Characterization of the mode of action of eCry1Gb.1Ig, a fall armyworm (Spodoptera frugiperda) active protein, with a novel site of action.

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.

Safety attributes of Pseudomonas sp. P26, an environmental microorganism with potential application in contaminated environments.

Currently, the selection of non-pathogenic microorganisms that lack clinically relevant antimicrobial resistance is crucial to bioaugmentation strategies. Pseudomonas sp. P26 (P26) is an environmental bacterium of interest due to its ability to remove aromatic compounds from petroleum, but its safety characteristics are still unknown. The study aimed to: a) determine P26 sensitivity to antimicrobials, b) investigate the presence of quinolone and ß-lactam resistance genes, c) determine the presence of virulence factors, and d) evaluate the effect of P26 on the viability of Galleria mellonella (an invertebrate animal model). P26 antimicrobial sensitivity was determined in vitro using the Kirby-Bauer agar diffusion method and the VITEK 2 automated system (BioMerieux®). Polymerase Chain Reaction was employed for the investigation of genes associated with quinolone resistance, extended-spectrum ß-lactamases, and carbapenemases. Hemolysin and protease production was determined in human blood agar and skimmed-milk agar, respectively. In the in vivo assay, different doses of P26 were injected into Galleria mellonella larvae and their survival was monitored daily. Control larvae injected with Pseudomonas putida KT2440 (a strain considered as safe) and Pseudomonas aeruginosa PA14 (a pathogenic strain) were included. Pseudomonas sp. P26 was susceptible to most evaluated antimicrobials, except for trimethoprim-sulfamethoxazole. No epidemiologically relevant genes associated with quinolone and ß-lactam resistance were identified. Hemolysin and protease production was only evidenced in the virulent strain (PA14). Furthermore, the results obtained in the in vivo experiment demonstrated that inocula less than 108 CFU/mL of P26 and P. putida KT2440 did not significantly affect larval survival, whereas larvae injected with the lowest dose of the pathogenic strain P. aeruginosa PA14 experienced instant mortality. The results suggest that Pseudomonas sp. P26 is a safe strain for its application in environmental bioremediation processes. Additional studies will be conducted to ensure the safety of this bacterium against other organisms.

Involvement of miR-8510a-3p in response to Cry1Ac protoxin by regulating PxABCG3 in Plutella xylostella.

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.

Induction of human-fetal-membrane remodeling in-vitro by the alpha hemolysin of Escherichia coli.

INTRODUCTION: Almost 80% of urinary tract infections during pregnancy are caused by uropathogenic strains of Escherichia coli. Alpha-hemolysin, toxin secreted by them, has a fundamental role in this pathology development. Considering that urinary tract infections are related with premature rupture of fetal membranes, we proposed to evaluate the effects that alpha-hemolysin induces on human-fetal-membranes. METHODS: Thirteen fetal membranes obtained from elective cesarean sections (>37 weeks) were mounted in a transwell-device generating two independent chambers. To mimic an ascendant-urinary-tract infection, membranes were incubated with different concentrations of pure alpha-hemolysin from the choriodecidual side during 24h. Extensive histological analyses were performed and transepithelial electrical-resistance were determined. Cell viability, metalloproteinase activity and cyclooxygenase-2- gene expression was estimated by lactate-dehydrogenase-release assay, zymography and RT-qPCR, respectively. Finally, four fetal membranes were treated with hemolysin preincubated with polyclonal anti-hemolysin antibodies. Cell viability and metalloproteinase activity were monitored. RESULTS: After 24 h of treatment, fetal membranes evidenced a structural damage and a decrease in membrane resistance that progressed as the concentration of alpha hemolysin increased. While the amniotic-epithelial-layer remained practically unaffected, the chorion cells manifested an increase in vacuolization and necrosis. In addition, the extracellular matrix exhibited collagen-fiber disorganization, a marked decrease in fiber content, and became thicker in presence of the toxin. Cyclooxigenase-2 expression and metalloproteinase activity were also higher in the treated groups than in untreated ones. Finally, a preincubation of hemolysin with specific antibodies prevented the cytotoxicity on the chorion cells and the increase in metalloproteinase activity. DISCUSSION: Hemolysin induces structural and molecular changes associated with the remodeling of human-fetal-membranes in-vitro.

