Ovary Dissection Is a Sensitive Measure of Sterility in Anopheles gambiae Exposed to the Insect Growth Regulator Pyriproxyfen

SIMPLE SUMMARY: Malaria is transmitted by Anopheles mosquitoes. An important control tool against malaria is insecticidal bed nets containing a pyrethroid insecticide and another active ingredient, such as pyriproxyfen (PPF), which sterilizes adult mosquitoes by disrupting egg development. To evaluate the impact of PPF-treated bed nets, there are two potential techniques available to measure mosquito sterility: (1) observing egg-laying rates and (2) ovarial dissection. Our aim was to show which technique was most effective by exposing adult mosquitoes to PPF-treated or untreated nets and comparing sterility outcomes. When identifying the true percentage of mosquitoes sterilized from PPF exposure (i.e., true positives), both techniques showed similar sensitivity (egg-laying: 99% vs. dissection: 100%). For identifying non-exposed mosquitoes in the untreated group (i.e., true negatives), dissection had a higher specificity than egg-laying (53% vs. 19%). To show that ovary dissection remains effective under different conditions, we exposed mosquitoes to insecticides using two different exposure methods, including nets treated with pyrethroids, and blinded the investigator to reduce bias when scoring. The investigator predicted the exposure status of dissected females across all conditions with >90% accuracy. We conclude that ovary dissection is a more sensitive and specific tool for measuring sterility in Anopheles mosquitoes exposed to PPF. ABSTRACT: Pyriproxyfen (PPF) is an insect growth regulator used in the co-treatment of long-lasting insecticidal nets for its ability to sterilize female mosquitoes. To evaluate the efficacy of PPF-treated nets on mosquito reproductivity, most studies observe oviposition (egg-laying) rates in the laboratory. This technique has several technical disadvantages. Our study assessed if ovarial dissection could serve as an effective proxy for evaluating sterility in Anopheles gambiae mosquitoes. Blood-fed females were exposed to untreated or PPF-treated nets in cylinder assays and followed over several days to observe oviposition rates or egg development by dissection. For identifying PPF-exposed mosquitoes, both techniques demonstrated high sensitivity (oviposition: 99.1%; dissection: 100.0%), but for identifying non-exposed mosquitoes, specificity was significantly higher in the dissection group (52.5% vs. 18.9%). To assess whether dissection could be applied to nets treated with a pyrethroid or co-treated with a pyrethroid and PPF in tunnel tests, a blinded investigator performed dissections to predict the PPF exposure status across different treatment groups. The exposure status of dissected females was predicted with >90% accuracy. We report that dissection is a sensitive technique to assess sterility in female Anopheles gambiae mosquitoes and can be used as a predictor of PPF exposure.