Laboratory evaluation of the contact irritancy of a clothianidin solo formulation vs. clothianidin-deltamethrin mixture formulations for indoor residual spraying against pyrethroid-resistant Anopheles gambiae sensu lato.

BACKGROUND: Clothianidin-based indoor residual spraying (IRS) formulations have become available for malaria control as either solo formulations of clothianidin or a mixture of clothianidin with the pyrethroid deltamethrin. While both formulations have been successfully used for malaria control, studies investigating the effect of the pyrethroid in IRS mixtures may help improve our understanding for development of future IRS products. It has been speculated that the irritant effect of the pyrethroid in the mixture formulation may result in shorter mosquito contact times with the treated walls potentially leading to a lower impact. METHODS: We compared contact irritancy expressed as the number of mosquito take-offs from cement surfaces treated with an IRS formulation containing clothianidin alone (SumiShield® 50WG) to clothianidin-deltamethrin mixture IRS formulations against pyrethroid-resistant Anopheles gambiae sensu lato under controlled laboratory conditions using a modified version of the World Health Organisation cone bioassay. To control for the pyrethroid, comparison was made with a deltamethrin-only formulation. Both commercial and generic non-commercial mixture formulations of clothianidin and deltamethrin were tested. RESULTS: The clothianidin solo formulation did not show significant contact irritancy relative to the untreated control (3.5 take-offs vs. 3.1 take-offs, p = 0.614) while all deltamethrin-containing IRS induced significant irritant effects. The number of take-offs compared to the clothianidin solo formulation (3.5) was significantly higher with the commercial clothianidin-deltamethrin mixture (6.1, p = 0.001), generic clothianidin-deltamethrin mixture (7.0, p < 0.001), and deltamethrin-only (8.2, p < 0.001) formulations. The commercial clothianidin-deltamethrin mixture induced similar contact irritancy as the generic clothianidin-deltamethrin mixture (6.1 take-offs vs. 7.0 take-offs, p = 0.263) and deltamethrin-only IRS (6.1 take-offs vs. 8.2, p = 0.071), showing that the irritant effect in the mixture was attributable to its deltamethrin component. CONCLUSIONS: This study provides evidence that the enhanced contact irritancy of the pyrethroid in clothianidin-deltamethrin IRS mixtures can shorten mosquito contact times with treated walls compared to the clothianidin solo formulation. Further trials are needed to directly compare the efficacy of these formulation types under field conditions and establish the impact of this enhanced contact irritancy on the performance of IRS mixture formulations containing pyrethroids.

Mutations and intron polymorphisms in voltage-gated sodium channel genes of different geographic populations of Culex pipiens pallens/Culex pipiens quinquefasciatus in China.

BACKGROUND: Culex pipiens pallens and Culex pipiens quinquefasciatus are the dominant species of Culex mosquitoes in China and important disease vectors. Long-term use of insecticides can cause mutations in the voltage-gated sodium channel (vgsc) gene of mosquitoes, but little is known about the current status and evolutionary origins of vgsc gene in different geographic populations. Therefore, this study aimed to determine the current status of vgsc genes in Cx. p. pallens and Cx. p. quinquefasciatus in China and to investigate the evolutionary inheritance of neighboring downstream introns of the vgsc gene to determine the impact of insecticides on long-term evolution. METHODS: Sampling was conducted from July to September 2021 in representative habitats of 22 provincial-level administrative divisions in China. Genomic DNA was extracted from 1308 mosquitoes, the IIS6 fragment of the vgsc gene on the nerve cell membrane was amplified using polymerase chain reaction, and the sequence was used to evaluate allele frequency and knockdown resistance (kdr) frequency. MEGA 11 was used to construct neighbor-joining (NJ) tree. PopART was used to build a TCS network. RESULTS: There were 6 alleles and 6 genotypes at the L1014 locus, which included the wild-type alleles TTA/L and CTA/L and the mutant alleles TTT/F, TTC/F, TCT/S and TCA/S. The geographic populations with a kdr frequency less than 20.00% were mainly concentrated in the regions north of 38° N, and the geographic populations with a kdr frequency greater than 80.00% were concentrated in the regions south of 30° N. kdr frequency increased with decreasing latitude. And within the same latitude, the frequency of kdr in large cities is relatively high. Mutations were correlated with the number of introns. The mutant allele TCA/S has only one intron, the mutant allele TTT/F has three introns, and the wild-type allele TTA/L has 17 introns. CONCLUSIONS: Cx. p. pallens and Cx. p. quinquefasciatus have developed resistance to insecticides in most regions of China. The neighboring downstream introns of the vgsc gene gradually decreased to one intron with the mutation of the vgsc gene. Mutations may originate from multiple mutation events rather than from a single origin, and populations lacking mutations may be genetically isolated.

