REL2 overexpression in theAnopheles gambiaemidgut causes major transcriptional changes but fails to induce an immune response

The NF-κB-like transcription factor, REL2, is a key player in the mosquito Immunodeficiency (Imd) pathway and holds promise for controlling malaria parasite infections in genetically modifiedAnopheles gambiaemosquitoes. We engineered transgenic mosquitoes overexpressing REL2 from within the bloodmeal-inducible zinc carboxypeptidase A1 (CP) host gene in the adult posterior midgut. Our results confirmed elevated REL2 expression in the posterior midgut following a bloodmeal, with the corresponding protein localized within epithelial cell nuclei. While this induced overexpression triggered substantial transcriptional changes, accompanied by notable fitness costs, the resultant reduction inPlasmodium falciparuminfection was modest. An in-depth analysis of regulatory regions of differentially regulated genes allowed us to identify direct REL2 target genes and revealed signatures indicative of potential transcriptional repressors. To account for potential impacts of host gene modification, we also created a CP knockout line that caused marginal effects on mosquito fitness. These findings shed light on the observed absence of transcriptional activation and, in some cases, induced repression of antimicrobial peptides (AMPs) presumed to be under Imd pathway control. In conclusion, our study suggests that elevated REL2 expression in the posterior midgut may induce the upregulation of negative immune regulators, facilitating control over an otherwise unrestrained immune response, and that concurrent transcriptional derepression may be needed to effectively induce the mosquito immune response. This work contributes valuable insights into the intricate regulation of midgut immunity in malaria vector mosquitoes.