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Understanding the molecular mechanisms underlying insect resistance to Bacillus thuringiensis (Bt) pesticidal proteins is crucial for sustainable pest management. Here, we found that downregulation of the Plutella xylostella ecdysone oxidase gene ( PxEO ) in the normal feeding stages contributes to increased 20-hydroxyecdysone (20E) titer and mediates resistance to the Bt Cry1Ac toxin. The PxEO gene was cloned and its expression was significantly downregulated in the midgut of Bt-resistant and Cry1Ac-selected P. xylostella . Silencing of the PxEO gene significantly reduced Cry1Ac susceptibility, and downregulation of the PxEO gene is closely linked to Cry1Ac resistance in P. xylostella . The PxEO protein metabolized ecdysone (E) and 20E in vitro , and its reduction elevated 20E titers and activated the MAPK-mediated trans -regulatory mechanism known to directly cause the resistance phenotype. Together with our recently reported 20E-degrading glucose dehydrogenase, this finding highlights a robust, multipronged, approach developed by this insect in its 20E-mediated defense against harmful agents.

Sepsis-induced acute lung injury represents a significant threat to human health and is frequently associated with pulmonary thrombosis due to dysregulation of the coagulofibrinolytic system. Plasmin, the major protease that degrades fibrin aggregates, is activated predominantly by tissue-plasminogen activator (tPA), whereas tPA is negatively regulated by plasminogen activator inhibitor (PAI-1). Under septic conditions, the imbalance between coagulation and fibrinolysis results in excessive microthrombosis. Pulmonary capillary endothelial cells serve as a primary source of tPA and PAI-1. The molecular pathways regulating their expression levels depend on the differential activity of transcription factors. In this study, we elucidated the role of the zinc-finger transcription factor GATA3 in response to sepsis-induced pulmonary embolism. Endothelial cell-specific GATA3-deficient mice (G3-ECKO) presented increased susceptibility to bacterial endotoxin-induced pulmonary embolism, which was associated with increased PAI-1 expression levels and decreased tPA expression levels in the lungs. Septic lung extracts from G3-ECKO mice consistently presented decreased plasmin activity, which likely underlies the increased coagulation. These results demonstrate that GATA3 plays a protective role against bacterial endotoxin-induced pulmonary vascular embolism. Our findings will contribute to understanding the molecular mechanisms involving GATA3 in preventing pulmonary embolism.

Farmers in North America face significant pressure from insects in their maize fields, particularly from corn rootworm ( Diabrotica spp.). Research into proteins capable of insecticidal activity has found several produced by ferns. One protein, IPD079Ea, was derived from Ophioglossum pendulum and has shown activity against corn rootworm. An environmental risk assessment was conducted for maize event DP-915635-4, which provides control of corn rootworms via expression of the IPD079Ea protein. This assessment focused on IPD079Ea and characterized potential exposure and hazard to non-target organisms (NTOs). For exposure, estimated environmental concentrations (EECs) were calculated. For hazard, laboratory dietary toxicity studies were conducted with IPD079Ea and surrogate non-target organisms. Environmental risk was characterized by comparing hazard and exposure to calculate the margin of exposure (MOE). Based on the MOE values for DP-915635-4 maize, the IPD079Ea protein is not expected to result in unreasonable adverse effects on beneficial NTO populations at environmentally relevant concentrations.

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, has evolved resistance to nearly every management tactic utilized in the field. This study investigated the resistance mechanisms in a WCR strain resistant to the Bacillus thuringiensis (Bt) protein eCry3.1Ab using dsRNA to knockdown WCR midgut genes previously documented to be associated with the resistance. ATP-binding cassette transporter (ABCC4), aminopeptidase-N, cadherin, and cathepsin-B were previously found to be differentially expressed in eCry3.1Ab-resistant WCR larvae when compared to susceptible larvae after feeding on maize expressing eCry3.1Ab and its near-isoline. Here we compared the susceptibility of resistant and susceptible WCR larvae to eCry3.1Ab protein in presence or absence of dsRNA targeting the above genes using 10-day diet overlay toxicity assays. Combining ABCC4 dsRNA with eCry3.1Ab protein increased susceptibility to Bt protein in WCR-resistant larvae, but the other three genes had no such effect. Among 65 ABC transport genes identified, several were expressed differently in resistant or susceptible WCR larvae, fed on eCry3.1Ab-expressing maize versus its isoline, that may be involved in Bt resistance. Our findings provide strong evidence that ABCC4 is indirectly involved in WCR resistance to eCry3.1Ab protein by enhancing the effects of Bt-induced toxicity., Competing Interests: Competing interests: Adriano Pereira is an employee of RNAiSSANCE AG, St. Louis, MO, USA. All other authors declare no competing interests. Ethics approval and consent to participate: The experimental research and collection of plant and insect materials of this study comply with the relevant institutional, national, and international guidelines and legislation., (© 2024. The Author(s).)

Bacillus thuringiensis (Bt) bacteria produce different pore forming toxins with insecticidal activity, including Cry and Vip3 proteins. While both Cry and Vip3 cause insect death by forming pores in susceptible lepidopteran larval midgut cells, their mechanisms of action differ. The Vip3Aa protoxin adopts a tetramer-structure, where each monomer has five distinct domains. Upon proteolytic activation, the Vip3 tetramer undergoes a large conformational change forming a syringe like structure that is ready for membrane insertion and pore formation. Here we show that Vip3Aa protoxin had low binding to Spodoptera frugiperda brush border membrane vesicles (BBMV) unlike the activated toxin that bound specifically in a concentration dependent way, suggesting that a structural change upon Vip3Aa proteolytic activation is required for efficient receptor binding. Consistently, the Vip3Aa protoxin showed no toxicity to Sf9 cells compared to the activated toxin. In contrast, Cry1Fa protoxin and its activated toxin, were both highly toxic to Sf9 cells. To identify the region of Vip3 involved in binding to BBMV proteins, different overlapping peptides from Vip3Aa covering domains III, IV and V were expressed, and binding analysis were performed against BBMV, showing that domain III is the primary binding domain. Additionally, domains III, IV and V amino acid residues that become exposed upon activation of Vip3Aa were identified. Mutagenesis of these exposed residues revealed three amino acids (K385, K526 and V529) located in two structural adjacent loops, domain III loop β5-β6 and loop α11-β16 that connects domains III and IV, that are crucial for binding to the midguts of S. frugiperda larvae and for toxicity. Our results demonstrate that proteolytic activation of Vip3Aa exposes a receptor binding region essential for its toxicity., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Infante et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The global imperative to enhance crop protection while preserving the environment has increased interest in the application of biological pesticides. Bacillus thuringiensis (Bt) is a Gram-positive bacterium that can produce nematicidal proteins and accumulate them in parasporal crystals. Root-knot nematodes are obligate root plant parasitic which are distributed worldwide, causing severe damages to the infested plants and, consequently, large yield reductions. In this work, we have evaluated the toxicity of the Bt crystal proteins Cry5, Cry21, App6, and Xpp55 against two root-knot nematodes belonging to the Meloidogyne genus (M. incognita and M. javanica). The results show that all four proteins, when solubilized, were highly toxic for both nematode species. To check the potential of using Bt strains producing nematicidal crystal proteins as biopesticides to control root-knot nematodes in the field, in planta assays were conducted, using two wild Bt strains which produced Cry5 or a combination of App6 and Cry5 proteins. The tests were carried out with cucumber or with tomato plants infested with M. javanica J2, irrigated with spore + cristal mixtures of the respective strains. The results showed that the effectiveness of the nematicidal activity was plant-dependent, as Bt was able to reduce emerged J2 in tomato plants but not in cucumber plants. In addition, the toxicity observed in the in planta assays was much lower than expected, highlighting the difficulty of the proteins supplied as crystals to exert their toxicity. This emphasizes the delivery of the Bt proteins as crucial for its use to control root-knot nematodes. KEY POINTS: • Solubilized Cry5, Cry21, App6 and Xpp55 Bt proteins are toxic to M. javanica. • Cry21 toxicity to M. incognita is similar to that of Cry5, App6, and Xpp55 proteins. • The Cry5 and App6 toxicities on M. javanica after Bt irrigation is crop dependent., Competing Interests: Declarations. Ethics approval: This work does not contain any studies with human participants or animals performed by any of the authors. Consent to participate: All authors consented to the publication of this work. Conflict of interests: The authors declare no competing interests., (© 2024. The Author(s).)

