Comparative transcriptional analysis between susceptible and resistant populations of Aedes (Stegomyia) aegypti (Linnaeus, 1762) after malathion exposure.

Aedes aegypti is an important vector of arboviruses, including dengue, chikungunya and Zika. The application of synthetic insecticides is a frequently used strategy to control this insect. Malathion is an organophosphate insecticide that was widely used in Brazil in the 1980s and 1990s to control the adult form of A. aegypti. In situations where resistance to currently used insecticides is detected, the use of malathion may be resumed as a control measure. Many studies have confirmed resistance to malathion, however, comparative studies of differential gene expression of the entire transcriptome of resistant and susceptible insects are scarce. Therefore, understanding the molecular basis of resistance to this insecticide in this species is extremely important. In this paper, we present the first transcriptomic description of susceptible and resistant strains of A. aegypti challenged with malathion. Guided transcriptome assembly resulted in 39,904 transcripts, where 2133 differentially expressed transcripts were detected, and three were validated by RT-qPCR. Enrichment analysis for these identified transcripts resulted in 13 significant pathways (padj < 0.05), 8 associated with down-regulated and 5 with up-regulated transcripts in treated resistant insects. It was possible to divide the transcripts according to the mechanism of action into three main groups: (i) genes involved in detoxification metabolic pathways; (ii) genes of proteins located in the membrane/extracellular region; and (iii) genes related to DNA integration/function. These results are important in advancing knowledge of genes related to resistance mechanisms in this insect, enabling the development of effective technologies and strategies for managing insecticide resistance.

Phenotypic evidence of deltamethrin resistance and identification of selective sweeps in Anopheles mosquitoes on the Bijagós Archipelago, Guinea-Bissau.

Vector control in the Bijagós Archipelago of Guinea-Bissau currently relies on pyrethroid insecticide-treated nets. However, data on insecticide resistance in Guinea-Bissau is limited. This study identified deltamethrin resistance in the Anopheles gambiae sensu lato complex on Bubaque island using WHO tube tests in November 2022. Whole genome sequencing of An. gambiae sensu stricto mosquitoes identified six single nucleotide polymorphisms (SNPs) previously associated with, or putatively associated with, insecticide resistance: T791M, L995F, N1570Y, A1746S and P1874L in the vgsc gene, and L119V in the gste2 gene. Twenty additional non-synonymous SNPs were identified in insecticide-resistance associated genes. Four of these SNPs were present at frequencies over 5% in the population: T154S, I126F and G26S in the vgsc gene and A65S in ace1. Genome wide selection scans using Garud's H12 statistic identified two selective sweeps: one in chromosome X and one in chromosome 2R. Both selective sweeps overlap with metabolic genes previously associated with insecticide resistance, including cyp9k1 and the cyp6aa/cyp6p gene cluster. This study presents the first phenotypic testing for deltamethrin resistance and the first whole genome sequence data for Anophelesgambiae mosquitoes from the Bijagós, contributing data of significance for vector control policy in this region.

Unveiling the Role of Two Rhodopsin-like GPCR Genes in Insecticide-Resistant House Flies, Musca domestica.

Insecticide resistance in insects, driven by the overexpression of P450 enzymes, presents a significant challenge due to the enhanced metabolic detoxification of insecticides. Although the transcriptional regulation of P450 genes is not yet fully understood, G-protein-coupled receptor (GPCR) genes have emerged as key regulators in this process. This study is the first to associate GPCR genes with insecticide resistance in Musca domestica. We identified two key rhodopsin-like GPCR genes, ALHF_02706.g1581 and ALHF_04422.g2918, which were significantly overexpressed in the resistant ALHF strain compared to sensitive strains. Notably, both ALHF_02706.g1581 and ALHF_04422.g2918 were mapped to autosome 2, where critical but unidentified regulatory factors controlling resistance and P450 gene regulation are located. This supports our hypothesis that GPCRs function as trans-regulatory factors for P450-mediated resistance. Functional analysis using transgenic Drosophila demonstrated that overexpression of these rhodopsin-like GPCR genes increased permethrin resistance by approximately two-fold. Specifically, ALHF_02706.g1581 overexpression significantly upregulated the Drosophila resistance-related P450 genes CYP12D1, CYP6A2, and CYP6A8, while ALHF_04422.g2918 increased CYP6G1 and CYP6A2 expression, thereby enhancing insecticide detoxification in rhodopsin-like GPCR transgenic Drosophila lines. These findings suggest that these rhodopsin-like GPCR genes on autosome 2 may act as trans-regulatory factors for P450-mediated resistance, underscoring their critical role in insecticide detoxification and resistance development in M. domestica.