Mapping malaria vectors and insecticide resistance in a high-endemic district of Haryana, India: implications for vector control strategies.

BACKGROUND: Achieving effective control and elimination of malaria in endemic regions necessitates a comprehensive understanding of local mosquito species responsible for malaria transmission and their susceptibility to insecticides. METHODS: The study was conducted in the highly malaria prone Ujina Primary Health Center of Nuh (Mewat) district of Haryana state of India. Monthly entomological surveys were carried out for adult mosquito collections via indoor resting collections, light trap collections, and pyrethrum spray collections. Larvae were also collected from different breeding sites prevalent in the region. Insecticide resistance bioassay, vector incrimination, blood meal analysis was done with the collected vector mosquitoes. RESULTS: A total of 34,974 adult Anopheles mosquitoes were caught during the survey period, out of which Anopheles subpictus was predominant (54.7%). Among vectors, Anopheles stephensi was predominant (15.5%) followed by Anopheles culicifacies (10.1%). The Human Blood Index (HBI) in the case of An. culicifacies and An. stephensi was 6.66 and 9.09, respectively. Vector incrimination results revealed Plasmodium vivax positivity rate of 1.6% for An. culicifacies. Both the vector species were found resistant to DDT, malathion and deltamethrin. CONCLUSION: The emergence of insecticide resistance in both vector species, compromises the effectiveness of commonly used public health insecticides. Consequently, the implementation of robust insecticide resistance management strategies becomes imperative. To effectively tackle the malaria transmission, a significant shift in vector control strategies is warranted, with careful consideration and adaptation to address specific challenges encountered in malaria elimination efforts.

Insights and challenges of insecticide resistance modelling in malaria vectors: a review.

BACKGROUND: Malaria is one of the most devastating tropical diseases, resulting in loss of lives each year, especially in children under the age of 5 years. Malaria burden, related deaths and stall in the progress against malaria transmission is evident, particularly in countries that have moderate or high malaria transmission. Hence, mitigating malaria spread requires information on the distribution of vectors and the drivers of insecticide resistance (IR). However, owing to the impracticality in establishing the critical need for real-world information at every location, modelling provides an informed best guess for such information. Therefore, this review examines the various methodologies used to model spatial, temporal and spatio-temporal patterns of IR within populations of malaria vectors, incorporating pest-biology parameters, adopted ecological principles, and the associated modelling challenges. METHODS: The review focused on the period ending March 2023 without imposing restrictions on the initial year of publication, and included articles sourced from PubMed, Web of Science, and Scopus. It was also limited to publications that deal with modelling of IR distribution across spatial and temporal dimensions and excluded articles solely focusing on insecticide susceptibility tests or articles not published in English. After rigorous selection, 33 articles met the review's elibility criteria and were subjected to full-text screening. RESULTS: Results show the popularity of Bayesian geostatistical approaches, and logistic and static models, with limited adoption of dynamic modelling approaches for spatial and temporal IR modelling. Furthermore, our review identifies the availability of surveillance data and scarcity of comprehensive information on the potential drivers of IR as major impediments to developing holistic models of IR evolution. CONCLUSIONS: The review notes that incorporating pest-biology parameters, and ecological principles into IR models, in tandem with fundamental ecological concepts, potentially offers crucial insights into the evolution of IR. The results extend our knowledge of IR models that provide potentially accurate results, which can be translated into policy recommendations to combat the challenge of IR in malaria control.

