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Multiple functions of caspases include normal cell turnover, proper development and function of the immune system, hormone-dependent atrophy, embryonic development and chemical-induced cell injury. During artificial propagation of Pacific cod, Gadus macrocephalus , high mortality occurred during early development stages. Here, we performed various analyses on the cDNA and protein sequences of six different G. macrocephalus caspases namely GmCasp3 , 6 , 7 , 8 , 9 and 10 , and tried to investigate the contributions of caspase family to the development and Nervous Necrosis Virus (NNV) resistance. Sequence analysis of GmCaspase proteins showed that each caspase shared conserved domains like “HG”, “QACXG (X for R, G or Q)” and “GSWF” except GmCasp10. Sequence alignment and phylogenetic tree showed that GmCasp8 and GmCasp10 were quite different from those of other fishes. 3-D models indicated that structure of GmCasp3 is very conservative, but GmCasp6 , 7 , 8 , 9 and 10 are less conservative. Tissue distribution analysis showed that six Gmcaspases mRNA transcripts were detected in tissues of intestine, gill, thymus, head-kidney and spleen with different abundance, but Gmcasp7 were not detected in the brain. GmCasp3 transcript was kept at very low level in the early development stages, while the expression levels of GmCasp6 , 7 , 8 , 10 were different at various development stages. GmCasp8 level seemed to be much higher than other caspases in the heads of 65dph and 75dph juveniles. To understand the role of caspases during NNV outbreak, modulation in expression of each Gmcaspases were investigated. The results showed that GmCasp3 transcript level increased significantly when NNV broke out, while GmCasp7 , 8 , 9 and 10 in cod heads decreased obviously at 69dph and 77dph. The results suggest that caspases in Pacific cod should be diverse in their structure and function, and their unique features and response to NNV outbreak add more evidences for the specificity of immune system in Pacific cod. [ABSTRACT FROM AUTHOR]

Mortality (>90%) is a big concern in larval rearing facilities of Pacific cod, Gadus macrocephalus , limiting its culture presently still in the experimental stages. Understanding the immune system development of G. macrocephalus is crucial to optimize the aquaculture of this species, to improve the use of economic resources and to avoid abuse of antibiotics. For the transcriptome analysis, using an Illumina sequencing platform, 61,775,698 raw reads were acquired. After a de novo assembly, 77,561 unigenes were obtained. We have classified functionally these transcripts by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). 27 genes mainly related to hematopoietic or lymphoid organ development and somatic diversification of immune receptors have been reported for the first time in Pacific cod, and 14 Ig heavy chain ( μ chain) locuses were assembled using Trinity. Based on our previous achievement, we have chosen Rag1 and Igμ as immune system development biomarkers. Full length cDNA of Rag1 and Igμ as biomarkers were obtained respectively using RACE PCR. Concerning Rag1 , the deduced amino acid of Rag1 and protein immunodetection revealed a Rag1 isoform of 69 kDa, significantly different from other fish orthologs, such as Oncorhynchus mykiss (121 kDa). Phylogenetic analysis reveals a unique immune system for the Gadus genre, not exclusive for Atlantic cod, among vertebrates. Meanwhile, full length cDNA of Igμ included an ORF of 1710 bp and the deduced amino acid was composed of a leader peptide, a variable domain, CH1, CH2, Hinge, CH3, CH4 and C-terminus, which was in accordance with most teleost. Absolute quantification PCR revealed that significant expression of Rag1 appeared earlier than Igμ , 61 and 95 dph compared to 95 dph, respectively. Here we report the first transcriptomic analysis of G. macrocephalus as the starting point for genetic research on immune system development towards improving the Pacific cod aquaculture. [ABSTRACT FROM AUTHOR]