Comparative multi‐tissue analyses identify testis‐specific serine/threonine protein kinase (TSSK) genes involved in male fertility in the melon fly Zeugodacus cucurbitae

BACKGROUND Zeugodacus cucurbitae is an agricultural pest species with robust reproductive capabilities capable of causing extensive damage. The advent of novel male fertility‐related pest control strategies has been an area of active entomological research focused on the sterile insect technique (SIT) strategy. RESULTS RNA‐sequencing analyses were conducted using 16 tissue samples from adult male Z. cucurbitae, leading to the identification of 5338 genes that were differentially expressed between the testes and three other analyzed tissue types. Of these genes, 808 exhibited high levels of testis expression. A quantitative polymerase chain reaction (qPCR) approach was used to validate the expression of ten of these genes selected at random, including ZcTSSK1 and ZcTSSK3, which are testis‐specific serine/threonine protein kinase (TSSK) genes. Evaluation via a loss‐of‐function‐based knockdown assay showed that the down‐regulation of either of these two genes in males was associated with significantly decreased egg hatching rates. In situ hybridization analyses revealed the expression of both of these transcripts in the transformation zone, and significant decreases in Z. cucurbitae sperm numbers were observed following double‐stranded RNA treatment. Together, these results suggested that inhibiting ZcTSSK1 and ZcTSSK3 expression was sufficient to alter male fertility in Z. cucurbitae. Conclusion These transcriptional sequencing results provide a foundation for further efforts to clarify the regulators of Z. cucurbitae male fertility. These preliminary analyses of the functions of ZcTSSK family genes as regulators of spermatogenesis underscore their importance in the processes integral to male fecundity and their potential as targets for pest control efforts centered on the genetic manipulation of males. © 2023 Society of Chemical Industry. The melon fly, Zeugodacus cucurbitae, is one of the most destructive pest of agriculture throughout multiple areas of the world. In this study, we identified 5343 genes differentially expressed between testis and other three tissues through RNA‐sequencing (RNA‐Seq) of 16 tissues of male adult Z. cucurbitae, including 801 genes with extremely high expression in testis. In these genes, two testis‐specific serine/threonine protein kinase genes, ZcTSSK1 and ZcTSSK3, were identified for the spatiotemporal expression analysis. Quantitative polymerase chain reaction (qPCR) and fluorescence in situ hybridization analysis of ZcTSSK