Signatures of adaptation at key insecticide resistance loci in Anopheles gambiae in Southern Ghana revealed by reduced-coverage WGS.

Resistance to insecticides and adaptation to a diverse range of environments present challenges to Anopheles gambiae s.l. mosquito control efforts in sub-Saharan Africa. Whole-genome-sequencing is often employed for identifying the genomic basis underlying adaptation in Anopheles, but remains expensive for large-scale surveys. Reduced coverage whole-genome-sequencing can identify regions of the genome involved in adaptation at a lower cost, but is currently untested in Anopheles mosquitoes. Here, we use reduced coverage WGS to investigate population genetic structure and identify signatures of local adaptation in Anopheles mosquitoes across southern Ghana. In contrast to previous analyses, we find no structuring by ecoregion, with Anopheles coluzzii and Anopheles gambiae populations largely displaying the hallmarks of large, unstructured populations. However, we find signatures of selection at insecticide resistance loci that appear ubiquitous across ecoregions in An. coluzzii, and strongest in forest ecoregions in An. gambiae. Our study highlights resistance candidate genes in this region, and validates reduced coverage WGS, potentially to very low coverage levels, for population genomics and exploratory surveys for adaptation in Anopheles taxa.

A chromosome-level genome assembly of the soybean pod borer: insights into larval transcriptional response to transgenic soybean expressing the pesticidal Cry1Ac protein.

BACKGROUND: Genetically modified (GM) crop plants with transgenic expression of Bacillus thuringiensis (Bt) pesticidal proteins are used to manage feeding damage by pest insects. The durability of this technology is threatened by the selection for resistance in pest populations. The molecular mechanism(s) involved in insect physiological response or evolution of resistance to Bt is not fully understood. RESULTS: To investigate the response of a susceptible target insect to Bt, the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), was exposed to soybean, Glycine max, expressing Cry1Ac pesticidal protein or the non-transgenic parental cultivar. Assessment of larval changes in gene expression was facilitated by a third-generation sequenced and scaffolded chromosome-level assembly of the L. glycinivorella genome (657.4 Mb; 27 autosomes + Z chromosome), and subsequent structural annotation of 18,197 RefSeq gene models encoding 23,735 putative mRNA transcripts. Exposure of L. glycinivorella larvae to transgenic Cry1Ac G. max resulted in prediction of significant differential gene expression for 204 gene models (64 up- and 140 down-regulated) and differential splicing among isoforms for 10 genes compared to unexposed cohorts. Differentially expressed genes (DEGs) included putative peritrophic membrane constituents, orthologs of Bt receptor-encoding genes previously linked or associated with Bt resistance, and those involved in stress responses. Putative functional Gene Ontology (GO) annotations assigned to DEGs were significantly enriched for 36 categories at GO level 2, respectively. Most significantly enriched cellular component (CC), biological process (BP), and molecular function (MF) categories corresponded to vacuolar and microbody, transport and metabolic processes, and binding and reductase activities. The DEGs in enriched GO categories were biased for those that were down-regulated (≥ 0.783), with only MF categories GTPase and iron binding activities were bias for up-regulation genes. CONCLUSIONS: This study provides insights into pathways and processes involved larval response to Bt intoxication, which may inform future unbiased investigations into mechanisms of resistance that show no evidence of alteration in midgut receptors.

Heterologous expression, biochemical characterization and prospects for insecticide biosensing potential of carboxylesterase Ha006a from Helicoverpa armigera.

