Expansive and diverse phenotypic landscape of field Aedes aegypti larvae with differential susceptibility to temephos: beyond metabolic detoxification

Arboviruses including dengue, Zika and chikungunya are amongst the most significant public health concerns worldwide and their control relies heavily on the use of insecticides to control the vector mosquito Aedes aegypti. The success of controlling these vector-pathogen systems is threatened by widespread insecticide resistance. The work presented here profiled the gene expression of the larvae from two field populations of Ae. aegypti with differential susceptibility to temephos. The contrasting phenotypes originated from two Colombian urban locations, Bello and Cúcuta, that we have previously reported to have distinctive disease incidence, socioeconomics, and climate. The closeness of the geographical origin of the study populations was suspected to be highly influential in the profiling of the gene expression of resistance since the mosquito’s resistance levels themselves are highly dependent upon environmental variables. We demonstrated that an exclusive field-to-lab (Ae. aegypti reference strain New Orleans) comparison generates an over estimation of differential gene expression (DGE) and that the inclusion of a geographically relevant field control, as used here, yields a more discrete, and likely, more specific set of genes. The composition of the obtained DGE profiles is varied, with commonly reported resistance associated genes such as detoxifying enzymes having only a small representation. We identify cuticle biosynthesis, ion exchange homeostasis, an extensive number of long non-coding RNAs, and chromatin modelling among the specifically and differentially expressed genes in field resistant Ae. aegypti larvae. It was also shown that temephos resistant larvae undertake further gene expression responses when temporarily exposed to this insecticide. The results from the sampling triangulation approach undertaken here contributes a discrete DGE profiling with reduced noise that permitted the observation of a greater gene diversity. This deeper gene granularity significantly increases the number of potential targets for the control of insecticide resistant mosquitoes and widens our knowledge base on the complex phenotypic network of the Ae. aegypti mosquito responses to insecticides. Author Summary Aedes aegypti mosquitoes are vectors for several significant human viruses including dengue, Zika and chikungunya. The lack of widely available vaccines and specific antiviral treatments for these viruses means that the principal method for reducing disease burden is through controlling the vector mosquitoes. Mosquito control relies heavily on the use of insecticides and successful vector control is threatened by widespread insecticide resistance in Ae. aegypti. Here, we examined changes in gene expression that occur in temephos resistant populations of Ae. aegypti from two field populations in Colombia. We compare gene expression in resistant larvae from Cúcuta with susceptible larvae from Bello and a susceptible laboratory strain of Ae. aegypti (New Orleans). We also compare mosquitoes from Cúcuta with and without temephos exposure. We report several differentially expressed genes beyond those usually reported in resistant mosquitoes. We also demonstrate the over estimation in differential gene expression that can occur when field resistant populations are compared against lab susceptible populations only. The identification of new mechanisms involved in the development of insecticide resistance is crucial to fully understanding how resistance occurs and how best it can be reduced.

calculated using probit analysis. Resistance ratios (RR) calculated as a ratio of the lethal Orleans) strain. Bello and New Orleans are both susceptible to temephos whilst larvae from 2 6 5 Cúcuta are resistant. SE = standard error. total genes in the reference genome.  of DE transcripts detected in the field resistant samples.

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We also sought to investigate the gene expression changes in insecticide resistant larvae 2 9 9 under transient exposure to insecticide. There were only 19 transcripts significantly 3 0 0 differentially expressed in larvae within the resistant population which were exposed to 3 0 1 temephos when compared to unexposed larvae from the same population ( Fig 4E). All 19 of 3 0 2 those transcripts were overexpressed in the exposed group with no significant down regulated 3 0 3 gene expression detected ( Fig 4C).

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We addressed the issue of potential misrepresentation of gene expression metrics by ubiquinone and other terpenoid-quinone biosynthesis (path:00130) (Fig 5).  The overexpressed transcriptome of field temephos resistant Aedes aegypti larvae 3 3 7 The transcriptomic overview provided by the GO and KEGG enrichment models was represented genes (Fig 7). The former allowed the visualisation of the data's granularity by expression profiles between field and lab populations (here FR and LS) rather than between 3 5 0 field to field (e.g., FR and FS). Differences in gene expression were also visualised using  Table).

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Other over expressed genes included the hydrocarbon biosynthesis pathway enzyme acetyl- The under expressed transcriptome of field resistant Aedes aegypti larvae The transcript profiles of the 75 annotated protein coding genes significantly under expressed variation between genes and between resistance status.

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Genes encoding detoxification enzymes were also presented in this set of under expressed   The expression of several ion and solute membrane transporters were also down regulated.

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These included the sodium-coupled cation-chloride cotransporter AAEL009886 (aeCC3), the 3 8 9 sodium/chloride dependent amino acid transporter AAEL000298, the sodium/solute expressed genes were also 30 lncRNA genes in the temephos resistant larvae (S1 Table). Gene expression profile of temephos exposed larvae from the resistant population 3 9 3 Gene expression in the field resistant population following the controlled exposure to 3 9 4 temephos was compared with gene expression of samples from the same population without 3 9 5 insecticide exposure. The exposed samples showed 19 significantly (FC >2, p-value <0.05)  Table) in comparison to the non-exposed samples.