Galleria mellonella, the greater wax moth has always been an important pest against honeybees and has remained a nightmare for beekeeping farmers. Management of G. mellonella in live honeybee colonies is very difficult because most current management practices can destroy whole honeybee colonies. In the present study, experiments were conducted to isolate and characterize Bacillus thuringiensis from infected greater wax moth cadavers and to evaluate their biocontrol ability against G. mellonella. The bioefficacy of these isolates has been evaluated against greater wax moth along with the standard strain HD-1. Among all the strains tested, NBAIR BtGa demonstrated higher efficacy compared to other strains, with an LC 50 value of 125.17 µg/ml, whereas HD-1 exhibited a significantly higher LC 50 value of 946.61 µg/ml. Considering the economic importance of NBAIR BtGa we performed whole genome sequencing of this strain resulting in the identification of a genome size of 5.96 Mb consisting of 6888 protein-coding genes. Gene ontology analysis categorized these genes into three groups based on their roles, i.e., biological functions (2169 genes), cellular components (1900 genes), and molecular functions (2774 genes). Through insecticidal toxicity-related genes (ITRG) profiling of our strain across the genome by Bt toxin scanner and cry processor resulted in the identification of several Cry proteins namely Cry1Ab11, Cry1Ia44, Cry1Aa2, Cry2Af1, Cry1Da2, Cry1Eb1, Cry1Ab5, Cry1Cb2, Cry1Ac2. Besides Cry proteins, other ITRG genes, viz. Vip3Bb2, Zwittermicin A resistance proteins, Chitinase C, Mpp46Ab1, immune inhibitor A, Bmp1, Vpb4Ca1, and Spp1Aa1 were also reported, which show toxicity against lepidopteran pests. The studies were also conducted to test the biosafety of Bt toxins against honeybee larvae and adults, which showed strain NBAIR BtGa was more than 99% safer for honeybee larvae as well as adults. Thus, the data generated ascertains its effectiveness as a biocontrol agent and it can be used further for the development of bio formulation for the management of G. mellonella in honeybee colonies., Competing Interests: Declarations. Ethical approval: This article does not contain any studies conducted on human participants or animals. The authors consent to the publication of the manuscript in its current form. Consent to participate: Not applicable. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Uridine diphosphate-glycosyltransferases (UGTs) catalyze sugar conjugation of endogenous and exogenous molecules in insects. In this study, 45 putative UGT genes in 11 families were identified from the genome of S. litura. Exposure to Bt toxins in 5th-instar larvae of the WT strain led to a significant upregulation of midgut UGT40 expression, particularly of SlUGT40D20, SlUGT40D22, and SlUGT40F25. This upregulation was not observed following exposure to chemical pesticides. Knockout of the UGT genes SlUGT40D20 and SlUGT40D22 in S. litura (mutant strains SlUGT40D20-KO and SlUGT40D22-KO) via CRISPR/Cas9-mediated mutagenesis increased susceptibility of S. litura to Bacillus thuringiensis (Bt) insecticidal proteins. However, in comparison with the wild-type (WT) strain, the mutants did not change susceptibility to chemical pesticides. Observations of 5th-instar larval midgut by electron microscopy revealed severe damage to the midgut epithelium caused by Cry1Ac toxin at 10 μg/g in the SlUGT40D20-KO strain compared to the WT. SDS-PAGE and LC MS/MS analyses identified a specific protein band corresponding to putative proteoglycans in the peritrophic matrix of the WT strain, which was absent in the SlUGT40D20-KO strain. Our study suggests an inverse correlation between expression of some UGTs and the susceptibility of S. litura larvae to some Bt toxins., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Transgenic crops producing crystalline (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) have been used extensively to control some major crop pests. However, many populations of the noctuid moth Helicoverpa zea , one of the most important crop pests in the United States, have evolved practical resistance to several Cry proteins including Cry1Ac. Although mutations in single genes that confer resistance to Cry proteins have been identified in lab-selected and gene-edited strains of H. zea and other lepidopteran pests, the genetic basis of field-evolved resistance to Cry proteins in H. zea has remained elusive. We used a genomic approach to analyze the genetic basis of field-evolved resistance to Cry1Ac in 937 H. zea derived from 17 sites in seven states of the southern United States. We found evidence for extensive gene flow among all populations studied. Field-evolved resistance was not associated with mutations in 20 single candidate genes previously implicated in resistance or susceptibility to Cry proteins in H. zea or other lepidopterans. Instead, resistance in field samples was associated with increased copy number of a cluster of nine trypsin genes. However, trypsin gene amplification occurred in a susceptible sample and not in all resistant samples, implying that this amplification does not always confer resistance and mutations in other genes also contribute to field-evolved resistance to Cry1Ac in H. zea . The mismatch between lab-generated and field-evolved resistance in H. zea is unlike other cases of Bt resistance and reflects challenges for managing this pest., Competing Interests: Competing interests statement:J.A.F. and B.E.T. are coauthors of patents on engineering Bacillus thuringiensis (Bt) toxins to counter resistance (US10704059) and potentiating Bt toxins (US20090175974A1), respectively. Badische Anilin und Soda Fabrik (BASF), Corteva Agriscience, Cotton Incorporated, Syngenta, and the Agricultural Biotechnology Stewardship Technical Committee (representing a consortium of agricultural biotechnology companies) did not provide funding to support this work but have funded other work by some of the authors.

The resistance to the insecticidal protein Cry1Ac from the bacterium Bacillus thuringiensis (Bt) in the cabbage looper, Trichoplusia ni , has previously been identified to be associated with a frameshift mutation in the ABC transporter ABCC2 gene and with altered expression of the aminopeptidase N (APN) genes APN1 and APN6 , shown as missing of the 110-kDa APN1 (phenotype APN1¯) in larval midgut brush border membrane vesicles (BBMV). In this study, genetic linkage analysis identified that the APN1¯ phenotype and the ABCC2 mutation in Cry1Ac-resistant T. ni segregated independently, although they were always associated under Cry1Ac selection. The ABCC2 mutation and APN1¯ phenotype were separated into two T. ni strains respectively. Bioassays of the T. ni strains with Cry1Ac determined that the T. ni with the APN1¯ phenotype showed a low level resistance to Cry1Ac (3.5-fold), and the associated resistance is incompletely dominant in the background of the ABCC2 mutation. Whereas the ABCC2 mutation-associated resistance to Cry1Ac is at a moderate level, and the resistance is incompletely recessive in the genetic background of downregulated APN1. Analysis of Cry1Ac binding to larval midgut BBMV indicated that the midgut in larvae with the APN1¯ phenotype had reduced binding affinity for Cry1Ac, but the number of binding sites remained unchanged, and the midgut in larvae with the ABCC2 mutation had both reduced binding affinity and reduced number of binding sites for Cry1Ac. The reduced Cry1Ac binding to BBMV from larvae with the ABCC2 mutation or APN1¯ phenotype correlated with the lower levels of resistance.IMPORTANCEThe soil bacterium Bacillus thuringiensis (Bt) is an important insect pathogen used as a bioinsecticide for pest control. Bt genes coding for insecticidal proteins are the primary transgenes engineered into transgenic crops (Bt crops) to confer insect resistance. However, the evolution of resistance to Bt proteins in insect populations in response to exposure to Bt threatens the sustainable application of Bt biotechnology. Cry1Ac is a major insecticidal toxin utilized for insect control. Genetic mechanisms of insect resistance to Cry1Ac are complex and require to be better understood. The resistance to Cry1Ac in Trichoplusia ni is associated with a mutation in the ABCC2 gene and also associated with the APN expression phenotype APN1¯. This study identified the genetic independence of the APN1¯ phenotype from the ABCC2 mutation and isolated and analyzed the ABCC2 mutation-associated and APN1¯ phenotype-associated resistance traits in T. ni to provide new insights into the genetic mechanisms of Cry1Ac resistance in insects., Competing Interests: The authors declare no conflict of interest.

