Cathepsin D （CTSD） is a lysosomal acidic endoproteinase that plays an important role in immune response, In this study, we obtained sweetfish （Plecoglossus altivelis） CTSD （PaCTSD） via de-novo transcriptome sequencing of sweetfish macrophages. The full length cDNA sequence of PaCTSD was 1955 bp encoding a propeptide of 397 amino acids. The deduced protein had a calculated molecular weight of 43.17x 103. Multiple alignment with other known CTSD amino acid sequences revealed amino acid conservation through the teleosts. Phylogenetic tree analysis showed that PaCTSD grouped tightly with other fish CTSD, and was close to that of Atlantic salmon and rainbow trout. Subsequently, PaCTSD was prokaryotically expressed and refolded by the urea gradient method on a nickel-nitrilotriacetic acid column. Enzyme activity analysis showed that PaCTSD exhibited pH-dependent proteolytic activity. Quantitative real-time PCR showed that PaCTSD mRNA was expressed in all detected tissues in healthy sweetfish. The highest expression was observed in the spleen and white blood cells, followed by liver, head-kidney, kidney, intestine, gill, and muscle. After Listonella anguillarum infection, PaCTSD transcripts were up-regulated significantly in liver, spleen, white blood cells, and head-kidney of sweetfish. In summary, PaCTSD has proteolytic activity and is closely involved in the immune response of sweetfish.
Ayu （Plecoglossus altivelis） fish, which are an amphidromous species distributed in East Asia, live in brackish water （BW） during their larval stage and in fresh water （FW） during their adult stage. In this study, we found that FW-acclimated ayu larvae exhibited a slower growth ratio compared with that of BW-acclimated larvae. However, the mechanism underlying FW acclimation on growth suppression is poorly known. We employed transcriptome analysis to investigate the differential gene expression of FW acclimation by RNA sequencing. We identified 158 upregulated and 139 downregulated transcripts in FW-acclimated ayu larvae compared with that in BW-acclimated larvae. As determined by Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway mapping, functional annotation of the genes covered diverse biological functions and processes, and included neuroendo- crinology, osmotic regulation, energy metabolism, and the cytoskeleton. Transcriptional expression of several differentially expressed genes in response to FW acclimation was further confirmed by real-time quantitative PCR. In accordance with transcriptome analysis, iodothyronine deiodinase （ID）, pro-opiom- elanocortin （POMC）, betaine-homocysteine S-meth- yltransferase 1 （BHMT）, fructose-bisphosphate aldolase B （aldolase B）, tyrosine aminotransferase （TAT）, and Na＋-K＋ ATPase （NKA） were upregulated after FW acclimation. Furthermore, the mRNA expressions of b-type natriurefic peptide （BNP） and transgelin were downregulated after FW acclimation. Our data indicate that FW acclimation reduced the growth rate of ayu larvae, which might result from the expression alteration of genes related to endocrine hormones, energy metabolism, and direct osmoregulation.
