凡纳滨对虾(Litopenaeus vannamei)盐度调控相关基因CLCA1的克隆及表达分析

钙激活氯离子通道调节剂(calcium-activated chloride channel regulator,CLCA)为一类金属蛋白酶依赖家族,在哺乳动物上皮组织杯状细胞的粘液产生和粘液平衡中起重要作用.为了探究CLCA1基因在凡纳滨对虾渗透压中的作用机制,本研究采用RACE技术在凡纳滨对虾中克隆出了CLCA1基因,该基因总长度为3129 bp,5'端非编码区长度为175 bp,3'端非编码区长度为107 bp,开放阅读框ORF长度为2847 bp,共编码984个氨基酸,有1个跨膜区域,膜外有VMA结构域.与其他物种氨基酸序列比对结果显示,凡纳滨对虾CLCA1氨基酸与其他物种相似性都较低,相似度最高的为刀额新对虾(Procambarus clarkii) CLCA2氨基酸序列,为47.83％.RT-qPCR结果显示,CLCA1基因在凡纳滨对虾各组织中均有表达,其中肠道、肝胰腺和鳃中的表达量较高;对不同盐度下凡纳滨对虾4个组织中CLCA1基因的定量结果显示,随着盐度的下降,CLCA1基因的表达量在4个组织中呈下调趋势,表明CLCA1基因与凡纳滨对虾的渗透压调节相关.本研究初步阐明了CLCA1基因的理化性质,对CLCA1基因在凡纳滨对虾体内各组织和不同盐度下各组织的表达定量可为CLCA1基因在今后甲壳动物的渗透压调节及免疫调节的研究提供基础支持.     

Transmission of white spot syndrome virus (WSSV) to Litopenaeus vannamei from infected cephalothorax, abdomen, or whole shrimp cadaver.

Shrimp viruses can remain infectious in frozen shrimp tissue and have been found in frozen commodity shrimp. Therefore, the threat of viral outbreaks in wild and cultured shrimp via frozen commodity shrimp exists. Because frozen shrimp are imported with and without the cephalothorax, more knowledge is needed concerning the infectivity of a cephalothorax relative to that of an abdomen. We compared the mortality rates from shrimp exposed to a WSSV-infected cephalothorax, abdomen, or whole shrimp cadaver. Estimates of transmission coefficients from the exposures to the infected cephalothorax, abdomen, or whole shrimp were also calculated because the transmission coefficients account for differences in the initial doses. In addition, we compared the variability in infectivity of pieces of shrimp by feeding 24 equal-sized pieces of cephalothorax and abdomen to 24 individually isolated shrimp. In Expt 1, susceptible shrimp did not completely consume the infected abdomen, and a significant difference was detected among shrimp exposed to the abdomen (mortality rate = 0.40), cephalothorax (mortality rate = 0.75), and whole shrimp cadaver (mortality rate = 0.67). The calculated transmission coefficients were 0.95 from an infected cephalothorax, 0.59 from an infected abdomen, and 0.69 from an infected whole shrimp cadaver. In Expt 2, susceptible shrimp were starved to ensure complete ingestion of each dose. No significant difference was observed in the estimated mortality rates from an infected cephalothorax (0.58), abdomen (0.63), or whole shrimp (0.67). The calculated transmission coefficients were 0.84 from an infected cephalothorax, 0.83 from an infected abdomen, and 0.60 from an infected whole shrimp cadaver. In Expt 3, no difference was observed in the mortality rates resulting from exposures to pieces of infected cephalothorax (0.57) or abdomen (0.58). Our results suggested that there was no difference in the viral loads of a WSSV-infected cephalothorax or abdomen, but that the cephalothorax was more infectious, probably because it was more palatable. In addition, our results are inconsistent with some assumptions of pathogen transmission used in epidemiological models. Some shrimp may be less aggressive feeders; therefore, susceptible shrimp are differentially contacting the dead infected shrimp in the exposure tanks, violating the random mixing assumption. Moreover, virus is probably not homogeneously distributed throughout an infected shrimp, suggesting that contacts between susceptible and infected shrimp are not equally likely to result in transmission.     

