The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of β-pore-forming toxins (β-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin\'s mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.

The comment titled \"Factors related to Bacillus thuringiensis and gut physiology\" disputes some of the inferences in the paper \"An Alkaline Foregut Protects Herbivores from Latex in Forage, but Increases Their Susceptibility to Bt Endotoxin\" published in this journal. The key points in the dissent are the following: 1. Bt is generally safe to non-target species. 2. Transgenic Bt crops provide additional ecological benefits due to reductions in conventional pesticide use. 3. Susceptibility to Bt does not indicate alkalinity, nor vice versa. My response is summarized as follows: 1. Bt can form non-specific pores at concentrations of 100 ng/mL in culture, and so is potentially unsafe for animals with gut environments in which Bt persists at or above this level. 2. Initial reductions in insecticide applications have not been sustained and are even increasing in areas planted with transgenic Bt cotton. 3. Acidic guts degrade Bt more efficiently, but I concede that gut alkalinity does not imply susceptibility to Bt due to many factors including resistance in target species, toxin heterogeneity and variable modes of action. However, the susceptibility of foregut-fermenting herbivores with alkaline guts to Bt intoxication cannot be invalidated without further study.

A recent article has proposed that alkaline guts may lead to a general susceptibility to the biological control agent Bacillus thuringiensis and the pesticidal proteins derived from it. An analysis of the literature presented here clarifies our knowledge on the activity and safety of these agents, indicating that alkaline guts are not determinant of sensitivity and that the generalized conclusions proposed in the previous article cannot be substantiated.

Entomopathogenic nematodes are used as biological control agents against a broad range of insect pests. We ascribed the pathogenicity of these organisms to the excretory/secretory products (ESP) released by the infective nematode. Our group characterized different virulence factors produced by Steinernema carpocapsae that underlie its success as an insect pathogen. A novel ShK-like peptide (ScK1) from this nematode that presents high sequence similarity with the ShK peptide from a sea anemone was successfully produced recombinantly in Escherichia coli. The secondary structure of ScK1 appeared redox-sensitive, exhibiting a far-UV circular dichroism spectrum consistent with an alpha-helical secondary structure. Thermal denaturation of the ScK1 allowed estimating the melting temperature to 59.2 ± 0.1 °C. The results from toxicity assays using Drosophila melanogaster as a model show that injection of this peptide can kill insects in a dose-dependent manner with an LD50 of 16.9 µM per adult within 24 h. Oral administration of the fusion protein significantly reduced the locomotor activity of insects after 48 h (p < 0.05, Tukey\'s test). These data show that this nematode expresses insecticidal peptides with potential as next-generation insecticides.

Drosophila nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that represent a target for insecticides. Peptide neurotoxins are known to block nAChRs by binding to their target subunits, however, a better understanding of this mechanism is needed for effective insecticide design. To facilitate the analysis of nAChRs we used a CRISPR/Cas9 strategy to generate null alleles for all ten nAChR subunit genes in a common genetic background. We studied interactions of nAChR subunits with peptide neurotoxins by larval injections and styrene maleic acid lipid particles (SMALPs) pull-down assays. For the null alleles, we determined the effects of α-Bungarotoxin (α-Btx) and ω-Hexatoxin-Hv1a (Hv1a) administration, identifying potential receptor subunits implicated in the binding of these toxins. We employed pull-down assays to confirm α-Btx interactions with the Drosophila α5 (Dα5), Dα6, Dα7 subunits. Finally, we report the localisation of fluorescent tagged endogenous Dα6 during Drosophila CNS development. Taken together, this study elucidates native Drosophila nAChR subunit interactions with insecticidal peptide toxins and provides a resource for the in vivo analysis of insect nAChRs.

