Deciphering the regulatory landscape of venom gene expression in a parasitoid wasp at single‐cell resolution

The venom gland is a remarkable organ of the parasitoid wasps, which produces venom that controls the host and is important for the survival and reproduction of the parasitoid. But little is known about how venom gland cells precisely regulate venom synthesis. Here, we used single-nucleus RNA sequencing to construct the cell atlases of the venom gland and ovary of the Pteromalus puparum, a natural enemy of vegetable pests. The cells in the venom gland were clustered into six subsets, and the main cell types in the ovary were follicle cells, germline cells and nurse cells. The cellular function of venom gland subsets named VgC1 to VgC6 was enriched in the production and secretion of venom. In addition, the expression pattern of identified venom genes that 77 genes expressed across all six VgCs and 46 genes in five VgCs, demonstrated that most venom genes expressed widely in VgCs. Finally, hdWGCNA analysis of venom gland cells identified a set of co-expressed venom and non-venom genes that include putative regulators of venom production in P. puparum. Our analysis of transcription factor (TF) binding sites within the venom modules revealed that TFs such as AP-1 and EHF are potential regulators of venom genes. This work reveals the cellular heterogeneity, venom gene expression patterns, and transcriptional regulatory networks in the venom gland of P. puparum at single-cell resolution. These findings provide fundamental insights into the biology of venom-producing organs and establish a molecular foundation for developing novel biopesticides based on venom proteins.