Antimicrobial Peptides (AMPs): Roles,Functions and Mechanism of Action

International Journal of Peptide Research and Therapeutics - Antimicrobial peptides (AMPs) are a crucial part of innate immunity that exist in the most of living organisms. In fact, AMPs have...     

Pathogenesis of a Thai strain of white spot syndrome virus (WSSV) in juvenile, specific pathogen-free Litopenaeus vannamei

White spot syndrome virus (WSSV) causes disease and mortality in cultured and wild shrimp. A standardized WSSV oral inoculation procedure was used in specific pathogen-free (SPF) Litopenaeus vannamei (also called Penaeus vannamei) to determine the primary sites of replication (portal of entry), to analyze the viral spread and to propose the cause of death. Shrimp were inoculated orally with a low (10(1.5) shrimp infectious dose 50% endpoint [SID50]) or a high (10(4) SID50) dose. Per dose, 6 shrimp were collected at 0, 6, 12, 18, 24, 36, 48 and 60 h post inoculation (hpi). WSSV-infected cells were located in tissues by immunohistochemistry and in hemolymph by indirect immunofluorescence. Cell-free hemolymph was examined for WSSV DNA using 1-step PCR. Tissues and cell-free hemolymph were first positive at 18 hpi (low dose) or at 12 hpi (high dose). With the 2 doses, primary replication was found in cells of the foregut and gills. The antennal gland was an additional primary replication site at the high dose. WSSV-infected cells were found in the hemolymph starting from 36 hpi. At 60 hpi, the percentage of WSSV-infected cells was 36 for the epithelial cells of the foregut and 27 for the epithelial cells of the integument; the number of WSSV-infected cells per mm2 was 98 for the gills, 26 for the antennal gland, 78 for the hematopoietic tissue and 49 for the lymphoid organ. Areas of necrosis were observed in infected tissues starting from 48 hpi (low dose) or 36 hpi (high dose). Since the foregut, gills, antennal gland and integument are essential for the maintenance of shrimp homeostasis, it is likely that WSSV infection leads to death due to their dysfunction.     

凡纳对虾SWD抗菌肽基因的克隆与性质研究

目的:为了进一步探索抗菌肽分子的作用机制,我们以凡纳对虾具有单个WAP结构域的抗菌肽基因作为研究对象,进行分子生物学方面的分析。方法:通过cDNA全长序列的扩增,以及氨基酸序列的比较分析、系统进化树的分析以及分子结构的初步预测,我们对凡纳对虾SWD分子的结构进行了详细分析。结果:Lv-SWD的cDNA序列全长为434bp,编码92个氨基酸;Lv-SWD存在由24个氨基酸残基形成的信号肽序列,以及由8个保守存在的半胱氨酸残基形成的WAP结构域和一段富含脯氨酸的结构基序。结论:通过这些分子结构的研究,以及与其他SWD分子的比较,作者推测Lv-SWD分子是一种具有抑菌活性的抗菌肽分子,它在凡纳对虾的先天免疫系统中应该发挥着重要的免疫功能。     

White spot syndrome virus (WSSV) in Litopenaeus vannamei captured from the Gulf of California near an area of extensive aquaculture activity

For the shrimp farming industry of Mexico, disease outbreaks caused by white spot syndrome virus (WSSV) are relatively recent. Efforts to control the virus are assisted by monitoring for its prevalence in aquaculture systems, but few attempts have been made to search for it in carriers from coastal waters. To search for WSSV carriers in the Gulf of California, we made surveys off the coast of Sinaloa, Mexico, in March 2001, November 2001, and September 2003 using polymerase chain reaction (PCR) assays and histopathology. WSSV-positive shrimp were detected only in November 2001, after hurricane Julliete. This suggested possible dispersal of WSSV to the marine environment from infected shrimp farms.     

