Potential of MALDI-TOF MS biotyping to detect deltamethrin resistance in the dengue vector Aedes aegypti.

Insecticide resistance in mosquitoes is spreading worldwide and represents a growing threat to vector control. Insecticide resistance is caused by different mechanisms including higher metabolic detoxication, target-site modification, reduced penetration and behavioral changes that are not easily detectable with simple diagnostic methods. Indeed, most molecular resistance diagnostic tools are costly and labor intensive and then difficult to use for routine monitoring of insecticide resistance. The present study aims to determine whether mosquito susceptibility status against the pyrethroid insecticides (mostly used for mosquito control) could be established by the protein signatures of legs and/or thoraxes submitted to MALDI-TOF Mass Spectrometry (MS). The quality of MS spectra for both body parts was controlled to avoid any bias due to unconformity protein profiling. The comparison of MS profiles from three inbreeds Ae. aegypti lines from French Guiana (IRF, IR03, IR13), with distinct deltamethrin resistance genotype / phenotype and the susceptible reference laboratory line BORA (French Polynesia), showed different protein signatures. On both body parts, the analysis of whole protein profiles revealed a singularity of BORA line compared to the three inbreeding lines from French Guiana origin, suggesting that the first criteria of differentiation is the geographical origin and/or the breeding history rather than the insecticide susceptibility profile. However, a deeper analysis of the protein profiles allowed to identify 10 and 11 discriminating peaks from leg and thorax spectra, respectively. Among them, a specific peak around 4870 Da was detected in legs and thoraxes of pyrethroid resistant lines compared to the susceptible counterparts hence suggesting that MS profiling may be promising to rapidly distinguish resistant and susceptible phenotypes. Further work is needed to confirm the nature of this peak as a deltamethrin resistant marker and to validate the routine use of MS profiling to track insecticide resistance in Ae. aegypti field populations.

Overexpression of an Integument Esterase Gene LbEST-inte4 Infers the Malathion Detoxification in Liposcelis bostrychophila (Psocoptera: Liposcelididae).

Liposcelis bostrychophila, commonly known as booklouse, is an important stored-product pest worldwide. Studies have demonstrated that booklices have developed resistance to several insecticides. In this study, an integument esterase gene, LbEST-inte4, with upregulated expression, was characterized in L. bostrychophila. Knockdown of LbEST-inte4 resulted in a substantial increase in the booklice susceptibility to malathion. Overexpression of LbEST-inte4 in Drosophila melanogaster significantly enhanced its malathion tolerance. Molecular modeling and docking analysis suggested potential interactions between LbEST-inte4 and malathion. When overexpressed LbEST-inte4 in Sf9 cells, a notable elevation in esterase activity and malathion tolerance was observed. HPLC analysis indicated that the LbEST-inte4 enzyme could effectively degrade malathion. Taken together, the upregulated LbEST-inte4 appears to contribute to malathion tolerance in L. bostrychophila by facilitating the depletion of malathion. This study elucidates the molecular mechanism underlying malathion detoxification and provides the foundations for the development of effective prevention and control measures against psocids.

Integrated de novo transcriptome of Culex pipiens mosquito larvae as a resource for genetic control strategies.

We present a de novo transcriptome of the mosquito vector Culex pipiens, assembled by sequences of susceptible and insecticide resistant larvae. The high quality of the assembly was confirmed by TransRate and BUSCO. A mapping percentage until 94.8% was obtained by aligning contigs to Nr, SwissProt, and TrEMBL, with 27,281 sequences that simultaneously mapped on the three databases. A total of 14,966 ORFs were also functionally annotated by using the eggNOG database. Among them, we identified ORF sequences of the main gene families involved in insecticide resistance. Therefore, this resource stands as a valuable reference for further studies of differential gene expression as well as to identify genes of interest for genetic-based control tools.

Contribution of the transcription factor SfGATAe to Bt Cry toxin resistance in Spodoptera frugiperda through reduction of ABCC2 expression.

Insect resistance evolution poses a significant threat to the advantages of biopesticides and transgenic crops utilizing insecticidal Cry-toxins from Bacillus thuringiensis (Bt). However, there is limited research on the relationship between transcriptional regulation of specific toxin receptors in lepidopteran insects and their resistance to Bt toxins. Here, we report the positive regulatory role of the SfGATAe transcription factor on the expression of the ABCC2 gene in Spodoptera frugiperda. DNA regions in the SfABCC2 promoter that are vital for regulation by SfGATAe, utilizing DAP-seq technology and promoter deletion mapping. Through yeast one-hybrid assays, DNA pull-down experiments, and site-directed mutagenesis, we confirmed that the transcription factor SfGATAe regulates the core control site PBS2 in the ABCC2 target gene. Tissue-specific expression analysis has revealed that SfGATAe is involved in the regulation and expression of midgut cells in the fall armyworm. Silencing SfGATAe in fall armyworm larvae resulted in reduced expression of SfABCC2 and decreased sensitivity to Cry1Ac toxin. Overall, this study elucidated the regulatory mechanism of the transcription factor SfGATAe on the expression of the toxin receptor gene SfABCC2 and this transcriptional control mechanism impacts the resistance of the fall armyworm to Bt toxins.

