RNA-seq transcriptome analysis and identification of the theromacin antimicrobial peptide of the copepod Apocyclops royi.

Copepods, including Apocyclops royi, are small aquatic crustaceans and one of the important foods for fish and shellfish larvae. However, studies of the host-pathogen interactions and understanding of infectious disease in copepods are still very limited, yet they are likely to be a significant factor in the sustainable development of copepod aquaculture. In the present study, we performed de novo RNA sequence analysis of A. royi-TH (a Thai isolate of A. royi), which yielded 4.80 Gb bases of clean data and a total of 29,786 unigenes. Annotation was then performed by comparison against seven functional databases, yielding 17,617 (NR: 59.15%), 2,969 (NT: 9.97%), 15,023 (SwissProt: 50.44%), 14,543 (KOG: 48.82%), 15,077 (KEGG: 50.62%), 6,763(GO: 22.71%), and 15,841 (InterPro: 53.18%) unigenes. In comparison to the components of the shrimp Toll pathway, LGBP, Spätzle, Toll receptors, MyD88, Pelle, TRAF6, Dorsal, and Cactus homologs were successfully identified in A. royi-TH. Additionally, a novel antimicrobial peptide (Theromacin-like) was characterized in A. royi (ArTM-like). The ArTM-like ORF was 279 bp and predicted to encode for 92 amino acid residues, with a mature peptide of 75 amino acids and a molecular mass of 8.56 kDa. The genomic organization of the ArTM-like gene consisted of three exons and two introns. Expression analysis indicated that ArTM-like mRNA was abundantly expressed in copepodid and adult stages as an immune responsive gene after infection with the pathogenic Vibrio parahaemolyticus-(AHPND)-causing strain. Altogether, the knowledge obtained in this study will provide a basis for future functional studies of the molecular mechanisms in copepod immunity that may eventually be applied for disease prevention in copepod aquaculture.