Peritrophins are involved in the defense against Bacillus thuringiensis and nucleopolyhedrovirus formulations in Spodoptera littoralis (Lepidoptera: Noctuidae).

The peritrophic matrix (or peritrophic membrane, PM) is present in most insects where it acts as a barrier to mechanical insults and pathogens, as well as a facilitator of digestive processes. The PM is formed by the binding of structural PM proteins, referred to as peritrophins, to chitin fibrils and spans the entire midgut in lepidopterans. To investigate the role of peritrophins in a highly polyphagous lepidopteran pest, namely the cotton leafworm (Spodoptera littoralis), we generated Insect Intestinal Mucin (IIM-) and non-mucin Peritrophin (PER-) mutant strains via CRISPR/Cas9 mutagenesis. Both strains exhibited deformed PMs and retarded developmental rates. Bioassays conducted with Bacillus thuringiensis (Bt) and nucleopolyhedrovirus (SpliNPV) formulations showed that both the IIM- and PER- mutant larvae were more susceptible to these bioinsecticides compared to the wild-type (WT) larvae with intact PM. Interestingly, the provision of chitin-binding agent Calcofluor (CF) in the diet lowered the toxicity of Bt formulations in both WT and IIM- larvae and the protective effect of CF was significantly lower in PER- larvae. This suggested that the interaction of CF with PER is responsible for Bt resistance mediated by CF. In contrast, the provision of CF caused increased susceptibility to SpliNPV in both mutants and WT larvae. The study showed the importance of peritrophins in the defense against pathogens in S. littoralis and revealed novel insights into CF-mediated resistance to Cry toxin.

JAK/STAT signaling regulated intestinal regeneration defends insect pests against pore-forming toxins produced by Bacillus thuringiensis.

A variety of coordinated host-cell responses are activated as defense mechanisms against pore-forming toxins (PFTs). Bacillus thuringiensis (Bt) is a worldwide used biopesticide whose efficacy and precise application methods limits its use to replace synthetic pesticides in agricultural settings. Here, we analyzed the intestinal defense mechanisms of two lepidopteran insect pests after intoxication with sublethal dose of Bt PFTs to find out potential functional genes. We show that larval intestinal epithelium was initially damaged by the PFTs and that larval survival was observed after intestinal epithelium regeneration. Further analyses showed that the intestinal regeneration caused by Cry9A protein is regulated through c-Jun NH (2) terminal kinase (JNK) and Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. JAK/STAT signaling regulates intestinal regeneration through proliferation and differentiation of intestinal stem cells to defend three different Bt proteins including Cry9A, Cry1F or Vip3A in both insect pests, Chilo suppressalis and Spodoptera frugiperda. Consequently, a nano-biopesticide was designed to improve pesticidal efficacy based on the combination of Stat double stranded RNA (dsRNA)-nanoparticles and Bt strain. This formulation controlled insect pests with better effect suggesting its potential use to reduce the use of synthetic pesticides in agricultural settings for pest control.

MiR-2b-3p Downregulated PxTrypsin-9 Expression in the Larval Midgut to Decrease Cry1Ac Susceptibility of the Diamondback Moth, Plutella xylostella (L.).

Crystal (Cry) toxins, produced by Bacillus thuringiensis, are widely used as effective biological pesticides in agricultural production. However, insects always quickly evolve adaptations against Cry toxins within a few generations. In this study, we focused on the Cry1Ac protoxin activated by protease. Our results identified PxTrypsin-9 as a trypsin gene that plays a key role in Cry1Ac virulence in Plutella xylostella larvae. In addition, P. xylostella miR-2b-3p, a member of the micoRNA-2 (miR-2) family, was significantly upregulated by Cry1Ac protoxin and targeted to PxTrypsin-9 downregulated its expression. The mRNA level of PxTrypsin-9, regulated by miR-2b-3p, revealed an increased tolerance of P. xylostella larvae to Cry1Ac at the post-transcriptional level. Considering that miR-2b and trypsin genes are widely distributed in various pest species, our study provides the basis for further investigation of the roles of miRNAs in the regulation of the resistance to Cry1Ac and other insecticides.

