Expression reduction and a variant of a P450 gene mediate chlorpyrifos resistance in Tetranychus urticae Koch.

INTRODUCTION: Understanding how insects and mites develop resistance to chlorpyrifos is crucial for effective field management. Although extensive research has demonstrated that T. urticae exhibits high resistance to chlorpyrifos, the specific resistance mechanism remains elusive. Investigating this mechanism could provide valuable insights for pest control strategies. OBJECTIVES: This study aimed to reveal the mechanism of chlorpyrifos resistance in T. urticae. METHODS: The expression level of the CYP392D8 gene in T. urticae strains were analyzed using real- time quantitative PCR and western blot techniques. Functional validation of CYP392D8 was conducted through RNAi and heterogeneous expression. The production of chlorpyrifos-oxon in both resistant and susceptible strains were quantified using LC-MS/MS. Furthermore, the metabolic capabilities of CYP392D8 variants were verified using HPLC-MS and molecular docking. RESULTS: The results showed the expression of CYP392D8 was reduced in some Chinese resistant populations and mites with knocked down CYP392D8 showed decreased susceptibility to chlorpyrifos. Chlorpyrifos-oxon, the active metabolite of chlorpyrifos, was generated when chlorpyrifos was incubated with recombinant CYP392D8 protein in vitro. And a higher efficiency of chlorpyrifos-oxon formation was observed with the CYP392D8-S variant from susceptible mites compared to the CYP392D8-R variant from resistant mites. After treatment with low doses of chlorpyrifos, susceptible mite extracts produced substantial amounts of chlorpyrifos-oxon, while resistant mites only showed trace amounts. In addition, molecular docking studies showed that CYP392D8-S had a higher binding capacity to chlorpyrifos than the CYP392D8-R variant. CONCLUSION: This study reveals a mechanism of insecticide resistance due to the bioactivation reduction in combination with the sequence variation in a pest mite. These findings provide an important theoretical bias for management strategies of mites in the field and comprehensive control.

A midgut transcriptional regulatory loop favors an insect host to withstand a bacterial pathogen.

Mounting evidence suggests that insect hormones associated with growth and development also participate in pathogen defense. We have discovered a previously undescribed midgut transcriptional control pathway that modulates the availability of 20-hydroxyecdysone (20E) in a worldwide insect pest (Plutella xylostella), allowing it to defeat the major virulence factor of an insect pathogen Bacillus thuringiensis (Bt). A reduction of the transcriptional inhibitor (PxDfd) increases the expression of a midgut microRNA (miR-8545), which in turn represses the expression of a newly identified ecdysteroid-degrading glucose dehydrogenase (PxGLD). Downregulation of PxGLD reduces 20E degradation to increase 20E titer and concurrently triggers a transcriptional negative feedback loop to mitigate 20E overproduction. The moderately elevated 20E titer in the midgut activates a MAPK signaling pathway to increase Bt tolerance/resistance. These findings deepen our understanding of the functions attributed to these classical insect hormones and help inform potential future strategies that can be employed to control insect pests.

A Shared Receptor Suggests a Common Ancestry between an Insecticidal Bacillus thuringiensis Cry Protein and an Anti-Cancer Parasporin.

Cry toxins, produced by the bacterium Bacillus thuringiensis, are of significant agronomic value worldwide due to their potent and highly specific activity against various insect orders. However, some of these pore-forming toxins display specific activity against a range of human cancer cells whilst possessing no known insecticidal activity; Cry41Aa is one such toxin. Cry41Aa has similarities to its insecticidal counterparts in both its 3-domain toxic core structure and pore-forming abilities, but how it has evolved to target human cells is a mystery. This work shows that some insecticidal Cry toxins can enhance the toxicity of Cry41Aa against hepatocellular carcinoma cells, despite possessing no intrinsic toxicity themselves. This interesting crossover is not limited to human cancer cells, as Cry41Aa was found to inhibit some Aedes-active Cry toxins in mosquito larval assays. Here, we present findings that suggest that Cry41Aa shares a receptor with several insecticidal toxins, indicating a stronger evolutionary relationship than their divergent activities might suggest.

Identification of a biomarker for Bacillus thuringiensis strains with high toxicity against Spodoptera frugiperda based on insecticidal gene linkage analysis.

