Role In Insecticide Resistance Research Articles

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.

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.

Association of a rapidly selected 4.3kb transposon-containing structural variation with a P450-based resistance to pyrethroids in the African malaria vector Anopheles funestus.

Deciphering the evolutionary forces controlling insecticide resistance in malaria vectors remains a prerequisite to designing molecular tools to detect and assess resistance impact on control tools. Here, we demonstrate that a 4.3kb transposon-containing structural variation is associated with pyrethroid resistance in central/eastern African populations of the malaria vector Anopheles funestus. In this study, we analysed Pooled template sequencing data and direct sequencing to identify an insertion of 4.3kb containing a putative retro-transposon in the intergenic region of two P450s CYP6P5-CYP6P9b in mosquitoes of the malaria vector Anopheles funestus from Uganda. We then designed a PCR assay to track its spread temporally and regionally and decipher its role in insecticide resistance. The insertion originates in or near Uganda in East Africa, where it is fixed and has spread to high frequencies in the Central African nation of Cameroon but is still at low frequency in West Africa and absent in Southern Africa. A marked and rapid selection was observed with the 4.3kb-SV frequency increasing from 3% in 2014 to 98% in 2021 in Cameroon. A strong association was established between this SV and pyrethroid resistance in field populations and is reducing pyrethroid-only nets' efficacy. Genetic crosses and qRT-PCR revealed that this SV enhances the expression of CYP6P9a/b but not CYP6P5. Within this structural variant (SV), we identified putative binding sites for transcription factors associated with the regulation of detoxification genes. An inverse correlation was observed between the 4.3kb SV and malaria parasite infection, indicating that mosquitoes lacking the 4.3kb SV were more frequently infected compared to those possessing it. Our findings highlight the underexplored role and rapid spread of SVs in the evolution of insecticide resistance and provide additional tools for molecular surveillance of insecticide resistance.

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.

Insight into Hyalomma anatolicum biology by comparative genomics analyses

Hyalomma anatolicum is an obligatory blood-sucking ectoparasite and contributes to the transmission of Crimean–Congo haemorrhagic fever (CCHF) virus, Theileria spp. and Babesia spp. Progress in exploring the adaptive strategy of this ectoparasite and developing tools to fight it has been hindered by the lack of a complete genome. Herein, we assembled the genome using diverse sources of data from multiple sequencing platforms and annotated the 1.96 Gb genome of Hy. anatolicum. Comparative genome analyses and the predicted protein encoding genes reveal unique facets of this genome, including gene family expansion associated with blood feeding and digestion, multi-gene families involved in detoxification, a great number of neuropeptides and corresponding receptors regulating tick growth, development, and reproduction, and glutathione S-transferase genes playing roles in insecticide resistance and detoxification of multiple xenobiotic factors. This high quality reference genome provides fundamental data for obtaining insights into a variety of aspects of tick biology and developing novel strategies to fight notorious tick vectors of human and animal pathogens.

RNA interference of NADPH-cytochrome P450 reductase increases the susceptibility of Aphis gossypii Glover to sulfoxaflor

NADPH-cytochrome P450 reductase (CPR) is essential for the detoxification of endogenous and exogenous substances mediated by cytochrome P450. While several insect CPRs have been found to be associated with insecticide resistance, the CPR of Aphis gossypii has not been characterized, and its functional role in insecticide resistance remains undefined. In this study, we cloned and characterized the full-length sequence of A. gossypii CPR (AgCPR). The deduced amino acid sequence of AgCPR contains all conserved domains of CPR, which shows high similarity to other insect CPRs and was clustered into a same branch of aphids according to phylogenetic analysis. The transcript of AgCPR was present in all developmental stages, with the highest expression in the adult stage. Furthermore, the expression of AgCPR could be induced by sulfoxaflor, a commonly used insecticide, in a time- and dose-dependent manner. Further silencing of AgCPR by feeding dsRNA significantly increased the susceptibility of A. gossypii to this insecticide. These findings suggest that AgCPR may play a significant role in the susceptibility of A. gossypii to sulfoxaflor and in the development of P450-mediated resistance to sulfoxaflor.

