High-throughput analysis of insecticides on malaria vectors using liquid chromatography tandem mass spectrometry

Astrid Spielmeyer; Marc F Schetelig; Josiane Etang

doi:10.1371/journal.pone.0211064

High-throughput analysis of insecticides on malaria vectors using liquid chromatography tandem mass spectrometry

PLoS One. 2019 Feb 7;14(2):e0211064. doi: 10.1371/journal.pone.0211064. eCollection 2019.

Authors

Astrid Spielmeyer¹, Marc F Schetelig², Josiane Etang^{2

3

4}

Affiliations

¹ Justus Liebig University Giessen, Institute for Food Chemistry and Food Biotechnology, Giessen, Germany.
² Justus Liebig University Giessen, Institute for Insect Biotechnology, Giessen, Germany.
³ Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale (OCEAC), Institut de Recherche de Yaoundé (IRY), Yaoundé, Cameroun.
⁴ University of Douala, Faculty of Medicine and Pharmaceutical Sciences, Douala, Cameroon.

Abstract

Background: Different setups and protocols have been developed for investigating insecticide effects on Anopheles (An.) mosquitoes, vectors of malaria. However, chemical uptake resulting from their tarsal contact with insecticide-treated material has seldom been investigated. To address the challenges encountered in the interpretation of bioassay data, a high throughput method for chemical analysis on malaria vectors was developed and validated for five selected insecticides including alpha-cypermethrin (aCYP), deltamethrin (DM), etofenprox (EPX), permethrin (PM), pirimiphos-methyl (PPM).

Methods: The method includes a single chemical extraction step via an ultrasound probe on mosquito samples and analysis via liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-MS/MS). The protocol was established for two malaria vector species, Anopheles gambiae senso stricto (s.s.) and An. stephensi, both males and females. Recovery rates ranged from 70 to 100% without any influence of sex or species. The method was efficiently applied to female An. gambiae s.s. of the KISUMU1 reference strain, after susceptibility tests using the World Health Organization's standard protocol.

Results: Susceptibility tests revealed 13.4-18.4 minutes knockdown times for 50% mosquitoes during exposure to EPX and pyrethroids. The mortality rates 24 hours post-exposure to insecticides were mostly 99-100%, except in two PM and three PPM assays suggesting possible or confirmed resistance to these insecticides. The mean insecticide uptake in dead mosquitoes ranged from 23 pg (aCYP) to 1812 pg (EPX) per specimen. However, the mean uptake in survivors to PM and PPM was reduced by at least 25%, suggesting that acute doses were not achieved in these specimens during bioassays.

Conclusions: The developed and validated UHPLC-MS/MS method could be used to address some limitations of bioassays or to assess the penetration of insecticides in mosquito matrix with reference to cuticle thickness and other insecticide resistance mechanisms.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Anopheles / growth & development*
Biological Assay / methods*
Female
Insecticides / pharmacology*
Malaria*
Male
Mosquito Vectors / growth & development*

Substances

Insecticides

Grants and funding

This work was funded by the Alexander von Humboldt-Stiftung (grant number 3.4 – CMR – 1164160) in the framework of the Georg Forster Research Fellowship (HERMES) for Experienced Researchers (to JE). Funding was also provided by the LOEWE Center for Insect Biotechnology & Bioresources of the Hessian State Ministry for Higher Education, Research and the Arts (MFS). The authors are grateful to the Deutsche Forschungsgemeinschaft (DFG) and the State of Hesse for funding the TripleTOF mass spectrometer (INST 162/490-1 FUGG, Institute of Food Chemistry, Prof. Hamscher). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.