Draft genome of neotropical Bacillus thuringiensis UFT038 and its potential against lepidopteran soybean pests.

Bacillus thuringiensis (Bt) is known for its Cry and Vip3A pesticidal proteins with high selectivity to target pests. Here, we assessed the potential of a novel neotropical Bt strain (UFT038) against six lepidopteran pests, including two Cry-resistant populations of fall armyworm, Spodoptera frugiperda. We also sequenced and analyzed the genome of Bt UFT038 to identify genes involved in insecticidal activities or encoding other virulence factors. In toxicological bioassays, Bt UFT038 killed and inhibited the neonate growth in a concentration-dependent manner. Bt UFT038 and HD-1 were equally toxic against S. cosmioides, S. frugiperda (S_Bt and R_Cry1 + 2Ab populations), Helicoverpa zea, and H. armigera. However, larval growth inhibition results indicated that Bt UFT038 was more toxic than HD-1 to S. cosmioides, while HD-1 was more active against Chrysodeixis includens. The draft genome of Bt UFT038 showed the cry1Aa8, cry1Ac11, cry1Ia44, cry2Aa9, cry2Ab35, and vip3Af5 genes. Besides this, genes encoding the virulence factors (inhA, plcA, piplC, sph, and chi1-2) and toxins (alo, cytK, hlyIII, hblA-D, and nheA-C) were also identified. Collectively, our findings reveal the potential of the Bt UFT038 strain as a source of insecticidal genes against lepidopteran pests, including S. cosmioides and S. frugiperda.

Cell wall-localized Bt protein endows rice high resistance to Lepidoptera pests.

BACKGROUND: The commercialized Bt (Bacillus thuringiensis) crops accumulate Bt protein within cells, but the intracellular interactions of foreign protein with endogenous protein inevitably result in large or small unintended effects. In this study, the Bt gene Cry1Ca was linked with the sequences of extracellular secretion signal peptide and carbohydrate binding module 11 to constitute a fusion gene SP-Cry1Ca-CBM11, and the fusion gene driven by constitutive promoters was used for secreting and anchoring onto the cell wall to minimize unintended effects. RESULTS: The transient expression in tobacco leaves demonstrated that the fusion protein was anchored on cell walls. The Cry1Ca contents of five homozygous rice transformants of single-copy insertion were different and descended in the order leaf > root > stem. The maximum content of Cry1Ca was 17.55 µg g-1 in leaves of transformant 21H037. The bioassay results revealed that the transformants exhibited high resistance to lepidopteran pests. The corrected mortality of pink stem borer (Sesamia inferens) and striped stem borer (Chilo suppressalis) ranged from 96.33% to 100%, and from 83.32% to 100%, respectively, and the corrected mortality of rice leaf roller (Cnaphalocrocis medinalis) was 92.53%. Besides, the agronomic traits of the five transformants were normal and similar to that of the recipient, and the transformants were highly resistant to glyphosate at the germination and seedling stages. CONCLUSION: The fusion Bt protein was accumulated on cell walls and endowed the rice with high resistance to lepidopteran pests without unintended effects in agronomic traits. © 2023 Society of Chemical Industry.

Alpha hemolysin of E. coli induces hemolysis of human erythrocytes independently of toxin interaction with membrane proteins.

Alpha hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic strains of E. coli. The role of glycophorins (GPs) as putative receptors for HlyA binding to red blood cells (RBCs) has been debated. Experiments using anti-GPA/GPB antibodies and a GPA-specific epitope nanobody to block HlyA-GP binding on hRBCs, showed no effect on hemolytic activity. Similarly, the hemolysis induced by HlyA remained unaffected when hRBCs from a GPAnull/GPBnull variant were used. Surface Plasmon Resonance experiments revealed similar values of the dissociation constant between GPA and either HlyA, ProHlyA (inactive protoxin), HlyAΔ914-936 (mutant of HlyA lacking the binding domain to GPA) or human serum albumin, indicating that the binding between the proteins and GPA is not specific. Although far Western blot followed by mass spectroscopy analyses suggested that HlyA interacts with Band 3 and spectrins, hemolytic experiments on spectrin-depleted hRBCs and spherocytes, indicated these proteins do not mediate the hemolytic process. Our results unequivocally demonstrate that neither glycophorins, nor Band 3 and spectrins mediate the cytotoxic activity of HlyA on hRBCs, thereby challenging the HlyA-receptor hypothesis. This finding holds significant relevance for the design of anti-toxin therapeutic strategies, particularly in light of the growing antibiotic resistance exhibited by bacteria.

