Insect biomass is declining globally, likely driven by climate change and pesticide use, yet systematic studies on the effects of various chemicals remain limited. In this work, we used a chemical library of 1024 molecules—covering insecticides, herbicides, fungicides, and plant growth inhibitors—to assess the impact of sublethal pesticide doses on insects. In Drosophila melanogaster , 57% of chemicals affected larval behavior, and a higher proportion compromised long-term survivability. Exposure to sublethal doses also induced widespread changes in the phosphoproteome and changes in development and reproduction. The negative effects of agrochemicals were amplified when the temperature was increased. We observed similar behavioral changes across multiple insect species, including mosquitoes and butterflies. These findings suggest that widespread sublethal pesticide exposure can alter insect behavior and physiology, threatening long-term population survival.

Increasing insecticide resistance in malaria-transmitting vectors represents a public health threat, but underlying mechanisms are poorly understood. Here, a data integration approach is used to analyse transcriptomic data from comparisons of insecticide resistant and susceptible Anopheles populations from disparate geographical regions across the African continent. An unbiased, integrated analysis of this data confirms previously described resistance candidates but also identifies multiple novel genes involving alternative resistance mechanisms, including sequestration, and transcription factors regulating multiple downstream effector genes, which are validated by gene silencing. The integrated datasets can be interrogated with a bespoke Shiny R script, deployed as an interactive web-based application, that maps the expression of resistance candidates and identifies co-regulated transcripts that may give clues to the function of novel resistance-associated genes.

Malaria control in Africa is dependent upon the use insecticides but intensive use of a limited number of chemicals has led to resistance in mosquito populations. Increased production of enzymes that detoxify insecticides is one of the most potent resistance mechanisms. Several metabolic enzymes have been implicated in insecticide resistance but the processes controlling their expression have remained largely elusive. Here, we show that the transcription factor Maf-S regulates expression of multiple detoxification genes, including the key insecticide metabolisers CYP6M2 and GSTD1 in the African malaria vector Anopheles gambiae. Attenuation of this transcription factor through RNAi induced knockdown reduced transcript levels of these effectors and significantly increased mortality after exposure to the pyrethroid insecticides and DDT (permethrin: 9.2% to 19.2% (p = 0.015), deltamethrin: 3.9% to 21.6% (p = 0.036) and DDT: 1% to 11.7% (p = <0.01), whilst dramatically decreasing mortality induced by the organophosphate malathion (79.6% to 8.0% (p = <0.01)). Additional genes regulated by Maf-S were also identified providing new insight into the role of this transcription factor in insects. Maf-S is a key regulator of detoxification genes in Anopheles mosquitoes. Disrupting this transcription factor has opposing effects on the mosquito’s response to different insecticide classes providing a mechanistic explanation to the negative cross resistance that has been reported between pyrethroids and organophosphates.

The role of ATP‐binding cassette (ABC) transporters in conferring insecticide resistance has received much attention recently. Here we identify ABC transporters differentially expressed in insecticide‐resistant populations of the malaria vector, Anopheles gambiae . Although we found little evidence that the orthologues of the multidrug resistance proteins described in other species are associated with resistance in An. gambiae we did identify a subset of ABC proteins consistently differentially expressed in pyrethroid‐resistant populations from across Africa. We present information on the phylogenetic relationship, primary sites of expression and potential role of ABC transporters in mediating the mosquito's response to insecticides. Furthermore we demonstrate that a paralogous group of eight ABCG transporters, clustered on chromosome 3R, are highly enriched in the legs of An. gambiae mosquitoes, consistent with a proposed role for this ABC subfamily in transport of lipids to the outer surface of the cuticle. Finally, antibodies raised against one of the most highly expressed ABC transporters in adult females, ABCG7 (AGAP009850), localized this transporter to the pericardial cells. These data will help prioritize members of this gene family for further localization and functional validation studies to identify the in vivo function of these transporters in the mosquito and determine whether elevated expression of members of this family contribute to insecticide resistance.

The elevated expression of enzymes with insecticide metabolism activity can lead to high levels of insecticide resistance in the malaria vector, Anopheles gambiae. In this study, adult female mosquitoes from an insecticide susceptible and resistant strain were dissected into four different body parts. RNA from each of these samples was used in microarray analysis to determine the enrichment patterns of the key detoxification gene families within the mosquito and to identify additional candidate insecticide resistance genes that may have been overlooked in previous experiments on whole organisms. A general enrichment in the transcription of genes from the four major detoxification gene families (carboxylesterases, glutathione transferases, UDP glucornyltransferases and cytochrome P450s) was observed in the midgut and malpighian tubules. Yet the subset of P450 genes that have previously been implicated in insecticide resistance in An gambiae, show a surprisingly varied profile of tissue enrichment, confirmed by qPCR and, for three candidates, by immunostaining. A stringent selection process was used to define a list of 105 genes that are significantly (p ≤0.001) over expressed in body parts from the resistant versus susceptible strain. Over half of these, including all the cytochrome P450s on this list, were identified in previous whole organism comparisons between the strains, but several new candidates were detected, notably from comparisons of the transcriptomes from dissected abdomen integuments. The use of RNA extracted from the whole organism to identify candidate insecticide resistance genes has a risk of missing candidates if key genes responsible for the phenotype have restricted expression within the body and/or are over expression only in certain tissues. However, as transcription of genes implicated in metabolic resistance to insecticides is not enriched in any one single organ, comparison of the transcriptome of individual dissected body parts cannot be recommended as a preferred means to identify new candidate insecticide resistant genes. Instead the rich data set on in vivo sites of transcription should be consulted when designing follow up qPCR validation steps, or for screening known candidates in field populations.