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.

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.

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.

Survey of Penaeus monodon Post Larvae in Semi Saline Zone of Kholpatua River of Satkhira Near Sundarbans Reserved Forest Bangladesh

This study monitored the present status ofPenaeus monodon post larvae （PL.） of Kholpatua river in Satkhira. The survey was conducted in Kholpatua river from January 2014 to December 2014 and was compared with the ten years old secondary data. The results showed that there was a 41.57% decrease in number of catches ofP. monodon PL. over the course of time from ten years ago to the year of 2014. Although the number of active fishermen＇s boats have increased from 250 to 360 in ten years. The study also showed that the severe shrimp seed collection activities reduced the availability ofP. monodon PL. at Satkhira district, which has a severe negative impact on the availability of total catches. To control such decrease of P. monodon PL. in Bangladesh, more intensive study should be done.

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.

盐碱地池塘斑节对虾养殖适宜放养密度的研究试验

为探索盐碱地池塘养殖斑节对虾的适宜放养密度,在条件、面积相同的盐碱地池塘,消毒、肥水后,斑节对虾标粗苗放养密度分别为10.5万、15万、19.5万、22.5万尾/hm^(2),养殖3个月后,以养成规格、成活率、生长速度、产量和效益等作为评价指标加以比较分析。研究结果表明,在10.5万至22.5万尾/hm^(2)放养密度范围内,随着密度增加,对虾规格逐渐变小,成活率降低,虽然产量有所提高,但因小规格对虾售价偏低,养殖利润较少。10.5万尾/hm^(2)密度条件下,对虾成活率高、规格大、售价高,利润最高;15万尾/hm^(2)密度条件下,产量较高,规格和价格在当地市场接受度高,利润略低,养殖业主也可根据当地市场情况考虑选择适宜养殖密度。

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.

Verification of Drainage Sterilization System Uses Low-Voltage Pulsed Electric Field in a Prawn Farm

It is necessary to take measures against infectious diseases in the Southeast Asian prawn farming industry. In giant tiger prawn （Penaeus monodon） aquaculture, diseases caused by viruses such as white spot syndrome virus （WSSV） and bacteria such as Vibrio have become a menace. Appropriate treatments of prawn culture pond＇s waters are advocated for preventing and controlling pathogens. The purpose of this study was to conduct an antimicrobial water treatment using a low-voltage pulsed electric field sterilization. Here we prepared a mechanical low-voltage pulsed electric field system with copper wire coiling around a titanium ring. The viability of WSSV in seawater was examined by prawn infectivity experiments. We inoculated healthy prawns （approximately 10-15 g） with the WSSV master sample that passed 0-3 times through the system. WSSV infection in prawns decreased according to the number of passes through the system. Healthy prawns survived for〉 10 days without feeding, where as prawns inoculated with the WSSV master sample showed symptoms of white spot and died in about 4 days. Two-thirds of the prawns inoculated with the WSSV master sample that was passed once through the system developed WSSV symptoms and were polymerase chain reaction （PCR） positive. However, no symptoms were observed and prawns were PCR negative when the WSSV master sample was passed three times through the system. Based on these results, we purpose that a low-voltage pulsed electric field system will serve as an efficient pond drainage sterilization system and will replace conventional treatments using chemicals such as sodium hypochlorite.

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.