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These 19 transcripts were mapped to 13 genes (Fig 10). The products of the over expressed overexpressed genes had uncharacterised products in Ae. aegypti (S2 Table). reduces the effectivity of vector control [70][71][72]. In this study we report resistance to susceptible. Bello is an area of relatively low arbovirus incidence , whilst Cúcuta is an area of high arbovirus incidence 4 0 8 which has seen routine use of temephos for Ae. aegypti control over four decades . The 4 0 9 reported resistance in Cúcuta is consistent with previous reports of temephos resistance in Ae. . Whilst the resistance to temephos appears to have reduced in Cúcuta The triangulation of differential gene expression against two unrelated susceptible 4 1 8 populations, one lab and one field, was selected to reduce confounding effects of phenotypic 4 1 9 differences between populations unrelated to insecticide resistance. Whilst this experimental 4 2 0 design does reduce these confounding effects it is not possible to mitigate this entirely and 4 2 1 therefore some of the differences in gene expression which are observed here may not be 4 2 2 related to temephos resistance but resistance to other insecticides and other phenotypic 4 2 3 differences between populations. The differential gene expression reported here could be the  Genes which were found to be differentially expressed in the current study may also be the (oxidoreductase activity (GO:0016491) and oxidation-reduction process (GO:0055114)) were 4 5 8 also found to be enriched in the temephos resistant larvae. Thus, by cross examining the data 4 5 9 in field-to-field and field-to-lab population comparison, we observed genes representing these and 11 CEs would have been reported as differentially expressed (S3 Table). This suggests 4 6 4 that large overexpression of detoxification genes may be partly related to differences between 4 6 5 field and lab mosquitoes rather than associated with the insecticide resistant phenotype. Large overexpression of detoxification in mosquitoes may also only be observed in mosquitoes Chitin biosynthesis 4 7 0 The thickness and composition of the cuticle has been identified as a critical determinant of temephos resistant larvae in the current study, also has roles in the innate immune response to The expression of several ion coupled solute membrane transporters was down regulated in The aquatic life of the Ae. aegypti larval stages demands an ion exchange homeostasis that 5 1 8 differs from that of the adult mosquitoes. Due to their freshwater habitat Ae. aegypti larvae 5 1 9 must excrete water gained by osmosis, reabsorb salt prior to excreting urine, and absorb salt 5 2 0 from their surroundings [110]. Whilst the opposite is true in adults where water retention is 5 2 1 needed due to constant loss through evaporation. A key process in this is Na+-dependent co-5 2 2 transport which is typically down the large inward (extracellular to intracellular) Na+ further role for this protein in mediating insecticide resistance.

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There were 55 over expressed and 30 under expressed genes encoding for long non-coding RNA (lncRNA) in the temephos resistant larvae (S1 Table). Non-coding RNAs (ncRNA) are have also been associated with insect's response to xenobiotics, with reports of differential in protein coding genes, the development of next generation techniques have now provided 5 6 0 an opportunity to also study noncoding RNA. Whilst work has been conducted into Ae. aegypti [126] there have been no studies that have aimed to investigate the role of In the study we also tracked gene expression in insecticide resistant larvae following direct 5 6 9 response to temephos exposure. Thirteen genes were found to have a significantly increased 5 7 0 expression following a controlled exposure to temephos. Among those 13 genes were two 5 7 1 serine proteases: trypsin -1 (AAEL016975) and serine protease stubble (AAEL020367), a 5 7 2 cysteine protease: cathepsin-1 (AAEL011167), a sodium/chloride dependent amino acid Ae. aegypti from Cúcuta [17]. Serine proteases have also been shown to degrade insecticides performance and fitness disadvantage.

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Deleterious effects of insecticide resistance can affect a wide range of life-history traits (e.g.

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longevity, biting behaviour, and vector competence) [140,141]. Although the cost of resistance genes is believed to gradually decrease due to subsequent modifier mutations patterns that resistant mosquitoes further undergo when exposed to the insecticide could be a source for novel assets for vector control. The study of such targets for insecticide 5 9 2 development is a strategy that, to our knowledge, has not yet been explored.

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Conclusion 5 9 4 This study found differential insecticide responses from Ae. aegypti field samples of two 5 9 5 previously epidemiologically characterised sites in Colombia. Using these contrasting Ae. strains. The two overexpressed P450s in resistant Ae. aegypti larvae represent some ten-fold presented here contributed to what seems to be an expansive and varied phenotypic landscape 6 0 7 in the Ae. aegypti responses to insecticides of current importance.   functional annotation for the DEG sets was carried with several different repositories: VectorBase, Gene Ontology (GO) and KEGG Enrichment Analysis. Susceptible) separated from both field samples. The orthogonal dispersion of these samples 6 4 1 allowed for the triangulation of the data as described in the main text. those over expressed (red), under expressed (blue) and with no significant differential  indicate the number of differentially expressed transcripts in each category. Culicidae) to temephos from three districts of Tamil Nadu, India. J Vector Borne Dis. in aedes aegypti populations from DHF-endemic areas in Padang, Indonesia. the co-circulation of dengue, zika and chikungunya in three different ecosystems in Evidence of multiple pyrethroid resistance mechanisms in the malaria vector