Identifying suitable methods for evaluating the sterilizing 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: May 2024

Abstract
Background
Nets containing pyriproxyfen, an insect growth regulator that sterilizes 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 sterilizing effects of pyriproxyfen on adult malaria vectors can be assessed by measuring oviposition or by dissecting mosquito ovaries to determine damage by pyriproxyfen (ovary dissection).

Method
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 (100 μg and 200 μg) and to unwashed and washed pieces of a pyriproxyfen long-lasting net in cone bioassays. Survivors were assessed for the sterilizing 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 by trained laboratory technicians.

Results
The total number of An. gambiae s.l. mosquitoes assessed for the sterilizing effects of pyriproxyfen were 1745 for the oviposition method and 1698 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 sensu stricto Kisumu (18–34% vs. 58–76%, P < 0.05). Sterilization rates of the Kisumu strain in bottle bioassays with the pyriproxyfen diagnostic dose (100 μg) were 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 compared to the oviposition method in both bottle bioassays (99–100% vs. 34–48%) and cone tests (100% vs.18–76%). Ovary dissection was also more sensitive for 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. 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 sterilizing effects of pyriproxyfen on adult malaria vectors in susceptibility bioassays and for evaluating the efficacy of pyriproxyfen-treated nets.