Helicoverpa armigera ATP-binding cassette transporter ABCA2 is a functional receptor of Bacillus thuringiensis Cry2Ab toxin.

Crystalline (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) are widely used in transgenic crops to control important insect pests. Bt crops have many benefits compared with traditional broad-spectrum insecticides, including improved pest control with reduced negative impacts on off-target organisms and fewer environmental consequences. Transgenic corn and cotton producing Cry2Ab Bt toxin are used globally to control several major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Resistance to the Cry2Ab toxin and to Bt crops producing Cry2Ab is associated with mutations in the midgut ATP-binding cassette transporter ABCA2 gene in several lepidopterans. Gene-editing knockout has further shown that ABCA2 plays an important functional role in Cry2Ab intoxication. However, the precise role of ABCA2 in the mode of action of Cry2Ab has yet to be reported. Here, we used two in vitro expression systems to study the roles of the H. armigera ABCA2 (HaABCA2) protein in Cry2Ab intoxication. Cry2Ab bound to cultured Sf9 insect cells producing HaABCA2, resulting in specific and dose-dependent susceptibility to Cry2Ab. In contrast, Sf9 cells expressing recombinant mutant proteins missing at least one of the extracellular loop regions 1, 3, 4, and 6 or the intracellular loop containing nucleotide-binding domain 1 lost susceptibility to Cry2Ab, indicating these regions are important for receptor function. Consistent with these results, Xenopus laevis oocytes expressing recombinant HaABCA2 showed strong ion membrane flux in the presence of Cry2Ab, suggesting that HaABCA2 is involved in promoting pore formation during Cry2Ab intoxication. Together with previously published data, our results support HaABCA2 being an important receptor of Cry2Ab where it functions to promote intoxication in H. armigera.

Repurposing rabeprazole sodium as an anti-Clostridium perfringens drug by inhibiting perfringolysin O.

AIMS: Clostridium perfringens infections affect food safety, human health, and the development of the poultry feed industry. Anti-virulence is an alternative strategy to develop new drug. Perfringolysin O (PFO) is an exotoxin of C. perfringens that has been demonstrated to play critical roles in the pathogenesis of this organism, promising it an attractive target to explore drugs to combat C. perfringens infection. METHODS AND RESULTS: Based on an activity-based screening, we identified six PFO inhibitors from the Food and Drug Administration (FDA)-approved drug library, among which rabeprazole sodium (RS) showed an optimal inhibitory effect with an IC50 of 1.82 ± 0.746 µg ml-1. The GLY57, ASP58, SER190, SER193-194, ASN199, GLU204, ASN377, THR379, and ALA200 in PFO interacted with RS during binding based on an energy analysis and H-bond analysis. This interaction blocked the oligomer formation of PFO, thereby inhibiting its cytotoxicity. RS treatment significantly increased the survival rate and alleviated pathological damage in C. perfringens or PFO-treated Galleria mellonella. CONCLUSIONS: RS could potentially be used as a candidate drug for treating C. perfringens infection.

A soybean trypsin inhibitor reduces the resistance to transgenic maize in a population of Spodoptera frugiperda (Lepidoptera: Noctuidae).

