Identification of genes expressed in lymphoid organs of the black tiger shrimp Penaeus monodon and in response to severe pathogens

Abstract

Expressed sequence tag (EST) analysis was employed to identify genes from the two CDNA libraries of lymphoid organs of unchallenged and Vibrio harveyi challenged shrimps, Penaeus monodon. The number of clones were approximately 3.2x10 in the normal library and 3.2x10 in the challenged library. The 446 clones of the normal library and 642 clones of the challenged library were randomly picked and partially sequenced. One hundred and eighty (40.4%) EST clones of the normal library and 286 (44.5%) EST clones of the challenged library matched significantly with the deposited genes. Matched EST clones (466) of both libraries represented 283 different proteins. Sixty nine clones (6.34% of the total sequenced clones) representing 28 different genes were putative immune genes. These genes coded for the enzymes and proteins of the proPO system, the antimicrobial peptides, the proteinases and proteinase inhibitors, the heat shock proteins and the other immune molecules. To study the gene expression in haemocytes of white spot syndrome virus (WSSV) and V. harveyi challenged shrimps, the cDNA microarray analysis was employed. The CDNA chips composing of 718 unique genes from the black tiger shrimp and 308 unique genes from the kuruma shrimp were used in this study. The gene expression profiles were determined at 6, 24 48 and 72 hours post-injection of the pathogens. The highest number of genes in response to WSSV challenge was observed (135 genes) at 24 hpi whereas those in response to V. harveyi challenge were observed (156 genes) at 6 hpi. Calmodulin (CaM), asialoglycoprotein receptor (ASGPR) and tubulin were the products of up regulated genes. The up regulation of these genes were further confirmed by real time RT-PCR along with the other genes including calcineurin, CDC like kinase 2 and protein phosphatase 1 genes, CaM binding and CaM candidate binding proteins. The results showed differential expression of them in P. monodon haemocytes upon pathogen challenge. The temporal expression of CaM in response to WSSV and V. harveyi challenge was studied by in situ hybridization. The result showed that the percentages of the CaM positive cells of WSSV and V.harveyi challenged shrimp haemocytes were higher than those of the control groups, suggesting that the invading pathogens somehow trigger an increase of CaM transcript in shrimp haemocytes. Moreover, immunohistochemistry showed that bacterial invasion resulted in the distribution of CaM expressing haemocytes throughout the shrimp cepharothorax, especially in gill and hepatopancreas. The CaM protein was also observed in shrimp epithelium cells, such as lymphoid organ, gill and hepatopancreas. The results obtained from the present study provided basic clues for further study on shrimp disease control and management.