The association between knockdown resistance and treatment outcome of 1% permethrin lotion in head lice infestations in Nonthaburi province, Thailand.

Head lice infestations significantly impact schoolchildren, with permethrin being the primary treatment. The escalation in the prevalence of the knockdown resistance (kdr) mutation, potentially affecting treatment efficacy, is highly concerning. This study examined head lice infestation prevalence, the efficacy of 1% permethrin lotion, kdr mutation frequency, and the association between kdr genotype and permethrin treatment outcomes in schoolchildren in Nonthaburi province, Thailand. Participants and lice samples were collected from six primary schools using cluster sampling. Clinical outcomes and adverse events were assessed on days 7 and 14 post-treatment. Kdr mutations (T917I) in head lice samples were analyzed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Head lice infestation prevalence was 6.1%, with a kdr mutation frequency of 63.2% and a permethrin cure rate of approximately 50%. Multivariate regression analysis revealed the association between kdr genotype and clinical response to permethrin treatment (Odds Ratio (OR) = 13.15, p = 0.005). The presence of resistance genes hinders the cure rate (presence 35.9% versus absence 85.7%). These findings highlight the importance of the kdr genotype in predicting permethrin treatment responses in head lice infestations. Furthermore, in regions with documented reduced permethrin efficacy and a high prevalence of kdr mutations, alternative non-pyrethroid-based pediculicides should be considered. This research could help improve the control of head lice infestations, especially in problematic areas of Thailand.

Differences in seasonal dynamics and pyrethroid resistance development among Anopheles Hyrcanus group species.

BACKGROUND: The Anopheles Hyrcanus group, which transmits Plasmodium vivax, consists of six confirmed species in South Korea. An epidemiological study revealed differences in the seasonal occurrence patterns of each species. Pyrethroid resistance in An. sinensis dates back to the early 2000s, whereas information on pyrethroid resistance in other species is lacking despite their greater significance in malaria epidemiology. METHODS: Anopheles mosquitoes were collected from two malaria-endemic regions in South Korea for 2 years and their knockdown resistance (kdr) mutations were genotyped. The larval susceptibility to λ-cyhalothrin was compared in six Anopheles species and its seasonal changes in three species were investigated. The full-length sequences of the voltage-sensitive sodium channel (VSSC) were compared across six species to evaluate potential target-site insensitivity. The contribution of the kdr mutation to phenotypic resistance was confirmed by comparing median lethal time (LT50) to λ-cyhalothrin between populations of Anopheles belenrae with distinct genotypes. RESULTS: The composition and seasonal occurrence of rare species (Anopheles kleini, Anopheles lestri, and Anopheles sineroides) varied considerably, whereas An. sinensis occurs continuously throughout the season. A kdr mutation in the form of heterozygous allele was newly identified in An. belenrae, An. lesteri, An. pullus, and An. sineroides. The baseline susceptibility to λ-cyhalothrin was the highest in An. belenrae, followed by An. lesteri, An. sineroides, An. kleini, An. pullus, and An. sinensis, with median lethal concentration (LC50) values ranging from 6.0- to 73.5-fold higher than that of An. belenrae. The susceptibility of An. sinensis and An. pullus varied by season, whereas that of An. belenrae remained stable. The kdr-heterozygous An. belenare population exhibited 5.1 times higher LT50 than that of the susceptible population. Species-specific VSSC sequence differences were observed among the six species. CONCLUSIONS: Our findings suggest that the status and extent of pyrethroid resistance vary among Anopheles Hyrcanus group species. While An. sinensis, the predominant species, developed a considerable level of pyrethroid resistance through kdr mutation, the resistance levels of other species appeared to be less pronounced. Large-scale monitoring is crucial to fully understand species-specific seasonal occurrence and resistance status for effective management strategies, considering the ongoing impact of climate change on their vectorial capacity.

Symbiont community assembly shaped by insecticide exposure and feedback on insecticide resistance of Spodoptera frugiperda.

