Key gene modules and hub genes associated with pyrethroid and organophosphate resistance in Anopheles mosquitoes: a systems biology approach.

Indoor residual spraying (IRS) and insecticide-treated nets (ITNs) are the main methods used to control mosquito populations for malaria prevention. The efficacy of these strategies is threatened by the spread of insecticide resistance (IR), limiting the success of malaria control. Studies of the genetic evolution leading to insecticide resistance could enable the identification of molecular markers that can be used for IR surveillance and an improved understanding of the molecular mechanisms associated with IR. This study used a weighted gene co-expression network analysis (WGCNA) algorithm, a systems biology approach, to identify genes with similar co-expression patterns (modules) and hub genes that are potential molecular markers for insecticide resistance surveillance in Kenya and Benin. A total of 20 and 26 gene co-expression modules were identified via average linkage hierarchical clustering from Anopheles arabiensis and An. gambiae, respectively, and hub genes (highly connected genes) were identified within each module. Three specific genes stood out: serine protease, E3 ubiquitin-protein ligase, and cuticular proteins, which were top hub genes in both species and could serve as potential markers and targets for monitoring IR in these malaria vectors. In addition to the identified markers, we explored molecular mechanisms using enrichment maps that revealed a complex process involving multiple steps, from odorant binding and neuronal signaling to cellular responses, immune modulation, cellular metabolism, and gene regulation. Incorporation of these dynamics into the development of new insecticides and the tracking of insecticide resistance could improve the sustainable and cost-effective deployment of interventions.

Transcriptomic analysis of Anopheles gambiae from Benin reveals overexpression of salivary and cuticular proteins associated with cross-resistance to pyrethroids and organophosphates.

BACKGROUND: Insecticide resistance (IR) is one of the major threats to malaria vector control programs in endemic countries. However, the mechanisms underlying IR are poorly understood. Thus, investigating gene expression patterns related to IR can offer important insights into the molecular basis of IR in mosquitoes. In this study, RNA-Seq was used to characterize gene expression in Anopheles gambiae surviving exposure to pyrethroids (deltamethrin, alphacypermethrin) and an organophosphate (pirimiphos-methyl). RESULTS: Larvae of An. gambiae s.s. collected from Bassila and Djougou in Benin were reared to adulthood and phenotyped for IR using a modified CDC intensity bottle bioassay. The results showed that mosquitoes from Djougou were more resistant to pyrethroids (5X deltamethrin: 51.7% mortality; 2X alphacypermethrin: 47.4%) than Bassila (1X deltamethrin: 70.7%; 1X alphacypermethrin: 77.7%), while the latter were more resistant to pirimiphos-methyl (1.5X: 48.3% in Bassila and 1X: 21.5% in Djougou). RNA-seq was then conducted on resistant mosquitoes, non-exposed mosquitoes from the same locations and the laboratory-susceptible An. gambiae s.s. Kisumu strain. The results showed overexpression of detoxification genes, including cytochrome P450s (CYP12F2, CYP12F3, CYP4H15, CYP4H17, CYP6Z3, CYP9K1, CYP4G16, and CYP4D17), carboxylesterase genes (COEJHE5E, COE22933) and glutathione S-transferases (GSTE2 and GSTMS3) in all three resistant mosquito groups analyzed. Genes encoding cuticular proteins (CPR130, CPR10, CPR15, CPR16, CPR127, CPAP3-C, CPAP3-B, and CPR76) were also overexpressed in all the resistant groups, indicating their potential role in cross resistance in An. gambiae. Salivary gland protein genes related to 'salivary cysteine-rich peptide' and 'salivary secreted mucin 3' were also over-expressed and shared across all resistant groups. CONCLUSION: Our results suggest that in addition to metabolic enzymes, cuticular and salivary gland proteins could play an important role in cross-resistance to multiple classes of insecticides in Benin. These genes warrant further investigation to validate their functional role in An. gambiae resistance to insecticides.

Whole transcriptomic analysis reveals overexpression of salivary gland and cuticular proteins genes in insecticide-resistant Anopheles arabiensis from Western Kenya.

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 in Anopheles arabiensis which is one of the most widespread malaria vectors in the country. We investigated the gene expression profiles of insecticide resistant An. arabiensis populations 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-susceptible An. arabiensis Dongola 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 in An. arabiensis in Western Kenya and suggests that salivary gland proteins and cuticular proteins are associated with resistance to multiple classes of insecticides.

Postintervention Immunological and Entomological Survey of Lymphatic Filariasis in the City of Olinda, Brazil, 2015-2016.