Bacillus thuringiensis (Bt) has been successfully used commercially for more than 60 years for biocontrol of insect pests. Since 1996, transgenic plants expressing Bt crystal (Cry) proteins have been used commercially to provide protection against insects that predate on corn and cotton. More recently, Bt Cry proteins that target nematodes have been discovered. One of these, Cry14Ab, has been expressed in transgenic soybean plants and found to provide significant protection against the soybean cyst nematode, Heterodera glycines. However, to date there has been no description of high-level resistance to any Cry14A family protein in nematodes. Here, we describe forward genetic screens to identify such mutants using the nematode Caenorhabditis elegans. Although non-conditional screens failed to identify highly resistant C. elegans, a conditional (temperature-sensitive) genetic screen identified one mutant, bre-6(ye123) (for Bt protein resistant), highly resistant to both Cry14Aa and Cry14Ab. The mutant comes at a high fitness cost, showing significant delays in growth and development and reduced fecundity. bre-6(ye123) hermaphrodites are only weakly resistant to copper intoxication, indicating that the mutant is not highly resistant to all insults. Backcrossing-whole genome sequencing was used to identify the gene mutated in ye123 as the nuclear hormone receptor nhr-31. RNAi, DNA rescue, and CRISPR analyses confirm that resistance to Cry14Aa intoxication in bre-6(ye123) is due to mutation of nhr-31 and was renamed nhr-31(ye123). As predicted for a mutation in this gene, nhr-31(ye123) animals showed significantly reduced expression of most of the subunits of the C. elegans vacuolar ATPase (vATPase). Mutants in the vATPase subunits unc-32 and vha-7 also show resistance to Cry14Aa and/or Cry14Ab. These data demonstrate that nhr-31 and the vATPase play a significant role in the intoxication of C. elegans by Cry14A family proteins, that reduction in vATPase levels result in high resistance to Cry14A family proteins, and that such resistance comes at a high fitness cost. Based on the relative difficulty of finding resistant mutants and the fitness cost associated with the vATPase pathway, our data suggest that transgenic Cry14Ab plants may hold up well to resistance by nematode parasites., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Root-knot nematodes (RKNs) are plant pests that infect the roots of host plants. Bacillus thuringiensis (Bt) nematicidal proteins exhibited toxicity to nematodes. However, the application of nematicidal proteins for plant protection is hampered by the lack of effective delivery systems in transgenic plants. In this study, we discovered the accumulation of leucoplasts (root plastids) in galls and RKN-induced giant cells. RKN infection causes the degradation of leucoplasts into small vesicle-like structures, which are responsible for delivering proteins to RKNs, as observed through confocal microscopy and immunoelectron microscopy. We showed that different-sized proteins from leucoplasts could be taken up by Meloidogyne incognita female. To further explore the potential applications of leucoplasts, we introduced the Bt crystal protein Cry5Ba2 into tobacco and tomato leucoplasts by fusing it with a transit peptide. The transgenic plants showed significant resistance to RKNs. Intriguingly, RKN females preferentially took up Cry5Ba2 protein when delivered through plastids rather than the cytosol. The decrease in progeny was positively correlated with the delivery efficiency of the nematicidal protein. In conclusion, this study offers new insights into the feeding behavior of RKNs and their ability to ingest leucoplast proteins, and demonstrates that root leucoplasts can be used for delivering nematicidal proteins, thereby offering a promising approach for nematode control., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

Great interest exists in developing a transgenic trait that controls the economically important soybean ( Glycine max ) pest, soybean cyst nematode (SCN, Heterodera glycines ), due to its adaptation to native resistance. Soybean plants expressing the Bacillus thuringiensis delta-endotoxin, Cry14Ab, were recently demonstrated to control SCN in both growth chamber and field testing. In that communication, ingestion of the Cry14Ab toxin by SCN second stage juveniles (J2s) was demonstrated using fluorescently labeled Cry14Ab in an in vitro assay. Here, we show that consistent with expectations for a Cry toxin, Cry14Ab has a mode of action unique from the native resistance sources Peking and PI 88788. Further, we demonstrate in planta the ingestion and localization of the Cry14Ab toxin in the midgut of nematodes feeding on roots expressing Cry14Ab using immunogold labeling and transmission electron microscopy. We observed immunolocalization of the toxin and resulting intestinal damage primarily in the microvillus-like structure (MvL)-containing region of the midgut intestine but not in nematodes feeding on roots lacking toxin. This demonstrated that Cry14Ab was taken up by the J2 SCN, presumably through the feeding tube within the plant root cell that serves as its feeding site. This suggests that relatively large proteins can be taken up through the feeding tube. Electron microscopy showed that Cry14Ab caused lysis of the midgut MvL membrane and eventual degradation of the MvL and the lysate, forming particulate aggregates. The accumulated electron-dense aggregate in the posterior midgut intestine was not observed in SCN in nonCry14Ab-expressing plants. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: R.H.B. worked on this project as a paid consultant for the BASF Corporation (BASF). M.T.M., J.W., and J.D. are employed by BASF, whose products are evaluated in this study. The remaining authors declare no conflict of interest.

Tobacco Etch virus (TEV) protease is one of the most common tools for removing fusion tags, but no study has shown that TEV can be expressed at high levels in the GRAS host strain Bacillus subtilis and purified for further application. In this study, the fusion protein BsLysSN-TEV C/S-His-TEV consisting of a fusion tag, N-terminal domain of a lysyl-tRNA synthetase (BsLysSN) coded by B. subtilis lysS gene, placed at the N-terminus followed by an endoprotease TEV cleavage site and then the expression of this fusion protein in the cytoplasm of B. subtilis was investigated. The SDS-PAGE and Western-blot analysis demonstrated that His-TEV was overexpressed under the induction of IPTG. This result infers that His-TEV protease showed promising activity in the B. subtilis cytoplasm by the cleavage of the fusion protein. These cleavage products could be purified using the Ni-NTA column, which effectively cleaved the purified recombinant protein substrate, which can be applied in the protein purification process to remove the fusion tag. Significantly, since both His-TEV protease and the fusion recombinant protein substrate are expressed in the endotoxin-free host strain, the tag removal and purified product should be theoretically endotoxin-free, which could be a promising approach for producing therapeutic proteins and also for other relevant biomedical applications., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Synergistic factors can enhance the toxicity of Bt toxins and delay the development of Bt resistance. Previous research has demonstrated that a Helicoverpa armigera cadherin fragment (HaCad-TBR) increased the toxicity of Cry1Ac in Plutella xylostella larvae but did not have a synergistic effect on Cry1B, Cry1C, and Cry1F toxins. In this study, a fusion protein (HaCad-TBR-2D3 V L ) derived from HaCad-TBR and a Bt Cry1-specific antibody peptide was expressed in Escherichia coli . The HaCad-TBR-2D3 V L enhanced Cry1Ac toxicity more efficiently in insects and Sf9 cells than HaCad-TBR and also significantly increased the toxicity of Cry1B, Cry1C, and Cry1F toxins in insects. Further investigation indicated that the improved stability in insect midguts and higher binding capacity with Bt toxins contributed to the enhanced synergism of HaCad-TBR-2D3 V L over HaCad-TBR. This study suggested that Bt antibody fragments can potentially broaden the synergistic range of Bt receptor fragments, providing a theoretical foundation for developing broad-spectrum synergists for other biopesticides.

The extensive use of Bacillus thuringiensis (Bt) in pest management has driven the evolution of pest resistance to Bt toxins, particularly Cry1Ac. Effective management of Bt resistance necessitates a good understanding of which pest proteins interact with Bt toxins. In this study, we screened a Helicoverpa armigera larval midgut cDNA library and captured 208 potential Cry1Ac-interacting proteins. Among these, we further examined the interaction between Cry1Ac and a previously unknown Cry1Ac-interacting protein, HaDALP ( H. armigera death-associated LIM-only protein), as well as its role in toxicology. The results revealed that HaDALP specifically binds to both the Cry1Ac protoxin and activated toxin, significantly enhancing cell and larval tolerance to Cry1Ac. Additionally, HaDALP was overexpressed in a Cry1Ac-resistant H. armigera strain. These findings reveal a greater number of Cry1Ac-interacting proteins than previously known and demonstrate, for the first time, that HaDALP reduces Cry1Ac toxicity by sequestering both the protoxin and activated toxin.