BACKGROUND: Bacillus thuringiensis (Bt) is a Gram-positive bacterium that produces various insecticidal proteins used to control insect pests. Spodoptera frugiperda is a global insect pest which causes serious damage to crops, but bio-insecticides currently available to control this pest have limited activity and so new ones are always being sought. In this study we have tested the hypothesis that a biomarker for strain toxicity could be found that would greatly facilitate the identification of new potential products. RESULTS: Using genomic sequencing data we constructed a linkage network of insecticidal genes from 1957 Bt genomes and found that four gene families, namely cry1A, cry1I, cry2A and vip3A, showed strong linkage. For 95 strains isolated from soil samples we assayed them for toxicity towards S. frugiperda and for the presence of the above gene families. All of the strains that showed high toxicity also contained a member of the vip3A gene family. Two of them were more toxic than a commercially available strain and genomic sequencing identified a number of potentially novel toxin-encoding genes. CONCLUSIONS: The presence of a vip3A gene in the genome of a Bt strain proved to be a strong indicator of toxicity towards S. frugiperda validating this biomarker approach as a strategy for future discovery programs. © 2024 Society of Chemical Industry.

Characterization of the individual domains of the Bacillus thuringiensis Cry2Aa implicates Domain I as a possible binding site to Helicoverpa armigera.

Bacillus thuringiensis (Bt) Cry2Aa is a member of the Cry pore-forming, 3-domain, toxin family with activity against both lepidopteran and dipteran insects. Although domains II and III of the Cry toxins are believed to represent the primary specificity determinant through specific binding to cell receptors, it has been proposed that the pore-forming domain I of Cry2Aa also has such a role. Thus, a greater understanding of the functions of Cry2Aa's different domains could potentially be helpful in the rational design of improved toxins. In this work, cry2Aa and its domain fragments (DI, DII, DIII, DI-II and DII-DIII) were subcloned into the vector pGEX-6P-1 and expressed in Escherichia coli. Each protein was recognized by anti-Cry2Aa antibodies and, except for the DII fragment, could block binding of the antibody to Cry2Aa. Cry2Aa and its DI and DI-II fragments bound to brush border membrane vesicles (BBMV) from H. armigera and also to a ca 150 kDa BBMV protein on a far western (ligand) blot. In contrast the DII, DIII and DII-III fragments bound to neither of these. None of the fragments were stable in H. armigera gut juice nor showed any toxicity towards this insect. Our results indicate that contrary to the general model of Cry toxin activity domain I plays a role in the binding of the toxin to the insect midgut.

N-terminal proteolysis determines the differential activity of Bacillus thuringiensis Cry2A toxins towards Aedes aegypti.

It has long been known that while both the Bacillus thuringiensis pesticidal proteins Cry2Aa and Cry2Ab have wide-ranging activities against lepidopteran insects only the former has activity against the mosquito Aedes aegypti. We have previously shown that this differential specificity is influenced by the N-terminal region of these proteins and here demonstrate that this is due to these sections affecting proteolytic activation. Enzymes from the midgut of A. aegypti cleave Cry2Aa at the C-terminal side of amino acid 49 resulting in a 58 kDa fragment whereas these enzymes do not cleave Cry2Ab at this position. The 58 kDa, but not the protoxin, form of Cry2Aa is capable of interacting with brush border membrane vesicles from A. aegypti.

Throwing Brazilian strains into the melting pot of P. xylostella resistance to Bacillus thuringiensis.

The resistance of pest insects to biopesticides based on the bacterium Bacillus thuringiensis (Bt) is normally associated with changes to the receptors involved in the mechanism of action of the pesticidal proteins produced by Bt. In some strains of Plutella xylostella (the diamondback moth) resistance has evolved through a signalling mechanism in which the genes encoding the receptor proteins are downregulated whereas in others it has been linked to structural changes in the receptors themselves. One such well characterized mutation is in the ABCC2 gene indicating that changes to this protein can result in resistance. However other studies have found that knocking out this protein does not result in a significant level of resistance. In this study we wanted to test the hypothesis that constitutive receptor downregulation is the major cause of Bt resistance in P. xylostella and that mutations in the now poorly expressed receptor genes may not contribute significantly to the phenotype. To that end we investigated the expression of a receptor (ABCC2) and the major regulator of the signalling pathway (MAP4K4) in two resistant and four susceptible strains. No correlation was found between expression levels and susceptibility; however, a frameshift mutation was identified in the ABCC2 receptor in a newly characterized resistant strain.

