Standard operating procedure for testing the susceptibility of adult sand flies to insecticides in WHO bottle bioassays
Overview
This SOP describes the process for evaluating the susceptibility of adult sand fly vectors (Phlebotomus spp. and Lutzomyia longipalpis) to insecticides in the WHO bottle bioassay. The WHO bottle bioassay is a direct response-to-exposure test, in which sand fly mortality is measured after 1 h of exposure to a known standard concentration (e.g., the discriminating concentration, DC) of an insecticide. The mortality of test sand flies is recorded at 24 h (or more for slow-acting compounds) after the 1-h exposure period. Instructions are provided on preparing and coating bottles with insecticide, exposing sand flies, and recording and interpreting test results.
The WHO bottle bioassay was first developed for testing mosquitoes by modifying the bottle bioassay of the United States Centers for Disease Control and Prevention to harmonize the test end-points with those of the WHO tube tests, so that vector mortality can be evaluated and recorded at the same time after exposure. The bottle bioassay SOP for mosquitoes has now been further modified to test the susceptibility of sand flies to insecticides that are unstable or cannot be impregnated on usual Whatman No. 1 filter papers.
The initial draft of the SOP was prepared by Dr Vincent Corbel (IRD) and Dr Rajpal S. Yadav (WHO) and discussed at a WHO consultation on 29 June 2022 that was convened to review results of a WHO multi-centre study on determining discriminating concentrations of insecticides for monitoring resistance in sand flies. Thereafter, a revised draft of the SOP incorporating the technical comments of study investigators, experts to WHO and other stakeholders listed above was peer reviewed. The peer reviewed SOP was finalized in consultation with the main contributors and the experts who had previously participated in the WHO consultation.
Although the WHO bottle bioassays for mosquitoes are also used to test vector susceptibility to insect growth regulators (e.g., juvenile hormone mimic compound pyriproxyfen), certain additional steps are required to measure the sterilizing properties of juvenile hormone mimics (i.e., oviposition inhibition and reduced fecundity) postexposure. An SOP is available for testing insect growth regulators with mosquitoes, but it has not yet been validated for sand flies. Therefore, this SOP should not be used for testing the susceptibility of sand flies to insecticide growth regulators that reduce insect fecundity and fertility. For insecticides that can be impregnated on filter papers, the SOP for WHO tube tests with sand flies should be followed.