Exploring the mechanism of microbiota assembly and its ecological consequences is crucial for connecting microbiome variation to ecosystem function. However, the influencing factors underlying microbiota assembly in the host-microbe system and their impact on the host phenotype remain unclear. Through investigating the prevalent and worsening ecological phenomenon of insecticide resistance in global agriculture, we found that insecticide exposure significantly changed the gut microbiota assembly patterns of a major agricultural invasive insect pest, Spodoptera frugiperda. The relative importance of various microbiota assembly processes significantly varied with habitat heterogeneity and heterogeneous selection serving as a potential predictor of the host's insecticide resistance in field populations. Moreover, disturbance of the gut microbiota assembly through antibiotics was revealed to significantly affect the rate and heritability of insecticide resistance evolution, leading to a delay in insecticide resistance evolution in this insect pest. These findings indicate that the gut microbiota assembly process of the insect host is influenced by persistent exposure to habitat conditions, particularly insecticides. This variation in insecticide exposure-related community assembly process subsequently influences the insect host's insecticide resistance phenotype. This study provides insights into gut microbiota assembly processes from a symbiotic perspective and underscores the significant impact of symbiotic community changes on host phenotypic variation.

Study on the cross-resistance of Aedes albopictus (Skuse) (Diptera: Culicidae) to deltamethrin and pyriproxyfen.

BACKGROUND: Insecticide resistance poses a significant challenge in the implementation of vector-borne disease control strategies. We have assessed the resistance levels of Aedes albopictus to deltamethrin and pyriproxyfen (PPF) in Fujian Province (China) and investigated the correlation between these resistance levels and mutations in the voltage-gated sodium channel (VGSC). METHODS: The WHO bioassay protocol was used to evaluate the resistance coefficient of Ae. albopictus to deltamethrin and PPF, comparing a susceptible population from the Foshan (FS) area with wild populations from the Sanming (SM), Quanzhou (QZ), Zhangzhou (ZZ), Putian (PT) and Fuzhou (FZ) areas in Fujian Province. Genomic DNA was analyzed by PCR and sequencing to detect knockdown resistance (kdr) in the VGSC, specifically at the pyrethroid resistance alleles V1016V, I1532I and F1534F. Molecular docking was also performed to analyze the binding interactions of PPF and its metabolite 4'-OH-PPF to cytochrome P450 (CYP) 2C19, 2C9 and 3A4 and Ae. albopictus methoprene-tolerant receptors (AeMet), respectively. RESULTS: The analysis of resistance to deltamethrin and PPF among Ae. albopictus populations from the various regions revealed that except for the sensitive population in FS and the SM population, the remaining four regional populations demonstrated resistance levels ranging from 4.31- to 18.87-fold for deltamethrin and from 2.85- to 3.62-fold for PPF. Specifically, the FZ and PT populations exhibited high resistance to deltamethrin, whereas the ZZ and QZ populations approached moderate resistance levels. Also, the resistance of the FZ, PT and ZZ populations to PPF increased slowly but consistently with the increasing trend of deltamethrin resistance. Genomic analysis identified multiple non-synonymous mutations within the VGSC gene; the F1534S and F1534L mutations showed significant resistance to deltamethrin in Ae. albopictus. Molecular docking results revealed that PPF and its metabolite 4'-OH-PPF bind to the Ae. albopictus AeMet receptor and CYP2C19. CONCLUSIONS: The wild Ae. albopictus populations of Fujian Province showed varying degrees of resistance to deltamethrin and PPF and a trend of cross-resistance to deltamethrin and PPF. Increased vigilance is needed for potential higher levels of cross-resistance, especially in the PT and FZ regions.

Polygenic, autosomal, and stable spirotetramat resistance in Chrysoperla carnea resulting in increased fitness.