Lepidopteran pests have been successfully managed by the adoption of insect resistant transgenic plants expressing Cry and/or Vip insecticidal proteins derived from Bacillus thuringiensis (Bt plants). Among such pests, Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae) is highlighted for its destructive potential in maize crops and for cases of field-evolved resistance to Bt plants. Cry insecticidal proteins expressed in Bt plants are known for their interaction with insect midgut receptors and subsequent midgut cell disruption that leads to target pest death. In the midgut of lepidopteran larval pests such as S. frugiperda, serine proteases are important in dietary protein digestion and activation or degradation of insecticidal proteins. This work was conducted to evaluate if the use of a soybean trypsin inhibitor (SBTI) could disrupt the development of a Bt-susceptible and a Bt-resistant population of S. frugiperda ingesting Bt (expressing Cry1F, Cry1A.105, and Cry2Ab2 Cry proteins) and non-Bt maize plants. The SBTI was produced and purified using recombinant expression in E. coli followed by purification in Ni-Sepharose. Bioassays using non-Bt maize leaves indicated that the development of susceptible and resistant populations of S. frugiperda was not influenced by the ingestion of SBTI. However, when the resistant population consumed Bt maize plants amended with SBTI, high mortality along with a reduction in larval weight and reduced activity of digestive trypsins were observed. Although the mode of action was not elucidated, it is possible that the consumption of SBTI increased susceptibility to Bt maize in the resistant population of S. frugiperda.

Reduced expression of the P-glycoprotein gene HaABCB1 is linked to resistance to Bacillus thuringiensis Cry1Ac toxin but not Cry2Ab toxin in Helicoverpa armigera.

Rapid evolution of pest resistance to Bt insecticidal proteins presents a serious threat to the sustainable use of Bt crops. The cotton bollworm has been extensively exposed to Bt cotton worldwide and has evolved resistance in laboratory and field. Previous studies have highlighted the significant roles played by the ABC transporter proteins in Bt resistance. In this study, the ORF of HaABCB1 was cloned and analyzed. The expression of HaABCB1 was detected in all developmental stages and tissues, with the highest expression in third instar larvae stage and hindgut tissue. Compared with susceptible strain, a remarkable decrease of HaABCB1 expression in Cry1Ac resistant strain while no significant change in Cry2Ab resistant strain were found. The HaABCB1 expression reduced after susceptible larvae induced by Cry1Ac, but no obvious expression changes after Cry2Ab exposure. RNAi-mediated down-regulation of HaABCB1 could lead to a significant reduction in larval susceptibility to Cry1Ac, but not to Cry2Ab, in susceptible strain. Genetic linkage analysis confirmed that decreased expression of the HaABCB1 mediates resistance to Cry1Ac, but not Cry2Ab resistance. This knowledge contributes to better understanding of the complex molecular mechanisms underlying Bt resistance and provide theoretical foundation for the development of new strategies for pest resistance management.

Analysis of Synergism between Extracellular Polysaccharide from Bacillus thuringensis subsp. kurstaki HD270 and Insecticidal Proteins.

Bacillus thuringiensis (Bt) is the most widely used biopesticide worldwide and can produce several insecticidal crystal proteins and vegetative insecticidal proteins (Vips) at different growth stages. In our previous study, extracellular polysaccharides (EPSs) of Bt strain HD270 were found to enhance the insecticidal activity of Cry1Ac protoxin against Plutella xylostella (L.) and promote the binding of Cry1Ac to the intestinal brush border membrane vesicles (BBMVs). Whether the synergistic activity of Bt EPSs is common to other Cry1-type or Vip proteins is unclear, as is the potential synergistic mechanism. In this study, crude EPS-HD270 was found to increase the toxicity of Cry1-type toxins and Vip3Aa11 against different lepidopteran pests by approximately 2-fold. The purified EPS-HD270 also possessed synergistic activity against the toxicity of Cry1Ac and Vip3Aa11 against Spodoptera frugiperda (J.E. Smith) and Helicoverpa armigera (Hübner). Furthermore, we found that EPS-HD270 had a strong binding ability with Vip3Aa11 and promoted the binding of Vip3Aa11 to the BBMVs of H. armigera and S. frugiperda. Bt EPS-HD270 also protected Vip3Aa11 from proteolytic processing in larval midgut juice. Bt EPSs had universal synergistic effects on Cry1-type or Vip toxins against S. frugiperda and H. armigera. Bt EPS-HD270 exhibited synergistic activity with Vip3Aa through promotion of binding to BBMVs and protection from digestion by midgut protease. The results indicated that synergistic activity with Bt toxins was an important function of Bt EPSs, which was very different from other Bacillus spp.