Lymphatic filariasis (LF) is a leading cause of disability due to infectious disease worldwide. The Recife Metropolitan Region (RMR) is the only remaining focus of LF in Brazil, where the parasite Wuchereria bancrofti is transmitted solely by the mosquito Culex quinquefasciatus. This study reports the results of transmission assessment surveys and molecular xenomonitoring in the city of Olinda, RMR, after nearly 15 years (2015-2016) of interventions for LF elimination. Participants were screened for W. bancrofti antigen via immunochromatographic card tests (ICT) in: 1) door-to-door surveys conducted for all children aged 5-7 years from 4 out of 17 intervention areas treated with at least five annual doses of mass drug administration (MDA), and 2) a two-stage cluster sampling survey of residents aged 5 years and older in non-MDA areas. Mosquitoes were collected via handheld aspirators in four MDA areas, differentiated by species, sex, and physiological status, pooled into groups of up to 10 blood-fed, semigravid, and gravid mosquitoes, and screened for W. bancrofti infection by real-time quantitative polymerase chain reaction (RT-qPCR). All 1,170 children from MDA areas and the entire population sample of 990 residents in non-MDA areas were ICT negative. In MDA areas, a total of 3,152 female Cx. quinquefasciatus mosquitoes in 277 households (range, 0-296 mosquitoes per house) were collected via aspiration. RT-qPCR of 233 pools of mosquitos were negative for W. bancrofti RNA; an independent reference laboratory confirmed these results. These results provide evidence that LF transmission has been halted in this setting.

Comparative Transcriptomic Analysis of Insecticide-Resistant Aedes aegypti from Puerto Rico Reveals Insecticide-Specific Patterns of Gene Expression.

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.

Pyrethroid resistance in the New World malaria vector Anopheles albimanus is mediated by cytochrome P450 CYP6P5.

Pyrethroid resistance in the malaria vector Anopheles albimanus presents an obstacle to malaria elimination in the Americas. Here, An. albimanus CYP6P5 (the most overexpressed P450 in a Peruvian population) was functionally characterized. Recombinant CYP6P5 metabolized the type II pyrethroids, deltamethrin and α-cypermethrin with comparable affinities (KM of 3.3 µM ± 0.4 and 3.6 µM ± 0.5, respectively), but exhibited a 2.7-fold higher catalytic rate for α-cypermethrin (kcat of 6.02 min-1 ± 0.2) versus deltamethrin (2.68 min-1 ± 0.09). Time-course assays revealed progressive depletion of the above pyrethroids with production of four HPLC-detectable metabolites. Low depletion was obtained with type I pyrethroid, permethrin. Transgenic expression in Drosophila melanogaster demonstrated that overexpression of CYP6P5 alone conferred type II pyrethroid resistance, with only 16% and 55.3% mortalities in flies exposed to 0.25% α-cypermethrin and 0.15% deltamethrin, respectively. Synergist bioassays using P450 inhibitor piperonylbutoxide significantly recovered susceptibility (mortality = 73.6%, p < 0.001) in synergized flies exposed to 4% piperonylbutoxide, plus 0.25% α-cypermethrin, compared to non-synergized flies (mortality = 4.9%). Moderate resistance was also observed towards 4% DDT. These findings established the preeminent role of CYP6P5 in metabolic resistance in An. albimanus, highlighting challenges associated with deployment of insecticide-based control tools in the Americas.

Fitness Cost of Sequential Selection with Deltamethrin in Aedes aegypti (Diptera: Culicidae).

In Mexico, Aedes aegypti (L.) is the primary dengue vector, chikungunya, and Zika viruses. The continued use of synthetic pyrethroids has led to the development of resistance in target populations, which has diminished the effectiveness of vector control programs. Resistance has been associated with disadvantages that affect the biological parameters of resistant mosquitoes compared to susceptible ones. In the present study, the disadvantages were evaluated by parameters related to survival and reproduction ('fitness cost') after selection with deltamethrin for five generations. The parameters analyzed were the length of the development cycle, sex ratio, survival, longevity, fecundity, egg viability, preoviposition, oviposition and postoviposition periods, and growth parameters. In the deltamethrin-selected strain, there was a decrease in the development cycle duration, the percentage of pupae, the oviposition period, and eggs viability. Although mean daily fecundity was not affected after the selection process, this, together with the decrease in the survival and fecundity levels by specific age, significantly affected the gross reproductive rate (GRR), net reproductive rate (Ro), and intrinsic growth rate (rm) of the group selected for five generations with deltamethrin compared to the group without selection. Identifying the 'cost' of resistance in biological fitness represents an advantage if it is desired to limit the spread of resistant populations since the fitness cost is the less likely that resistant individuals will spread in the population. This represents an important factor to consider in designing integrated vector management programs.

A whole transcriptomic approach provides novel insights into the molecular basis of organophosphate and pyrethroid resistance in Anopheles arabiensis from Ethiopia.

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.

Development of molecular assays to detect target-site mechanisms associated with insecticide resistance in malaria vectors from Latin America.