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.

The entomopathogenic fungus Beauveria bassiana provides an eco-friendly substitute to chemical insecticides for mosquito control. Nevertheless, its widespread application has been hindered by its comparatively slow efficacy in eliminating mosquitoes. To augment the potency of B. bassiana against Aedes mosquitoes, a novel recombinant strain, Bb -Cyt1Aa, was developed by incorporating the Bacillus thuringiensis toxin gene Cyt1Aa into B. bassiana . The virulence of Bb -Cyt1Aa was evaluated against Aedes aegypti and Aedes albopictus using insect bioassays. Compared to the wild-type (WT) strain, the median lethal time (LT 50 ) for A. aegypti larvae infected with Bb -Cyt1Aa decreased by 33.3% at a concentration of 1 × 10 8 conidia/mL and by 22.2% at 1 × 10 7 conidia/mL. The LT 50 for A. aegypti adults infected with Bb -Cyt1Aa through conidia ingestion was reduced by 37.5% at 1 × 10 8 conidia/mL and by 33.3% at 1 × 10 7 conidia/mL. Likewise, the LT 50 for A. aegypti adults infected with Bb -Cyt1Aa through cuticle contact decreased by 33.3% and 30.8% at the same concentrations, respectively. Furthermore, the Bb -Cyt1Aa strain also demonstrated increased toxicity against both larval and adult A. albopictus , when compared to the WT strain. In conclusion, our study demonstrated that the expression of B. thuringiensis toxin Cyt1Aa in B. bassiana enhanced its virulence against Aedes mosquitoes. This suggests that B. bassiana expressing Cyt1Aa has potential value for use in mosquito control., Importance: Beauveria bassiana is a naturally occurring fungus that can be utilized as a bioinsecticide against mosquitoes. Cyt1Aa is a delta-endotoxin protein produced by Bacillus thuringiensis that exhibits specific and potent insecticidal activity against mosquitoes. In our study, the expression of this toxin Cyt1Aa in B. bassiana enhances the virulence of B. bassiana against Aedes aegypti and Aedes albopictus , thereby increasing their effectiveness in killing mosquitoes. This novel strain can be used alongside chemical insecticides to reduce dependence on harmful chemicals, thereby minimizing negative impacts on the environment and human health. Additionally, the potential resistance of B. bassiana against mosquitoes in the future could be overcome by acquiring novel combinations of exogenous toxin genes. The presence of B. bassiana that expresses Cyt1Aa is of significant importance in mosquito control as it enhances genetic diversity, creates novel virulent strains, and contributes to the development of safer and more sustainable methods of mosquito control., Competing Interests: The authors declare no conflict of interest.

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a leading cause of end-stage liver disease associated with increased mortality and cardiovascular disease. Obesity and diabetes are the most important risk factors of MASLD. It is well-established that obesity-associated insulin resistance leads to a situation of tissue lipotoxicity characterized by an accumulation of excess fat in non-fat tissues such as the liver, promoting the development of MASLD, and its progression into metabolic dysfunction-associated steatohepatitis., Methods: Here, we aimed to review the impact of disrupted intestinal permeability, antimicrobial proteins and bacterial endotoxin in the development and progression of MASLD., Results and Conclusion: Recent studies demonstrated that obesity- and obesogenic diets-associated alterations of intestinal microbiota along with the disruption of intestinal barrier integrity, the alteration in antimicrobial proteins and, in consequence, an enhanced translocation of bacterial endotoxin into bloodstream might contribute to this pathological process through to impacting liver metabolism and inflammation., (© 2024 The Authors. European Journal of Clinical Investigation published by John Wiley & Sons Ltd on behalf of Stichting European Society for Clinical Investigation Journal Foundation.)

Background: Bacillus thuringiensis (Bt) and its crystal toxin or δ-endotoxins (Cry) offer great potential for the efficient control of crop pests. A vast number of pests can potentially infect the same host plant, either simultaneously or sequentially. However, no effective Bt-Cry protein has been reported to control both aphids and plant parasitic nematodes due to its highly specific activity., Results: Our study indicated that the Cry5Ba2 protein was toxic to the green peach aphid Myzus persicae, which had a median lethal concentration (LC 50 ) of 9.7 ng μL -1 and fiducial limits of 3.1-34.6 ng μL -1 . Immunohistochemical localization of Cry5Ba2 revealed that it could bind to the apical tip of microvilli in midgut regions. Moreover, transgenic tobacco plants expressing Cry5Ba2 exhibited significant resistance to Myzus persicae, as evidenced by reduced insect survival and impaired fecundity, and also intoxicated the Meloidogyne incognita as indicated by a decrease in galls and progeny reproduction., Conclusion: In sum, we identified a new aphicidal Bt toxin resource that could simultaneously control both aboveground and belowground pests, thus extending the application range of Bt-based strategy for crop protection. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

The incidence of perturbed gastrointestinal integrity, as well as resulting systemic immune responses and gastrointestinal symptoms, otherwise known as exercised-induced gastrointestinal syndrome (EIGS), is common among individuals who partake in prolonged exercise. EIGS may cause the translocation of pathogenic material, including whole bacteria and bacterial endotoxins, from the lumen into circulation, which may progress into clinical consequences such as sepsis, and potentially subsequent fatality. However, further investigation is warranted to assess the possibility of food allergen and/or digestive enzyme luminal to circulatory translocation in response to exercise, and the clinical consequences. Findings from this narrative literature review demonstrate evidence that whole bacteria and bacterial endotoxins translocation from the gastrointestinal lumen to systemic circulation occurs in response to exercise stress, with a greater propensity of translocation occurring with accompanying heat exposure. It has also been demonstrated that food allergens can translocate from the lumen to systemic circulation in response to exercise stress and initiate anaphylaxis. To date, no research investigating the effect of exercise on the translocation of digestive enzymes from the lumen into systemic circulation exists. It is evident that EIGS and consequential pathogenic translocation presents life-threatening clinical implications, warranting the development and implementation of effective management strategies in at-risk populations., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

The dynamic interplay between intramammary IgG, formation of antigen-IgG complexes and effector immune cell function is essential for immune homeostasis within the bovine mammary gland. We explore how changes in the recognition and binding of anti-LPS IgG to the glycolipid "functional" core in milk from healthy or clinically diagnosed Escherichia coli (E. coli) mastitis cows' controls endotoxin function. In colostrum, we found a varied anti-LPS IgG repertoire and novel soluble LPS/IgG complexes with direct IgG binding to the LPS glycolipid core. These soluble complexes, absent in milk from healthy lactating cows, were evident in cows diagnosed with E. coli mastitis and correlated with endotoxin-driven inflammation. E. coli mastitis milk displayed a proportional reduction in anti-LPS glycolipid core IgG compared to colostrum. Milk IgG extracts showed that only colostrum IgG attenuated LPS induced endotoxin activity. Furthermore, LPS-stimulated reactive oxygen species (ROS) in milk granulocytes was only suppressed by colostrum IgG, while IgG extracts of neither colostrum nor E. coli mastitis milk influenced N-formylmethionine-leucyl-phenylalanine (fMLP)-stimulated ROS in LPS primed granulocytes. Our findings support bovine intramammary IgG diversity in health and in response to E. coli infection generate milk anti-LPS IgG repertoires that coordinate appropriate LPS innate-adaptive immune responses essential for animal health., Competing Interests: Declaration of conflicting interestsThe authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: The authors, SH, DF and RK are or were (DF) employees of Koru Diagnostics Ltd, confirm there is no individual personal financial relationship and declare that research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest. AP is a shareholder of Koru Diagnostics Ltd and as CSO was involved in conceptualisation, supervision, investigation and writing of the original draft. No payment or services from a third party was received for any aspect of the submitted work. In addition, selective concepts and data described in this manuscript are part of a patent submission by Koru Diagnostics Ltd, entitled “Method for Detecting Microorganisms and Uses Thereof” in November 2022. Patent Ref: PCT/NC2022/050136, #KDL1007PC. The raw data supporting the conclusions of this study are not readily available as they are proprietary and part of a pending patent submission. However, requests to access specific data should be directed to Koru Diagnostics Ltd info@korudiagnostics.com.