Functional characterization of SlGSTD3 and SlGSTD4 associated with phoxim and chlorpyrifos resistance in Spodoptera litura

Glutathione S-transferases (GSTs) is one of the main detoxification enzyme systems in insects and play important roles in insecticide resistance by direct metabolism, sequestration and antioxidant activity. Several GSTs genes in Spodoptera litura, a polyphagous agricultural pest, have been demonstrated to be overexpressed and involved in organophosphates and pyrethroids resistance. Previous studies have indicated the significant overexpression of two delta class GSTs genes (SlGSTd3 and SlGSTd4) in organophosphates and pyrethroids resistant populations. Here, they were heterologous expressed, and their metabolism activity and antioxidant activity were determined. Results indicated that the recombinant protein SlGSTD3 and SlGSTD4 both showed metabolism activity to phoxim and chlorpyrifos, but not to fenvalerate, cyhalothrin or beta cypermethrin. The metabolism activity of SlGSTD3 to phoxim and chlorpyrifos is higher than that of SlGSTD4. The recombinant vector of SlGSTD3 and SlGSTD4 both showed antioxidant activity after exposure to cumene hydroperoxide. Further modeling and docking analysis indicated that the 3D structure of SlGSTD3 and SlGSTD4 were well shaped for phoxim and chlorpyrifos, and the binding affinity for phoxim was stronger than that of chlorpyrifos. Our work provides evidence that SlGSTd3 and SlGSTd4 both play roles in phoxim and chlorpyrifos resistance in S. litura.

Functional analysis of an overexpressed glutathione S-transferase BdGSTd5 involved in malathion and malaoxon detoxification in Bactrocera dorsalis

Glutathione S-transferases (GSTs) are one of the three detoxification enzyme families. The constitutive and inducible overexpression of GSTs genes plays an important role in insecticide resistance. Previous study showed that malathion resistance was polygenic, and elevated GSTs activity was one of the important factor participating in malathion resistance of Bactrocera dorsalis (Hendel), a serious economic pest worldwide. BdGSTd5 overexpression was inducible upon exposure to malathion. However, the involvement of BdGSTd5 in malathion resistance has not been clarified. In this study, we found that BdGSTd5 sequence harbored the conserved region of delta class GSTs, which were overexpressed in malathion resistant strain of B. dorsalis compared to malathion susceptible strain. The highest mRNA expression level of BdGSTd5 was found in 1-day-old adult, and the levels decreased with aging. The dsBdGSTd5 injection effectively silenced (73.4% reduction) the expression of BdGSTd5 and caused significant increase in susceptibility to malathion with a cumulative mortality increasing of 13.5% at 72 h post malathion treatment (p < 0.05). Cytotoxicity assay demonstrated that BdGSTd5 was capable of malathion detoxification. Molecular docking analysis further indicated the interactive potential of BdGSTd5 with malathion and its toxic oxide malaoxon. The recombinant BdGSTd5 exhibited glutathione-conjugating activity toward 1-chloro-2, 4-dinitrobenzene and malathion and malaoxon metabolic capacity with significant reduction (p < 0.05) of the peak areas by 90.0% and 73.1%, respectively. Taken together, the overexpressed BdGSTd5 contributes to malathion metabolism and resistance, which detoxify the malathion in B. dorsalis via directly depleting malathion and malaoxon.

Transposable elements and xenobiotic resistance.

Transposable elements or TEs are well known drivers of adaptive change in plants and animals but their role in insecticide resistance remains poorly documented. This review examines the potential role of transposons in resistance and identifies key areas where our understanding remains unclear. Despite well-known model systems such as upregulation of Drosophila Cyp6g1, many putative examples lack functional validation. The potential types of transposon-associated changes that could lead to resistance are reviewed, including changes in up-regulation, message stability, loss of function and alternative splicing. Where potential mechanisms appear absent from the resistance literature examples are drawn from other areas of biology. Finally, ways are suggested in which transgenic expression could be used to validate the biological significance of TE insertion. In the absence of such functional expression studies many examples of the association of TEs and resistance genes therefore remain as correlations.