In vivo titration of white spot syndrome virus (WSSV) in specific pathogen-free Litopenaeus vannamei by intramuscular and oral routes

White spot syndrome virus (WSSV) is a devastating pathogen in shrimp aquaculture. Standardized challenge procedures using a known amount of infectious virus would assist in evaluating strategies to reduce its impact. In this study, the shrimp infectious dose 50% endpoint (SID50 ml(-1)) of a Thai isolate of WSSV was determined by intramuscular inoculation (i.m.) in 60 and 135 d old specific pathogen-free (SPF) Litopenaeus vannamei using indirect immunofluorescence (IIF) and 1-step polymerase chain reaction (PCR). Also, the lethal dose 50% endpoint (LD50 ml(-1)) was determined from the proportion of dead shrimp. The median virus infection titers in 60 and 135 d old juveniles were 10(6.8) and 10(6.5) SID50 ml(-1), respectively. These titers were not significantly different (p > or = 0.05). The titration of the WSSV stock by oral intubation in 80 d old juveniles resulted in approximately 10-fold reduction in virus titer compared to i.m. inoculation. This lower titer is probably the result of physical and chemical barriers in the digestive tract of shrimp that hinder WSSV infectivity. The titers determined by infection were identical to the titers determined by mortality in all experiments using both i.m. and oral routes at 120 h post inoculation (hpi), indicating that every infected shrimp died. The determination of WSSV titers for dilutions administered by i.m. and oral routes constitutes the first step towards the standardization of challenge procedures to evaluate strategies to reduce WSSV infection.     

南美白对虾传染性皮下及造血组织坏死病毒抗性相关基因的微卫星标记研究

传染性皮下及造血组织坏死病毒(infectious hypodermal and hematopoietic necrosis virus,IHHNV)是南美白对虾养殖中最普遍的虾病毒之一.它能引起生长缓慢和畸形,通称矮小畸形综合症(RDS).分子标记是研究抗病基因的有效手段之一,微卫星(microsatellite)标记是新发展的遗传标记.介绍用微卫星技术对南美白对虾抗病和感病群体筛选出的IHHNV抗性相关基因进行研究.     

Expression and applications of recombinant crustacean hyperglycemic hormone from eyestalks of white shrimp (Litopenaeus vannamei) against bacterial infection

Crustacean hyperglycemic hormone (CHH) has many functions to regulate carbohydrate metabolism, ecdysis and reproduction including ion transport in crustaceans. The cDNA encoding CHH peptides containing 369 bp open reading frame encoding 122 amino acids was cloned from eyestalk of white shrimp (Litopenaeus vannamei) and was produced by a bacterial expression system. The biological activity of recombinant L. vannamei crustacean hyperglycemic hormone (rLV-CHH) was tested. The hemolymph glucose level of shrimp increased two-fold at 1h after the rLV-CHH injection and then returned to normal after 3h. In addition to the effect of rLV-CHH administration (25 μg/shrimp) on immunological responses of white shrimp against pathogenic bacteria, Vibrio harveyi was studied. Results showed that the blood parameters of shrimp injected with rLV-CHH; the THC, PO activity, serum protein level and clearance ability to V. harveyi, were also higher than those of Neg-protein and PBS-injected shrimp. The survival of shrimp injected with rLV-CHH was significantly higher (66.0%) than shrimp that injected with Neg-protein (33.3%) and PBS (28.9%) after 14 days. It is possible that the administration of rLV-CHH in L. vannamei exhibited a higher immune response related to resistance against V. harveyi infection.     

Embryonic Expression of Vasoactive Intestinal Peptide (VIP) and VIP Receptor Genes

Abstract: Vasoactive intestinal peptide (VIP) exhibits pronounced effects on the growth rate of cultured mouse embryonic day (E) 9.5 embryos and acts in tissue culture as a potent glial mitogen and neuron survival factor. However, previous studies using immunohistochemistry or in situ hybridization in the rat have not revealed the presence and location of VIP or VIP mRNA in the early developing embryo CNS. Using a sensitive in situ hybridization assay with a ³³P-labeled riboprobe, we show here that the VIP gene is expressed at least as early as E11 in the mouse hindbrain. Northern blot analysis on RNA from brain dissected from mouse embryos beginning at E14 confirmed that a correct-size mRNA for VIP was present by E14 and at later time points. Expression of the VIP2 receptor gene was also detected by northern analysis in E14 mouse brains. These studies support the hypothesis that VIP produced by the embryo exerts important effects on embryonic nervous system development.     