Green lacewing, Chrysoperla carnea (Stephens) is a generalist predator used as a biological control agent in agro ecosystems. In order to use chemical and biological control in an integrated way, it is advantageous to know about natural enemy resistance response to a selected chemical. To determine C. carnea spirotetramat resistance potential, a population collected from the field was selected in the laboratory. Then we determined how spirotetramat resistance was inherited and how much it impacts the fitness of C. carnea. After eighteen selections with spirotetramat, the selected population (Spiro-Sel) of C. carnea had a 47-fold of resistance when compared to an UNSEL population. Inheritance results showed that spirotetramat resistance was inherited as an autosomal, incompletely dominant and polygenic trait. The values of effective dominance decreased from 0.87 (incomplete dominant) to 0.00 (complete recessive) as the concentration of spirotetramat increased from 625 mg/L to 10000 mg/L. The Spiro-Sel strain had no cross resistance to chlorfenapyr (1.10-fold), deltamethrin (1.26-fold) and chlorpyrifos (1.27-fold). After 7 generations without selection pressure resistance to all experimental insecticides in the Spiro-Sel strain was stable. Fitness data of the Spiro-Sel, Cross A, Cross B, UNSEL and susceptible strains of C. carnea showed that spirotetramat resistance increased the fitness of the selected green lacewing population. Life history parameters like fecundity, net reproductive rate, and relative fitness of the Spiro-Sel strain significantly increased when compared to the susceptible or unselected strains of C. carnea. These findings show that C. carnea is a perfect candidate for integrated pest management (IPM) programmes that combine biological control methods with selective pesticide applications to manage a variety of insect pests. Additionally, it would reduce the possibility of pests developing pesticide resistance despite repeated applications. It would be an excellent choice for widespread releases and be effective in most spray programs.

Identification and expression patterns of voltage-gated sodium channel genes with intron retentions in different strains of Bactrocera dorsalis.

Pyrethroid are the primary insecticides used for controlling of Bactricera dorsalis, a highly destructive and invasive fruit pest. Field populations have developed serious resistance, especially to ß-cypermethrin. While mutations in the voltage-gated sodium channel (Vgsc) are a common mechanism of pyrethroid resistance, variations in BdVgsc associated with ß-cypermethrin resistance remain unclear. Here, we reported the resistance levels of five field populations from China, with resistance ratio ranging from 1.54 to 21.34-fold. Cloning the full length of BdVgsc revealed no specific or known amino acid mutations between the most resistant population and the susceptible strain. However, three types of partial intron retention (IRE4-5, IRE19-f and IREL-24) were identified in BdVgsc transcripts, with these intron retentions containing stop codons. The expression of IRE4-5 transcripts and total BdVgsc showed different trends across developmental stages and tissues. Exposure to ß-cypermethrin led to increased expression of IRE4-5. Comparison of genomic and transcriptional sequences reveled that IRE4-5 transcripts had two types (IRE4-5.5 T and IRE4-5.6 T) caused by genomic variations. Both field and congenic strains indicated that homozygotes for IRE4-5.5 T had lower IRE4-5 transcript levels than homozygotes for IRE4-5.6 T. However, congenic and field strains exhibited inconsistent results about the association of expression levels of IRE4-5 transcripts with sensitivity to ß-cypermethrin. In summary, this study is the first to identify intron retention transcripts in the Vgsc gene from B. dorsalis and to examine their expression patterns across different developmental stages, tissues, and strains with varying sensitivities to ß-cypermethrin. The potential role of the intron retentions of BdVgsc in insecticide toxicity is also discussed.

piggyBac-based transgenic Helicoverpa armigera expressing the T92C allele of the tetraspanin gene HaTSPAN1 confers dominant resistance to Bacillus thuringiensis toxin Cry1Ac.

Transgenic crops producing insecticidal proteins from Bacillus thuringiensis (Bt) have revolutionized pest control. However, the evolution of resistance by target pests poses a significant threat to the long-term success of Bt crops. Understanding the genetics and mechanisms underlying Bt resistance is crucial for developing resistance detection methods and management tactics. The T92C mutation in a tetraspanin gene (HaTSPAN1), resulting in the L31S substitution, is associated with dominant resistance to Cry1Ac in a major pest, Helicoverpa armigera. Previous studies using CRISPR/Cas9 technique have demonstrated that knockin of the HaTSPAN1 T92C mutation confers a 125-fold resistance to Cry1Ac in the susceptible SCD strain of H. armigera. In this study, we employed the piggyBac transposon system to create two transgenic H. armigera strains based on SCD: one expressing the wild-type HaTSPAN1 gene (SCD-TSPANwt) and another expressing the T92C mutant form of HaTSPAN1 (SCD-TSPANmt). The SCD-TSPANmt strain exhibited an 82-fold resistance to Cry1Ac compared to the recipient SCD strain, while the SCD-TSPANwt strain remained susceptible. The Cry1Ac resistance followed an autosomal dominant inheritance mode and was genetically linked with the transgene locus in the SCD-TSPANmt strain of H. armigera. Our results further confirm the causal association between the T92C mutation of HaTSPAN1 and dominant resistance to Cry1Ac in H. armigera. Additionally, they suggest that the piggyBac-mediated transformation system we used in H. armigera is promising for functional investigations of candidate Bt resistance genes from other lepidopteran pests.