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.

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.

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.

MDR49 coding for both P-glycoprotein and TMOF transporter functions in ivermectin resistance, trypsin activity inhibition, and fertility in the yellow fever mosquito, Aedes aegypti.

This study investigated the function of the MDR49 gene in Aedes aegypti. MDR49 mutants were constructed using CRISPR/Cas9 technology; the mutation led to increased sensitivity to ivermectin (LC50: from 1.3090 mg L-1 to 0.5904 mg L-1), and a reduction in midgut trypsin activity. These findings suggest that the P-gp encoded by MDR49 confers resistance to ivermectin and impacts the reproductive function in Ae. aegypti. RNA interference technology showed that knockdown of MDR49 gene resulted in a significant decrease in the expression of VGA1 after a blood meal, as well as a decrease in the number of eggs laid and their hatching rate. LC-MS revealed that following ivermectin treatment, the MDR493d+2s/3d+2s strain larvae exhibited significantly higher drug concentrations in the head and fat body compared to the wild type. Modeling of inward-facing P-gp and molecular docking found almost no difference in the affinity of P-gp for ivermectin before and after the mutation. However, modeling of the outward-facing conformation demonstrated that the flexible linker loop between TM5 and TM6 of P-gp undergoes changes after the mutation, resulting in a decrease in trypsin activity and an increase in sensitivity to ivermectin. These results provide useful insights into ivermectin resistance and the other roles played by the MDR49 gene.

Knockdown of one cytochrome P450 gene CYP6DW4 increases the susceptibility of Bemisia tabaci to dimpropyridaz, a novel pyridazine pyrazolecarboxamide insecticide.

Bemisia tabaci is a formidable insect pest worldwide, and it exhibits significant resistance to various insecticides. Dimpropyridaz is a novel pyridazine pyrazolecarboxamide insecticide used against sucking insect pests, but there is little information regarding its metabolic detoxification in arthropods or cross-resistance with other insecticides. In this study, we found that dimpropyridaz shows no cross-resistance with three other popular insecticides, namely abamectin, cyantraniliprole, and flupyradifurone. After treatment of B. tabaci adults with a high dose of dimpropyridaz, higher cytochrome P450 monooxygenase (P450) activity was detected in the survivors, and the expression of the P450 gene CYP6DW4 was highly induced. Cloning and characterization of the full-length amino acid sequence of CYP6DW4 indicated that it contains conserved domains typical of P450 genes, phylogenetic analysis revealed that it was closely related to a B. tabaci protein, CYP6DW3, known to be involved in detoxification of imidacloprid. Silencing of CYP6DW4 by feeding insects with dsRNA significantly increased the susceptibility of B. tabaci to dimpropyridaz. In addition, homology modeling and molecular docking analyses showed the stable binding of dimpropyridaz to CYP6DW4, with binding free energy of -6.65 kcal/mol. Our findings indicate that CYP6DW4 plays an important role in detoxification of dimpropyridaz and possibly promotes development of resistance in B. tabaci.

Glutathione S-transferase directly metabolizes imidacloprid in the whitefly, Bemisia tabaci.

The whitefly Bemisia tabaci poses a significant threat to various crops and ornamental plants and causes severe damage to the agricultural industry. Over the past few decades, B. tabaci has developed resistance to several pesticides, including imidacloprid. Therefore, elucidating the mechanism that leads to insecticide detoxification is very important for controlling B. tabaci and managing whitefly resistance to neonicotinoid insecticides. Among insect detoxification enzymes, glutathione S-transferase (GST) is an important phase II detoxification enzyme that helps detoxify exogenous toxic substances. In this study, we cloned the BtGSTz1 gene and observed that its expression level was greater in imidacloprid-resistant populations than sensitive populations of B. tabaci. By silencing BtGSTz1 via RNA interference, we found a significant increase in the mortality of imidacloprid-resistant B. tabaci. Additionally, prokaryotic expression and in vitro metabolism studies revealed that the recombinant BtGSTz1 protein could metabolize 36.36% of the total imidacloprid, providing direct evidence that BtGSTz1 plays a crucial role in the detoxification of imidacloprid. Overall, our study elucidated the role of GSTs in physiological activities related to insecticide resistance, which helps clarify the resistance mechanisms conferred by GSTs and provides useful insights for sustainable integrated pest management.