RNA m6 A Methylation Suppresses Insect Juvenile Hormone Degradation to Minimize Fitness Costs in Response to A Pathogenic Attack.

Bioinsecticides and transgenic crops based on the bacterial pathogen Bacillus thuringiensis (Bt) can effectively control diverse agricultural insect pests, nevertheless, the evolution of resistance without obvious fitness costs has seriously eroded the sustainable use of these Bt products. Recently, it has been discovered that an increased titer of juvenile hormone (JH) favors an insect host (Plutella xylostella) to enhance fitness whilst resisting the Bt pathogen, however, the underlying regulatory mechanisms of the increased JH titer are obscure. Here, the involvement of N6 -methyladenosine (m6 A) RNA modification in modulating the availability of JH in this process is defined. Specifically, it is found that two m6 A methyltransferase subunit genes, PxMettl3 and PxMettl14, repress the expression of a key JH-degrading enzyme JH esterase (JHE) to induce an increased JH titer, mitigating the fitness costs associated with a robust defense against the Bt pathogen. This study identifies an as-yet uncharacterized m6 A-mediated epigenetic regulator of insect hormones for maintaining fitness during pathogen defense and unveils an emerging Bt resistance-related m6 A methylation atlas in insects, which further expands the functional landscape of m6 A modification and showcases the pivotal role of epigenetic regulation in host-pathogen interactions.

Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX.

The Lysinibacillus sphaericus proteins Tpp49Aa1 and Cry48Aa1 can together act as a toxin toward the mosquito Culex quinquefasciatus and have potential use in biocontrol. Given that proteins with sequence homology to the individual proteins can have activity alone against other insect species, the structure of Tpp49Aa1 was solved in order to understand this protein more fully and inform the design of improved biopesticides. Tpp49Aa1 is naturally expressed as a crystalline inclusion within the host bacterium, and MHz serial femtosecond crystallography using the novel nanofocus option at an X-ray free electron laser allowed rapid and high-quality data collection to determine the structure of Tpp49Aa1 at 1.62 Å resolution. This revealed the packing of Tpp49Aa1 within these natural nanocrystals as a homodimer with a large intermolecular interface. Complementary experiments conducted at varied pH also enabled investigation of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals-a crucial step in its mechanism of action. To better understand the cooperation between the two proteins, assays were performed on a range of different mosquito cell lines using both individual proteins and mixtures of the two. Finally, bioassays demonstrated Tpp49Aa1/Cry48Aa1 susceptibility of Anopheles stephensi, Aedes albopictus, and Culex tarsalis larvae-substantially increasing the potential use of this binary toxin in mosquito control.

Characterization of a novel cell wall hydrolase CwlE involved in Bacillus thuringiensis subsp. israelensis mother cell lysis.

Cell wall hydrolases are ubiquitous among spore-form bacteria and essential for mother cell lysis. In this study, a novel cell wall hydrolase gene cwlE involved in mother cell lysis was characterized from Bacillus thuringiensis subsp. israelensis (Bti) strain Bt-59. cwlE was specifically expressed in Bti and located in the large plasmid carrying the insecticidal genes. The encoded CwlE protein consists of a MurNAc-LAA domain and two highly conserved catalytic residues (E26 and E151). The recombinant CwlE-His protein was able to digest the cell wall of Bti, indicating that CwlE is an N-acetylmuramoyl-L-alanine amidase. Transcriptional analysis indicated that cwlE began to express at the early stage of stationary phase and was controlled by SigE. Single mutation of cwlE gene delayed Bti mother cell lysis, while double mutation of cwlE and sigK completely blocked Bti mother cell lysis. After exposure to UV light to deactivate the crystal proteins, the level of decrease of insecticidal activity against mosquito larvae of Bt-59 (ΔcwlE-sigK) was less than that observed for Bt-59. This study elucidates the mechanism of Bti mother cell lysis and provides an effective strategy for mosquito control using Bt products with increased persistence.

