A big anti-lipopolysaccharide factor showing sexual dimorphism is involved in anti-WSSV immunity in crayfish

Anti-lipopolysaccharide factor (ALF) is an important active substance in crustacean innate immunity with broad-spectrum antimicrobial and antiviral effects. In this study, we identified a big ALF from Procambarus clarkii, named PcbALF. The full-length cDNAs of two PcbALF isoforms (PcbALF1 and PcbALF2) were 2619 bp and 2142 bp with an open reading frame of 1074 bp and 1317 bp that encodes 357 and 438 amino acids, respectively. Intron retention of PcbALF1 resulted in the loss of the DUF3254 domain. PcbALF1 and PcbALF2 were widely distributed in multiple tissues and their expression in different tissues exhibits sexual dimorphism. Further study found that PcbALF2 expression levels were up-regulated in the intestine and gills of male and female P. clarkii after stimulation with white spot syndrome virus (WSSV) from 0 to 48 h. Furthermore, RNA interference analysis showed that the knockdown of PcbALF2 inhibited PcbALF2 transcription and promoted VP28 expression during WSSV infection. Injection of rPcbALF with DUF3254 domain suppressed VP28 expression, suggesting that PcbALF has a negative role in WSSV replication. Our study shows that PcbALF is involved in anti-WSSV immunity in P. clarkii and exhibits sexual dimorphism.