The development of insecticide resistance in malaria vectors is of increasing concern in Ethiopia because of its potential implications for vector control failure. To better elucidate the specificity of resistance mechanisms and to facilitate the design of control strategies that minimize the likelihood of selecting for cross-resistance, a whole transcriptomic approach was used to explore gene expression patterns in a multi-insecticide resistant population of Anopheles arabiensis from Oromia Region, Ethiopia. This field population was resistant to the diagnostic doses of malathion (average mortality of 71.9%) and permethrin (77.4%), with pools of survivors and unexposed individuals analyzed using Illumina RNA-sequencing, alongside insecticide susceptible reference strains. This population also demonstrated deltamethrin resistance but complete susceptibility to alpha-cypermethrin, bendiocarb and propoxur, providing a phenotypic basis for detecting insecticide-specific resistance mechanisms. Transcriptomic data revealed overexpression of genes including cytochrome P450s, glutathione-s-transferases and carboxylesterases (including CYP4C36, CYP6AA1, CYP6M2, CYP6M3, CYP6P4, CYP9K1, CYP9L1, GSTD3, GSTE2, GSTE3, GSTE4, GSTE5, GSTE7 and two carboxylesterases) that were shared between malathion and permethrin survivors. We also identified nineteen highly overexpressed cuticular-associated proteins (including CYP4G16, CYP4G17 and chitinase) and eighteen salivary gland proteins (including D7r4 short form salivary protein), which may be contributing to a non-specific resistance phenotype by either enhancing the cuticular barrier or promoting binding and sequestration of insecticides, respectively. These findings provide novel insights into the molecular basis of insecticide resistance in this lesser well-characterized major malaria vector species.

Aedes aegypti transmits major arboviruses of public health importance, including dengue, chikungunya, Zika, and yellow fever. The use of insecticides represents the cornerstone of vector control; however, insecticide resistance in Ae. aegypti has become widespread. Understanding the molecular basis of insecticide resistance in this species is crucial to design effective resistance management strategies. Here, we applied Illumina RNA-Seq to study the gene expression patterns associated with resistance to three widely used insecticides (malathion, alphacypermethrin, and lambda-cyhalothrin) in Ae. aegypti populations from two sites (Manatí and Isabela) in Puerto Rico (PR). Cytochrome P450s were the most overexpressed detoxification genes across all resistant phenotypes. Some detoxification genes (CYP6Z7, CYP28A5, CYP9J2, CYP6Z6, CYP6BB2, CYP6M9, and two CYP9F2 orthologs) were commonly overexpressed in mosquitoes that survived exposure to all three insecticides (independent of geographical origin) while others including CYP6BY1 (malathion), GSTD1 (alpha-cypermethrin), CYP4H29 and GSTE6 (lambda-cyhalothrin) were uniquely overexpressed in mosquitoes that survived exposure to specific insecticides. The gene ontology (GO) terms associated with monooxygenase, iron binding, and passive transmembrane transporter activities were significantly enriched in four out of six resistant vs. susceptible comparisons while serine protease activity was elevated in all insecticide-resistant groups relative to the susceptible strain. Interestingly, cuticular-related protein genes (chinase and chitin) were predominantly downregulated, which was also confirmed in the functional enrichment analysis. This RNA-Seq analysis presents a detailed picture of the candidate detoxification genes and other pathways that are potentially associated with pyrethroid and organophosphate resistance in Ae. aegypti populations from PR. These results could inform development of novel molecular tools for detection of resistance-associated gene expression in this important arbovirus vector and guide the design and implementation of resistance management strategies.

Background: Effective vector control is key to malaria prevention. However, this is now compromised by increased insecticide resistance due to continued reliance on insecticide-based control interventions. In Kenya, we have observed heterogenous resistance to pyrethroids and organophosphates inAnopheles arabiensiswhich is one of the most widespread malaria vectors in the country. We investigated the gene expression profiles of insecticide resistantAn. arabiensispopulations from Migori and Siaya counties in Western Kenya using RNA-Sequencing. Centers for Disease Control and Prevention (CDC) bottle assays were conducted using deltamethrin (DELTA), alphacypermethrin (ACYP) and pirimiphos-methyl (PMM) to determine the resistance status in both sites.Results: Mosquitoes from Migori had average mortalities of 91%, 92% and 58% while those from Siaya had 85%, 86%, and 30% when exposed to DELTA, ACYP and PMM, respectively. RNA-Seq analysis was done on pools of mosquitoes which survived exposure (‘resistant’), mosquitoes that were not exposed, and the insecticide-susceptibleAn. arabiensisDongola strain. Gene expression profiles of resistant mosquitoes from both Migori and Siaya showed an overexpression mainly of salivary gland proteins belonging to both the short and long form D7 genes, and cuticular proteins (including CPR9, CPR10, CPR15, CPR16). Additionally, the overexpression of detoxification genes including cytochrome P450s (CYP9M1, CYP325H1, CYP4C27, CYP9L1 and CYP307A1), 2 carboxylesterases and a glutathione-S-transferase (GSTE4) were also shared between DELTA, ACYP, and PMM survivors, pointing to potential contribution to cross resistance to both pyrethroid and organophosphate insecticides.Conclusion: This study provides novel insights into the molecular basis of insecticide resistance inAn. arabiensisin Western Kenya and suggests that salivary gland proteins and cuticular proteins are associated with resistance to multiple classes of insecticides.