Assessment of the role of an ABCC transporter TuMRP1 in the toxicity of abamectin to Tetranychus urticae.

The rapid evolution of pest resistance threatens the sustainable utilization of bioinsecticides such as abamectin, and so deciphering the molecular mechanisms affecting toxicity and resistance is essential for their long-term application. Historical studies of abamectin resistance in arthropods have mainly focused on mechanisms involving the glutamate-gated chloride channel (GluCl) targets, with the role of metabolic processes less clear. The two-spotted spider mite, Tetranychus urticae, is a generalist herbivore notorious for rapidly developing resistance to pesticides worldwide, and abamectin has been widely used for its control in the field. After reanalyzing previous transcriptome and RNA-seq data, we here identified an ABC transporter subfamily C gene in T. urticae named multidrug resistance-associated protein 1 (TuMRP1), whose expression differed between susceptible and resistant populations. Synergism bioassays with the inhibitor MK-571, the existence of a genetic association between TuMRP1 expression and susceptibility to abamectin, and the effect of RNA interference mediated silencing of TuMRP1 were all consistent with a direct role of this transporter protein in the toxicity of abamectin. Although ABC transporters are often involved in removing insecticidal compounds from cells, our data suggest either an alternative role for these proteins in the mechanism of action of abamectin or highlight an indirect association between their expression and abamectin toxicity.

Development of an Online Genome Sequence Comparison Resource for Bacillus cereus sensu lato Strains Using the Efficient Composition Vector Method.

An automated method was developed for differentiating closely related B. cereus sensu lato (s.l.) species, especially biopesticide Bacillus thuringiensis, from other human pathogens, B. anthracis and B. cereus sensu stricto (s.s.). In the current research, four typing methods were initially compared, including multi-locus sequence typing (MLST), single-copy core genes phylogenetic analysis (SCCGPA), dispensable genes content pattern analysis (DGCPA) and composition vector tree (CVTree), to analyze the genomic variability of 23 B. thuringiensis strains from aizawai, kurstaki, israelensis, thuringiensis and morrisoni serovars. The CVTree method was the best option to be used for typing B. thuringiensis strains since it proved to be the fastest method, whilst giving high-resolution data about the strains. In addition, CVTree agrees well with ANI-based method, revealing the relationship between B. thuringiensis and other B. cereus s.l. species. Based on these data, an online genome sequence comparison resource was built for Bacillus strains called the Bacillus Typing Bioinformatics Database to facilitate strain identification and characterization.

Group Selection as a Basis for Screening Mutagenized Libraries of Public Goods (Bacillus thuringiensis Cry Toxins).

The pesticidal toxins of Bacillus thuringiensis (Bt) supply the active proteins for genetically modified insect-resistant crops. There is therefore keen interest in finding new toxins, or improving known toxins, in order to increase the mortality of various targets. The production and screening of large libraries of mutagenized toxins are among the means of identifying improved toxins. Since Cry toxins are public goods, and do not confer advantages to producers in competition, conventional directed evolution approaches cannot be used here. Instead, thousands of individual mutants have to be sequenced and assayed individually, a costly and time-consuming process. In this study, we tested a group selection-based approach that could be used to screen an uncharacterized pool of Cry toxin mutants. This involved selecting for infectivity between subpopulations of Bt clones within metapopulations of infected insects in three rounds of passage. We also tested whether additional mutagenesis from exposure to ethyl methanesulfonate could increase infectivity or supply additional Cry toxin diversity during passage. Sequencing of pools of mutants at the end of selection showed that we could effectively screen out Cry toxin variants that had reduced toxicity with our group selection approach. The addition of extra mutagenesis during passage decreased the efficiency of selection for infectivity and did not produce any additional novel toxin diversity. Toxins with loss-of-function mutations tend to dominate mutagenized libraries, and so a process for screening out these mutants without time-consuming sequencing and characterization steps could be beneficial when applied to larger libraries. IMPORTANCE Insecticidal toxins from the bacterium Bacillus thuringiensis are widely exploited in genetically modified plants. This application creates a demand for novel insecticidal toxins that can be used to better manage resistant pests or control new or recalcitrant target species. An important means of producing novel toxins is via high-throughput mutagenesis and screening of existing toxins, a lengthy and resource-intensive process. This study describes the development and testing of an efficient means of screening a test library of mutagenized insecticidal toxins. Here, we showed that it is possible to screen out loss-of-function mutations with low infectivity within a pool without the need to characterize and sequence each mutant individually. This has the potential to improve the efficiency of processes used to identify novel proteins.

