Immune role of MrNFκBI-α, an IκB family member characterized in prawn M. rosenbergii

NF kappa B inhibitor alpha (MrNFκBI-α) was sequenced from a freshwater prawn Macrobrachium rosenbergii. The MrNFκBI-α protein contains a long ankyrin repeat region circular domain between 193 and 413 along with its 6 repeats (ankyrin repeat 1,2,3,4,5 and 6). An IκB degradation motif and a putative PEST motif is present at 37-64 and 418-471 of the N- and C-terminal regions of MrNFκBI-α respectively. The gene expressions of MrNFκBI-α in healthy and infectious hematopoietic and hypodermal necrosis virus (IHHNV), poly I:C, Aeromonas hydrophila and Enterococcus faecium injected M.?rosenbergii were examined using quantitative real time PCR. The MrNFκBI-α is expressed in all the tissues taken for examination and the highest is observed in hemocytes. The MrNFκBI-α gene expression is strongly up-regulated in hemocytes of prawn after IHHNV, poly I:C, A.?hydrophila and E.?faecium infection. This result indicates an important role of MrNFκBI-α in M.?rosenbergii immune system. This, however, remains to be verified by further studies.     

Hepatopancreas is the extraovarian site of vitellogenin synthesis in black tiger shrimp, Penaeus monodon.

The site of yolk protein synthesis in crustaceans has long been a subject of controversy. The vitellogenin gene structure was partially reported only very recently in Macrobrachium rosenbergii, after which the hepatopancreas was confirmed as the extraovarian site of vitellogenin synthesis in that species. Ovaries are the most frequently reported as the site of yolk protein synthesis in penaeid shrimp. Using cDNA reversed-transcribed from mRNA isolated from the hepatopancreas of vitellogenic female shrimp, Penaeus monodon, we found that its deduced amino acid sequence had high identity of 48% with that from M. rosenbergii vitellogenin. A similar location of the intron in the sequenced region of genomic DNA was also found between these two species. We therefore concluded that the hepatopancreas the extraovarian site of vitellogenin synthesis in P. monodon in vivo. The partial structure of vitellogenin gene is presented in this study.     

两种沼虾溶菌酶基因ORF的克隆和罗氏沼虾溶菌酶基因的组织表达(英文)

分别提取罗氏沼虾和日本沼虾血细胞总RNA,RT-PCR扩增获得特异性cDNA片段,纯化后克隆到T载体上。序列测定表明所克隆的两种沼虾溶菌酶基因的开放阅读框(ORF)为477bp,共编码158个氨基酸,包括溶菌酶成熟肽140个氨基酸残基和信号肽18个氨基酸残基。同源性分析表明,罗氏沼虾和日本沼虾溶菌酶基因的碱基序列及推测氨基酸序列高度同源,分别为99.4%和98.1%。两种沼虾溶菌酶基因的碱基序列和推测氨基酸序列与Gen-Bank上其他对虾溶菌酶的同源性达83.0%和80.0%以上。两种沼虾溶菌酶都具有c-型溶菌酶典型的两个酶活性位点(Glu51)和(Asp68),以及8个保守结构氨基酸残基Cys,且在101、106和107位上缺少Asp,因而推测本实验所克隆的两种沼虾溶菌酶基因属c-型溶菌酶基因的非钙结合亚型。以PCR法制备罗氏沼虾溶菌酶基因的生物素标记探针,斑点杂交检测感染弧菌后溶菌酶基因mRNA在各组织中的转录水平,结果表明受感染6h后在眼、肌肉、鳃、肝胰腺、肠管中的表达量均有升高,其中在肝胰腺中的表达量最高,约为对照组的560%。在不同感染时间里,肝胰腺中该基因表达量有较大的变化:感染后3h表达量最低,24h后表达量升至最高,大约为对照组的430%,48h时的表达量又有所下降,但仍明显高于对照组(约为330%)。受弧菌感染后罗氏沼虾溶菌酶基因转录的上调证明溶菌酶基因在非特异性免疫中的直接作用,同时表明肝胰腺可能在沼虾的免疫防御过程起重要作用。       

Production of monoclonal antibodies specific to Macrobrachium rosenbergii nodavirus using recombinant capsid protein