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 (LC 50 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 T 0 and T 1 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 LC 50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T 0 and T 1 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., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Metabolic endotoxemia is a severe health problem for residents in developed countries who follow a Western diet, disrupting intestinal microbiota and the whole organism's homeostasis. Although the effect of endotoxin on the human immune system is well known, its long-term impact on the human body, lasting many months or even years, is unknown. This is due to the difficulty of conducting in vitro and in vivo studies on the prolonged effect of endotoxin on the central nervous system. In this article, based on the available literature, we traced the path of endotoxin from the intestines to the blood through the intestinal epithelium and factors promoting the development of metabolic endotoxemia. The presence of endotoxin in the bloodstream and the inflammation it induces may contribute to lowering the blood-brain barrier, potentially allowing its penetration into the central nervous system; although, the theory is still controversial. Microglia, guarding the central nervous system, are the first line of defense and respond to endotoxin with activation, which may contribute to the development of neurodegenerative diseases. We traced the pro-inflammatory role of endotoxin in neurodegenerative diseases and its impact on the epigenetic regulation of microglial phenotypes.

The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Pacheco et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Pesticide resistance in arthropods threatens agricultural productivity and the control of vector-borne diseases. The ATP-binding cassette (ABC) transporters have emerged as important factors in the toxicity of synthetic pesticides, as well as for Bacillus thuringiensis insecticidal Cry protein binding. Depending on the localization of expression, both higher and lower expression of ABCs have been linked with pesticide resistance. The recent development of genetic-based approaches such as RNAi and CRISPR/Cas9 gene editing in nonmodel species, has greatly contributed to unveil their functional importance in pesticide toxicity and resistance. Using these tools, we are now poised to further unravel the molecular genetic mechanisms of gene regulation uncovering more elusive regulatory resistance genes., Competing Interests: Declaration of Competing Interest Nothing declared., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Genetically engineered (GE) cotton event MON 88702, producing Mpp51Aa2 (previously mCry51Aa2) from Bacillus thuringiensis (Bt), controls sucking pests, such as Lygus spp. (Hemiptera: Miridae) and thrips (Thysanoptera). Ingesting high doses of the insecticidal protein resulted in adverse effects on life table parameters of beneficial, predatory Orius spp. (Hemiptera: Anthocoridae). This triggered laboratory studies with more realistic food treatments, including different combinations of prey types with and without Bt protein to further characterize risks to this important group of non-target organisms. In this work, exclusive feeding of frozen spider mites (Tetranychus urticae, Acari: Tetranychidae) from Bt cotton confirmed adverse effects on longevity and fecundity of O. majusculus adults. Alternate feeding of Bt protein-containing spider mites and Bt-free Ephestia kuehniella (Lepidoptera: Pyralidae) eggs mitigated effects on longevity, but not on fecundity. When living larvae of Spodoptera littoralis (Lepidoptera: Noctuidae) from Bt cotton were fed to the predators, however, no effects on longevity and reproduction of female O. majusculus were observed, despite the fact that Bt protein concentrations in larvae were almost as high as concentrations in spider mites. When a diverse mix of prey species with various Bt protein concentrations is consumed in the field, it is unlikely that exposure of Orius spp. to Mpp51Aa2 is high enough to exert adverse effects on predator populations. MON 88702 cotton may thus be a valuable tool for integrated management of sucking pests., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Competing Interests: Declaration of competing interest No financial support was provided for the preparation of this article.

A novel group of biocidal compounds are the Crystal 3D (Cry) and Cytolytic (Cyt) proteins produced by Bacillus thuringiensis (Bt). Some Bt Cry proteins have a selective nematocidal activity, with Cry5B being the most studied. Cry5B kills nematode parasites by binding selectively to membrane glycosphingolipids, then forming pores in the cell membranes of the intestine leading to damage. Cry5B selectively targets multiple species of nematodes from different clades and has no effect against mammalian hosts. Levamisole is a cholinergic anthelmintic that acts by selectively opening L-subtype nicotinic acetylcholine receptor ion-channels (L-AChRs) that have been found on muscles of nematodes. A synergistic nematocidal interaction between levamisole and Cry5B at the whole-worm level has been described previously, but the location, mechanism and time-course of this synergism is not known. In this study we follow the timeline of the effects of levamisole and Cry5B on the Ca2+ levels in enterocyte cells in the intestine of Ascaris suum using fluorescence imaging. The peak Ca2+ responses to levamisole were observed after approximately 10 minutes while the peak responses to activated Cry5B were observed after approximately 80 minutes. When levamisole and Cry5B were applied simultaneously, we observed that the responses to Cry5B were bigger and occurred sooner than when it was applied by itself. It is proposed that the synergism is due to the cytoplasmic Ca2+ overload that is induced by the combination of levamisole opening Ca2+ permeable L-subtype nAChRs and the Ca2+ permeable Cry5B toxin pores produced in the enterocyte plasma membranes. The effect of levamisole potentiates and speeds the actions of Cry5B that gives rise to bigger Ca2+ overloads that accelerates cell-death of the enterocytes., Competing Interests: The authors declare no competing interests., (Copyright: © 2024 Williams et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Insect pests cause immense agronomic losses worldwide. One of the most destructive of major crops is the Fall Armyworm (Spodoptera frugiperda, FAW). The ability to migrate long distances, a prodigious appetite, and a demonstrated ability to develop resistance to insecticides, make it a difficult target to control. Insecticidal proteins, for example those produced by the bacterium Bacillus thuringiensis, are among the safest and most effective insect control agents. Genetically modified (GM) crops expressing such proteins are a key part of a successful integrated pest management (IPM) program for FAW. However, due to the development of populations resistant to commercialized GM products, new GM traits are desperately needed. Herein, we describe a further characterization of the newly engineered trait protein eCry1Gb.1Ig. Similar to other well characterized Cry proteins, eCry1Gb.1Ig is shown to bind FAW midgut cells and induce cell-death. Binding competition assays using trait proteins from other FAW-active events show a lack of competition when binding FAW brush border membrane vesicles (BBMVs) and when utilizing non-pore-forming versions as competitors in in vivo bioassays. Similarly, insect cell lines expressing SfABCC2 and SfABCC3 (well characterized receptors of existing commercial Cry proteins) are insensitive to eCry1Gb.1Ig. These findings are consistent with results from our previous work showing that eCry1Gb.1Ig is effective in controlling insects with resistance to existing traits. This underscores the value of eCry1Gb.1Ig as a new GM trait protein with a unique site-of-action and its potential positive impact to global food production., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Mg(OH) 2 was used as the nanocarrier of the Bacillus thuringiensis (Bt) Cry1Ac protein, and the synthesized Cry1Ac-Mg(OH) 2 composites were regular and uniform nanosheets. Nano-Mg(OH) 2 could effectively improve the insecticidal effect of the Cry1Ac protein toward Ectropis obliqua . It could enhance the damage degree of the Cry1Ac protein to intestinal epithelial cells and microvilli, induce and enrich the production of reactive oxygen species (ROS) in the midgut, and enhance the degradation of the Cry1Ac protein into active fragments. Furthermore, an anti-rinsing assay showed that the Cry1Ac-Mg(OH) 2 composites were bound to the notch structure of the tea leaf surface. The retention of the Cry1Ac protein increased by 11.45%, and sprayed nano-Mg(OH) 2 was rapidly absorbed by different tissues of tea plants. Moreover, nano-Mg(OH) 2 and composites did not significantly affect non-target organisms. These results show that nano-Mg(OH) 2 can serve as a safe and effective biopesticide carrier, which provides a new approach for stable and efficient Bt preparation.

Juvenile hormone binding protein (JHBP) is a key regulator of JH signaling, and crosstalk between JH and 20-hydroxyecdysone (20E) can activate and fine-tune the mitogen-activated protein kinase cascade, leading to resistance to insecticidal proteins from Bacillis thuringiensis (Bt). However, the involvement of JHBP in the Bt Cry1Ac resistance of Plutella xylostella remains unclear. Here, we cloned a full-length cDNA encoding JHBP, and quantitative real-time PCR (qPCR) analysis showed that the expression of the PxJHBP gene in the midgut of the Cry1Ac-susceptible strain was significantly higher than that of the Cry1Ac-resistant strain. Furthermore, CRISPR/Cas9-mediated knockout of the PxJHBP gene significantly increased Cry1Ac susceptibility, resulting in a significantly shorter lifespan and reduced fertility. These results demonstrate that PxJHBP plays a critical role in the resistance to Cry1Ac protoxin and in the regulation of physiological metabolic processes associated with reproduction in adult females, providing valuable insights to improve management strategies of P. xylostella .