Cryptic population structure and insecticide resistance in Anopheles gambiae from the southern Democratic Republic of Congo.

The Democratic Republic of Congo (DRC) suffers from one of the highest malaria burdens worldwide, but information on its Anopheles vector populations is relatively limited. Preventative malaria control in DRC is reliant on pyrethroid-treated nets, raising concerns over the potential impacts of insecticide resistance. We sampled Anopheles gambiae from three geographically distinct populations (Kimpese, Kapolowe and Mikalayi) in southern DRC, collecting from three sub-sites per population and characterising mosquito collections from each for resistance to pyrethroids using WHO tube bioassays. Resistance to each of three different pyrethroids was generally high in An. gambiae with < 92% mortality in all tests, but varied between collections, with mosquitoes from Kimpese being the most resistant. Whole genome sequencing of 165 An. gambiae revealed evidence for genetic differentiation between Kimpese and Kapolowe/Mikalayi, but not between the latter two sample sites despite separation of approximately 800 km. Surprisingly, there was evidence of population structure at a small spatial scale between collection subsites in Kimpese, despite separation of just tens of kilometres. Intra-population (H12) and inter-population (FST) genome scans identified multiple peaks corresponding to genes associated with insecticide resistance such as the voltage gated sodium channel (Vgsc) target site on chromosome 2L, a Cyp6 cytochrome P450 cluster on chromosome arm 2R, and the Cyp9k1 P450 gene on chromosome X. In addition, in the Kimpese subsites, the P450 redox partner gene Cpr showed evidence for contemporary selection (H12) and population differentiation (FST) meriting further exploration as a potential resistance associated marker.

Whole genome sequence analysis of population structure and insecticide resistance markers in Anopheles melas from the Bijagós Archipelago, Guinea-Bissau.

BACKGROUND: Anopheles melas is an understudied malaria vector with a potential role in malaria transmission on the Bijagós Archipelago of Guinea-Bissau. This study presents the first whole-genome sequencing and population genetic analysis for this species from the Bijagós. To our knowledge, this also represents the largest population genetic analysis using WGS data from non-pooled An. melas mosquitoes. METHODS: WGS was conducted for 30 individual An. melas collected during the peak malaria transmission season in 2019 from six different islands on the Bijagós Archipelago. Bioinformatics tools were used to investigate the population structure and prevalence of insecticide resistance markers in this mosquito population. RESULTS: Insecticide resistance mutations associated with pyrethroid resistance in Anopheles gambiae s.s. from the Bijagós were absent in the An. melas population, and no signatures of selective sweeps were identified in insecticide resistance-associated genes. Analysis of structural variants identified a large duplication encompassing the cytochrome-P450 gene cyp9k1. Phylogenetic analysis using publicly available mitochondrial genomes indicated that An. melas from the Bijagós split into two phylogenetic groups because of differentiation on the mitochondrial genome attributed to the cytochrome C oxidase subunits COX I and COX II and the NADH dehydrogenase subunits 1, 4, 4L and 5. CONCLUSIONS: This study identified an absence of insecticide-resistant SNPs common to An. gambiae in the An. melas population, but did identify structural variation over insecticide resistance-associated genes. Furthermore, this study presents novel insights into the population structure of this malaria vector using WGS analysis. Additional studies are required to further understand the role of this vector in malaria transmission.

Comparative Genomics Uncovers the Evolutionary Dynamics of Detoxification and Insecticide Target Genes Across 11 Phlebotomine Sand Flies.