A relaxin receptor gene RpGPCR41 is involved in the resistance of Rhopalosiphum padi to pyrethroids.

Rhopalosiphum padi is a global pest that poses a significant threat to wheat crops and has developed resistance to various insecticides. G protein-coupled receptors (GPCRs), known for their crucial role in signaling and biological processes across insect species, have recently gained attention as a potential target for insecticides. GPCR has the potential to contribute to insect resistance through the regulation of P450 gene expression. However, GPCRs in R. padi remained unexplored until this study. We identified a total of 102 GPCRs in R. padi, including 81 receptors from family A, 10 receptors from family B, 8 receptors from family C, and 3 receptors from family D. Among these GPCR genes, 16 were up-regulated in both lambda-cyhalothrin and bifenthrin-resistant strains of R. padi (LC-R and BIF-R). A relaxin receptor gene, RpGPCR41, showed the highest up-regulated expression in both the resistant strains, with a significant increase of 14.3-fold and 22.7-fold compared to the susceptible strain (SS). RNA interference (RNAi) experiments targeting the relaxin receptor significantly increase the mortality of R. padi when exposed to the LC50 concentration of lambda-cyhalothrin and bifenthrin. The expression levels of five P450 genes (RpCYP6CY8, RpCYP6DC1, RpCYP380B1, RpCYP4CH2, and RpCYP4C1) were significantly down-regulated following knockdown of RpGPCR41 in LC-R and BIF-R strains. Our results highlight the involvement of GPCR gene overexpression in the resistance of R. padi to pyrethroids, providing valuable insights into the mechanisms underlying aphid resistance and a potential target for aphid control.

Characteristics of Trypsin genes and their roles in insecticide resistance based on omics and functional analyses in the malaria vector Anopheles sinensis.

Trypsin is one of the most diverse and widely studied protease hydrolases. However, the diversity and characteristics of the Trypsin superfamily of genes have not been well understood, and their role in insecticide resistance is yet to be investigated. In this study, a total of 342 Trypsin genes were identified and classified into seven families based on homology, characteristic domains and phylogenetics in Anopheles sinensis, and the LY-Domain and CLECT-Domain families are specific to the species. Four Trypsin genes, (Astry2b, Astry43a, Astry90, Astry113c) were identified to be associated with pyrethroid resistance based on transcriptome analyses of three field resistant populations and qRT-PCR validation, and the knock-down of these genes significantly decrease the pyrethroid resistance of Anopheles sinensis based on RNAi. The activity of Astry43a can be reduced by five selected insecticides (indoxacarb, DDT, temephos, imidacloprid and deltamethrin); and however, the Astry43a could not directly metabolize these five insecticides, like the trypsin NYD-Tr did in earlier reports. This study provides the overall information frame of Trypsin genes, and proposes the role of Trypsin genes to insecticide resistance. Further researches are necessary to investigate the metabolism function of these trypsins to insecticides.

Sphingomonas bacteria could serve as an early bioindicator for the development of chlorantraniliprole resistance in Spodoptera frugiperda.

The fall armyworm (Spodoptera frugiperda) was found to have invaded China in December 2018, and in just one year, crops in 26 provinces were heavily affected. Currently, the most effective method for emergency control of fulminant pests is to use of chemical pesticides. Recently, most fall armyworm populations in China were begining to exhibite low level resistance to chlorantraniliprole. At present, it is not possible to sensitively reflect the low level resistance of S. frugiperda by detecting target mutation and detoxification enzyme activity. In this study we found that 12 successive generations of screening with chlorantraniliprole caused S. frugiperda to develop low level resistance to this insecticide, and this phenotype was not attribute to genetic mutations in S. frugiperda, but rather to a marked increase in the relative amount of the symbiotic bacteria Sphingomonas. Using FISH and qPCR assays, we determined the amount of Sphingomonas in the gut of S. frugiperda and found Sphingomonas accumulation to be highest in the 3rd-instar larvae. Additionally, Sphingomonas was observed to provide a protective effect to against chlorantraniliprole stress to S. frugiperda. With the increase of the resistance to chlorantraniliprole, the abundance of bacteria also increased, we propose Sphingomonas monitoring could be adapted into an early warning index for the development of chlorantraniliprole resistance in S. frugiperda populations, such that timely measures can be taken to delay or prevent the widespread propagation of resistance to this highly useful agricultural chemical in S. frugiperda field populations.

Rifampicin synergizes the toxicity of insecticides against the green peach aphid, Myzus persicae.