Enzymes have attracted considerable scientific attention for their crucial role in detoxifying a wide range of harmful compounds. In today's global context, the extensive use of insecticides has emerged as a significant threat to the environment, sparking substantial concern. Insects, including economically important pests like Helicoverpa armigera, have developed resistance to conventional pest control methods through enzymes like carboxyl/cholinesterases. This study specifically focuses on a notable carboxyl/cholinesterase enzyme from Helicoverpa armigera (Ha006a), with the goal of harnessing its potential to combat environmental toxins. A total of six insecticides belonging to two different classes displayed varying inhibitory responses towards Ha006a, thereby rendering it effective in detoxifying a broader spectrum of insecticides. The significance of this research lies in discovering the bioremediation property of Ha006a, as it hydrolyzes synthetic pyrethroids (fenvalerate, λ-cyhalothrin and deltamethrin) and sequesters organophosphate (paraoxon ethyl, profenofos, and chlorpyrifos) insecticides. Additionally, the interaction studies between organophosphate insecticides and Ha006a helped in the fabrication of a novel electroanalytical sensor using a modified carbon paste electrode (MCPE). This sensor boasts impressive sensitivity, with detection limits of 0.019 µM, 0.15 µM, and 0.025 µM for paraoxon ethyl, profenofos, and chlorpyrifos, respectively. This study provides a comprehensive biochemical and biophysical characterization of the purified esterase Ha006a, showcasing its potential to remediate different classes of insecticides.

Aberrant splicing of a nicotinic acetylcholine receptor alpha 6 subunit is associated with spinosad tolerance in the thrips predator Orius laevigatus.

Susceptibility to insecticides is one of the limiting factors preventing wider adoption of natural enemies to control insect pest populations. Identification and selective breeding of insecticide tolerant strains of commercially used biological control agents (BCAs) is one of the approaches to overcome this constraint. Although a number of beneficial insects have been selected for increased tolerance to insecticides the molecular mechanisms underpinning these shifts in tolerance are not well characterised. Here we investigated the molecular mechanisms of enhanced tolerance of a lab selected strain of Orius laevigatus (Fieber) to the commonly used biopesticide spinosad. Transcriptomic analysis showed that spinosad tolerance is not a result of overexpressed detoxification genes. Molecular analysis of the target site for spinosyns, the nicotinic acetylcholine receptor (nAChR), revealed increased expression of truncated transcripts of the nAChR α6 subunit in the spinosad selected strain, a mechanism of resistance which was described previously in insect pest species. Collectively, our results demonstrate the mechanisms by which some beneficial biological control agents can evolve insecticide tolerance and will inform the development and deployment of insecticide-tolerant natural enemies in integrated pest management strategies.

An optical system to detect, surveil, and kill flying insect vectors of human and crop pathogens.

Sustainable and effective means to control flying insect vectors are critically needed, especially with widespread insecticide resistance and global climate change. Understanding and controlling vectors requires accurate information about their movement and activity, which is often lacking. The Photonic Fence (PF) is an optical system that uses machine vision, infrared light, and lasers to identify, track, and interdict vectors in flight. The PF examines an insect's outline, flight speed, and other flight parameters and if these match those of a targeted vector species, then a low-power, retina-safe laser kills it. We report on proof-of-concept tests of a large, field-sized PF (30 mL × 3 mH) conducted with Aedes aegypti, a mosquito that transmits dangerous arboviruses, and Diaphorina citri, a psyllid which transmits the fatal huanglongbing disease of citrus. In tests with the laser engaged, < 1% and 3% of A. aegypti and D. citri, respectfully, were recovered versus a 38% and 19% recovery when the lacer was silenced. The PF tracked, but did not intercept the orchid bee, Euglossa dilemma. The system effectively intercepted flying vectors, but not bees, at a distance of 30 m, heralding the use of photonic energy, rather than chemicals, to control flying vectors.

CRISPR/Cas9-Mediated Knockout of the PxJHBP Gene Resulted in Increased Susceptibility to Bt Cry1Ac Protoxin and Reduced Lifespan and Spawning Rates in Plutella xylostella.

Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella.

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.

Adult mosquitoes of the sibling species Anopheles gambiae and Anopheles coluzzii exhibit contrasting patterns of susceptibility to four neonicotinoid insecticides along an urban-to-rural gradient in Yaoundé, Cameroon.

