Cloning and functional characterization of Pcficolin1 and Pcficolin2 in red swamp crayfish (Procambarus clarkii)

To explore the molecular characteristics, tissue expression patterns and biological functions of Ficolin1 and Ficolin2 in red swamp crayfish (Procambarus clarkii), this study cloned the coding sequences (CDS) of the two genes via PCR amplification technology, analyzed their expression profiles in different tissues of the crayfish by qPCR assay, and further investigated the regulatory effects of Ficolin1 and Ficolin2 on immune-related genes. The results showed that the CDS of Ficolin1 (PcFCN1) and Ficolin2 (PcFCN2) in red swamp crayfish were 969 bp and 783 bp in length, encoding 322 and 260 amino acids, respectively. Phylogenetic tree analysis and multiple amino acid sequence alignment revealed high conservation of both proteins. Tissue expression profile detection indicated that PcFCN1 and PcFCN2 were constitutively expressed in all 7 tested tissues: PcFCN1 exhibited the highest expression level in the heart and the lowest in the gills, while PcFCN2 showed the highest expression level in the gonads and the lowest in the hepatopancreas. After stimulation with Aeromonas salmonicida, PcFCN1 was continuously upregulated in the hepatopancreas and gills and PcFCN2 was significantly upregulated in the hepatopancreas, gills, and hindgut. Treatment of the hindgut of bacterially infected red swamp crayfish with Agaricus bisporus polysaccharide (ABPs) and curcumin (Cur) resulted in a trend of first increasing and then decreasing in the expression of PcFCN1 and PcFCN2. Additionally, direct stimulation of red swamp crayfish hindgut with PcFCN1 and PcFCN2 proteins could induce the expression of inflammatory factors and antimicrobial peptides. In conclusion, PcFCN1 and PcFCN2 were likely involved in key regulatory processes of the anti-pathogen immune response of red swamp crayfish. Anti-inflammatory substances such as ABPs and Cur can effectively downregulate the overexpression of PcFCN1 and PcFCN2 in bacterial infection, thereby alleviating the inflammatory response of the organism. The regulatory effects of ABPs and Cur on PcFCN1/PcFCN2 may be associated with their broad-spectrum anti-inflammatory properties, and further studies are needed to verify the underlying direct or indirect mechanisms.