Sand flies infect more than 1 million people annually with Leishmania parasites and other bacterial and viral pathogens. Progress in understanding sand fly adaptations to xenobiotics has been hampered by the limited availability of genomic resources. To address this gap, we sequenced, assembled, and annotated the transcriptomes of 11 phlebotomine sand fly species. Subsequently, we leveraged these genomic resources to generate novel evolutionary insights pertaining to their adaptations to xenobiotics, including those contributing to insecticide resistance. Specifically, we annotated over 2,700 sand fly detoxification genes and conducted large-scale phylogenetic comparisons to uncover the evolutionary dynamics of the five major detoxification gene families: cytochrome P450s (CYPs), glutathione-S-transferases (GSTs), UDP-glycosyltransferases (UGTs), carboxyl/cholinesterases (CCEs), and ATP-binding cassette (ABC) transporters. Using this comparative approach, we show that sand flies have evolved diverse CYP and GST gene repertoires, with notable lineage-specific expansions in gene groups evolutionarily related to known xenobiotic metabolizers. Furthermore, we show that sand flies have conserved orthologs of (i) CYP4G genes involved in cuticular hydrocarbon biosynthesis, (ii) ABCB genes involved in xenobiotic toxicity, and (iii) two primary insecticide targets, acetylcholinesterase-1 (Ace1) and voltage gated sodium channel (VGSC). The biological insights and genomic resources produced in this study provide a foundation for generating and testing hypotheses regarding the molecular mechanisms underlying sand fly adaptations to xenobiotics.

Whole-genome sequencing of major malaria vectors reveals the evolution of new insecticide resistance variants in a longitudinal study in Burkina Faso.

BACKGROUND: Intensive deployment of insecticide based malaria vector control tools resulted in the rapid evolution of phenotypes resistant to these chemicals. Understanding this process at the genomic level is important for the deployment of successful vector control interventions. Therefore, longitudinal sampling followed by whole genome sequencing (WGS) is necessary to understand how these evolutionary processes evolve over time. This study investigated the change in genetic structure and the evolution of the insecticide resistance variants in natural populations of Anopheles gambiae over time and space from 2012 to 2017 in Burkina Faso. METHODS: New genomic data have been generated from An. gambiae mosquitoes collected from three villages in the western part of Burkina Faso between 2012 and 2017. The samples were whole-genome sequenced and the data used in the An. gambiae 1000 genomes (Ag1000G) project as part of the Vector Observatory. Genomic data were analysed using the analysis pipeline previously designed by the Ag1000G project. RESULTS: The results showed similar and consistent nucleotide diversity and negative Tajima's D between An. gambiae sensu stricto (s.s.) and Anopheles coluzzii. Principal component analysis (PCA) and the fixation index (FST) showed a clear genetic structure in the An. gambiae sensu lato (s.l.) species. Genome-wide FST and H12 scans identified genomic regions under divergent selection that may have implications in the adaptation to ecological changes. Novel voltage-gated sodium channel pyrethroid resistance target-site alleles (V402L, I1527T) were identified at increasing frequencies alongside the established alleles (Vgsc-L995F, Vgsc-L995S and N1570Y) within the An. gambiae s.l. POPULATIONS: Organophosphate metabolic resistance markers were also identified, at increasing frequencies, within the An. gambiae s.s. populations from 2012 to 2017, including the SNP Ace1-G280S and its associated duplication. Variants simultaneously identified in the same vector populations raise concerns about the long-term efficacy of new generation bed nets and the recently organophosphate pirimiphos-methyl indoor residual spraying in Burkina Faso. CONCLUSION: These findings highlighted the benefit of genomic surveillance of malaria vectors for the detection of new insecticide resistance variants, the monitoring of the existing resistance variants, and also to get insights into the evolutionary processes driving insecticide resistance.

CYP4CE1 Metabolized Nitenpyram through Two Types of Oxidation Reaction, Hydroxylation, and N-Demethylation.

Nitenpyram, taking the place of imidacloprid, is a widely used neonicotinoid insecticide to control Nilaparvata lugens in Asia. Two P450s, CYP4CE1 and CYP6ER1, are key factors in the metabolic resistance against nitenpyram and imidacloprid. In this study, we found that CYP4CE1 expression was strongly associated with nitenpyram resistance in 8 field-collected populations, whereas CYP6ER1 expression correlated with imidacloprid resistance. Hence, we focused on nitenpyram metabolism by CYP4CE1, due to that imidacloprid metabolism by CYP6ER1 has intensively investigated. Mass spectrometry analysis revealed that recombinant CYP4CE1 metabolized nitenpyram into three products, N-desmethyl nitenpyram, hydroxy-nitenpyram, and N-desmethyl hydroxy-nitenpyram, with a preference for hydroxylation. In contrast, CYP6ER1 metabolized nitenpyram into a single product, N-desmethyl nitenpyram. These results provide new insights into the specific catalytic mechanisms of P450 enzymes in neonicotinoid metabolism and underscore the importance of different catalytic reactions in neonicotinoid insecticide resistance.

