Effect of dietary supplement of inactivated Lactobacillus plantarum Ep-M 17 on growth performance,immune response,disease resistance,and intestinal microbiota in Penaeus vannamei

Our previous study found that feeding with Lactobacillus plantarum Ep-M17 could effectively affect the growth performance,immune response,and gut microbiota of Penaeus vannamei.However,high temperature and pressure during feed pelletizing is the main problem that can lead to a decrease in the activity of probiotics or cause their inactivation.Further investigation needs to investigate whether inactivated Ep-M17 can exert similar effects as live Ep-M17.Therefore,we evaluated the effects of inactivated L.plantarum Ep-M17 on growth performance,immune response,disease resistance,and gut microbiota in P.vannamei.Results show that adding inactivated Ep-M17 to the feed also promoted body weight gain and increased relative immune protection in shrimp.Also,histological examination revealed that the administration of inactivated Ep-M17 led to improvements in the density and distribution of microvilli in the intestines and enhancements in the abundance of B and R cells in the hepatopancreas.Additionally,the inactivated Ep-M17 supplementation resulted in increased activity levels of nutrient immune-related enzymes in both the shrimp hepatopancreas and intestines.Moreover,it stimulated the expression of Lvlec,PEN-3a,Crustin,LGBP,Lysozyme,and proPo genes in both the hepatopancreas and intestines.Furthermore,the inactivated Ep-M17 also increased bacterial diversity in the gut of shrimp and promoted the abundance of specific flora,facilitating the host organism’s metabolism and immunity to improve the disease resistance of shrimp.Therefore,supplementation of inactivated L.plantarum Ep-M17 in shrimp diets can exert similar effects as live L.plantarum Ep-M17 effectively improving growth performance,gut microbiota,immune response,and disease resistance in P.vannamei.

Insights into sensitizing and eliciting capacity of gastric and gastrointestinal digestion products of shrimp(Penaeus vannamei)proteins in BALB/c mice

Shrimp(Penaeus vannamei)proteins have been shown an allergenic potential;however,little information is available on the sensitizing and eliciting capacity of shrimp protein digestion products.In this study,a BALB/c mice model was used to explore the allergenicity of shrimp protein sample(SPS)and their gastric and gastrointestinal digestion products(GDS/GIDS).As compared with the SPS groups,the GDS/GIDS groups caused lower specific immunoglobulins(Ig E/Ig G1)levels(P<0.05),but higher than the control groups,indicating that the digestion products sensitized the mice.Meanwhile,spleen index,mouse mast cell protease-1(m MCP-1)concentration and proportion of degranulated mast cells were significantly reduced in the GDS/GIDS groups(P<0.05);simultaneously,allergic symptoms,vascular permeability and histopathological changes of tissues were alleviated.Nevertheless,the allergenicity of digestion products cannot be eliminated and still cause systemic allergic reactions in mice.The study showed that the digestion products of shrimp still had high sensitizing and eliciting capacity.

Virulence changes in Vibrio parahaemolyticus during the freezing of Penaeus chinensis

Although Vibrio parahaemolyticus has become the most common pathogen in fresh and frozen seafood,its virulence changes have often been ignored during the processing of seafood.To investigate these potential risks,we used frozen Penaeus chinensis as examples,and the most virulent factors of V.parahaemolyticus,including amounts,viable but nonculturable(VBNC)status,toxins TDH and TRH,and virulence genes tdh and trh,were determined.Bacterial quantities were signifi cantly reduced during drain and sprinkling phases,but caused by different factors.By SYTO9 and PI staining showed that washing was the main reason for the bacterial reduction at the drain phase,while the strain entering VBNC state was another reason at sprinkling phase.Their hemolysis toxicity,produced by TDH and TRH,became stronger after inoculation on shrimp,and could be detected throughout the process.Moreover,tdh and trh also exhibited trends similar to that of the hemolysis toxicity test.tdh was almost to a two-fold expression level during ice-glazing phase,while trh only express at a low level,less than half of the expression level before inoculation.These results demonstrated that the strains were not dead during freezing process,but became VBNC cells,which still produced and accumulated toxins,especially TDH,the most virulent factor.

