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.

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.

Construction of full length cDNA expression library of hepatopancreas of Penaeus monodon

mRNA was isolated from the hepatopancrease of shrimp Penaeus monodon with a PolyAT-tract System 1000 Kit. By using mRNA as template, double - strand cDNA with EcoR I/Xho I ends was synthesized by using a ZAP Express cDNA Synthesis Kit. The cDNA was inserted into the lambda ZAP Express vector predigested with EcoR l/Xho Ⅰ, and the recombinant DNA was in vitro packaged into lambda phage with GigapackⅢ Gold packaging extracts. These recombinant phages were then used to transfect E. coli XL1 - Blue MRF', and finally a cDNA expression library was constructed. The library is 7.2 × 10~5pfu in capacity and its recombination ratio is higher than 99 % . The size of the inserted cDNAs was determined by EcoR l/Xho I digestion of 9 phagemids prepared by in vivo excision of plaques selected randomly from amplified cDNA library . The longest inserted cDNA is about 1.6 kb in length. The complete sequence (about 1.2 kb) of actin cDNA was amplified from the library by PCR reveals that this library contains full-length cDNAs of Penaeus monodon hepatopancreas and is available for screening and expression of shrimp genes.

Changes in oviduct structure in the black tiger shrimp,Penaeus monodon,during ovarian maturation

Objective:To examine the structure of the oviduct of the shrimp Penaeus monodon.Methods:The oviducts of P.monodon with three different major groups of ovarian development(Group(Gr.) 1:Stages I & V;Gr.2:Stages II & III;and Gr.3:Stage IV) were examined by light,transmission electron,and scanning electron microscopies,respectively.Results:The epithelium of the oviduct in Gr.1 was composed of tall simple columnar cells with their basal nuclei located on the basement membrane and its thick collagen fibers.In Gr.2,the oviduct seemed to produce some substances and their epithelial cells became transitional with centrally located nuclei and formed some vacuoles.Obviously,the epithelial cells in Gr.3(at Stage IV) were disorganized,disrupted,and shed accumulated spherical secretory substances including some cellular contents into the lumen.Conclusions:The structural changes of the P.monodon oviduct were related to ovarian maturation stages(Grs.1-3).Prior to spawning,only the oviduct epithelium at ovary Stage IV produced and secreted a number of spherical secretion substances into the lumen.These substances may act as the oviductal lubricants to facilitate the spawning process.

Effects of extraction parameters on physicochemical and functional characteristics of chitosan from Penaeus monodon shell

Objective:To investigate the effect of extraction parameters with particular interest during the microwave deacetylation process on the characteristics of chitosan produced from Penaeus monodon(P.monodon)sourced within the coastal region of Lagos,Nigeria for applications of controlled release systems for pharmaceutical industries.Methods:Chitosan was extracted from shrimp(P.monodon)shell and evaluated as a controlled release system for curcumin.Effects of relevant processing parameters on physicochemical and functional characteristics of the extracted chitosan were assessed.The crude chitosan was purified and used to prepare controlled release formulations for curcumin via ionic gelation with tripolyphosphate.Results:Data from the study showed that increasing time and temperature during deproteinization significantly improved the removal of protein bound to the shell matrix.Also,the ratio of the weight of the deproteinized sample to the volume of HCl used for demineralization influenced the process.During microwave-assisted production of chitosan from chitin,increase in the concentration of the deacetylating medium significantly increased solubility,viscosity and degree of deacetylation,whereas increasing temperature and time during deacetylation of chitin degraded the biopolymer to give low molecular weight chitosan.Optimized extraction and purification process yielded absolutely soluble medium to low molecular weight chitosan.The encapsulation efficiency,loading capacity,percentage yield,release efficiencies in simulated gastric and intestinal fluids of curcumin loaded in the formulations of chitosan from P.monodon were compared favorably to encapsulation and release characteristics of the encapsulated curcumin in commercially available chitosan used as the reference.Conclusions:Valorization of shrimp waste into pharmaceutically graded medium molecular weight chitosan was achieved.The chitosan obtained can be used as a controlled release system for phyto-pharmaceuticals.

Revealing localization and regulation of GTPase PmRab7 in lymphoid cells of Penaeus monodon after WSSV infection

Objective:To identify white spot syndrome virus(WSSV)entry into the host-cells of the cultured shrimp Penaeus monodon,we have attempted to localize PmRab7(Ras-related in brain)which is playing a vital role in the WSSV internalization.Methods:In this study,we have cloned PmRab7 and expressed in Escherichia coli,further purified rPmRab7 was used for antibody production,isolation of lysosomal sub-cellular fractions and western blot against lysosomal protein.Moreover,high fold-change in PmRab7 regulation with increasing copy number of WSSV has been studied by using real-time PCR.Results:651 bp amplicon size gene was successfully amplified,ligated amplicon with pTZ T-tail vector confirmed by colony PCR and retriction enzyme digestion on agarose gel.Subcloned(pRSET-B)651 bp gene transformed successfully in Rosetta and after 6 h of induction expressed rPmRab7 was on SDS page,furthermore soluble fraction of rPmRab7(26 kDa)was purified by ni-NTA column.AntiPmRab7 antibody was received by Merk Pvt.Ltd.,and western blot analysis revealed that PmRab7 is present in the lysosomal sub-cellular fraction.Copy number of WSSV was increased 5 fold in 24 h and 20 fold in 72 h of infection and subsequently transcrtipt of PmRab7 was Ct=1.0 to Ct=8.5.Conclusions:Presence of PmRab7 on lysosome clearly indicating PmRab7 participating in lysosomal maturation,other hand WSSV may follow the same route of entry.WSSV internalization has directly linked with regulation of PmRab7.