Overexpression of Two Odorant Binding Proteins Confers Chlorpyrifos Resistance in the Green Peach Aphid Myzus persicae.

The green peach aphid, Myzus persicae, is a worldwide agricultural pest. Chlorpyrifos has been widely used to control M. persicae for decades, thus leading to a high resistance to chlorpyrifos. Recent studies have found that insect odorant binding proteins (OBPs) play essential roles in insecticide resistance. However, the potential resistance mechanism underlying the cross-link between aphid OBPs and chlorpyrifos remains unclear. In this study, two OBPs (MperOBP3 and MperOBP7) were found overexpressed in M. persicae chlorpyrifos-resistant strains (CRR) compared to chlorpyrifos-sensitive strains (CSS); furthermore, chlorpyrifos can significantly induce the expression of both OBPs. An in vitro binding assay indicated that both OBPs strongly bind with chlorpyrifos; an in vivo RNAi and toxicity bioassay confirmed silencing either of the two OBPs can increase the susceptibility of aphids to chlorpyrifos, suggesting that overexpression of MperOBP3 and MperOBP7 contributes to the development of resistance of M. persicae to chlorpyrifos. Our findings provide novel insights into insect OBPs-mediated resistance mechanisms.

The Fat Body-Specific GST Gene SlGSTe11 Enhances the Tolerance of Spodoptera litura to Cyantraniliprole and Nicotine.

Spodoptera litura is a significant agricultural pest, and its glutathione S-transferase (GST) plays a crucial role in insecticide resistance. This study aimed to investigate the relationship between the SlGSTe11 gene of S. litura and resistance to cyantraniliprole and nicotine. Transcriptome analysis revealed that SlGSTe11 is highly expressed mainly in fat bodies, with a significant increase in SlGSTe11 gene expression under induction by cyantraniliprole and nicotine. The ectopic expression of the SlGSTe11 gene in transgenic fruit flies resulted in a 5.22-fold increase in the tolerance to cyantraniliprole. Moreover, compared to the UAS-SlGSTe11 line, the Act5C-UAS>SlGSTe11 line laid more eggs and had a lower mortality after nicotine exposure. RNAi-mediated inhibition of SlGSTe11 gene expression led to a significant increase in the mortality of S. litura under cyantraniliprole exposure. In vitro metabolism experiments demonstrated that the recombinant SlGSTe11 protein efficiently metabolizes cyantraniliprole. Molecular docking results indicated that SlGSTe11 has a strong affinity for both cyantraniliprole and nicotine. These findings suggest that SlGSTe11 is involved in the development of resistance to cyantraniliprole and nicotine in S. litura.

Mitochondrial Variation in Anopheles gambiae and Anopheles coluzzii: Phylogeographic Legacy and Mitonuclear Associations With Metabolic Resistance to Pathogens and Insecticides.

Mitochondrial DNA has been a popular marker in phylogeography, phylogeny, and molecular ecology, but its complex evolution is increasingly recognized. Here, we investigated mitochondrial DNA variation in Anopheles gambiae and Anopheles coluzzii, in relation to other species in the Anopheles gambiae complex, by assembling the mitogenomes of 1,219 mosquitoes across Africa. The mitochondrial DNA phylogeny of the Anopheles gambiae complex was consistent with previously reported highly reticulated evolutionary history, revealing important discordances with the species tree. The three most widespread species (An. gambiae, An. coluzzii, and Anopheles arabiensis), known for extensive historical introgression, could not be discriminated based on mitogenomes. Furthermore, a monophyletic clustering of the three saltwater-tolerant species (Anopheles merus, Anopheles melas, and Anopheles bwambae) in the Anopheles gambiae complex also suggested that introgression and possibly selection shaped mitochondrial DNA evolution. Mitochondrial DNA variation in An. gambiae and An. coluzzii across Africa revealed significant partitioning among populations and species. A peculiar mitochondrial DNA lineage found predominantly in An. coluzzii and in the hybrid taxon of the African "far-west" exhibited divergence comparable to the interspecies divergence in the Anopheles gambiae complex, with a geographic distribution matching closely An. coluzzii's geographic range. This phylogeographic relict of the An. coluzzii and An. gambiae split was associated with population and species structure, but not with the rare Wolbachia occurrence. The lineage was significantly associated with single nucleotide polymorphisms in the nuclear genome, particularly in genes associated with pathogen and insecticide resistance. These findings underline potential mitonuclear coevolution history and the role played by mitochondria in shaping metabolic responses to pathogens and insecticides in Anopheles.