The gene encoding the capsid protein of Macrobrachium rosenbergii nodavirus (MrNV) was cloned into pGEX-6P-1 expression vector and then transformed into the Escherichia coli strain BL21. After induction, capsid protein-glutathione-S-transferase (GST-MrNV; 64 kDa) was produced. The recombinant protein was separated using SDS-PAGE, excised from the gel, electro-eluted and then used for immunization for monoclonal antibody (MAb) production. Four MAbs specific to the capsid protein were selected and could be used to detect natural MrNV infections in M. rosenbergii by dot blotting, Western blotting and immunohistochemistry without cross-reaction with uninfected shrimp tissues or other common shrimp viruses. The detection sensitivity of the MAbs was 10 fmol μl-1 of the GST-MrNV, as determined using dot blotting. However, the sensitivity of the MAb on dot blotting with homogenate from naturally infected M. rosenbergii was approximately 200-fold lower than that of 1-step RT-PCR. Immunohistochemical analysis using these MAbs with infected shrimp tissues demonstrated staining in the muscles, nerve cord, gill, heart, loose connective tissue and inter-tubular tissue of the hepatopancreas. Although the positive reactions occurred in small focal areas, the immunoreactivity was clearly demonstrated. The MAbs targeted different epitopes of the capsid protein and will be used to develop a simple immunoassay strip test for rapid detection of MrNV.     

Localization of vitellogenin mRNA expression and vitellogenin uptake during ovarian maturation in the giant freshwater prawn Macrobrachium rosenbergii

In situ hybridization and immunohistochemical techniques were used to investigate the dynamics of vitellogenin (Vg) mRNA expression and Vg uptake during ovarian maturation in the hepatopancreas and ovary at differing stages of ovarian maturation in both intact and eyestalk ablated female Macrobrachium rosenbergii. In the hepatopancreas of intact animals, Vg mRNA expression was detected faintly two days after ecdysis, and signals showed a gradual increase as the molt cycle advanced to the premolt stages, but decreased at the late premolt stage. Vg mRNA was detected in the R-cells of the hepatopancreas, indicating that these cells are responsible for synthesizing Vg. No Vg mRNA expression was observed in the ovary. Immunohistochemistry results for the hepatopancreas showed a pattern of staining intensity similar to that of in situ hybridization. Increases in the accumulation of yolk protein in the oocytes occurred concomitantly with increasing Vg mRNA expression. In eyestalk ablated animals, Vg mRNA expression and Vg uptake showed similar but accelerated patterns to those of intact animals. This study has confirmed on the cellular level previous results that Vg synthesis is intrinsically correlated to ovarian maturation and the molt cycle in M. rosenbergii.     

Dynamics of vitellogenin mRNA expression and changes in hemolymph vitellogenin levels during ovarian maturation in the giant freshwater prawn Macrobrachium rosenbergii

The dynamics of vitellogenin mRNA expression during ovarian maturation in Macrobrachium rosenbergii were examined by measuring hemolymph vitellogenin (Vg) levels and Vg mRNA expression in the hepatopancreas and ovary at differing reproductive stages in both intact and eyestalk ablated animals. Vg mRNA was quantified using real-time RT-PCR and hemolymph Vg was measured by enzyme immunoassay. In intact animals, Vg mRNA levels in the hepatopancreas and hemolymph Vg levels showed a gradual increase during the molt cycle concomitant with increasing gonadosomatic index (GSI), with Vg levels decreasing prior to ecdysis although GSI continued to increase. Eyestalk ablation was seen to accelerate Vg synthesis as well as ovarian maturation, although it did not alter the overall pattern of Vg expression. Vg mRNA expression was negligible in the ovary of both intact and eyestalk ablated animals, confirming that the hepatopancreas is the principal site of Vg synthesis in M. rosenbergii with the ovary being only a minor contributor. This study has shown that Vg synthesis is correlated to ovarian maturation and the molt cycle in M. rosenbergii.     