中纬度地区内陆盐碱地大规格斑节对虾养殖技术

在中纬度盐碱地池塘,开展了斑节对虾(Penaeus monodon)大规格养成试验。提前一个月将对虾苗种在温室大棚内标粗,气温升到22℃以上在外塘进行接续养殖,通过对盐碱水离子检测与调配,将Na^(+)∶K^(+)调整为28~30∶1,Ga^(2+)∶Mg^(2+)调节至1∶2.8~3.0。池塘产大规格斑节对虾3809.5 kg/hm^(2),平均规格34尾/kg,利润11.55万元/hm^(2)。

北方斑节对虾工厂化养殖成功经验

近几年,北方工厂化养殖车间由于夏季炎热没有适宜养殖的水产品种,多数空置。2022年5月引进斑节对虾(Penaeus Monodon)虾苗100万尾进行工厂化养殖。经过180 d的养殖,共计出虾2.25万千克,成活率达到50%。产值190万元,总获利62万元。

Study on genetic diversity of cultivated populations in 4 species of shrimps

Genetic diversity of cultivated populations was investigated using assay of vertical slabpolyacrylamide gel electrophoresis in 4 species of shrimps, which were Penaeus japonicus, Penaeus monodon , Penaeus vannamei and Metapenaeus ensis, The results showed that the mean number of alle-les per locus (A) is 1.3±0.2, 1.3±0.1, 1.3±0.1 and 1.3 ±0.1 respectively; the percentage of polymorphic loci (P0.95) is 12.5, 6.7, 20 and 23.5 respectively; the expected heterozygosity (He) is 0.042±0.034,0.042±0.031, 0.094±0.042 and 0.097±0.047 respectively; and the observed heterozygosity (H0)is 0.029±0.024, 0.028±0.023,0.154±0.082 and 0.150±0.084 respectively. The difference of genetic diversity is obvious in 4 species of shrimps. The degree of genetic diversity is M. ensis > P. vannamei > P. japonicus > P. monodon. In short, the lower level of genetic diversity is estimated in 4 species of shrimps.

盐度对非洲斑节对虾幼体培育成活率的影响

试验对不同盐度非洲斑节对虾(Penaeus monodon)幼体培育总成活率及各阶段成活率与变态时间进行了研究,旨在为非洲斑节对虾人工育苗的成功提供基础数据。采用卤水调节,设置盐度25,26,27,28四个梯度试验组,每组3个平行,经15 d培育,结果表明:盐度变化对非洲斑节对虾幼体总成活率影响差异具统计学意义(P<0.05),盐度25~27时,随着盐度升高,幼体成活率从22%上升到41.3%,盐度27和28试验组差异不具统计学意义(P>0.05)。结果还表明盐度对无节幼体(N)、蚤状幼体(Z)、糠虾幼体(M)以及仔虾幼体(P)各阶段成活率影响不同,N与Z阶段:盐度影响显著,盐度在25~27,随着盐度升高,成活率随之增加,当盐度27与28时差异不具统计学意义;M阶段:盐度在25时成活率最低,26~28时成活率无差异统计学意义;P阶段,盐度对各试验组成活率影响差异不具统计学意义(P>0.05)。说明非洲斑节对虾幼体对盐度的耐受性强弱为N<Z<M<P。不同盐度对非洲斑节对虾幼体各阶段变态发育时间有影响,但差异不具统计学意义(P>0.05)。综上所述,非洲斑节对虾幼体培育的盐度不宜低于27。

Ecology of antibiotic resistant vibrios in traditional shrimp farming system(bhery)of West Bengal,India

Objective:To study the ecology of antibiotic resistant bacteria with emphasis on sucrose negative vibrios in water and sediments samples of traditional shrimp farming system(bhery)in West Bengal,India.Methods:The vibrios were isolated from traditional shrimp farm samples on thiosulphate citrate bile salt sucrose agar and sucrose negative bacterial strains were used as biomarkers to assess the frequency of antibiotic resistance.Results:The incoming water brought presumptive vibrios ranging from 5.50×10^(1)to 1.00×10^(3)mL in to the bhery,and there appeared to build up vibrios in the culture system with days of culture,as there was about 9 fold increase in vibrios.The levels of vibrios were observed to be moderately higher in outlet water and ranged between 4.15×10^(2)and 4.15x10^(3)mL.The counts of vibrios in pond sediment was found to be 1.00x10^(2)-4.90×10^(3)g;while in inlet(2.00×10^(2)-4.20×10^(4)g)and outlet(3.00×10^(2)-6.85×10^(3)g)their levels were observed to be higher than the pond sediment.Thirteen different Vibrio species were encountered in traditional shrimp culture system and all vibrios were sensitive to chloramphenicol,followed by ciprofloxacin and gatifloxacin(98.24%),gentamicin(95.61%)and other antibiotics.The multiple antibiotic resistance(MAR),i.e.,resistance to at least two antibiotics,was noticed among 43.85%of the sucrose negative vibrios and 41.86%of the sucrose negative non--vibrios.All vibrios harveyi strains exhibited MAR.Although no antibiotic was used in the bhery,the prevalence of MAR in 44%of the sucrose negative vibrios and nonvibrios is a cause of concern.The MAR index was higher in inlet water and sediment samples.The MAR observed in biomarker strains of pond water and sediment(40%)was comparable to those of inlet samples,thus confirming the fact that incoming water was the major source of antibiotic resistant bacteria.Conclusions:It seems that the shrimp culture in bhery does not favour the proliferation and spread of antibiotic resistant bacteria.