Resistance of Aedes albopictus (Diptera: Culicidae) larvae and adults to insecticides based on bioassays and knockdown resistance (kdr) mutations in Zhejiang Province, China.

Aedes albopictus, a common mosquito in Zhejiang Province, is a carrier of more than twenty arboviruses. There are dozens or even hundreds of imported cases of dengue fever every year in Zhejiang Province, and there have also been many local outbreaks caused by imported cases of dengue fever. The objectives were to assess the resistance of larvae and adults of several Ae. albopictus strains in Zhejiang Province to commonly used pyrethroid insecticides (beta-cypermethrin, deltamethrin and permethrin), and detect mutations in the sodium channel gene, to further analyse the relationship between phenotypic resistance and the frequency of mutations. The resistance of eight field strains of Ae. albopictus larvae to beta-cypermethrin, deltamethrin and permethrin ranged from 8.17 to 36.06, 12.12-107.3 and 1.55-81.9, respectively, and there was a significant positive correlation of interaction resistance among the three insecticides. The mutation frequencies of I1532T and F1534S in the larvae of Ae. albopictus were 0-6.25 % and 42.19-100.00 %. Moreover, the diagnostic doses of the three pyrethroids for adult Ae. albopictus mosquitoes were 0.2510 g/L, 0.1562 g/L, and 0.9072 g/L. Except for the Zhoushan strain, which was suspected to be resistant to beta-cypermethrin, the other field strains were resistant to the three pyrethroids, and there was a significant positive correlation of cross-resistance among the three insecticides. The mutation frequencies of I1532T and F1534S of adult Ae. albopictus were 0-1.56 % and 62.50-100.00 %. In addition, the LC50 of the larvae and the mortality rate of adult Ae. albopictus after treatment with the three pyrethroids were significantly and positively correlated with the frequency of the F1534S mutation. F1534S mutation occurred earlier than I1532T mutation in both larvae and adult Ae. albopictus. F1534S mutation in the sodium channel gene may be a particular biomolecular detection marker for resistance to pyrethroid insecticides in Ae. albopictus in Zhejiang Province.

Anatomy of a pest control failure: introgression of cytochrome P450 337B3 alleles from invasive old-world bollworm into native corn earworm (Lepidoptera: Noctuidae).

The establishment of invasive species populations can threaten the ecological balance in naïve habitats and impact agricultural production practices. Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) (old-world bollworm, OWBW) and Helicoverpa zea (corn earworm, CEW) were geographically separated prior to the 2013 report of OWBW invasion into South America. Introgression of OWBW-specific cytochrome P450 337B3 (CYP337B3) gene into CEW was repeatedly detected across South America and the Caribbean. Two hybrids were documented from Texas in 2019. In this study, screening insects collected in Olathe, CO, USA, where a failure of pyrethroids to control CEW damage to conventional sweetcorn in 2023 detected 28.6% of insects with the OWBW-specific CYP337B3 marker. Nucleotide sequencing of the CYP337B3 gene identified 73.1% and 26.9% of insects carried CYP337B3v2 and CYP337B3v6 alleles, respectively, and 0.15 overall frequency of CYP337B3 alleles. Based on prior data for distinct phylogeographic origins of CYP337B3v2 and v6 alleles, our results indicate Olathe samples were derived from 2 different introductions: An uncertain source of the v6 allele that was initially reported in West Africa and possibly South American or Caribbean origin of the globally distributed v2 allele. One of the 1618 individuals screened also carried a ribosomal RNA internal transcribed spacer 1 derived from OWBW. Local selection pressures at the Olathe location imposed by repeated pyrethroid exposures are likely attributed to the prevalence of CYP337B3, where control practices hasten the accumulation of phenotypic resistance by adaptive introgression. Pyrethroid and other resistance factors carried by invasive OWBW may continue to impact CEW management tactics across the Americas.