the GFP-Fortilin treatment. The present in vitro study indicated that TAT fusion protein improved the immune effect of fortilin.