Genome-wide analyses of Glutathione S-transferase gene family and expression profiling under deltamethrin exposure in non-biting midge Propsilocerus akamusi

Glutathione S-transferases (GSTs) are major enzymes in detoxification phase II, and have been functioned in resistance to various insecticides or oxidative stress. Herein, we selected the non-biting midge, Propsilocerus akamusi, widespread in Asian aquatic ecosystems, to uncover the gene location, structure, and phylogenetics relationship of GSTs at genome scale first time. Thirty-three cytosolic and four microsomal GST genes were identified and located on the four chromosomes. The cytosolic GSTs involved in the eight subclasses and five GST genes were unclassified. The expansion of GST genes in P. akamusi experienced duplication events on the delta, theta, xi, iota, and unclassified subclasses. The RNA-Seq analyses and RT-qPCR validation showed that the expression of PaGSTt2 gene is significantly elevated, with deltamethrin concentration increasing. The tertiary structure of PaGSTt2 enzyme was reconstructed, which was different from the other theta gene in the active site. In addition, the GST genes of six chironomids were first described based on the assembled genomes to explore the difference of those in the adaptation to kinds of environments. The GST frame for P. akmusi and its expression profiles provide valuable resources to understand their role in insecticide resistance of this species, as well as those of other biting midges.

Genome-Wide Identification and Expression Analysis of the Cytochrome P450 Gene Family in Bemisia tabaci MED and Their Roles in the Insecticide Resistance

The whitefly, Bemisia tabaci MED (Hemiptera: Aleyrodidae), is an omnivorous agricultural pest, which causes huge economic losses to agriculture and is highly resistant to many pesticides. The overexpression of cytochrome P450 may play an important role in host adaptation and insecticide resistance in B. tabaci MED. Therefore, the present study systematically analyzed the cytochrome P450 gene family at the genome-wide level to understand its function in B. tabaci MED. Our analysis identified 58 cytochrome P450 genes in B. tabaci MED, among which 24 were novel. Phylogenetic analysis revealed broad functional and species-specific diversification in B. tabaci MED P450, suggesting the role of multiple P450 genes in detoxifying. Reverse transcription-real time quantitative PCR (RT-qPCR) showed that CYP4CS2, CYP4CS5, CYP4CS6, CYP4CS8, CYP6DW4, CYP6DW5, CYP6DW6, CYP6DZ8, and CYP6EN1 genes increased significantly after two days of exposure to imidacloprid. Interestingly, all nine genes belonged to the CYP4 and CYP6 families. A decrease in the expression of five genes (CYP6DW4, CYP6DW5, CYP6DW6, CYP6DZ8, and CYP4CS6) via RNA interference (RNAi) resulted in a significant increase in the mortalities of whiteflies when exposed to imidacloprid. These results indicate that the overexpression of the P450 genes may play an essential role in imidacloprid tolerance of B. tabaci MED. Thus, the present study provides basic information on P450 genes in B. tabaci MED, which will further help elucidate the insecticide resistance mechanism in the agricultural pest whitefly.

CRISPR/Cas9-mediated knockout of NlCYP6CS1 gene reveals its role in detoxification of insecticides in Nilaparvata lugens (Hemiptera: Delphacidae).

