Expression analysis of immune related genes in the black tiger shrimp Penaeus monodon and potential application in broodstock selection

Abstract

The semi-quantitative RT-PCR technique was used to examine the expression levels of 11 immune-related genes from hemocytes of the black tiger shrimp in response to Vibrio harveyi and WSSV challenges at different time courses after injection. These defense-related genes obtained from the Penaeus monodon EST libraries, consist of prophenoloxidase activating factor (PPAF), cytosolic manganese superoxide dismutase precursor (SOD), glutathione peroxidase (GPx), transglutaminase (TG), whey acidic protein (WAP), apoptosis inhibitor survivin (survivin), P 109 protein, cyclophilin 18, peptidyI-prolyl cis-trans isomerase 5 (PPI), chaperonin containing t-complex polypeptide-1 (CCTP) and heat shock protein 60 (HSP60). The mRNA expression in response to bacterial challenge during the time points of injection showed significant increase (p<0.05) of PPAF, GPx, TG, WAP and PPI however, the significant decrease (p<0.05) of SOD and surviving expression were observed. Upon the viral challenge, the expression levels of PPAF, WAP and HSP60 were significantly increased (p<0.05), whereas those of GPx, SOD and survivin were significantly decreased. Interestingly, both pathogens caused up-regulation of PPAF and WAP genes which were then relatively quantified by Real-time RT-PCR. The results showed that the highest expression level of PPAF gene after V. harveyi and WSSV challenges was 4.13 and 2.53 fold at 24 hpi, respectively. Furthermore, the highest expression level of WAP gene after V. harveyi and WSSV challenges was 4.23 and 6.68 fold at 24 hpi, respectively. The mRNA levels of PPAF, GPx, WAP, TG, PPI and HSP 60 were up-regulated after bacterial or viral challenge, indicating the probability of those genes in host defense against invading pathogens. In addition, the expression of the 6 genes were determine in the early-larval stages (Nauplius III, Zoea II, Mysis II,) post-larvae (PL5 and 15) and juvenile (1 month) of the shrimp P. monodon. RT-PCR analysis showed that transcripts of PPAF, GPx. TG, PPI and HSP60 were already present in the early stage of Nauplius III, whereas WAP mRNA were observed in the Zoea II and later through developmental stages tested. Interestingly, the expression levels of almost genes in post-larval stages were higher than in early-larval stage. The expression levels of 6 immune-related genes were examined by a semi-quantitative RT-PCR from hemocytes of juvenile P. monodon from 3 different shrimp farms: commercial farms (Suphunburi, Chachoengsao) and a domesticated farm (Nakhonsithammarat, specific-pathogen-free (SPF) shrimp). The results showed that the transcription levels of PPAF, GPx, WAP, PPI and TG were significantly higher (p<0.05) in the domesticated P. monodon than those from the commercial farms, whereas that of HSP60 was not significant difference. Consequently, it was suggested that, the domesticated P. monodon might be healthy than commercial shrimp farms based on the level of immune-related genes. From expression analysis some immune related genes such as PPAF, WAP, PPI and TG can be candidate biomarkers for health monitoring and have potential used for broodstock selection.