Isolation and characterization of microsatellite markers from Pacific white shrimp (Litopenaeus vannamei)

We report the development of 11 polymorphic microsatellite loci in pacific white shrimp (Litopenaeus vannamei) using an unenriched genomic library. The number of the alleles ranged from two to 18 and observed hererozygosity ranged from 0.0286 to 0.9429, indicating that these markers will be useful for population studies and mapping in pacific white shrimp. Seven loci were detected deviated from Hardy–Weinberg, caused by deficiency of heterozygote, suggesting population genetic structure across the sampled population. No evidence for linkage disequilibrium was found.     

Recovery of protein,chitin, carotenoids and glycosaminoglycans from Pacific white shrimp (Litopenaeus vannamei) processing waste

Shrimp head waste is a major byproduct of crustacean processing in North-eastern Brazil and represents an interesting source of bioactive molecules. Additionally, its use increases the sustainability of processing fishery products. The present study reports a process developed for recovering bioactive molecules from shrimp heads through autolysis. A protein hydrolysate (120 ± 0.4 g) formed by a 9% (w/v) solution was recovered and lyophilized from 1 kg of shrimp heads. Approximately 195 ± 0.5 mg of carotenoids was recovered as an ethanolic extract. The recovery of chitin and chitosan were 25 ± 2 g kg^?1 and 17 ± 4 g kg^?1 wet processing waste, respectively. Chitosans were characterized by ¹³C NMR, and FT-IR analysis and exhibited a variable degree of deacetylation (60–80%). Sulfated glycosaminoglycans that exhibited electrophoretic migration similar to mammalian standards were also recovered (79 ± 2 mg kg^?1 wet processing waste), and their degradation products suggested the presence of C6-sulfated heparan sulfate. These data point to the feasibility of an integrated process for isolating highly bioactive molecules, such as sulfated- and amino-polysaccharides, with a broad spectrum of applications from shrimp processing waste.     

The function of mitochondrial F(O)F(1) ATP-synthase from the whiteleg shrimp Litopenaeus vannamei muscle during hypoxia

The effect of hypoxia and re-oxygenation on the mitochondrial complex F(O)F(1)-ATP synthase was investigated in the whiteleg shrimp Litopenaeus vannamei. A 660 kDa protein complex isolated from mitochondria of the shrimp muscle was identified as the ATP synthase complex. After 10h at hypoxia (1.5-2.0 mg oxygen/L), the concentration of L-lactate in plasma increased significantly, but the ATP amount and the concentration of ATPβ protein remained unaffected. Nevertheless, an increase of 70% in the ATPase activity was detected, suggesting that the enzyme may be regulated at a post-translational level. Thus, during hypoxia shrimp are able to maintain ATP amounts probably by using some other energy sources as phosphoarginine when an acute lack of energy occurs. During re-oxygenation, the ATPase activity decreased significantly and the ATP production continued via the electron transport chain and oxidative phosphorylation. The results obtained showed that shrimp faces hypoxia partially by hydrolyzing the ATP through the reaction catalyzed by the mitochondrial ATPase which increases its activity.     

Gene expression and protein levels of thioredoxin in the gills from the whiteleg shrimp (Litopenaeus vannamei) infected with two different viruses: the WSSV or IHHNV

The thioredoxin (TRX) system in crustaceans has demonstrated to act as a cell antioxidant being part of the immune response by dealing with the increased production of reactive oxygen species during bacterial or viral infection. Since the number of marine viruses has increased in the last years significantly affecting aquaculture practices of penaeids, and due to the adverse impact on wild and cultured shrimp populations, it is important to elucidate the dynamics of the shrimp response to viral infections. The role of Litopenaeus vannamei thioredoxin (LvTRX) was compared at both, mRNA and protein levels, in response to two viruses, the white spot syndrome virus (WSSV) and the infectious hypodermal and hematopoietic necrosis virus (IHHNV). The results confirmed changes in the TRX gene expression levels of WSSV-infected shrimp, but also demonstrated a more conspicuous response of TRX to WSSV than to IHHNV. While both the dimeric and monomeric forms of LvTRX were detected by Western blot analysis during the WSSV infection, the dimer on its reduced form was only detected through the IHHNV infectious process. These findings indicate that WSSV or IHHNV infected shrimp may induce a differential response of the LvTRX protein.