Yersinia (Y.) enterocolitica, an etiological agent of yersiniosis, is a bacterium whose pathogenicity is determined, among other things, by its ability to produce toxins. The aim of this article was to present the most important toxins that are produced by biotype 1A strains of Y. enterocolitica, and to discuss their role in the pathogenesis of yersiniosis. Y. enterocolitica biotype 1A strains are able to synthesize variants of thermostable YST enterotoxin and play a key role in the pathogenesis of yersiniosis. Biotype 1A strains of Y. enterocolitica also produce Y. enterocolitica pore-forming toxins, YaxA and YaxB. These toxins form pores in the cell membrane of host target cells and cause osmotic lysis, which is of particular importance in systemic infections. Insecticidal toxin complex genes have been detected in some clinical biotype 1A strains of Y. enterocolitica. However, their role has not yet been fully elucidated. Strains belonging to biotype 1A have long been considered non-pathogenic. This view is beginning to change due to the emerging knowledge about the toxigenic potential of these bacteria and their ability to overcome the defense barriers of the host organism.

The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents.

BACKGROUND: The pine weevil (Hylobius abietis) is a major forest regeneration pest causing high levels of seedling mortality and economic losses. Current management relies on silviculture, stem coatings and insecticides. Here we evaluated for the first time the effects of Bacillus thuringiensis (Bt) strains on H. abietis adults: two producing the Coleoptera-targeted toxins Cry3Aa (Bt tenebrionis NB-176) and Cry8Da (Bt galleriae SDS-502), and one producing the Diptera-targeted Cry10A (Bt israelensis AM65-52). Choice and nonchoice assays using individual and mixtures of Bt formulations, containing these strains respectively, were conducted.
RESULTS: We found that Bt had toxic and lethal effects on H. abietis, but effects varied with strain and formulation concentration. The Diptera-targeted Bt israelensis had the most negative effects on weevil weight, feeding and mortality (70-82% feeding reduction, 65-82% greater mortality than control), whereas the effect was lower for the Coleoptera-specific Bt tenebrionis (38-42%; 37-42%) and Bt galleriae (11-30%; 15-32%). Reduced weevil feeding was observed after 3 days, and the highest mortality occurred 7-14 days following Bt exposure. However, we found no synergistic toxic effects, and no formulation combination was better than Bt israelensis alone at reducing consumption and survival. Also, pine weevils were not deterred by Bt, feeding equally on Bt-treated and non-Bt treated food.
CONCLUSIONS: There is potential to develop forest pest management measures against H. abietis that include Bt, but only the Diptera-targeted Bt israelensis would provide effective seedling protection. Its Diptera-specificity may need reconsideration, and evaluation of other Bt strains/toxins against H. abietis would be of interest. © 2020 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Photorhabdus luminescens is a gram-negative bacterium that symbiotically associates with insect-parasitic nematode, Heterorhabditis indica. Herein, we have characterized an insecticidal gene, Txp40 (1008 bp) from the indigenous isolates of P. luminescens, and tested its bioefficacy against Galleria mellonella via injectable and oral bioassay. The recombinant protein characterized from P. luminescens strain H3 exhibited comparatively greater insect toxicity than strain H1 in terms of LD50 and LT50 values. Txp40 holds great potential to replace Bt toxins in global agriculture.

Bacterial plasmids can vary from small selfish genetic elements to large autonomous replicons that constitute a significant proportion of total cellular DNA. By conferring novel function to the cell, plasmids may facilitate evolution but their mobility may be opposed by co-evolutionary relationships with chromosomes or encouraged via the infectious sharing of genes encoding public goods. Here, we explore these hypotheses through large-scale examination of the association between plasmids and chromosomal DNA in the phenotypically diverse Bacillus cereus group. This complex group is rich in plasmids, many of which encode essential virulence factors (Cry toxins) that are known public goods. We characterized population genomic structure, gene content and plasmid distribution to investigate the role of mobile elements in diversification. We analysed coding sequence within the core and accessory genome of 190 B. cereus group isolates, including 23 novel sequences and genes from 410 reference plasmid genomes. While cry genes were widely distributed, those with invertebrate toxicity were predominantly associated with one sequence cluster (clade 2) and phenotypically defined Bacillus thuringiensis. Cry toxin plasmids in clade 2 showed evidence of recent horizontal transfer and variable gene content, a pattern of plasmid segregation consistent with transfer during infectious cooperation. Nevertheless, comparison between clades suggests that co-evolutionary interactions may drive association between plasmids and chromosomes and limit wider transfer of key virulence traits. Proliferation of successful plasmid and chromosome combinations is a feature of specialized pathogens with characteristic niches (Bacillus anthracis, B. thuringiensis) and has occurred multiple times in the B. cereus group.