The brown planthopper (Nilaparvata lugens) is one of the major rice insect pests in Asia. Recently, high levels of insecticide resistance have been frequently reported and cytochrome P450 monooxygenase (P450)-mediated metabolic detoxification is a common resistance mechanism in N. lugens. However, there has been no persuasive genetic method to prove the role of P450s in insecticide resistance in N. lugens. Here, CRISPR/Cas9 system was used to disrupt the P450 gene NlCYP6CS1 to elucidate its role in insecticide resistance in field populations of N. lugens. We successfully constructed a homozygous strain (Nl6CS1-KO) with a 5-bp deletion and 1-bp insertion mutation of NlCYP6CS1. Compared with a background resistant strain (Nl-R), the susceptibility of knockout strain Nl6CS1-KO to imidacloprid, nitenpyram, thiamethoxam, dinotefuran, and pymetrozine was increased by 2.3-, 3.4-, 7.0-, 4.2- and 3.9-fold, respectively, but not significantly changed to triflumezopyrim, chlorpyrifos and buprofezin. Life table analysis demonstrated that the Nl6CS1-KO strain resembled the Nl-R strain in terms of egg and nymph developmental duration and adult lifespan, but differed from the Nl-R strain in the survival rate of eggs and nymphs, reproduction, and body weight. Our study demonstrates the effect of functional deletion of NlCYP6CS1 on multiple insecticide resistance in N. lugens. For the first time, we applied CRISPR/Cas9 system to reveal the mechanism of insecticide resistance in N. lugens, which may shed light on similar studies in other hemipteran insects. © 2023 Society of Chemical Industry.

CRISPR/Cas9-mediated D472N substitution in the Rdl1 of Plutella xylostella confers low resistance to abamectin.

Abamectin is one of the main insecticides used for the control of Plutella xylostella, a destructive pest of cruciferous crops. Target-site mutation plays an important role in insecticide resistance. A point mutation (D472N) has been reported in the Rdl1 γ-aminobutyric acid receptor (GABAR) in P. xylostella, but its roles in insecticide resistance remain unknown. In this study, the D472N mutation of the Rdl1 GABAR was detected in several field populations of P. xylostella and showed a positive correlation with abamectin resistance. A knock-in homozygous mutation strain (D472N-KI) of P. xylostella was successfully constructed using CRISPR/Cas9 coupled with homology-directed repair, and the bioassay results demonstrated that compared with the susceptible strain, the D472N-KI strain had 11.1- and 3.7-fold increased resistance to abamectin and endosulfan, respectively. There was no difference in resistance to fipronil, broflanilide or isocycloseram, which also target the GABAR. In addition, the total fecundity of the D472N-KI strain was significantly reduced by 50.0%. Our results suggest that the homozygous D472N mutation in Rdl1 confers a low level of resistance to abamectin in P. xylostella but causes significant fecundity disadvantages, which may delay the development of resistance to some extent. These results lay a foundation for further understanding the mechanisms of abamectin resistance in insect pests. © 2022 Society of Chemical Industry.

MiRNA novel_268 targeting NlABCG3 is involved in nitenpyram and clothianidin resistance in Nilaparvata lugens

The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most destructive pests that seriously threatens the high-quality and safe production of rice. However, due to the unscientific use of chemical insecticides, N. lugens has developed varying levels of resistance to insecticides, including nitenpyram and clothianidin. The ATP-binding cassette (ABC) transporter plays a nonnegligible role in phase III of the detoxification process, which may play an important role in insecticide resistance. In the present study, NlABCG3 was significantly overexpressed in both the NR and CR populations compared with susceptible populations. Silencing NlABCG3 significantly increased the susceptibility of BPH to nitenpyram and clothianidin. In addition, RNAi-mediated knockdown of three key genes in the miRNA biogenesis pathway altered the level of NlABCG3. Subsequently, the luciferase reporter assays demonstrated that novel_268 binds to the NlABCG3 coding region and downregulates its expression. Furthermore, injection of miRNA inhibitors or mimics of novel_268 significantly altered the susceptibility of N. lugens to nitenpyram and clothianidin. These results suggest that miRNA novel_268 targeting NlABCG3 is involved in nitenpyram and clothianidin resistance in N. lugens. These findings may help to enhance our knowledge of the transcriptional regulation of the ABC transporter that mediate insecticide resistance in N. lugens.