Baseline Susceptibility of the Field Populations of Ostrinia furnacalis in Indonesia to the Proteins Cry1A.105 and Cry2Ab2 of Bacillus thuringiensis.

Genetically modified MON 89034 corn (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins, viz. Cry1A.105 and Cry2Ab2, is a biotechnological option being considered for the management of the major corn pest in Indonesia, the Asian corn borer (Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae)). As a part of a proactive resistance-management program for MON 89034 corn in Indonesia, we assessed the baseline susceptibility of field-collected populations of O. furnacalis to Cry1A.105 and Cry2Ab2 proteins. Dose-response bioassays using the diet-dipping method indicated that the lethal concentration (LC50) values of Cry1A.105 and Cry2Ab2 in 24 different field populations of O. furnacalis ranged from 0.006 to 0.401 µg/mL and from 0.044 to 4.490 µg/mL, respectively, while the LC95 values ranged from 0.069 to 15.233 µg/mL for Cry1A.105 and from 3.320 to 277.584 µg/mL for Cry2Ab2. The relative resistance ratios comparing the most tolerant field populations and an unselected laboratory population were 6.0 for Cry1A.105 and 2.0 for Cry2Ab2 based on their LC50 values. Some field populations were more susceptible to both proteins than the unselected laboratory population. The LC99 and its 95% fiducial limits across the field populations were calculated and proposed as candidate diagnostic concentrations. These data provide a basis for resistance monitoring in Bt Corn and further support building resistance-management strategies in Indonesia.

Inheritance and fitness cost of laboratory-selected resistance to Vip3Aa in Helicoverpa zea (Lepidoptera: Noctuidae).

The polyphagous pest Helicoverpa zea (Lepidoptera: Noctuidae) has evolved practical resistance to transgenic corn and cotton producing Cry1 and Cry2 crystal proteins from Bacillus thuringiensis (Bt) in several regions of the United States. However, the Bt vegetative insecticidal protein Vip3Aa produced by Bt corn and cotton remains effective against this pest. To advance knowledge of resistance to Vip3Aa, we selected a strain of H. zea for resistance to Vip3Aa in the laboratory. After 28 generations of continuous selection, the resistance ratio was 267 for the selected strain (GA-R3) relative to a strain not selected with Vip3Aa (GA). Resistance was autosomal and almost completely recessive at a concentration killing all individuals from GA. Declines in resistance in heterogeneous strains containing a mixture of susceptible and resistant individuals reared in the absence of Vip3Aa indicate a fitness cost was associated with resistance. Previously reported cases of laboratory-selected resistance to Vip3Aa in lepidopteran pests often show partially or completely recessive resistance at high concentrations and fitness costs. Abundant refuges of non-Bt host plants can maximize the benefits of such costs for sustaining the efficacy of Vip3Aa against target pests.

Performance of cotton expressing Cry1Ac, Cry1F and Vip3Aa19 insecticidal proteins against Helicoverpa armigera, H. zea and their hybrid progeny, and evidence of reduced susceptibility of a field population of H. zea to Cry1 and Vip3Aa in Brazil.

The genetically modified cotton DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 expressing Cry1Ac, Cry1F and Vip3Aa19 from Bacillus thuringiensis Berliner (Bt) has been cultivated in Brazil since the 2020/2021 season. Here, we assessed the performance of DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton expressing Cry1Ac, Cry1F and Vip3Aa19 against Helicoverpa armigera (Hübner), Helicoverpa zea (Boddie), and their hybrid progeny. We also carried out evaluations with DAS-21023-5 × DAS-24236-5 cotton containing Cry1Ac and Cry1F. In leaf-disk bioassays, DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 was effective in controlling neonates from laboratory colonies of H. armigera, H. zea and the hybrid progeny (71.9%-100% mortality). On floral bud bioassays using L2 larvae, H. zea presented complete mortality, whereas H. armigera and the hybrid progeny showed <55% mortality. On DAS-21023-5 × DAS-24236-5 cotton, the mortality of H. armigera on leaf-disk and floral buds ranged from 60% to 73%, whereas mortality of hybrids was <46%. This Bt cotton caused complete mortality of H. zea larvae from a laboratory colony in the early growth stages, but mortalities were <55% on advanced growth stages and on floral buds. In field studies conducted from 2014 to 2019, DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton was also effective at protecting plants against H. armigera. In contrast, a population of H. zea collected in western Bahia in 2021/2022 on Bt cotton expressing Cry1 and Vip3Aa proteins, showed 63% mortality after 30 d, with insects developing into fifth and sixth instars, on DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton. We conclude that H. armigera, H. zea, and their hybrid progeny can be managed with DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton; however we found the first evidence in Brazil of a significant reduction in the susceptibility to DAS-21023-5 × DAS-24236-5 × SYN-IR102-7 cotton of a population of H. zea collected from Bt cotton in Bahia in 2021/2022.