The rapid global expansion of Aedes aegypti-borne diseases such as dengue, chikungunya, and Zika has positioned this mosquito as a key target for vector control programs. These programs rely heavily on insecticide use, leading to the widespread emergence of insecticide resistance. Understanding the molecular basis of resistance is essential for developing effective management strategies. In this study, we employed a whole-transcriptome (RNA-seq) approach to analyze gene expression in three Ae. aegypti populations from Mexico that underwent four generations of laboratory selection with deltamethrin. Several cytochrome P450 genes (CYP6AG4, CYP6M5, CYP307A1) and a chitin-binding peritrophin-like gene (Ae-Aper50) were significantly overexpressed following selection, supporting roles for both detoxification and midgut protection. We also observed a consistent downregulation of cuticular protein genes in deltamethrin-selected groups relative to the baseline populations, suggesting their involvement in baseline tolerance rather than induced resistance. Additionally, the overexpression of immune- and stress-related genes, including the RNA helicase MOV-10, indicates that insecticide selection may trigger broader physiological responses. These findings highlight complex, multi-pathway transcriptomic changes associated with resistance development in Ae. aegypti.

Aedes aegypti transmits major arboviruses of public health importance, including dengue, chikungunya, Zika, and yellow fever. The use of insecticides represents the cornerstone of vector control, however insecticide resistance in Ae. aegypti has become widespread. Understanding the molecular basis of insecticide resistance in this species is crucial to design effective resistance management strategies. Here, we applied Illumina RNA-Seq to study the gene expression patterns associated with resistance to three widely used insecticides (malathion, alpha-cypermethrin and lambda-cyhalothrin) in Ae. aegypti populations from 2 sites (Manatí and Isabela) in Puerto Rico (PR). Cytochrome P450s were the most over-expressed detoxification genes across all resistant phenotypes. Some detoxification genes (CYP6Z7, CYP28A5, CYP9J2, CYP6Z6, CYP6BB2, CYP6M9, and two CYP9F2 orthologs) were commonly over-expressed in mosquitoes that survived exposure to all three insecticides (independent of geographical origin) while others including CYP6BY1 (malathion), GSTD1 (alpha-cypermethrin), CYP4H29 and GSTE6 (lambda-cyhalothrin) were uniquely overexpressed in mosquitoes that survived exposure to specific insecticides. The gene ontology (GO) terms associated with monooxygenase, iron binding, and passive transmembrane transporter activities were significantly enriched in 4 out of 6 resistant vs susceptible comparisons while serine protease activity was elevated in all insecticide resistant groups relative to the susceptible strain. Interestingly, cuticular-related protein genes (chinase and chitin) were predominantly downregulated, which was also confirmed in the functional enrichment analysis. This RNA-Seq analysis presents a detailed picture of the candidate detoxification genes and other pathways that are potentially associated with pyrethroid and organophosphate resistance in Ae. aegypti populations from PR. These results could inform development of novel molecular tools for detection of resistance-associated gene expression in this important arbovirus vector and guide the design and implementation of resistance management strategies.

Anopheles albimanus is one of the principal malaria vectors in the Americas and exhibits phenotypic variation across its geographic distribution. High-quality reference genomes from geographically distant populations are essential to deepen our understanding of the biology, evolution, and genetic variation of this important malaria vector. In this study, we applied long-read PacBio and short-read Illumina sequencing technologies to assemble the complete genomes of two reference strains of An. albimanus, Stecla (originating from El Salvador), and Cartagena (originating from Colombia); and investigated the structural features of these genomes, including gene content, transposable elements (TEs), genetic variation, and structural rearrangements. Our hybrid assembly approach generated reference-quality genomes for each strain and recovered ~ 96% of the expected genome size. The genome assemblies of Stecla and Cartagena consisted of 109 and 149 scaffolds, with estimated genome sizes of 167.5 Mbp (N₅₀ = 88 Mbp) and 167.1 Mbp (N₅₀ = 87 Mbp), respectively. They exhibited a high level of completeness and contained a smaller number of gaps and ambiguous bases than either of the two previously published reference genomes for this species, suggesting a considerable improvement in the quality and completeness of the assemblies. A total of 12,082 and 12,120 protein-coding genes were predicted in Stecla and Cartagena, respectively. TE analyses indicated more repetitive content was captured in the long read assemblies. The assembled genomes shared 98.12% pairwise identity and synteny analyses suggested that gene position was conserved between both strains. These newly assembled genomes will serve as an important resource for future research in comparative genomics, proteomics, epigenetics, transcriptomics, and functional analysis of this important malaria vector.