Identifying suitable methods for evaluating the sterilising effects of pyriproxyfen on adult malaria vectors: a comparison of the oviposition and ovary dissection methods
Authors: Myers Alesha , Fagbohoun Josias , Houetohossou Georgine , Ndombidje Boris , Govoetchan Renaud , Todjinou Damien , Ngufor Corine 
Published: November 2023

Introduction
Nets containing pyriproxyfen, an insect growth regulator that sterilises adult mosquitoes, have become available for malaria control. Suitable methods for investigating vector susceptibility to pyriproxyfen and evaluating its efficacy on nets need to be identified. The sterilising effects of pyriproxyfen on adult malaria vectors can be assessed by holding mosquitoes in oviposition chambers to determine the impact on egg laying (oviposition) and by dissecting mosquito ovaries to determine whether they have been damaged by pyriproxyfen (ovary dissection).
Method
A series of laboratory bioassays were performed to compare the oviposition and ovary dissection methods for monitoring susceptibility to pyriproxyfen in wild malaria vectors using WHO bottle bioassays and for evaluating its efficacy on nets in cone bioassays. Blood-fed mosquitoes of susceptible and pyrethroid resistant strains of Anopheles gambiae sensu lato were exposed to pyriproxyfen-treated bottles at 100µg and 200µg per bottle and to unwashed and washed pieces of a pyriproxyfen long-lasting net in cone bioassays. Survivors were assessed for the sterilising effects of pyriproxyfen using both methods. The methods were compared in terms of their reliability, sensitivity, specificity, resources (cost and time) required and perceived difficulties and complexities by trained laboratory technicians.
Results
The total number of An. gambiae s.l. mosquitoes assessed for the sterilising effects of pyriproxyfen were 1,745 for the oviposition method and 1,698 for the ovary dissection method. Fertility rates of control unexposed mosquitoes were significantly higher with ovary dissection compared to oviposition in both bottle bioassays (99–100% vs. 34–59%, P < 0.05) and cone bioassays (99–100% vs. 18–33%, P < 0.001). Oviposition rates of control unexposed mosquitoes were lower with wild pyrethroid resistant An. gambiae s.l. Cové compared to the laboratory-maintained reference susceptible An gambiae s.s. Kisumu strain (18–34% vs. 58–76%, P < 0.05). Sterilisation rates of the Kisumu strain in bottle bioassays with the pyriproxyfen diagnostic dose (100µg) was suboptimal with the oviposition method (90%) but showed full susceptibility with ovary dissection (99%). Wild pyrethroid-resistant Cové mosquitoes were fully susceptible to pyriproxyfen in bottle bioassays using ovary dissection (> 99%) but not with the oviposition method (69%). Both methods showed similar levels of sensitivity (89–98% vs. 89–100%). Specificity was substantially higher with ovary dissection as it was more accurate than the oviposition method in detecting pyriproxyfen unexposed mosquitoes in both bottle bioassays (99–100% vs. 34–48%) and cone tests (100% vs.18–76%). Ovary dissection was also more capable of detecting the residual activity of pyriproxyfen in a washed net compared to oviposition. The oviposition method though cheaper, was less reliable and more time-consuming requiring 2–3 more days than ovary dissection. Laboratory technicians preferred ovary dissection mostly due to its reliability.
Conclusion
The ovary dissection method was more accurate, more reliable and more efficient compared to the oviposition method for evaluating the sterilising effects of pyriproxyfen on adult malaria vectors in bioassays. We recommend the use of ovary dissection for pyriproxyfen susceptibility bottle bioassays and for evaluating the efficacy of pyriproxyfen-treated nets in laboratory bioassays, semi-field trials and insecticidal durability studies.