Effects of Dietary Vitamin D_(3)on Growth Performance,Immune Status,and Calcium and Phosphorus Metabolism in Litopenaeus vannamei Under Different Salinity Conditions

Vitamin D_(3)(VD_(3)) plays a vital role in various physiological processes in addition to its role in regulating the homeostasis of calcium and phosphorus.The present study was conducted to investigate the effects of dietary VD_(3) on growth performance,immune status and the metabolism of calcium and phosphorus in Litopenaeus vannamei under the optimal salinity(25)and high salinity(35)conditions.Shrimp(2.1 g±0.1 g)were fed with experimental diets containing 0,1500,6000 and 12000IUkg^(−1)VD_(3) for 30 days under two salinity conditions respectively.The results showed that the growth performance and intestinal health were reduced,while oxidative stress was induced in the shrimp cultured at salinity 35 compared to those in the shrimp cultured at salinity 25.Interestingly,dietary supplementation of 1500 IU kg^(−1)VD_(3) at salinity 35 promoted the growth performance,improved the intestinal health,and enhanced the antioxidant capacity and non-specific immunity in shrimp.In addition,the content of inorganic phosphorus in shrimp serum was affected by dietary VD_(3) independent of salinity.In contrast,the expressions of calcium transport-related genes,including calmodulin and Ca2+-ATPase,were regulated by salinity,instead of dietary VD_(3).In conclusion,an appropriate supplementation of dietary VD_(3) under high-salt condition can promote the growth,alleviate intestinal inflammation,enhance antioxidant capacity and immunity,and contribute to phosphorus metabolism in L.vannamei.

不同糖源对南美白对虾Penaeus vannamei生长、成活率和虾体组成的影响

在饲料中分别添加w=20%的葡萄糖、小麦淀粉、玉米淀粉、马铃薯淀粉、蔗糖、糊精,饲养初始质量为(0 78±0 01)g的南美白对虾,经过56d的生长试验,观察不同糖源对于南美白对虾的生长、成活率、机体营养组成的影响。实验结果表明,南美白对虾对不同糖源的利用表现出差异性,蔗糖组的成活率、特定生长率、饲料效率均最高;其他各组差异不显著。不同糖源对南美白对虾生长的影响依次为蔗糖>玉米淀粉>小麦淀粉>糊精>葡萄糖>马铃薯淀粉;对南美白对虾成活率的影响依次为蔗糖>马铃薯淀粉>玉米淀粉>小麦淀粉>糊精>葡萄糖。不同糖源对南美白对虾全虾蛋白质含量和水分含量的影响差异不大,但是糊精和马铃薯淀粉组的全虾脂肪含量明显低于其他各组,蔗糖组的全虾灰分与其他各组都没有显著性差异,葡萄糖组最高,糊精组最低。

日本对虾(Penaeus japonicus)性腺差异表达基因GD13之cDNA5’端克隆

采用基于SMART(SwitchingMechanismAt5'endofRNATranscript)原理的cDNA末端快速扩增技术(RapidAmpli ficationofcDNAends:RACE),对日本对虾性腺差异表达基因cDNA5'端进行克隆,即在逆转录过程中通过SMARTⅡoligo的介导,在cDNA第1条链的3'端接上接头,然后根据已知序列设计的基因特异性引物(GSP1和GSP2)和接头引物(UPM和NUP)进行Nested PCR特异性扩增,并对特异产物进行克隆和序列分析,最终获得全长cDNA序列.测序结果表明,在卵巢内表达量高于精巢的GD13基因全长为656bp,编码164氨基酸,经NCBI检索,此基因与文昌鱼、家鼠的核糖体蛋白L24及黄牛的核糖体蛋白L30氨基酸序列同源性分别高达58%、56%和56%,可断定该基因为编码日本对虾核糖体蛋白L24亚基的基因.为进一步认识对虾生长发育和繁殖的调控机制奠定基础.

RAPD analysis of wild stock of penaeid shrimp (Penaeus chinensis )in Chinese coastal waters of the Huanghai Sea and coastal waters of the Bohai Sea

Genetic diversity of 32 individuals of P. chinensis in the Chinese coastal waters of the Huanghai and Bobal Seas was detected by RAPD technique. Twenty decamer primers of OPI were used for DNA amplification for each individual. The results showed that: Seventeen primers obtained reproducible fingerprints, and the bands were clear. Thirty--nine of 106 loci detected were polymorphic, amounting to 36. 8 %. Mean genetic distance was 0. 094 1 0. 020 6; 68 markers (63. 2 % of the total) showed stable homogeneity in all of the 32 individuals.

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.

Application of RAPD technology for identification in three different stocks of Penaeus chinensis

The genetic structure of three stocks (K, H and S) of Penaeus chinensis was detected by RAPD method which useed 41 random oligonucleotide primers with 10 bases. Stock K was from the spawning stock of the western coastal waters of the Korea Peninsula, H was from the Huanghai Sea-Bohai Sea stock and S was from the health cultured stock in Jimo, Shandong Province, China. Three genetic markers, F-02(540), OPN-13(800) and OPN-20(2200), which could differentiate the three different stocks, have been reported. The basic data for marker assisted selection (MAS) on genetic breeding of Penaeus chinensis are provided.