Selecting for infectivity across metapopulations can increase virulence in the social microbe Bacillus thuringiensis.

Passage experiments that sequentially infect hosts with parasites have long been used to manipulate virulence. However, for many invertebrate pathogens, passage has been applied naively without a full theoretical understanding of how best to select for increased virulence and this has led to very mixed results. Understanding the evolution of virulence is complex because selection on parasites occurs across multiple spatial scales with potentially different conflicts operating on parasites with different life histories. For example, in social microbes, strong selection on replication rate within hosts can lead to cheating and loss of virulence, because investment in public goods virulence reduces replication rate. In this study, we tested how varying mutation supply and selection for infectivity or pathogen yield (population size in hosts) affected the evolution of virulence against resistant hosts in the specialist insect pathogen Bacillus thuringiensis, aiming to optimize methods for strain improvement against a difficult to kill insect target. We show that selection for infectivity using competition between subpopulations in a metapopulation prevents social cheating, acts to retain key virulence plasmids, and facilitates increased virulence. Increased virulence was associated with reduced efficiency of sporulation, and possible loss of function in putative regulatory genes but not with altered expression of the primary virulence factors. Selection in a metapopulation provides a broadly applicable tool for improving the efficacy of biocontrol agents. Moreover, a structured host population can facilitate artificial selection on infectivity, while selection on life-history traits such as faster replication or larger population sizes can reduce virulence in social microbes.

Retrotransposon-mediated evolutionary rewiring of a pathogen response orchestrates a resistance phenotype in an insect host.

Ongoing host-pathogen interactions can trigger a coevolutionary arms race, while genetic diversity within the host can facilitate its adaptation to pathogens. Here, we used the diamondback moth (Plutella xylostella) and its pathogen Bacillus thuringiensis (Bt) as a model for exploring an adaptive evolutionary mechanism. We found that insect host adaptation to the primary Bt virulence factors was tightly associated with a short interspersed nuclear element (SINE - named SE2) insertion into the promoter of the transcriptionally activated MAP4K4 gene. This retrotransposon insertion coopts and potentiates the effect of the transcription factor forkhead box O (FOXO) in inducing a hormone-modulated Mitogen-activated protein kinase (MAPK) signaling cascade, leading to an enhancement of a host defense mechanism against the pathogen. This work demonstrates that reconstructing a cis-trans interaction can escalate a host response mechanism into a more stringent resistance phenotype to resist pathogen infection, providing a new insight into the coevolutionary mechanism of host organisms and their microbial pathogens.

Cyclosporin A acts as a novel insecticide against Cry1Ac-susceptible and -resistant Helicoverpa armigera.

Cotton bollworm (Helicoverpa armigera) is an economically important pest, which is difficult to manage due to its biological and ecological traits, and resistance to most insecticides. Alternative compounds for the sustainable management of H. armigera are needed. As a fungal metabolite, Cyclosporin A (CsA) has not been applied in agriculture pests. Here, CsA was evaluated as a propective insecticide for H. armigera. The results showed that CsA displayed high insecticidal activity against both Cry1Ac-susceptible and -resistant populations of H. armigera. Moreover, lower concentrations of CsA had clear effects, including significantly reduced pupal weight, pupation rate, emergence rate, ovary size, female fecundity and egg hatchability. Further study confirmed that CsA suppressed calcineurin activity and the subsequent expression of endogenous antimicrobial peptide genes (APMs), leading to impaired immunity, ultimately resulting in delayed development and increased mortality. Thus, CsA treatment could control the cotton bollworm population and even showed efficacy against those with Bt resistance. In addition, the morphological changes observed in insects fed CsA with lower concentrations provide insight into insect immunity, regulation of growth and development, regulation of body color, ovary development and sexual selection under external pressure. Overall, our study provides information on biological control potential of Cry1Ac-susceptible and -resistant populations of H. armigera to develop novel bioinsecticides.

