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The surface of insects is coated in cuticular hydrocarbons (CHCs); variations in the composition of this layer affect a range of traits including adaptation to arid environments and defence against pathogens and toxins. In the African malaria vector, Anopheles gambiae quantitative and qualitative variance in CHC composition have been associated with speciation, ecological habitat and insecticide resistance. Understanding how these modifications arise will inform us of how mosquitoes are responding to climate change and vector control interventions. CHCs are synthesised in sub-epidermal cells called oenocytes that are very difficult to isolate from surrounding tissues. Here we utilise a transgenic line with fluorescent oenocytes to purify these cells for the first time. Comparative transcriptomics revealed the enrichment of biological processes related to long chain fatty acyl-CoA biosynthesis and elongation of mono-, poly-unsaturated and saturated fatty acids and enabled us to delineate, and partially validate, the hydrocarbon biosynthetic pathway in An. gambiae. The bodies of insects are encased in an exoskeleton or cuticle that is key for their survival. The cuticle helps protect insects against damage, prevents water loss and can defend against pesticides. A better understanding of the role of the cuticle for survival in mosquitoes and other insects could lead to new ways to prevent the spread of diseases such as malaria. The cuticle is coated with various molecules from a group of chemicals called hydrocarbons. This coating is made by specialized cells called oenocytes and helps to protect insects. Hydrocarbons can also influence communications between certain insects by acting as recognition signals. In mosquitoes, oenocytes make several hydrocarbons using a set of processes that are not well understood, and the types of hydrocarbons they make can vary between individuals of the same species. It is unclear how this mixture of hydrocarbons is generated and how differences in the mixture can determine how mosquitoes adapt to their surroundings. Grigoraki et al. studied the genes that were active in isolated oenocytes from the mosquito Anopheles gambiae, which carries the parasite that causes malaria. The study revealed a set of genes which are highly active in oenocytes and control the production of fatty acids, a group of molecules used to make hydrocarbons. Other genes involved in creating hydrocarbons were also found. Grigoraki et al. further investigated a specific gene called FAS1899 and showed that loss of this gene reduces overall hydrocarbon production by 25%. Additionally, genes for transporting and recycling molecules and for producing fats were also shown to be active, which may indicate that oenocytes have a variety of unexplored roles besides making hydrocarbons. Grigoraki et al. identify the genes involved in producing the hydrocarbon coating of mosquitoes and demonstrate their significance. Further work is needed to understand the precise roles of each of these genes and how they are regulated to adapt the hydrocarbon coating to different situations. This can help explain how the hydrocarbon coating changes in mosquitoes, for example in response to the use of insecticides or climate change. This information is important to adapt and develop new tools to improve mosquito control.

Background Malaria vector control and research rely heavily on monitoring mosquito populations for the development of resistance to public health insecticides. One standard method for determining resistance in adult mosquito populations is the World Health Organization test (WHO bioassay). The WHO bioassay kit consists of several acrylic pieces that are assembled into a unit. Parts of the kit commonly break, reducing the capacity of insectaries to carry out resistance profiling. Since there is at present only a single supplier for the test kits, replacement parts can be hard to procure in a timely fashion. Methods Using computer-aided design software and widely available polylactic acid (PLA) filament as a printing material, we 3D designed and printed replacement parts for the WHO bioassay system. We conducted a comparison experiment between original WHO bioassay kits and 3D printed kits to assess congruence between results. The comparison experiment was performed on two Kenyan laboratory strains of Anopheles gambiae ( s.s. ), Kilifi and Mbita. Studentʼs t-tests were used to assess significant differences between tube types. Finally, we exposed the PLA filament to common solutions used with the bioassay kit. Results We were able to design and print functional replacements for each piece of the WHO bioassay kit. Replacement parts are functionally identical to and interchangeable with original WHO bioassay parts. We note no significant difference in mortality results obtained from PLA printed tubes and WHO acrylic tubes. Additionally, we observed no degradation of PLA in response to prolonged exposure times of commonly used cleaning solutions. Conclusions Our designs can be used to produce replacement parts for the WHO bioassay kit in any facility with a 3D printer, which are becoming increasingly widespread. 3D printing technologies can affordably and rapidly address equipment shortages and be used to develop bespoke equipment in laboratories.

Insecticide resistance is a major threat to gains in malaria control, which have been stalling and potentially reversing since 2015. Studies into the causal mechanisms of insecticide resistance are painting an increasingly complicated picture, underlining the need to design and implement targeted studies on this phenotype. In this study, we compare three populations of the major malaria vector An . coluzzii : a susceptible and two resistant colonies with the same genetic background. The original colonised resistant population rapidly lost resistance over a 6-month period, a subset of this population was reselected with pyrethroids, and a third population of this colony that did not lose resistance was also available. The original resistant, susceptible and re-selected colonies were subject to RNAseq and whole genome sequencing, which identified a number of changes across the transcriptome and genome linked with resistance. Firstly, an increase in the expression of genes within the oxidative phosphorylation pathway were seen in both resistant populations compared to the susceptible control; this translated phenotypically through an increased respiratory rate, indicating that elevated metabolism is linked directly with resistance. Genome sequencing highlighted several blocks clearly associated with resistance, including the 2Rb inversion. Finally, changes in the microbiome profile were seen, indicating that the microbial composition may play a role in the resistance phenotype. Taken together, this study reveals a highly complicated phenotype in which multiple transcriptomic, genomic and microbiome changes combine to result in insecticide resistance.

The surface of insects is coated in cuticular hydrocarbons (CHCs); variations in the composition of this layer affect a range of traits including adaptation to arid environments and defence against pathogens and toxins. In the African malaria vector, Anopheles gambiae quantitative and qualitative variance in CHC composition have been associated with speciation, ecological habitat and insecticide resistance. Understanding how these modifications arise will inform us of how mosquitoes are responding to climate change and vector control interventions. CHCs are synthesised in sub-epidermal cells called oenocytes that are very difficult to isolate from surrounding tissues. Here we utilise a transgenic line with fluorescent oenocytes to purify these cells for the first time. Comparative transcriptomics revealed the enrichment of biological processes related to long chain fatty acyl-CoA biosynthesis and elongation of mono-, poly-unsaturated and saturated fatty acids and enabled us to delineate, and partially validate, the hydrocarbon biosynthetic pathway in An gambiae.