Gut microbiota dysbiosis is a prominent determinant that significantly contributes to the disruption of lipid metabolism. Consequently, it is essential to the occurrence and development of non-alcoholic fatty liver disease (NAFLD). Nevertheless, the connection between diet and symbiotic gut microbiota in the progression of NAFLD remains uncertain. The purpose of this study was to explore the role of supplementing commensal Bacteroides fragilis ( B. fragilis ) on lipid metabolism, gut microbiota, and metabolites in high-fat diet (HFD)-fed mice, elucidating the impact of gut microbiota and metabolites on the development of NAFLD. Our study revealed that supplementation with B. fragilis exacerbated both weight gain and obesity in mice. B. fragilis exacerbated blood glucose levels and liver dysfunction in mice. Furthermore, an increase in liver lipid accumulation and the upregulation of genes correlated with lipid metabolism were observed in mice. Under an HFD, supplementation of commensal B. fragilis resulted in alterations in the gut microbiota, notably a significant increase in Desulfovibrionaceae , which led to elevated endotoxin levels and thereby influenced the progression of NAFLD. It was interesting that the simultaneous examination of gut microbiota metabolites revealed a more pronounced impact of diet on short-chain fatty acids. This study represented the pioneering investigation into the impact of B. fragilis on NAFLD. Our findings demonstrated that B. fragilis induced dysregulation in the intestinal microbiota, leading to elevated levels of lipopolysaccharide and dysfunction in glucose and lipid metabolism, thereby exacerbating NAFLD.IMPORTANCESome intestinal symbiotic microbes are involved in the occurrence of the metabolic disorders. Our study investigated the impact of supplementing commensal Bacteroides fragilis on host metabolism in high-fat diet-fed mice. Research results indicated that adding a specific bacterial strain to the complex intestinal microecology can worsen metabolic conditions. This effect mainly affects the structural diversity of intestinal microorganisms, the increase in harmful bacteria in the gut, and the elevation of endotoxin levels, blood glucose, and lipid metabolism, thereby impacting the progression of non-alcoholic fatty liver disease (NAFLD). Understanding the principles that govern the establishment of microbial communities comprising multiple species is crucial for preventing or repairing dysfunctions in these communities, thereby enhancing host health and facilitating disease treatment. This study demonstrated that gut microbiota dysbiosis could contribute to metabolic dysfunction and provides new insights into how to promote gut microbiota in the prevention and therapy of NAFLD., Competing Interests: The authors declare no conflict of interest.

By 2013, it had been shown that the genes cadherin-like receptor (Cad) and ATP-binding cassette transporter subfamily C2 (ABCC2) were responsible for insect resistance to several Cry1A toxins, acting as susceptibility-determining receptors, and many review articles have been published. Therefore, this review focuses on information about receptors and receptor-binding sites that have been revealed since 2014. Since 2014, studies have revealed that the receptors involved in determining susceptibility vary depending on the Cry toxin subfamily, and that binding affinity between Cry toxins and receptors plays a crucial role. Consequently, models have demonstrated that ABCC2, ABCC3, and Cad interact with Cry1Aa; ABCC2 and Cad with Cry1Ab and Cry1Ac; ABCC2 and ABCC3 with Cry1Fa; ABCB1 with Cry1Ba, Cry1Ia, Cry9Da, and Cry3Aa; and ABCA2 with Cry2Aa and Cry2Ba, primarily in the silkworm, Bombyx mori . Furthermore, since 2017, it has been suggested that the binding sites of BmCad and BmABCC2 on Cry1Aa toxin overlap in the loop region of domain II, indicating that Cry toxins use various molecules as receptors due to their ability to bind promiscuously in this region. Additionally, since 2017, several ABC transporters have been identified as low-efficiency receptors that poorly induce cell swelling in heterologously expressing cultured cells. In 2024, research suggested that multiple molecules from the ABC transporter subfamily, including ABCC1, ABCC2, ABCC3, ABCC4, ABCC10, and ABCC11, act as low-efficiency receptors for a single Cry toxin in the midgut of silkworm larvae. This observation led to the hypothesis that the presence of such low-efficiency receptors contributes to the evolution of Cry toxins towards the generation of highly functional receptors that determine the susceptibility of individual insects. Moreover, this evolutionary process is considered to offer valuable insights for the engineering of Cry toxins to overcome resistance and develop countermeasures against resistance.

Research Question: Can the addition of progesterone and neurotensin, molecular agents found in the female reproductive tract, after sperm washing increase the fertilization potential of human spermatozoa?, Design: (i) Cohort study of 24 men. Spermatozoa selected by swim-up were incubated in either progesterone or neurotensin (0.1-100 µM) for 1-4 h, and hyperactive motility and binding to hyaluronan (0.1-100 µM) were assessed. The effect of progesterone 10 µM on sperm function was assessed in a blinded manner, including: hyperactive motility, binding to hyaluronan, tyrosine phosphorylation, acrosome reaction and oxidative DNA damage. (i) Embryo safety testing [one-cell mouse embryo assay (MEA), endotoxin and sterility counts (n = 3)] in preclinical embryo models of IVF (murine and porcine, n = 7 each model) and a small preliminary human study (n = 4) of couples undergoing standard IVF with oocytes inseminated with spermatozoa ± 10 µM progesterone., Results: Progesterone 10 µM increased sperm binding to hyaluronan, hyperactive motility and tyrosine phosphorylation (all P < 0.05). Neurotensin had no effect (P > 0.05). Progesterone 10 µM in human embryo culture media passed embryo safety testing (MEA, endotoxin concentration and sterility plate count). In preclinical models of IVF, the exposure of spermatozoa to progesterone 10 µM and oocytes to progesterone 1 µM was not detrimental, and increased the fertilization rate in mice and the blastocyst cell number in mice and pigs (all P ≤ 0.03). In humans, every transferred blastocyst that had been produced from spermatozoa exposed to progesterone resulted in a live birth., Conclusion: The addition of progesterone to sperm preparation media shows promise as an adjunct to current methods for increasing fertilization potential. Randomized controlled trials are required to determine the clinical utility of progesterone for improving IVF outcomes., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Overuse of insecticides has accelerated the evolution of insecticide resistance and created serious environmental concerns worldwide, thus incentivizing development of alternative methods. Bacillus thuringiensis (Bt) is an insecticidal bacterium that has been developed as a biopesticide to successfully control multiple species of pests. It operates by secreting several insect toxins such as Cry1Ac. However, metabolic resistance based on ATP-binding cassette (ABC) transporters may play a crucial role in the development of metabolic resistance to Bt. Here, we characterized an ABCG gene from the agricultural pest Plutella xylostella (PxABCG3) and found that it was highly expressed in a Cry1Ac-resistant strain, up-regulated after Cry1Ac protoxin treatment. Binding miR-8510a-3p to the coding sequence (CDS) of PxABCG3 was then confirmed by a luciferase reporter assay and RNA immunoprecipitation. miR-8510a-3p agomir delivery markedly reduced PxABCG3 expression in vivo and consequently decreased the tolerance of P. xylostella to Cry1Ac, while reduction of miR-8510a-3p significantly increased PxABCG3 expression, accompanied by an increased tolerance to Cry1Ac. Our results suggest that miR-8510a-3p could potentially be used as a novel molecular target against P. xylostella or other lepidopterans, providing novel insights into developing effective and environmentally friendly pesticides., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024. Published by Elsevier B.V.)