An Extended Investigation of Unexpected Helicoverpa zea (Boddie) Survival and Ear Injury on a Transgenic Maize Hybrid Expressing Cry1A/Cry2A/Vip3A Toxins.

The wide occurrence of resistance to Cry1A and Cry2A insecticidal toxins from Bacillus thuringiensis (Bt) in the corn earworm/bollworm Helicoverpa zea (Boddie) leaves the Vip3A toxin produced during the vegetative stage of Bt as the only fully active toxin expressed in transgenic crops to control H. zea in the U.S.A. During 2021, the first unexpected survival of H. zea and injury (UXI) on a maize hybrid expressing Cry1A.105, Cry2Ab2, and Vip3Aa in Louisiana, U.S.A. were observed in two sentinel plots used for resistance monitoring. A follow-up intensive investigation was conducted with two H. zea populations established from larvae collected from the two UXI plots. The main goal of this study was to reveal if the unexpected damage was due to resistance development in the insect to the Bt toxins expressed in the maize hybrid. Diet-overlay bioassays showed that the two populations were highly resistant to Cry1A.105, moderately resistant to Cry2Ab2, but still highly susceptible to Vip3Aa when compared to a reference susceptible strain. In 10 d assays with detached ears, the larvae of the two UXI populations exhibited survival on ears expressing only Cry toxins but presented near 100% mortality on maize hybrids containing both cry and vip3A transgenes. Multiple field trials over three years demonstrated that natural H. zea populations in Louisiana were highly resistant to maize expressing only Cry toxins but remained susceptible to all tested hybrids containing cry and vip3A genes. Altogether, the results of this study suggest that the observed UXIs in Louisiana were associated with a resistance to Cry toxins but were not due to a resistance to Vip3A. The possible causes of the UXIs are discussed. The results generated and procedures adopted in this study help in determining thresholds for defining UXIs, assessing resistance risks, and documenting field resistance.

A novel Cry1A resistance allele of fall armyworm in the new invaded region.

The fall armyworm, Spodoptera frugiperda, is a devastating pest in its native range Western Hemisphere and has become a major invasive pest around the globe. Transgenic crops producing Bt toxins have been widely used to control S. frugiperda. However, the evolution of resistance threatens the sustainability of Bt crops. Field-evolved S. frugiperda resistance to Bt crops was observed in America, whereas, no case of field-resistance was reported in its newly invaded East Hemisphere. Here we investigated the molecular mechanism of a Cry1Ab-resistant LZ-R strain of S. frugiperda, which selected 27-generations using Cry1Ab after being collected in corn fields from China. Complementation tests between LZ-R strain and SfABCC2-KO strain, which have been knockout SfABCC2 gene and confer 174-fold resistance to Cry1Ab, showed a similar level of resistance in the F1-progeny as their parent stains, indicating that a common locus of SfABCC2 mutation in LZ-R stain. Sequencing of the full length of SfABCC2 cDNA from LZ-R strain, we characterize a novel mutation allele of SfABCC2. Cross-resistance results showed that Cry1Ab-resistance strain also confers >260-fold resistance to Cry1F, with no cross-resistance to Vip3A. These results provided evidence of a novel SfABCC2 mutation allele in the newly invaded East Hemisphere of S. frugiperda.