Study on purification and ultrastructure of a baculovirus in Penaeus chinensis

A kind of baculovirus was isolated from the cephalothorax homogenate of sick or morbid Penaeus chinensis by differential centrifugation and density gradient centrifugation. Electron microscopic examination of ultrathin section of the gills, stomach and mid-gut tissues also revealed the presence of rod-shaped baculoviral particles with the same size in the affected cell nuclei, where most of the virions arranging in cluster assembled and caused a series of cytopathic changes. The virion covered with bilaminal envelope was 320 ~ 400 nm × 100 ~ 130 nm in size, whereas the nucleocapsid ranged in size of 250~ 300 nm in length and 70 ~ 100 nm in breadth respectively. No nuclear polyhedron or granulin occlusion theies have been found in cells. According to the principle of viral classification, this newly found virus could probably belong to the non-occluded subgroup of insect baculoviridae, i. e., C subgroup baculovirus.

A STUDY ON PATHOGENS OF CHINESE PRAWN (PENAEUS CHINENSIS) VIRUS DISEASES

This pathogenic study shows that the viral diseases of Chinese prawns (Penaeus chinensis. O’sbeck)is due to three kinds of viruses: epithelium envelope baculovirus of Penaeus chinensis (EEBV-PC, de-tected by the authors in 1993), infections hypodermal and hematopoietic necrosis virus, andhepatopancreatic parvo-like virus, and that the first two viruses seem to be the main pathogens of theepidemic in the northern regions in 1993.

Genetic diversity and differentiation of three populations of Penaeus monodon Fabricus

Genetic diversity of two wild Penaeus monodon populations sampled from the coastal waters of Qinglan （ Hainan Province of China, HN） and Malaysia （KD）, and the F1 generation of a Thailand broodstock population （CP） were examined by vertical polyacrylamide gel electrophoresis. Of 21 loci encoded by ten enzymes, 11 were polymorphic. The mean proportions of polymorphic loci of HN, KD and CP were 36. 36%, 45.45% and 50.00% , with the average heterozygosities of 0.135, 0.181 and 0.191, and the effective numbers of alleles per loci were 1. 300, 1. 330 and 1. 329, respectively. The divergent indexes of HN, KD and CP were0.023, 0.124, and 0.117, respectively. The genetic distance between the two wild populations was 0.005, and the gene differentiation coefficient was also very low （0.014）. The results indicated that the F1 generation population had a higher genetic diversity than the two wild populations, and that the HN population had the lowest one. There was no significant differentiation between HN and KD populations and all populations existed with a slight heterozygote excess.

Effects of Rare Earths and Its Complex with Extract from Seaweed (Laminaria japonica) on Growth and Immunization of Penaeid Shrimp (Penaeus vannamei)

The effects of rare earths (RE) and its complex with extract from seaweed (Lamiaria japonica) on the growth and immunization of penaeid shrimp (Penaeus vannamei) were investigated. The results show that the survival rate, body length and weight of the shrimp treated by RE and its complex with extract from seaweed are higher than those of the control, and that the activities of the phenoloxidase (PO), lysozyme (LSZ), superoxide dismutase (SOD), peroxidase (POD), acid phosphatase (ACP) and alkaline phosphatase (ALP) in the shrimp treated by RE and the complex are also much higher than those from the control.

DETECTION OF WHITE SPOT SYNDROME VIRUS(WSSV) OF PENAEUS CHINENSIS BY IN SITU HYBRIDIZATION

White Spot Syndrome Virus (WSSV) was purified from hemolymph of infected shrimp. After nucleic acid extraction from the purified virus particles, EcoR I digested fragments of the WSSV genome were cloned; three of these fragments were used as non radioactive probes labeled with DIG 11 dUTP. The probes hybridized in situ, with sections located in the nuclei of all WSSV infected tissues. The virus was detected in the gill, stomach, epidermis, and connective tissue and so on, but not detected in healthy shrimp tissues and epithelial cells of hepatopancreatic tubules of diseased shrimp.