BACKGROUND: Neonicotinoids are potential alternatives for controlling pyrethroid-resistant mosquitoes, but their efficacy against malaria vector populations of sub-Saharan Africa has yet to be investigated. The aim of the present study was to test the efficacy of four neonicotinoids against adult populations of the sibling species Anopheles gambiae and Anopheles coluzzii sampled along an urban-to-rural gradient. METHODS: The lethal toxicity of three active ingredients for adults of two susceptible Anopheles strains was assessed using concentration-response assays, and their discriminating concentrations were calculated. The discriminating concentrations were then used to test the susceptibility of An. gambiae and An. coluzzii mosquitoes collected from urban, suburban and rural areas of Yaoundé, Cameroon, to acetamiprid, imidacloprid, clothianidin and thiamethoxam. RESULTS: Lethal concentrations of neonicotinoids were relatively high suggesting that this class of insecticides has low toxicity against Anopheles mosquitoes. Reduced susceptibility to the four neonicotinoids tested was detected in An. gambiae populations collected from rural and suburban areas. By contrast, adults of An. coluzzii that occurred in urbanized settings were susceptible to neonicotinoids except acetamiprid for which 80% mortality was obtained within 72 h of insecticide exposure. The cytochrome inhibitor, piperonyl butoxide (PBO), significantly enhanced the activity of clothianidin and acetamiprid against An. gambiae mosquitoes. CONCLUSIONS: These findings corroborate susceptibility profiles observed in larvae and highlight a significant variation in tolerance to neonicotinoids between An. gambiae and An. coluzzii populations from Yaoundé. Further studies are needed to disentangle the role of exposure to agricultural pesticides and of cross-resistance mechanisms in the development of neonicotinoid resistance in some Anopheles species.

Behavioral responses and life history traits of Taiwanese and Indonesian populations of Aedes aegypti surviving deltamethrin-clothianidin treatment.

BACKGROUND: Indoor residual spraying (IRS) capitalizes on the natural behavior of mosquitoes because Aedes aegypti commonly seeks indoor resting sites after a blood meal. This behavior allows mosquitoes to be exposed to insecticide-treated surfaces and subsequently killed. Combinations of deltamethrin and clothianidin with different modes of action have shown promise in IRS, effectively targeting both susceptible and pyrethroid-resistant malaria vectors. However, the effects of this approach on Aedes mosquitoes remain unclear. The present study tested the effects of deltamethrin-clothianidin mixture treatment on behavioral responses and life history traits of Taiwanese and Indonesian populations of Ae. aegypti. METHODS: We adopted an excito-repellent approach to explore the behavioral responses of pyrethroid-resistant Ae. aegypti populations from Indonesia and Taiwan to a deltamethrin-clothianidin mixture used in contact irritancy and non-contact repellency treatments. We further evaluated the life history traits of surviving mosquitoes (i.e., delayed mortality after 7-day post-treatment, longevity, fecundity, and egg hatching) and investigated the potential transgenerational hormetic effects of insecticide exposure (i.e., development rate and survival of immatures and adult mosquitos). RESULTS: All tested field populations of Ae. aegypti displayed strong contact irritancy responses; the percentage of escape upon insecticide exposure ranged from 38.8% to 84.7%. However, repellent effects were limited, with the escape percentage ranging from 4.3% to 48.9%. We did not observe immediate knockdown or mortality after 24 h, and less than 15% of the mosquitoes exhibited delayed mortality after a 7-day exposure period. However, the carryover effects of insecticide exposure on the survival of immature mosquitoes resulted in approximately 25% higher immature mortality than that in the control. By contrast, we further documented stimulated survivor reproduction and accelerated transgenerational immature development resulting from the sublethal effects of the insecticide mixture. In particular, the number of eggs laid by treated (both treatments) female mosquitoes increased by at least 60% compared with that of eggs laid by control female mosquitoes. CONCLUSIONS: IRS with deltamethrin-clothianidin effectively deters Aedes mosquitoes from entering residential areas and thereby reduces mosquito bites. However, the application rate (deltamethrin: 25 mg/m2; clothianidin: 200 mg/m2) may be insufficient to effectively kill Aedes mosquitoes. Insecticide response appears to vary across mosquito species; their behavioral and physiological responses to sublethal doses have crucial implications for mosquito control programs.

Key role of the ryanodine receptor I4790K mutation in mediating diamide resistance in Plutella xylostella.