Metabolism and antioxidant activity of SlGSTD1 in Spodoptera litura as a detoxification enzyme to pyrethroids

Glutathione S-transferase (GSTs) are members of multifunction enzymes in organisms and mostly known for their roles in insecticide resistance by conjugation. Spodoptera litura (Fabricius) is a voracious agricultural pest widely distributed in the world with high resistance to various insecticides. The function of GSTs in the delta group of S. litura is still lacking. Significantly up-regulation of SlGSTd1 was reported in four pyrethroids-resistant populations and a chlorpyrifos-selected population. To further explore its role in pyrethroids and organophosphates resistance, the metabolism and peroxidase activity of SlGSTD1 were studied by heterologous expression, RNAi, and disk diffusion assay. The results showed that Km and Vmax for 1-chloro-2,4-dinitrobenzene (CDNB) conjugating activity of SlGSTD1were 1.68 ± 0.11 mmol L−1 and 76.0 ± 2.7 nmol mg−1 min−1, respectively. Cyhalothrin, beta-cypermethrin, and chlorpyrifos had an obvious inhibitory effect on SlGSTD1 activity, especially for fenvalerate, when using CDNB as substrate. Fenvalerate and cyhalothrin can be metabolized by SlGSTD1 in E. coli and in vitro. Also, silencing of SlGSTd1 significantly increased the toxicity of fenvalerate and cyhalothrin, but had no significant effect on the mortality of larvae treated by beta-cypermethrin or chlorpyrifos. SlGSTD1 possesses peroxidase activity using cumene hydroperoxide as a stress inducer. The comprehensive results indicate that SlGSTD1 is involved in fenvalerate and cyhalothrin resistance of S. litura by detoxication and antioxidant capacity.

Comparative genomic analysis of ABC transporter genes in Tenebrio molitor and four other tenebrionid beetles (Coleoptera: Tenebrionidea).

ATP-binding cassette (ABC) transporters, one of the largest transmembrane protein families, transport a diverse number of substate across membranes. Details of their diverse physiological functions have not been established. Here, we identified 87 ABC transporter genes in the genomes of Tenebrio molitor along with those from Asbolus verrucosus (104), Hycleus cichorii (65), and Hycleus phaleratus (80). Combining these genes (336 in total) with genes reported in Tribolium castaneum (73), we analyzed the phylogeny of ABC transporter genes in all five Tenebrionids. They are assigned into eight subfamilies (ABCA-H). In comparison to other species, the ABCC subfamily in this group of beetles appears expanded. The expression profiles of the T. molitor genes at different life stages and in various tissues were also investigated using transcriptomic analysis. Most of them display developmental specific expression patterns, suggesting to us their possible roles in development. Most of them are highly expressed in detoxification-related tissues including gut and Malpighian tubule, from which we infer their roles in insecticide resistance. We detected specific or abundant expressions of many ABC transporter genes in various tissues such as salivary gland, ovary, testis, and antenna. This new information helps generate new hypotheses on their biological significance within tissues.

Characterization of carboxylesterase PxαE8 and its role in multi-insecticide resistance in Plutella xylostella (L.)

Carboxylesterase (CarE) was considered as important phase-I detoxifying enzymes which participated in detoxification of different types of insecticides. Up-regulation of CarE genes has been proved playing a major role in insecticide resistance in many pest insects, but its involvement in resistance to insecticides in Plutella xylostella has been rarely reported. In this study, a CarE cDNA named PxαE8 was identified in P. xylostella, which has an open reading frame of 1 599 nucleotides and putatively encodes 532 amino acids. The investigation of spatial expression profiles of PxαE8 revealed that it was expressed in all developmental stages, especially in larvae and adults. The body part/tissue-specific expression profiles showed that the PxαE8 mainly expressed in fat body, malpighian tubule and hemolymph of larvae. Further, the relative expression of PxαE8 in two multi-resistant field populations, Hainan (HN) and Guangdong (GD) populations, was found 24.4- and 15.5-fold higher than that in susceptible population, respectively. Knockdown of PxαE8 by RNA interference dramatically increased the mortalities of larvae of HN population treated with LC50 of beta-cypermethrin and phoxim by 25.3 and 18.3%, respectively. These results suggested that up-regulation of PxαE8 was involved in resistance to both beta-cypermethrin and phoxim in P. xylostella, which shed light on further understanding of molecular mechanisms of multi-insecticide-resistance in P. xylostella and other pest insects.