Lipopolysaccharide (LPS) constitutes much of the surface of Gram-negative bacteria, and if LPS enters the human body or brain can induce inflammation and act as an endotoxin. We outline the hypothesis here that LPS may contribute to the pathophysiology of Alzheimer's disease (AD) via peripheral infections or gut dysfunction elevating LPS levels in blood and brain, which promotes: amyloid pathology, tau pathology and microglial activation, contributing to the neurodegeneration of AD. The evidence supporting this hypothesis includes: i) blood and brain levels of LPS are elevated in AD patients, ii) AD risk factors increase LPS levels or response, iii) LPS induces Aβ expression, aggregation, inflammation and neurotoxicity, iv) LPS induces TAU phosphorylation, aggregation and spreading, v) LPS induces microglial priming, activation and neurotoxicity, and vi) blood LPS induces loss of synapses, neurons and memory in AD mouse models, and cognitive dysfunction in humans. However, to test the hypothesis, it is necessary to test whether reducing blood LPS reduces AD risk or progression. If the LPS endotoxin hypothesis is correct, then treatments might include: reducing infections, changing gut microbiome, reducing leaky gut, decreasing blood LPS, or blocking LPS response., (© 2024. The Author(s).)

The inflammasome components NLRP3 and ASC are cytosolic proteins, which upon sensing endotoxins or danger cues, form multimeric complexes to process interleukin (IL)-1β for secretion. Here we found that antigen (Ag)-triggered degranulation of IgE-sensitized mast cells (MCs) was mediated by NLRP3 and ASC. IgE-Ag stimulated NEK7 and Pyk2 kinases in MCs to induce the deposition of NLRP3 and ASC on granules and form a distinct protein complex (granulosome) that chaperoned the granules to the cell surface. MCs deficient in NLRP3 or ASC did not form granulosomes, degranulated poorly in vitro and did not evoke systemic anaphylaxis in mice. IgE-Ag-triggered anaphylaxis was prevented by an NLRP3 inhibitor. In endotoxin-primed MCs, pro-IL-1β was rapidly packaged into granules after IgE-Ag stimulation and processed within granule remnants by proteases after degranulation, causing lethal anaphylaxis in mice. During IgE-Ag-mediated degranulation of endotoxin-primed MCs, granulosomes promoted degranulation, combined with exteriorization and processing of IL-1β, resulting in severe inflammation., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Septic lung injury is characterized by uncontrollable inflammatory infiltrations and acute onset bilateral hypoxemia. Evidence has emerged of the beneficial effect of hydrogen in acute lung injury (ALI), but the underlying mechanism is unclear. In this research, the recovery action of hydrogen on lipopolysaccharide (LPS)-induced ALI in mice and A549 cells was investigated. The 7-day survival rate and body weight of mice were measured after intraperitoneal injection of LPS. Lung function was determined by a whole body plethysmography (WBP) system using the indicators respiratory rate and enhanced pause. Hematoxylin and eosin (HE) staining confirmed the signs of pulmonary edema and inflammatory ooze. Reverse transcription-polymerase chain reaction (RT-PCR) quantification was used to detect the expression of inflammatory factors. Western blotting analysis evaluated the expression levels of involved proteins in the AMP-activated protein kinase (AMPK) pathway. The experimental results confirmed that hydrogen provided an essential solution to the dissipative effects of LPS on survival rate, weight loss and lung function. The LPS-stimulated inflammatory factors, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were also suppressed by hydrogen in A549 cells. Western blot analysis showed that hydrogen significantly upregulated the levels of phosphorylated AMPK (p-AMPK) and lowered the LPS-induced increased expression of dynamin-related protein 1 (Drp1) and Caspase3. These findings prove that hydrogen attenuated LPS-treated ALI by activating the AMPK pathway, supporting the feasibility of hydrogen treatment for sepsis., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins. These active transporters are involved in the export of different substances such as xenobiotics. ABC transporters from subfamily C (ABCC) have also been described as functional receptors for different insecticidal proteins from Bacillus thuringiensis (Bt) in several lepidopteran species. Numerous studies have characterized the relationship between the ABCC2 transporter and Bt Cry1 proteins. Although other ABCC transporters sharing structural and functional similarities have been described, little is known of their role in the mode of action of Bt proteins. For Heliothis virescens , only the ABCC2 transporter and its interaction with Cry1A proteins have been studied to date. Here, we have searched for paralogs to the ABCC2 gene in H. virescens , and identified two new ABC transporter genes: HvABCC3 and HvABCC4 . Furthermore, we have characterized their gene expression in the midgut and their protein topology, and compared them with that of ABCC2. Finally, we discuss their possible interaction with Bt proteins by performing protein docking analysis.

The evolution of pest resistance to management tools reduces productivity and results in economic losses in agricultural systems. To slow its emergence and spread, monitoring and prevention practices are implemented in resistance management programs. Recent work suggests that genomic approaches can identify signs of emerging resistance to aid in resistance management. Here, we empirically examined the sensitivity of genomic monitoring for resistance management in transgenic Bt crops, a globally important agricultural innovation. Whole genome resequencing of wild North American Helicoverpa zea collected from non-expressing refuge and plants expressing Cry1Ab confirmed that resistance-associated signatures of selection were detectable after a single generation of exposure. Upon demonstrating its sensitivity, we applied genomic monitoring to wild H. zea that survived Vip3A exposure resulting from cross-pollination of refuge plants in seed-blended plots. Refuge seed interplanted with transgenic seed exposed H. zea to sublethal doses of Vip3A protein in corn ears and was associated with allele frequency divergence across the genome. Some of the greatest allele frequency divergence occurred in genomic regions adjacent to a previously described candidate gene for Vip3A resistance. Our work highlights the power of genomic monitoring to sensitively detect heritable changes associated with field exposure to Bt toxins and suggests that seed-blended refuge will likely hasten the evolution of resistance to Vip3A in lepidopteran pests., Competing Interests: Competing interests statement:This study was conducted while D.P. was a graduate student at NCSU and no funding from any company was used to perform this study. D.P. is currently employed by Bayer CropScience LP, Chesterfield, MO 63198. All other authors declare no competing interests.

In Nebraska USA, many populations of western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, now exhibit some level of resistance to all corn rootworm-active Bacillus thuringiensis Berliner (Bt) proteins expressed in commercial hybrids. Therefore, a study was conducted in northeast Nebraska from 2020-2022 to reevaluate current corn rootworm management options in continuous maize (consecutive planting for ≥2 years). Results from on-farm experiments to evaluate a standard soil-applied insecticide (Aztec® 4.67G) in combination with non-rootworm Bt or rootworm-active Bt pyramided maize (Cry3Bb1 + Gpp34Ab1/Tpp35Ab1) are reported within the context of WCR Bt resistance levels present. Corrected survival from Bt pyramid single-plant bioassays (<0.3, 0.3-0.49, >0.5) was used to place populations into 3 resistance categories. Variables evaluated included root injury, adult emergence, proportion lodged maize, and grain yield. Key results: A composite analysis of all populations across resistance levels indicated that addition of soil insecticide to Bt pyramid significantly reduced adult emergence and lodging but did not significantly increase root protection or yield. Within and among resistance category analyses of root injury revealed that the Bt pyramid remained highly efficacious at any non-rootworm Bt root injury level when resistance was absent or low. When corrected survival was >0.3, mean Bt pyramid root injury tracked more closely in a positive linear fashion with mean non-rootworm Bt root injury (rootworm density x level of resistance interaction). Similar trends were obtained for adult emergence but not yield. Mean Bt pyramid root injury rating was <0.75 in most populations with Bt resistance, which contributed to no significant yield differences among categories. Results are discussed within the context of IPM:IRM tradeoffs and the need to reduce WCR densities in this system to decrease the impact of the density x resistance interaction to bridge use of current pyramids with new technologies introduced over the next decade., Competing Interests: The authors have declared that no competing interests exist. LJM developed the research concept/study design to improve western corn rootworm IRM and IPM when Bt pyramids are deployed in a corn rootworm resistant landscape. LJM wrote/submitted proposals to Bayer CropScience and Pioneer Hi-Bred International (Corteva). Industry authors provided materials, information integral to the project, and review of initial manuscript draft., (Copyright: © 2024 Meinke et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Our study highlighted the immune changes by pro-inflammatory biomarkers in the gut-liver-axis-linked ROS-cell death mechanisms in chronic and acute inflammations when gut cells are exposed to endotoxins in patients with hepatic cirrhosis or steatosis. In duodenal tissue samples, gut immune barrier dysfunction was analyzed by pro-inflammatory biomarker expressions, oxidative stress, and cell death by flow cytometry methods. A significant innate and adaptative immune system reaction was observed as result of persistent endotoxin action in gut cells in chronic inflammation tissue samples recovered from hepatic cirrhosis with the A-B child stage. Instead, in patients with C child stage of HC, the endotoxin tolerance was installed in cells, characterized by T lymphocyte silent activation and increased Th1 cytokines expression. Interesting mechanisms of ROS-cell death were observed in chronic and acute inflammation samples when gut cells were exposed to endotoxins and immune changes in the gut-liver axis. Late apoptosis represents the chronic response to injury induction by the gut immune barrier dysfunction, oxidative stress, and liver-dysregulated barrier. Meanwhile, necrosis represents an acute and severe reply to endotoxin action on gut cells when the immune system reacts to pro-inflammatory Th1 and Th2 cytokines releasing, offering protection against PAMPs/DAMPs by monocytes and T lymphocyte activation. Flow cytometric analysis of pro-inflammatory biomarkers linked to oxidative stress-cell death mechanisms shown in our study recommends laboratory techniques in diagnostic fields.