The diamondback moth Plutella xylostella, a global insect pest of cruciferous vegetables, has evolved resistance to many classes of insecticides including diamides. Three point mutations (I4790M, I4790K, and G4946E) in the ryanodine receptor of P. xylostella (PxRyR) have been identified to associate with varying levels of resistance. In this study, we generated a knockin strain (I4790K-KI) of P. xylostella, using CRISPR/Cas9 to introduce the I4790K mutation into PxRyR of the susceptible IPP-S strain. Compared to IPP-S, the edited I4790K-KI strain exhibited high levels of resistance to both anthranilic diamides (chlorantraniliprole 1857-fold, cyantraniliprole 1433-fold) and the phthalic acid diamide flubendiamide (>2272-fold). Resistance to chlorantraniliprole in the I4790K-KI strain was inherited in an autosomal and recessive mode, and genetically linked with the I4790K knockin mutation. Computational modeling suggests the I4790K mutation reduces the binding of diamides to PxRyR by disrupting key hydrogen bonding interactions within the binding cavity. The approximate frequencies of the 4790M, 4790K, and 4946E alleles were assessed in ten geographical field populations of P. xylostella collected in China in 2021. The levels of chlorantraniliprole resistance (2.3- to 1444-fold) in these populations were significantly correlated with the frequencies (0.017-0.917) of the 4790K allele, but not with either 4790M (0-0.183) or 4946E (0.017-0.450) alleles. This demonstrates that the PxRyR I4790K mutation is currently the major contributing factor to chlorantraniliprole resistance in P. xylostella field populations within China. Our findings provide in vivo functional evidence for the causality of the I4790K mutation in PxRyR with high levels of diamide resistance in P. xylostella, and suggest that tracking the frequency of the I4790K allele is crucial for optimizing the monitoring and management of diamide resistance in this crop pest.

Monitoring Aedes populations for arboviruses, Wolbachia, insecticide resistance and its mechanisms in various agroecosystems in Benin.

Aedes mosquitoes are the main vectors of arboviruses in Benin. Cases of dengue have been reported in Benin with all four serotypes of the virus actively circulating in this region. Some agricultural settings are known to harbor Aedes vectors responsible for the transmission of arboviruses. The massive use of certain insecticides in agricultural settings has probably contributed to insecticide resistance in these vectors. In Benin, the susceptibility of arbovirus vectors to insecticides is poorly studied. In addition, the distribution of Wolbachia spp., which is used against some arboviruses is unknown. Moreover, there is limited information regarding the vectors responsible for the transmission of arboviruses in Benin. This present study monitored the species composition, arboviruses, and Wolbachia symbiont status, as well as the phenotypic and molecular insecticide resistance profile of Aedes populations from three agroecosystems in Benin. Aedes species identification was performed morphologically and confirmed using qPCR. (RT)-qPCR assay was applied for monitoring the presence of DENV, CHIKV, ZIKV, and WNV pathogens as well as for naturally occurring Wolbachia symbionts. Insecticide resistance was assessed phenotypically, by permethrin (0.75%) exposure of Adults (F0) using World Health Organization (WHO) bioassay protocols, and at the molecular level, using TaqMan (RT)-qPCR assays for assessing knock-down resistance (kdr) mutations (F1534C, V1016G/I, and S989P) and the expression levels of eight detoxification genes (P450s from the CYP9 and CYP6 families, carboxylesterases and glutathione-S-transferases). Aedes aegypti (Ae. aegypti) mosquitoes were the most abundant (93.9%) in the three agroecosystems studied, followed by Aedes albopictus (Ae. albopictus) mosquitoes (6.1%). No arboviruses were detected in the study's mosquito populations. Naturally occurring Wolbachia symbionts were present in 7 pools out of 15 pools tested. This could influence the effectiveness of vector control strategies based on exogenously introduced Wolbachia, all present in the three agroecosystems. Full susceptibility to permethrin was observed in all tested populations of Ae. albopictus. On the contrary, Ae. aegypti were found to be resistant in all three agroecosystem sites except for banana plantation sites, where full susceptibility was observed. Molecular analysis revealed that individual target site resistance kdr mutations F1534C and V1016G/I were detected in most Ae. aegypti populations. Additionally, double mutant (F1534C + V1016G/I) mosquitoes were found in some populations, and in one case, triple mutant (F1534C + V1016G/I + S989P) mosquitoes were detected. Metabolic resistance, as reflected by overexpression of three P450 genes (CYP6BB2, CYP9J26, and CYP9J32), was also detected in Ae. aegypti mosquitoes. Our study provides information that could be used to strategize future vector control strategies and highlights the importance of continuing vector surveillance. Future studies should assess the effect of piperonyl butoxide (PBO) on metabolic resistance and identify the different strains of Wolbachia spp., to choose the best vector control strategies in Benin.

