Role of chitin synthases CHS1 and CHS2 in biosynthesis of the cyst wall of Cryptocaryon irritans

Abstract

Cryptocaryon irritans, a protozoan parasite that infects marine fish, is characterized by a complex life cycle that includes a cyst-forming reproductive phase. However, the composition of the cyst wall and mechanism of its formation remain unclear. In this study, we identified chitin as a key component of the cyst wall using calcofluor white and wheat germ agglutinin, with Fourier-transform infrared spectroscopy confirming its β-form structure. Two chitin synthase genes, CHS1 and CHS2, were identified as being expressed throughout the life cycle and show close phylogenetic relationships with chitin synthase from ciliates. Incubation with specific anti-CHS1 and -CHS2 antibodies significantly reduced both the thickness and chitin content of the cyst wall, highlighting the critical role of these enzymes in chitin biosynthesis. Treatment with benzoylureas, which inhibit chitin synthesis, caused thinning of the cyst wall and downregulation of CHS gene expression, resulting in an 84 % reduction in the hatching rate after treatment with 0.01 mM CuSO4 compared with control tomonts. Western blot analysis demonstrated that recombinant CHS proteins are immunogenic, and tomonts from CHS-immunized grouper exhibited reduced size. These findings bridge a crucial knowledge gap in understanding of the C. irritans cyst wall and highlight promising targets for infection prevention and control strategies.