Paralipsa gularis (Zeller) is a storage pest; however, in recent years it has evolved into a considerable maize pest during the late growth stage in the border region between China and other Southeast Asian countries. Bt transgenic insect-resistant maize is an effective measure in controlling a wide range of lepidopteran pests, but there is a lack of research on the toxic effects of storage pests. We tested the toxicity of Bt-Cry1Ab, Vip3Aa, and their complex proteins against P. gularis via bioassay and investigated the efficiency of Bt-(Cry1Ab+Vip3Aa) maize in controlling P. gularis during the late growth stage of maize in the period 2022-2023. The bioassay results show that the susceptibilities of P. gularis to the two Bt proteins and their complex proteins were significantly different. The LC 50 values of DBNCry1Ab ("DBN9936" event), DBNVip3Aa ("DBN9501" event), DBN Cry1Ab+Vip3Aa ("DBN3601T" event), and Syngenta Cry1Ab+Vip3Aa ("Bt11" event × "MIR162" event) were 0.038 μg/g, 0.114 μg/g, 0.110 μg/g, and 0.147 μg/g, and the GIC 50 values were 0.014 μg/g, 0.073 μg/g, 0.027 μg/g, and 0.026 μg/g, respectively. Determination of the expression content of the insecticidal protein in different tissues of Bt-(Cry1Ab+Vip3Aa) maize shows that the total Bt protein content in different tissues was in the following order: stalk > bract > cob > kernel. However, the bioassay results show that the mortalities of P. gularis feeding on Bt-(Cry1Ab+Vip3Aa) maize in different tissues at different growth stages were all above 93.00%. The field trial indicates that the occurrence density of larvae and plant damage rate for conventional maize were 422.10 individuals/100 plants and 94.40%, respectively, whereas no larvae were found on Bt-(Cry1Ab+Vip3Aa) maize. In summary, this study implies that Bt-(Cry1Ab+Vip3Aa) maize has a high potential for control of P. gularis , providing a new technical measure for the management of the pest.

Bacillus thuringiensis Vip3 toxins form a tetrameric structure crucial for their insecticidal activity. Each Vip3Aa monomer comprises five domains. Interaction of the first four α-helices in domain I with the target cellular membrane was proposed to be a key step before pore formation. In this study, four N-terminal α-helix-deleted truncations of Vip3Aa were produced and, it was found that they lost both liposome permeability and insecticidal activity against Spodoptera litura . To further probe the role of domain I in membrane permeation, the full-length domain I and the fragments of N-terminal α-helix-truncated domain I were fused to green fluorescent protein (GFP), respectively. Only the fusion carrying the full-length domain I exhibited permeability against artificial liposomes. In addition, seven Vip3Aa-Cry1Ac fusions were also constructed by combination of α-helices from Vip3Aa domains I and II with the domains II and III of Cry1Ac. Five of the seven combinations were determined to show membrane permeability in artificial liposomes. However, none of the Vip3Aa-Cry1Ac combinations exhibited insecticidal activity due to the significant reduction in proteolytic stability. These results indicated that the N-terminal helix α1 in the Vip3Aa domain I is essential for both insecticidal activity and liposome permeability and that domain I of Vip3Aa preserved a high liposome permeability independently from domains II-V.

Besides being recognized by membrane receptor TLR4, lipopolysaccharide (LPS) can also be internalized into the cytosol and activate Caspase-4/11 pyroptotic pathways to further amplify inflammation in sepsis. The objective of this study was to investigate whether Galectin-3 (Gal3) could promote the uptake of LPS by governing RAGE or administering endocytosis, consequently activating Caspase 4/11 and mediating pyroptosis in sepsis-associated acute kidney injury (SA-AKI). By pinpointing Gal3, LPS, and EEA1 (endosome-marker) or LAMP1 (lysosome-marker) respectively, immunofluorescence discovered that Gal3 and LPS were mainly aggregated in early endosomes initially and translocated into lysosomes afterwards. In cells and animal models, Gal3 and the Caspase-4/11 pathways were simultaneously activated, and the overexpression of Gal3 could exacerbate pyroptosis, whereas inhibition of Gal3 or the knockdown of its expression could ameliorate pyroptosis, reduce the pathological changes of SA-AKI and improve the survival of the animals with SA-AKI. Silencing RAGE reduced pyroptosis in primary tubular epithelial cells (PTCs) activated by Gal3 and LPS but not in cells activated by Gal3 and outer membrane vesicles (with LPS inside), whereas pyroptosis in both was reduced by blockade of Gal3, indicating Gal3 promoted pyroptosis through both RAGE-dependent and RAGE-independent pathways. Our investigation further revealed a positive correlation between serum Gal3 and pyroptotic biomarkers IL-1 beta and IL-18 in patients with sepsis, and that serum Gal3 was an independent risk factor for mortality. Through our collective exploration, we unraveled the significant role of Gal3 in the internalization of LPS and the provocation of more intense pyroptosis, thus making it a vital pathogenic factor in SA-AKI and a possible therapeutic target. Gal3 enabled the internalization of endotoxin into endosomes and lysosomes via both RAGE-dependent (A) and RAGE-independent (B) pathways, leading to pyroptosis. The suppression of Gal3 curbed Caspase4/11 noncanonical inflammasomes and diminished sepsis and SA-AKI., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The bacterium Bacillus thuringiensis (Bt) is the most economically successful biopesticide to date, and Bt insecticidal proteins are produced in transgenic crops for pest control. However, relevant details in the Bt-mediated killing process remain undefined. In our previous research, we observed reduced larval susceptibility to Bt Cry1Ca in Chilo suppressalis, a major rice pest in China, after gut microbiota elimination. Here, we tested the hypothesis that gut microbiota, particularly abundant Enterococcus spp., influences C. suppressalis susceptibility to Cry1Ca. We isolated and identified four Enterococcus spp. from C. suppressalis gut microbiota and evaluated their impact on Cry1Ca toxicity. Among the four Enterococcus spp. identified, three of them (E. casseliflavus, E. faecalis, and E. mundtii) dramatically increased larval mortality when introduced in axenic C. suppressalis challenged with Cry1Ca. Gut epithelial damage by Cry1Ca promoted the translocation of Enterococcus spp. from the gut lumen into the hemocoel, where they proliferated and induced larval melanization and hemocyte apoptosis. Our combined findings demonstrate that the presence of specific gut microbiota can greatly affect susceptibility to Cry1Ca through melanization and apoptosis of hemocytes. Better understanding of the Bt intoxication process guides the development of bio-enhancers for Bt-based microbial biopesticides and potential improvement of transgenic crops., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 N 6 -methyladenosine (m 6 A) RNA modification in modulating the availability of JH in this process is defined. Specifically, it is found that two m 6 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 m 6 A-mediated epigenetic regulator of insect hormones for maintaining fitness during pathogen defense and unveils an emerging Bt resistance-related m 6 A methylation atlas in insects, which further expands the functional landscape of m 6 A modification and showcases the pivotal role of epigenetic regulation in host-pathogen interactions., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)