The SNP polymorphisms associated with WSSV-resistance of prophenoloxidase in red swamp crayfish (Procambarus clarkii) and its immune response against white spot syndrome virus (WSSV)

Abstract

Prophenoloxidase (proPO), a critical enzyme in innate immune, has been extensively studied in various crustacean species including red swamp crayfish Procambarus clarkii. However, little is known about the genetic structure of proPO from P. clarkii and its role in antivirals. In this study, the expression pattern of proPO upon white spot syndrome virus (WSSV) infection was investigated, and the proPO genomic DNA sequence was obtained from P. clarkii for the first time and analyzed. The results indicated that the mRNA level of proPO could be significantly upregulated in haemocytes, hepatopancreas, gill and midgut. The proPO genomic DNA sequence is 9085 bp in length, which includes five exons interrupted by four introns, an 82 bp 5′-flanking region, and a 1671 bp 3′-untranslated region. Two TATA boxes as well as three repeat sequences were predicted in the intron region of proPO. Subsequently, the single nucleotide polymorphisms (SNPs) of proPO were identified and genotyped in the crayfish population and the association between SNPs and anti-WSSV traits was investigated. Three SNPs in intron regions were identified, and the SNP 7081 T > C showed a significant association (P < 0.05) with anti-WSSV traits. In addition, the role of purified recombinant protein of proPO in anti-WSSV infection was explored. The results showed that recombinant proPO significantly reduced the amount of WSSV copy number in hepatopancreas and gill compared to the control group injected with bovine serum albumin (BSA). It was found that recombinant proPO could enhance the expression of proPO in hepatopancreas, peroxinectin in midgut, and the C-type lectin (ctl) in gill and midgut. Intramuscularly injection of juvenile crayfish with recombinant proPO could significantly decrease 65% mortality of WSSV-infected crayfish at 10 days as compared to the control group with BSA. The findings of this study suggested that the proPO is an important molecule in the antiviral immune system of crustacean and could provide theoretical for molecular marker-assisted WSSV resistance breeding of P. clarkii.