Immune response of white shrimp, Litopenaeus vannamei, after a concurrent infection with white spot syndrome virus and infectious hypodermal and hematopoietic necrosis virus

In the present study, we investigated immunological changes in viral-infected white shrimp, Litopenaeus vannamei. White shrimp were infected with white spot syndrome virus (WSSV) or co-infected with WSSV and infectious hypodermal and hematopoietic necrosis virus (IHHNV) as detected by polymerase chain reaction (PCR). The complete (100%) mortality rate of shrimp was caused by viral infection due to immune parameters being suppressed including decreases in phenoloxidase activity, total hemocyte counts, differential hemocyte counts, and the gene expressions of prophenoloxidase and peroxinectin. In addition, increases in lipopolysaccharide and beta-1,3-glucan-binding protein of hemocytes and the hepatopancreas, and respiratory bursts per cell, and a decrease in superoxide dismutase were found in viral-infected shrimp, which may have been related to the defense against viral infection.     

Rediscovery of the Australian strain of infectious hypodermal and haematopoietic necrosis virus

In this study we rediscovered and verified the presence of an infectious hypodermal and haematopoietic necrosis virus (IHHNV) strain amongst cultured penaeid prawns Penaeus monodon from 1993 in Australia on the basis of a PCR analysis with IHHNV specific primers and sequencing of the resulting amplicons. A total of 7 previously published diagnostic primers specific to IHHNV were tested against Australian penaeid prawns and only 1 elicited a positive IHHNV PCR result with 16 out of 20 Australian P. monodon samples examined. In comparison, all 7 primers produced IHHNV-positive amplicons from the New Caledonian control samples. Analysis and comparison of the 392 bp fragment derived from the Australian IHHNV strain (AY590120) with other geographical isolates revealed that the Madagascar isolate shared the highest nucleotide similarity (96.2%) and the Hawaiian and New Caledonian strain the highest nucleotide divergence (90.1 and 90.3% respectively). The high nucleotide variation observed between the Australian and Hawaiian strains provides an explanation for the lack of IHHNV detection amongst Australian prawns with published IHHNV PCRs and commercially available gene probes as they are primarily designed on the basis of the Hawaiian strain (AF218266). Results indicated that IHHNV has been present in Australia for a long (geological and contemporary) time and that the virus is endemic in penaeid prawns in the Australian environment.     

Appendage deformity syndrome--a nutritional disease of Macrobrachium rosenbergii

Culture of the freshwater prawn Macrobrachium rosenbergii as an alternative to penaeid shrimp has recently increased in coastal areas of southern India in order to avoid numerous problems, particularly with white spot syndrome virus (WSSV). However, M. rosenbergii culture is now threatened by a new disease, appendage deformity syndrome (ADS), that also results in high mortality. Analysis of ADS prawns for viruses such as WSSV, monodon baculovirus (MBV) and infectious hypodermal and hematopoeitic necrosis virus (IHHNV) gave negative results. ADS prawns were also negative for bacterial pathogens and affected animals did not respond to antibiotic therapy. A study of potential nutritional deficiency revealed that carotenoid supplementation in the diet led to a significant decrease in ADS prawns.     

Detection and genetic profiling of infectious hypodermal and haematopoietic necrosis virus (IHHNV) infections in wild berried freshwater prawn, <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> collected for hatchery production

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) has been detected widely in penaeid culture facilities in Asia and the Americas. IHHNV infection on sub-adult and postlarvae of the giant freshwater prawn, Macrobrachium rosenbergii which had caused up to 80% mortalities was first reported in Southeast Taiwan in 2006. In Malaysia, although, there has been no report on IHHNV infections in M. rosenbergii, preliminary work suggests that there is an urgent need to setup a screening protocol for IHHNV for both wild and cultured populations. In this study, polymerase chain reaction based screening was carried out on 30 randomly sampled berried wild M. rosenbergii before and after spawning. All samples did not showed any sign of IHHNV infection. However, the results showed that 20% of the samples were IHHNV positive. Sequence analysis of the amplified band using NCBI-BLAST showed that the putative IHHNV sequence had 98% nucleotide sequence (388 bp) identity with the IHHNV isolate AC-05-005 non-structural protein 1 gene and seven other IHHNV strains in the data bank further affirming the suggestion on the presence of IHHNV in wild freshwater prawn populations in Malaysia.