BACKGROUND: Malaria remains an important public health problem in Latin America, and the development of insecticide resistance in malaria vectors poses a major threat to malaria elimination efforts. Monitoring of insecticide susceptibility and the determination of the mechanisms involved in insecticide resistance are needed to effectively guide the deployment of appropriate vector control measures. Here, molecular assays have been developed to screen for mutations associated with insecticide resistance on the voltage-gated sodium channel (VGSC) and acetylcholinesterase-1 (Ace-1) genes in four malaria vectors from Latin America. METHODS: Degenerate primers were designed to amplify a partial fragment on the VGSC and Ace-1 genes. Wild-caught individuals for Anopheles albimanus (also historical samples and individuals from a laboratory strain), Anopheles darlingi, Anopheles vestitipennis and Anopheles pseudopunctipennis were used to optimize the PCR assays. All samples were sequenced to validate the PCR results and DNA alignments were constructed for each gene using the unique haplotypes observed. RESULTS: Primers designed successfully amplified the VGSC gene in An. albimanus, An. darlingi, An. vestitipennis and An. pseudopunctipennis, and the Ace-1 gene in both An. albimanus and An. darlingi. DNA sequencing revealed that compared with Anopheles gambiae, there were a total of 29, 28, 21 and 24 single nucleotide polymorphisms (SNPs) on the VGSC gene for An. albimanus (308 bp), An. darlingi (311 bp), An. pseudopunctipennis (263 bp) and An. vestitipennis (254 bp), respectively. On the 459 bp fragment of the Ace-1 gene, a total of 70 SNPs were detected in An. darlingi and 59 SNPs were detected in An. albimanus compared with An. gambiae. The SNPs detected on the VGSC gene were all synonymous. On the Ace-1 gene, non-synonymous substitutions were identified on three different codons. All species showed the homozygous wild-type kdr allele (coding for leucine) at codon 995 (formerly reported as codon 1014) on the VGSC gene, but one sample was heterozygous at codon 280 (formerly reported as codon 119) on the Ace-1 gene, coding for both the resistant (serine) and susceptible (glycine) amino acids. CONCLUSIONS: New molecular assays to amplify and screen the regions of the VGSC and Ace-1 genes associated with insecticide resistance are reported for An. albimanus, An. darlingi, An. vestitipennis, and An. pseudopunctipennis. The development of these PCR assays presents an important advance in the analysis of target-site resistance in malaria vectors in the Americas, and will further facilitate the characterization of insecticide resistance mechanisms in these species.

Contrasting patterns of gene expression indicate differing pyrethroid resistance mechanisms across the range of the New World malaria vector Anopheles albimanus.

Decades of unmanaged insecticide use and routine exposure to agrochemicals have left many populations of malaria vectors in the Americas resistant to multiple classes of insecticides, including pyrethroids. The molecular basis of pyrethroid resistance is relatively uncharacterised in American malaria vectors, preventing the design of suitable resistance management strategies. Using whole transcriptome sequencing, we characterized the mechanisms of pyrethroid resistance in Anopheles albimanus from Peru and Guatemala. An. albimanus were phenotyped as either deltamethrin or alpha-cypermethrin resistant. RNA from 1) resistant, 2) unexposed, and 3) a susceptible laboratory strain of An. albimanus was sequenced and analyzed using RNA-Seq. Expression profiles of the three groups were compared based on the current annotation of the An. albimanus reference genome. Several candidate genes associated with pyrethroid resistance in other malaria vectors were found to be overexpressed in resistant An. albimanus. In addition, gene ontology terms related to serine-type endopeptidase activity, extracellular activity and chitin metabolic process were also commonly overexpressed in the field caught resistant and unexposed samples from both Peru and Guatemala when compared to the susceptible strain. The cytochrome P450 CYP9K1 was overexpressed 14x in deltamethrin and 8x in alpha-cypermethrin-resistant samples from Peru and 2x in deltamethrin-resistant samples from Guatemala, relative to the susceptible laboratory strain. CYP6P5 was overexpressed 68x in deltamethrin-resistant samples from Peru but not in deltamethrin-resistant samples from Guatemala. When comparing overexpressed genes between deltamethrin-resistant and alpha-cypermethrin-resistant samples from Peru, a single P450 gene, CYP4C26, was overexpressed 9.8x (p<0.05) in alpha-cypermethrin-resistant samples. In Peruvian deltamethrin-resistant samples, the knockdown resistance mutation (kdr) variant alleles at position 1014 were rare, with approximately 5% frequency, but in the alpha-cypermethrin-resistant samples, the frequency of these alleles was approximately 15-30%. Functional validation of the candidate genes and the kdr mutation as a resistance marker for alpha-cypermethrin will confirm the role of these mechanisms in conferring pyrethroid resistance.