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

Date Published: February 7, 2019

Publisher: Public Library of Science

Author(s): Astrid Spielmeyer, Marc F. Schetelig, Josiane Etang, Pedro L. Oliveira.


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).

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.

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.

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.

Partial Text

Malaria remains one of the most deadly diseases in the world. In 2016, an estimated 216 million cases were recorded, leading to 445.000 deaths, mainly in children aged less than five years in Africa (92%) [1]. Since 2000, 663 million clinical cases have been averted by Insecticide Treated Nets (ITNs) and Indoor Residual Spraying (IRS) (jointly 78%) as well as Artemisinin Combination Therapies (ACTs) (22%) [2]. In few specific settings and circumstances, ITNs and IRS are supplemented by larval source management including larviciding, environmental measures or improvement of the housing to reduce the suitability of the environment as mosquito habitat or to restrict the human biting rates [3, 4]. This emphasizes the need for continued investments in malaria vector control, aiming at shortening the lifespan of mosquitoes near their human targets [5, 6]. Attempts to develop specific assays and new technologies that would sustain interventions against malaria or prompt the identification of public health insecticides with novel modes of action are therefore of high interest.

In the framework of malaria prevention, chemical analysis of insecticide-treated materials used for mosquito control interventions and insecticide resistance testing (e.g., mosquito nets, filter papers) is strongly recommended for quality control [34]. In parallel, data on chemical uptake by mosquitoes through tarsal contact with insecticide-treated material may improve the monitoring of insecticide effects on mosquito populations. To ensure its functionality, the UHPLC-MS/MS method described here has been developed and validated using a reference laboratory colony of malaria vector species (KISUM1 An. gambiae s.s.), five well known insecticides used in malaria vector control (DM, PM, EPX, PPM and aCYP) and the WHO’s standard protocol for susceptibility test on adult mosquitoes [29].

The UHPLC-MS/MS method described here has revealed the minimum uptake of each selected insecticide required to killed individual mosquitoes after tarsal contact with treated materials. Therefore, the standard bioassay protocols, while useful to monitor insecticide resistance or bio efficacy of treated material in field and laboratory studies, may not provide enough evidence for the failure in insecticide delivery from treated material to target mosquito populations. Further studies are needed to investigate the influence of mosquito sex, cuticle thickness and other insecticide resistance on the rate of insecticide uptake.




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