Resistance in mosquitos against novel insecticide

By Yeji Hong

Malaria is an infectious disease caused by Plasmodium parasites that are transmitted from one person to another through the bites of infected female Anopheles mosquitos. The estimated number of malaria deaths in 2018 stood at over 400,000, and the World Health Organisation (WHO) African region was home to over 90% of malaria cases and malaria deaths in that year (WHO, 2020). Being a preventable disease, research is ongoing to diversify malaria control techniques, including the use of malaria vector control programmes.

Despite research being carried out to devise new malaria control techniques in an attempt to steer away from using chemical insecticides, they remain the most widely used method of prevention. However, the reliance on chemical insecticides may not be long-lasting due to the rapid spread of insecticide resistance among mosquitos. Therefore, WHO highlights the critical need for all countries with ongoing malaria transmission to devise and apply effective insecticide resistance management strategies.

Recently, the neonicotinoid clothianidin was prequalified by WHO for indoor residual spraying, which refers to the coating of house surfaces with insecticide. Neonicotinoid pesticides are the most widely used class of insecticides, and they work by targeting the nicotinic acetylcholine receptor in the insect central nervous system to cause overstimulation, ultimately resulting in paralysis and death. As with all novel insecticides, an assessment was carried out to investigate susceptibility of mosquito populations to clothianidin. An initial study conducted by the U.S. President’s Malaria Initiative revealed that wild Anopheles populations from 16 African countries were fully susceptible to a dose of 2% w/v clothianidin, including the most important vectors of the Plasmodium parasite across the continent – An. gambiae, An. coluzzii and An. arabiensis (Oxborough et. al., 2019).

However, considering the widely documented association between agricultural use of pesticides and the development of insecticide resistance in Anopheles mosquito populations, a team from Cameroon’s Centre for Research in Infectious Diseases (CRID) assessed susceptibility in wild populations in Yaoundé (Fouet et. al., 2020). Five locations centred in Yaoundé were investigated, including a large farm in a suburban neighbourhood, two neighbourhoods located at the centre of Yaoundé and two rural sites at the outskirts of the city. An. gambiae, An. coluzzii and Culex mosquito larvae were collected from standing water and it was noted that An. coluzzii is found exclusively in the most urbanised neighbourhoods whilst An. gambiae is the only species present in rural areas. 

The CDC bottle bioassay was conducted, which involved releasing 3~5-day old females into test bottles containing clothianidin for 60 minutes. Following exposure, the mosquitoes were transferred and provided with 10% sugar solution, after which their mortality was monitored every day for a week. The team was then able to identify mosquitos displaying signals consistent with resistance. The assays revealed that all An. coluzzii and Culex populations were fully susceptible to the active dose of clothianidin, and mortality reached 100% within a week. However, An. gambiae mortality was only 58% in all 817 mosquitos, suggesting that populations of these species had resistance to clothianidin. Further bioassays were carried out with bottles coated with the synergist piperonyl butoxide (PBO) in addition to the clothianidin, which resulted in an increase in mortality to 90% in the resistant populations. PBO is an inhibitor of oxidases and esterases, giving an insight that Cytochrome P450 may play a primary role in clothianidin resistance in An. gambiae

The researchers speculated that this resistance to clothianidin was due to the use of agricultural neonicotinoids, considering the significant usage of neonicotinoids such as imidacloprid and acetamiprid in the agricultural hotspots in Cameroon. However, populations with resistance were still detected in rural sites without obvious evidence of neonicotinoid use, and it was further noted that Culex samples which were collected from breeding sites where resistant An. gambiae larvae thrive were still susceptible to clothianidin. This indicated that the resistance to neonicotinoids can be species-specific and may be just one of many mechanisms underlying the adaptation of insect populations to insecticides (Fouet et. al., 2020). Overall, the study carried out by CRID showed that resistance to the novel insecticide clothianidin is already strong in An. gambiae populations in agricultural areas of Cameroon, where low mortality rates are observed even with high doses of the active ingredient.

Currently, WHO guidelines for clothianidin resistance testing in mosquitos have yet to be established as many technical factors make it difficult to assess clothianidin susceptibility. The study carried out by CRID indicates the possibility of reduced efficacy of clothianidin in some agricultural regions of Sub-Saharan Africa due to pre-existing resistance amongst An. gambiae populations, despite preceding studies indicating complete susceptibility to clothianidin for 16 Anopheles species. This highlights the importance of having a thorough screening process for new insecticides being considered for inclusion in the malaria vector control programmes. WHO has yet to review the study carried out by CRID as it has not been published in a peer-reviewed journal (Makoni, 2020).  Despite the resistance detected in the An. gambiae mosquito populations from Yaoundé, clothianidin may still be an important asset in mosquito control as resistance to clothianidin is relatively low compared to resistance to alternative insecticides. However, it remains a great concern as to how long the insecticide will prove to be effective.


Fouet, C., Ayukarah, F.A., Marilene, M.A., Williams, T., Charles, S.W., Colince, K. (2020) Resistance of Anopheles gambiae to the new insecticide clothianidin associated with unrestricted use of agricultural neonicotinoids in Yaoundé, Cameroon. Available from: [Accessed 20th September 2020]

Makoni, M. (2020) Some mosquitos already have resistance to the latest weapon against Malaria. American Association for the Advancement of Science, viewed 19th September 2020, <;

Oxborough, R.M., Seyoum, A., Yihdego, Y., Dabire, R., Gnanguenon, V., Wat, F., et al. (2019) Susceptibility testing of Anopheles malaria vectors with the neonicotinoid insecticide clothianidin; results from 16 African countries, in preparation for indoor residual spraying with new insecticide formulations. Malar. J. BioMed Central. 18. Available from: [Accessed 20th September 2020]

World Health Organisation. (2020) Malaria, viewed 25th September 2020, <>&nbsp;

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