A single transcription factor facilitates an insect host combating Bacillus thuringiensis infection while maintaining fitness.

Maintaining fitness during pathogen infection is vital for host survival as an excessive response can be as detrimental as the infection itself. Fitness costs are frequently associated with insect hosts countering the toxic effect of the entomopathogenic bacterium Bacillus thuringiensis (Bt), which delay the evolution of resistance to this pathogen. The insect pest Plutella xylostella has evolved a mechanism to resist Bt toxins without incurring significant fitness costs. Here, we reveal that non-phosphorylated and phosphorylated forms of a MAPK-modulated transcription factor fushi tarazu factor 1 (FTZ-F1) can respectively orchestrate down-regulation of Bt Cry1Ac toxin receptors and up-regulation of non-receptor paralogs via two distinct binding sites, thereby presenting Bt toxin resistance without growth penalty. Our findings reveal how host organisms can co-opt a master molecular switch to overcome pathogen invasion with low cost, and contribute to understanding the underlying mechanism of growth-defense tradeoffs during host-pathogen interactions in P. xylostella.

Critical Analysis of Multi-Omic Data from a Strain of Plutella xylostella Resistant to Bacillus thuringiensis Cry1Ac Toxin.

Rapid evolution of resistance in crop pests to Bacillus thuringiensis (Bt) products threatens their widespread use, especially as pests appear to develop resistance through a range of different physiological adaptations. With such a diverse range of mechanisms reported, researchers have resorted to multi-omic approaches to understand the molecular basis of resistance. Such approaches generate a lot of data making it difficult to establish where causal links between physiological changes and resistance exist. In this study, a combination of RNA-Seq and iTRAQ was used with a strain of diamondback moth, Plutella xylostella (L.), whose resistance mechanism is well understood. While some of the causal molecular changes in the resistant strain were detected, other previously verified changes were not detected. We suggest that while multi-omic studies have use in validating a proposed resistance mechanism, they are of limited value in identifying such a mechanism in the first place.

Probing the Mechanism of Action of Cry41Aa on HepG2 through the Establishment of a Resistant Subline.

Cry41Aa, also called parasporin-3, belongs to a group of toxins from the entomopathogenic bacterium Bacillus thuringiensis that show activity against human cancer cells. Cry41Aa exhibits preferential cytocidal activity towards HL-60 (human promyelocytic leukaemia cells) and HepG2 (human liver cancer cells) cell lines after being proteolytically activated. To better understand the mechanism of action of Cry41Aa, we evolved resistance in HepG2 cells through repeated exposure to increasing doses of the toxin. Concentrations of Cry41Aa that killed over 50% of the parental HepG2 cells had no significant effect on the viability of the resistant cells and did not induce either pore formation or p38 phosphorylation (both characteristic features of pore-forming toxins). Preliminary RNA sequencing data identified AQP9 as a potential mediator of resistance, but extensive investigations failed to show a causal link and did not support an enhanced cell repair process as the resistance mechanism.

BPPRC database: a web-based tool to access and analyse bacterial pesticidal proteins.

Pesticidal proteins derived from the bacterium Bacillus thuringiensis, have provided the bases for a diverse array of pest management tools ranging from natural products used in organic agriculture, to modern biotechnological approaches. With advances in genome sequencing technologies and protein structure determination, an increasing number of pesticidal proteins from myriad bacterial species have been identified. The Bacterial Pesticidal Protein Resource Center (BPPRC) has been established to provide informational and analytical resources on the wide range of pesticidal proteins derived from bacteria that have potential utility for arthropod management. In association with a revised nomenclature for these proteins, BPPRC contains a database that allows users to browse and download sequences. Users can search the database for the best matches to sequences of interest and can incorporate their own sequences into basic informatic analyses. These analyses include the ability to draw and export guide trees from either whole protein sequences or, in the case of the three-domain Cry proteins, from individual domains. The associated website also provides a portal for users to submit protein sequences for naming. The BPPRC provides a single authoritative source of information to which all stakeholders can be referred including academics, government regulatory bodies and research and development personnel in the industrial sector. The database provides information on more than 1060 pesticidal proteins derived from 13 species of bacteria, including insecticidal activities for a subset of these proteins. Database URL: www.bpprc.org and www.bpprc-db.org/.