The frequency of kdr and ace-1 alleles in Anopheles gambiae s.l. before and during indoor residual spraying (IRS) implementation and four years after IRS withdrawal in three districts in Atacora, Benin.

BACKGROUND: Indoor residual spraying (IRS) was first implemented in the Atacora department, Benin from 2011 to 2012 using bendiocarb (carbamate) followed by annual spraying with pirimiphos-methyl (organophosphate) from 2013 to 2018. Before and after IRS implementation in Atacora, standard pyrethroid insecticide-treated bed nets were the main method of vector control in the area. This study investigated the knockdown resistance (kdr) gene (L1014F) and the acetylcholinesterase (ace-1) gene (G119S), before and during IRS implementation, and 4-years after IRS withdrawal from Atacora. This was done to assess how changes in insecticide pressure from indoor residual spraying may have altered the genotypic resistance profile of Anopheles gambiae s.l. METHOD: Identification of sibling species of An. gambiae s.l. and detection of the L1014F mutation in the kdr gene and G119S mutation in ace-1 genes was done using molecular analysis. Allelic and genotypic frequencies were calculated and compared with each other before and during IRS implementation and 4 years after IRS withdrawal. The Hardy-Weinberg equilibrium and genetic differentiation within and between populations were assessed. RESULTS: Prevalence of the L1014F mutation in all geographic An. gambiae s.l. (An. gambiae s.s., Anopheles. coluzzii, Anopheles. arabiensis, and hybrids of "An. gambiae s.s. and An. coluzzii") populations increased from 69% before IRS to 87% and 90% during and after IRS. The G119S allele frequency during IRS (20%) was significantly higher than before IRS implementation (2%). Four years after IRS withdrawal, allele frequencies returned to similar levels as before IRS (3%). Four years after IRS withdrawal, the populations showed excess heterozygosity at the ace-1 gene and deficit heterozygosity at the kdr gene, whereas both genes had excess heterozygosity before and during IRS (FIS < 0). No genetic differentiation was observed within the populations. CONCLUSIONS: This study shows that the withdrawal of IRS with bendiocarb and pirimiphos-methyl may have slowed down the selection of individual mosquitoes with ace-1 resistance alleles in contrast to populations of An. gambiae s.l. with the L1014F resistance allele of the kdr gene. This may suggest that withdrawing the use of carbamates or organophosphates from IRS or rotating alternative insecticides with different modes of action may slow the development of ace-1 insecticide-resistance mutations. The increase in the prevalence of the L1014F mutation of the kdr gene in the population, despite the cessation of IRS, could be explained by the growing use of pyrethroids and DDT in agriculture and for other domestic use. More observational studies in countries where carbamates or organophosphates are still being used as public health insecticides may provide additional insights into these associations.

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.

Long-term impact of rearing substrates on susceptibility to insecticides and metabolic enzyme activities in the house fly Musca domestica.