TRIPLOIDY INDUCTION WITH HEAT SHOCKS TO PENAEUS CHINENSIS AND THEIR EFFECTS ON GONAD DEVELOPMENT

Heat shocks effectively produced triploids in Penaeus chinensis . Fertilized eggs heat shocked (28-32℃) for 8 to 16 minutes, starting from 8 to 20 minutes after spawning, resulted in triploidy induction rates of 39%-75%. Several triploid populations were cultured to 10 cm. In a triploid population, two kinds of ovaries were observed. Histological examination showed apparent differences between these two kinds of ovaries, whereas among male shrimp, there were no such differences.

The Influences of Mg^(2+), Ca^(2+) and Mg ^(2+)/Ca ~ (2+) Ratio in Mixed Seawater on the Emergence Rate of Penaeus japonicus Postlarva

This paper reports the approprite ranges of Mg 2+, Ca 2+ and their ratio Mg 2+/Ca 2+ in mixed seawater for rearing of Penaeus japonicus larvae. The ranges for the above three indices are 1150-1450 mg/L, 360-440 mg/L and 2.8-3.4, respectively. The proper salinity range of mixed seawater is 22.1-33.8 obtained by mixing estuarine water and concentrated seawater.

Transcriptome Analysis Provides New Insights into Host Response to Hepatopancreatic Necrosis Disease in the Black Tiger Shrimp Penaeus monodon

The hepatopancreatic necrosis disease(HPND)is a common shrimp disease.Aquaculture of the black tiger shrimp Penaeus monodon has suffered with the frequent outbreak of HPND.Understanding the molecular mechanisms of host response to HPND is of pivotal importance for improving its farming performance.In this study,the RNA-Seq platform was utilized to investigate transcriptomic changes of the hepatopancreas in pond-cultured P.monodon with HPND symptoms.A total of 62071 genes with an average length of 953 bp were obtained,and a lot of simple sequence repeat(SSR)loci related to these genes were identified.Totally,5204 differentially expressed genes(DEGs)were detected between healthy shrimp and HPND shrimp,with 3399 genes upregulated and 1,805 genes down-regulated.These genes had a wide range of biological functions,and several well-known immunerelated genes including integrin alpha 5,integrin beta 1,C type lectin and catalase were among the DEGs.Cell signaling pathways including the extracellular matrix(ECM)receptor interaction,phagosome and lysosome pathways were significantly upregulated in HPND-infected shrimps,indicating their involvement in the immune responses of shrimp against HPND.The data obtained in this study offers new insights into the molecular mechanisms of shrimp host response to HPND disease,and provides a resource for molecular marker development and genetic breeding studies of P.monodon.

Computational identification and characterization of microRNAs and their targets in Penaeus monodon

Study on shrimp miRNAs was limited and just 7 mature miRNA sequences of Marsupenaeus japonicus are deposited in mir Base database. In this study, miRNAs and their target gene candidates were computationally identified from shrimp Penaeu s monodon and then experimentally validated. Using 39 908 expressed sequence tags(ESTs) and 21 124 genome survey sequences(GSSs) of P. monodon(pmo) as reference dataset, a comprehensive approach based on inter-species homolog search was employed to investigate the candidate miRNAs(i.e. pmo-miRNA). A total of eight miRNAs belonging to 7 families were computationally identified and five out of them were subsequently validated by PCR and sequencing. Of these, pmo-miR-4961a, pmo-miR-4961b, pmo-miR-4979 and pmo-miR-3819 were first identified from shrimps. Both the mature pmo-miRNAs and the corresponding precursors were conserved among different species. Based on perfect or near-perfect match to the target region, the target gene candidates of pmomiRNAs were predicted from 10 331 mRNA sequences of P. monodon. A total of 20 genes were predicted as the targets of pmo-miR-4961a, pmo-miR-4961b, pmo-miR-4979 and pmo-miR-6492. Experimental validation by dual luciferase reporter assay confi rmed the targeting between 3 pmo-miRNAs and one or two of their target genes, especially the pmo-miR-4979 which could significantly down-regulate the expression of target gene(JR226772). This study updates the miRNAs and their targets in P. monodon and lays a solid foundation for future RNAi study.

Distribution of Bacteria Injected in Body of Giant Black Shrimp, Penaeus Monodon

Distribution of injected Vibrio anguillarum in body of Penaeus monodon was studied with immunohistochemical method. Bacteria could be detected throughout the experiment in some individuals; however in lymphoid tissue, gill, heart and haemolymph of all vibrio injected shrimp, the bacteria could be observed only 5 min after injection. The bacteria density in haemolymph, haemolymph of the hepatopancreas and gills decreased with time. In the lymphoid organ and heart, the bacteria density was the highest 48 h after injection, then decreased. Nodules could be formed in the heart, lymphoid organ and injection site.