An OpIE2 -DsRed marker disrupts female blood-feeding and shortens lifespan in the malaria vector Anopheles gambiae

Anopheles gambiae is one of the principle vectors of human malaria. Over the past two decades, transgenic mosquito strains have been instrumental for studying mosquito biology and developing genetic control strategies such as gene drives. Mosquito transformants are typically identified using fluorescent markers which are assumed to be phenotypically neutral. While generating CRISPR-based gene drive strains with an OpIE2 -DsRed marker we unexpectedly found that transgenic females were unable to blood-feed and were consequently sterile. Males, in contrast, appeared physiologically normal and fertile and enabled maintenance of the strains. Recognizing the potential utility of dominant, female-specific sterility for mosquito control, we established new strains controlling for transgene content and integration site, which confirmed that the specific OpIE2 -DsRed marker was directly responsible for the blood-feeding defect. Behavioral assays showed that females exhibited normal attraction to a membrane feeder but failed to initiate blood-feeding, performing repeated cycles of probing and proboscis grooming in rapid succession before ultimately leaving the feeder unfed. Microscopic examination of OpIE2a females revealed a pronounced curvature of the proboscis. Similar malformations were also present in males and likely contributed to their reduced adult lifespan, overturning our preliminary conclusion of normal male fitness. A second promoter variant ( OpIE2b ), differing in flanking sequences at the IE-2 junction, drove bright marker expression without impairing blood-feeding or survival when integrated at the same genomic site. These findings demonstrate how minor differences in promoter architecture can elicit major, unexpected phenotypes. OpIE2b provides a robust, phenotypically neutral marker for An. gambiae research, while OpIE2a highlights the need for rigorous validation of transgenic components intended for research and applied releases.