Musca domestica Linnaeus is a devastating insect pest of medical and veterinary importance with reports of resistance development to commonly used insecticides worldwide. Rearing substrates usually play a crucial role in determining susceptibility to insecticides and control of insect pests. The aim of the present study was to investigate the effect of five rearing substrates of M. domestica on its susceptibility to different insecticides and activities of metabolic enzymes. After 30 generations of rearing, susceptibility of M. domestica to tested insecticides, viz., malathion, pirimiphos-methyl, alpha-cypermethrin, deltamethrin, methomyl, propoxur, spinetoram, and chlorfenapyr had evident differences. Musca domestica reared on hen liver exhibited reduced susceptibility to all insecticides followed by the strain reared on poultry manure. However, M. domestica reared on milk-based diet showed the highest susceptibility to tested insecticides followed by the strain reared on manures of buffalo and horse. In addition, M. domestica reared on different substrates exhibited significant differences (p < 0.01) in the activities of glutathione S-transferase (GST), cytochrome P450-dependent monooxygenase, and carboxylesterase (CarE). Overall, hen liver and poultry manure strains exhibited higher activities of metabolic enzymes than those of the milk-based diet, buffalo, and horse manure strains. In conclusion, the data of the present study exhibited a significant effect of rearing substrates on the susceptibility to insecticides and activities of metabolic enzymes in M. domestica. These results could be helpful for the sustainable management of M. domestica on different hosts by selecting appropriate insecticides.

Temperature, mosquito feeding status and mosquito density influence the measured bio-efficacy of insecticide-treated nets in cone assays.

BACKGROUND: The WHO cone bioassay is routinely used to evaluate the bioefficacy of insecticide-treated nets (ITNs) for product pre-qualification and confirmation of continued ITN performance during operational monitoring. Despite its standardized nature, variability is often observed between tests. We investigated the influence of temperature in the testing environment, mosquito feeding status and mosquito density on cone bioassay results. METHODS: Cone bioassays were conducted on MAGNet (alphacypermethrin) and Veeralin (alphacypermethrin and piperonyl butoxide (PBO)) ITNs, using laboratory-reared pyrethroid-resistant Anopheles funestus sensu stricto (FUMOZ strain) mosquitoes. Three experiments were conducted using standard cone bioassays following WHO-recommended test parameters, with one variable changed in each bioassay: (i) environmental temperature during exposure: 22-23 °C, 26-27 °C, 29-30 °C and 32-33 °C; (ii) feeding regimen before exposure: sugar starved for 6 h, blood-fed or sugar-fed; and (iii) mosquito density per cone: 5, 10, 15 and 20 mosquitoes. For each test, 15 net samples per treatment arm were tested with four cones per sample (N = 60). Mortality after 24, 48 and 72 h post-exposure to ITNs was recorded. RESULTS: There was a notable influence of temperature, feeding status and mosquito density on An. funestus mortality for both types of ITNs. Mortality at 24 h post-exposure was significantly higher at 32-33 °C than at 26-27 °C for both the MAGNet [19.33% vs 7%; odds ratio (OR): 3.96, 95% confidence interval (CI): 1.99-7.87, P < 0.001] and Veeralin (91% vs 47.33%; OR: 22.20, 95% CI: 11.45-43.05, P < 0.001) ITNs. Mosquito feeding status influenced the observed mortality. Relative to sugar-fed mosquitoes, The MAGNet ITNs induced higher mortality among blood-fed mosquitoes (7% vs 3%; OR: 2.23, 95% CI: 0.94-5.27, P = 0.068) and significantly higher mortality among starved mosquitoes (8% vs 3%, OR: 2.88, 95% CI: 1.25-6.63, P = 0.013); in comparison, the Veeralin ITNs showed significantly lower mortality among blood-fed mosquitoes (43% vs 57%; OR: 0.56, 95% CI: 0.38-0.81, P = 0.002) and no difference for starved mosquitoes (58% vs 57%; OR: 1.05, 95% CI: 0.72-1.51, P = 0.816). Mortality significantly increased with increasing mosquito density for both the MAGNet (e.g. 5 vs 10 mosquitoes: 7% vs 12%; OR: 1.81, 95% CI: 1.03-3.20, P = 0.040) and Veeralin (e.g. 5 vs 10 mosquitoes: 58% vs 71%; OR 2.06, 95% CI: 1.24-3.42, P = 0.005) ITNs. CONCLUSIONS: The results of this study highlight that the testing parameters temperature, feeding status and mosquito density significantly influence the mortality measured in cone bioassays. Careful adherence to testing parameters outlined in WHO ITN testing guidelines will likely improve the repeatability of studies within and between product testing facilities.