Cytochrome P450 Genes of the CYP4 Clan and Pyrethroid Resistance in Chagas Disease Vectors

Triatomine insects are vectors of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease. Although residual pyrethroid spraying has been a successful vector control strategy for many years, a growing number of pyrethroid-resistance foci is being documented, mainly in Triatoma infestans, that led to failures in vector elimination. Insecticide resistance is a multifactorial phenomenon that often implies a combination of three different mechanisms: increased insecticide detoxification, reduced affinity of the site of action, and reduced insecticide penetration through the cuticle. All three mechanisms were reported in pyrethroid-resistant T. infestans. Cytochrome P450s are enzymes involved in the metabolism of xenobiotics and endogenous chemicals. They are encoded by CYP genes and classified into different families and clans. In triatomines, the CYP4 clan is divided in two families, CYP3093 and CYP4, and both exhibit genome-wide, triatomine-specific gene expansions. Some members from each family have been reported to be involved in two of the mechanisms mentioned above, i.e., they participate in insecticide detoxification in different organs and tissues, and in the synthesis of cuticular hydrocarbons, which ultimately can contribute to a reduced insecticide penetration. The aim of this manuscript is to review the current state of knowledge of P450 genes belonging to the CYP4 clan in triatomines and to highlight their potential role in insecticide resistance.

Biodegradation of insecticides by gut bacteria isolated from stored grain beetles and its implication in host insecticide resistance

Stored grain pests have developed widespread resistance to currently available grain protectants. Although symbiotic microbes played an important role in insecticide resistance of insect hosts, it has not been reported in stored grain pests. In this study, five gut bacteria were excised from adult Sitophilus oryzae (L.) (Coleoptera: Curculionidae), Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae), and Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae). These bacteria can degrade malathion, pirimiphos-methyl, and deltamethrin and utilize these insecticides as the carbon source in vitro. When treated with 0.5–10 mg/kg of malathion, pirimiphos-methyl and 0.3–0.75 mg/kg of deltamethrin, gnotobiotic reinoculation of a given bacterial strain significantly enhanced survival rates of its hosts. The significance of gut bacteria in host insecticide tolerance was insecticide dosage-dependent. Furthermore, the in vitro insecticide biodegradability of gut bacteria was not completely consistent with their in vivo performance in host insecticide resistance. This suggests that there may be other physiological processes responsible for host insecticide tolerance mediated by symbiotic bacteria except for direct degradation of insecticides. These results provide insight into insecticide resistance management of stored grain pests and further study of the association between gut bacteria and insecticide resistance.

The Birth-and-Death Evolution of Cytochrome P450 Genes in Bees.

The birth-and-death model of multigene family evolution describes how gene families evolve and diversify through duplication and deletion. The cytochrome P450s are one of the most diverse and well-studied multigene families, involved in both physiological and xenobiotic functions. Extensive studies of insect P450 genes have demonstrated their role in insecticide resistance. Bees are thought to experience toxin exposure through their diet of nectar and pollen, as well as the resin-collecting behavior exhibited by some species. Here, we describe the repertoire of P450 genes in the orchid bee Euglossa dilemma. Male orchid bees form perfume bouquets used in courtship displays by collecting volatile compounds, resulting in exposure to compounds known to be toxic. In addition, we conducted phylogenetic and selection analyses across ten bee species encompassing three bee families. We find that social behavior and resin collection are not correlated with the repertoire of P450 present in a bee species. However, our analyses revealed that P450 clades can be classified as stable and unstable, and that genes involved in xenobiotic metabolism are more likely to belong to unstable clades. Furthermore, we find that unstable clades are under more dynamic evolutionary pressures and exhibit signals of adaptive evolution. This work highlights the complexity of multigene family evolution, revealing that multiple factors contribute to the diversification, stability, and dynamics of this gene family. Furthermore, we provide a resource for future detailed studies investigating the function of different P450s in economically important bee species.