Myzus persicae is an important pest that has developed resistance to nearly all currently used insecticidal products. The employment of insecticide synergists is one of the effective strategies that need to be developed for the management of this resistance. Our study showed that treatment with a combination of the antibiotic, rifampicin, with imidacloprid, cyantraniliprole, or clothianidin significantly increased their toxicities against M. persicae, by 2.72, 3.59, and 2.41 folds, respectively. Rifampicin treatment led to a noteworthy reduction in the activities of multifunctional oxidases (by 32.64%) and esterases (by 23.80%), along with a decrease in the expression of the CYP6CY3 gene (by 58.57%) in M. persicae. It also negatively impacted the fitness of the aphids, including weight, life span, number of offspring, and elongation of developmental duration. In addition, bioassays showed that the combination of rifampicin and a detoxification enzyme inhibitor, piperonyl butoxide, or dsRNA of CYP6CY3 further significantly improved the toxicity of imidacloprid against M. persicae, by 6.19- and 7.55-fold, respectively. The present study suggests that development of active ingredients such as rifampicin as candidate synergists, show promise to overcome metabolic resistance to insecticides in aphids.

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.

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.

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.

Lactic acid bacteria modulate the CncC pathway to enhance resistance to ß-cypermethrin in the oriental fruit fly.

The gut microbiota of insects has been shown to regulate host detoxification enzymes. However, the potential regulatory mechanisms involved remain unknown. Here, we report that gut bacteria increase insecticide resistance by activating the cap "n" collar isoform-C (CncC) pathway through enzymatically generated reactive oxygen species (ROS) in Bactrocera dorsalis. We demonstrated that Enterococcus casseliflavus and Lactococcus lactis, two lactic acid-producing bacteria, increase the resistance of B. dorsalis to ß-cypermethrin by regulating cytochrome P450 (P450) enzymes and α-glutathione S-transferase (GST) activities. These gut symbionts also induced the expression of CncC and muscle aponeurosis fibromatosis. BdCncC knockdown led to a decrease in resistance caused by gut bacteria. Ingestion of the ROS scavenger vitamin C in resistant strain affected the expression of BdCncC/BdKeap1/BdMafK, resulting in reduced P450 and GST activity. Furthermore, feeding with E. casseliflavus or L. lactis showed that BdNOX5 increased ROS production, and BdNOX5 knockdown affected the expression of the BdCncC/BdMafK pathway and detoxification genes. Moreover, lactic acid feeding activated the ROS-associated regulation of P450 and GST activity. Collectively, our findings indicate that symbiotic gut bacteria modulate intestinal detoxification pathways by affecting physiological biochemistry, thus providing new insights into the involvement of insect gut microbes in the development of insecticide resistance.

The Anopheles coluzzii range extends into Kenya: detection, insecticide resistance profiles and population genetic structure in relation to conspecific populations in West and Central Africa.

BACKGROUND: Anopheles coluzzii is a primary vector of malaria found in West and Central Africa, but its presence has hitherto never been documented in Kenya. A thorough understanding of vector bionomics is important as it enables the implementation of targeted and effective vector control interventions. Malaria vector surveillance efforts in the country have tended to focus on historically known primary vectors. The current study sought to determine the taxonomic status of samples collected from five different malaria epidemiological zones in Kenya as well as describe the population genetic structure and insecticide resistance profiles in relation to other An. coluzzii populations. METHODS: Mosquitoes were sampled as larvae from Busia, Kwale, Turkana, Kirinyaga and Kiambu counties, representing the range of malaria endemicities in Kenya, in 2019 and 2021 and emergent adults analysed using Whole Genome Sequencing (WGS) data processed in accordance with the Anopheles gambiae 1000 Genomes Project phase 3. Where available, historical samples from the same sites were included for WGS. Comparisons were made with An. coluzzii cohorts from West and Central Africa. RESULTS: This study reports the detection of An. coluzzii for the first time in Kenya. The species was detected in Turkana County across all three time points from which samples were analyzed and its presence confirmed through taxonomic analysis. Additionally, there was a lack of strong population genetic differentiation between An. coluzzii from Kenya and those from the more northerly regions of West and Central Africa, suggesting they represent a connected extension to the known species range. Mutations associated with target-site resistance to DDT and pyrethroids and metabolic resistance to DDT were found at high frequencies up to 64%. The profile and frequencies of the variants observed were similar to An. coluzzii from West and Central Africa but the ace-1 mutation linked to organophosphate and carbamate resistance present in An. coluzzii from coastal West Africa was absent in Kenya. CONCLUSIONS: These findings emphasize the need for the incorporation of genomics in comprehensive and routine vector surveillance to inform on the range of malaria vector species, and their insecticide resistance status to inform the choice of effective vector control approaches.