CRISPR/Cas9-Mediated Knockout of the Chorion Gene s36 Impairs Ovarian Development and Abolishes Egg Hatching in the Melon Fly Zeugodacus cucurbitae

The melon fly, Zeugodacus cucurbitae (Coquillett), is a globally invasive pest of cucurbit crops whose concealed larval feeding limits chemical control and has driven interest in reproduction-based management. The chorion gene s36 encodes an eggshell protein conserved among dipterans. However, whether s36 is required for female reproduction in Z. cucurbitae remains unknown. We generated a homozygous s36 mutant line by CRISPR/Cas9 and characterized its reproductive phenotype. Disruption of s36 strongly reduced transcript abundance and truncated the predicted protein. Mutant females showed elevated mortality after sexual maturation, fecundity was approximately halved, and none of the eggs laid hatched. Ovaries were markedly reduced in size and were scored as abnormal in every mutant female examined, and eggs were smaller, dull and shriveled rapidly in air, indicating compromised chorion integrity. Both vitellogenin genes were down-regulated in the ovary. Whole-body transcriptomics identified 179 differentially expressed genes enriched in lipid and hormone-related metabolism. Tissue-resolved RT-qPCR showed vitellogenin and yellow-g2 transcripts elevated in the fat body yet depleted in the ovary; whether this pattern reflects impaired delivery of yolk precursors to the oocyte or the secondary loss of the follicular tissue in which these genes are normally expressed remains to be determined. These findings identify s36 as required for normal ovarian maturation and egg viability in Z. cucurbitae, and as a candidate target for genetic control of this pest.