Genomic stability and differential expression of type V cif genes in Wolbachia with variable cytoplasmic incompatibility phenotypes in spider mites

Abstract The two-spotted spider mite, Tetranychus urticae, is a globally significant agricultural pest frequently infected by the endosymbiont Wolbachia. Wolbachia induces cytoplasmic incompatibility (CI) with varying intensity across geographic populations. Despite the importance of CI, its molecular mechanisms in T. urticae remain poorly understood. Here, we demonstrate that CI differences are driven by both Wolbachia (wTurt) strain variations and host nuclear background. The wTurt genome harbors two pairs of cif genes (cifA1-B1 and cifA2-B2), both phylogenetically classified as Type V. Genomic analysis revealed no SNP differences in both pair of cif genes in wTurt infecting different geographic populations of spider mites. While cifA1-B1 is barely expressed, cifA2-B2 exhibits high expression levels that vary significantly across multiple T. urticae populations, with expression levels positively correlated with wTurt density. Yeast two-hybrid experiments revealed physical interactions between CifA2 and the PD-(D/E)xK nuclease domain of CifB2, suggesting that this pair may contribute to CI modulation. Codon optimization revealed that the CifB2 nuclease domain is toxic in yeast, but this toxicity was not rescued by CifA2. Transgenic Drosophila assays likewise failed to reproduce CI-related phenotypes. Altogether, our findings advance the understanding of Wolbachia-host reproductive manipulation and provide insights for symbiont-based biocontrol strategies for agricultural mite pests.