Mthawanji, R. R. | Search bioRxiv

bioRxiv2026

Assessing the additional effects of piperonyl butoxide (PBO) exposure on Anopheles mosquitoes

Insecticide treated nets (ITNs) remain one the frontline tool in malaria control in Africa. The synergist piperonyl butoxide (PBO) has been incorporated into ITNs to combat pyrethroid resistance and these pyrethroid-PBO ITNs are now in widespread use across Africa. PBO is known to inhibit the degradation of pyrethroids and other insecticide classes, but little is known about other biological or behavioural effects of PBO exposure. This work assessed the impact of PBO on Anopheles gambiae s.l. male longevity as well as female host-seeking and blood feeding behaviours using laboratory assays. PBO had an immediate impact on blood feeding of female An. gambiae s.l. whereas the effect was not observed 24h post-exposure. Kaplan-Meier survival curves showed a significant difference in mosquito longevity post-exposure to PBO with male mosquitoes living nearly three times as long if exposed to control papers compared to PBO. In olfactometer assays, after exposure to control papers, control mosquitoes were significantly more attracted to host-odours, however, this attraction preference was reversed following PBO exposure. This study shows that in experimental assays, PBO has other effects on mosquito physiology and this is likely to contribute to the impact of pyrethroid - PBO ITNs in vector control programmes.

ecology↗

bioRxiv2026

Integrated behavioural, morphological, and reproductive responses reveal a trade-off during diapause in Culex pipiens

Diapause is a critical adaptive strategy that enables temperate mosquito species to survive adverse environmental conditions and maintain population persistence across seasons. In Culex pipiens, diapause plays a key role in overwintering and influences the seasonal dynamics of arbovirus transmission. However, diapause expression is often assessed using single traits, limiting our understanding of its integrated physiological basis and variation among populations. In this study, we investigated the behavioural, morphological, and reproductive signatures of diapause across three laboratory strains of Culex pipiens (Mogden, Pirbright, and Pirbright Hybrid) reared under diapause-inducing (10 {degrees}C), cold (14 {degrees}C), and control (26-27 {degrees}C) conditions. We quantified blood-feeding behaviour, wing size as a proxy for somatic growth, and spermatheca size as an indicator of reproductive development. Diapause-inducing conditions resulted in a coordinated phenotype characterised by strong suppression of blood-feeding, increased somatic size, and marked inhibition of reproductive development. Mosquitoes reared at 10 {degrees}C exhibited near-complete feeding inhibition and significantly reduced spermatheca size, consistent with reproductive arrest, while those reared at 14 {degrees}C showed intermediate phenotypes. In contrast, control mosquitoes displayed active feeding and fully developed reproductive structures. Wing size increased progressively with decreasing temperature, with the largest individuals observed under diapause-inducing conditions. When analysed together, wing size and spermatheca development exhibited opposing responses across temperature treatments, revealing a strong negative association and indicating a trade-off between somatic growth and reproductive investment. This integrated response supports the interpretation of diapause as a coordinated life-history strategy involving resource reallocation towards survival. Additionally, diapause expression varied among strains, with the Mogden strain showing reduced sensitivity compared with Pirbright and hybrid populations, highlighting the role of genetic background in diapause plasticity. These findings demonstrate that diapause in Culex pipiens is a multi-trait, plastic phenotype with important implications for overwintering success and the seasonal dynamics of arbovirus transmission in temperate regions.

ecology↗