Genotoxic evaluation in Oreochromis niloticus (Fish: Characidae) of recombinant spore–crystal complexes Cry1Ia,Cry10Aa and Cry1Ba6 from Bacillus thuringiensis

Bioinsecticides from Bacillus thuringiensis (Bt) are widely used around the world in biological control against larval stages of many insect species. Bt has been considered a biopesticide that is highly specific to different orders of insects, non-polluting and harmless to humans and other vertebrates, thus becoming a viable alternative for combating agricultural pests and insect vectors of diseases. The family of Bt δ-endotoxins are crystal-protein inclusions showing toxicity to insects’ midgut, causing cell lysis leading to starvation, septicemia and death. The aim of this study is to evaluate the genotoxic potential of recombinant Bt spore–crystals expressing Cry1Ia, Cry10Aa and Cry1Ba6 on peripheral erythrocyte cells of Oreochromis niloticus, through comet assay, micronucleus (MN) test and nuclear abnormalities (NA) analysis. Fish (n = 10/group) were exposed for 96 h at 10⁷ spores 30 l^?1, 10⁸ spores 30 l^?1 or 10⁹ spores 30 l^?1 of Bt spore–crystals. Cry1Ia showed a significant increase in comet cells at levels 1 and 2, but not at levels 3 and 4, so it was not mutagenic nor did it induce MN or NA. These three spore–crystals showed some fish toxicity at only the highest exposure level, which normally does not occur in the field.     

Hematotoxicity and genotoxicity evaluations in Swiss mice intraperitoneally exposed to Bacillus thuringiensis (var kurstaki) spore crystals genetically modified to express individually Cry1Aa,Cry1Ab,Cry1Ac,or Cry2Aa

Bacillus thuringiensis (Bt) has been widely used in foliar sprays as part of integrated pest management strategies against insect pests of agricultural crops. Since the advent of genetically modified plants expressing Bt δ‐endotoxins, the bioavailability of Cry proteins has increased, and therefore for biosafety reasons their adverse effects should be studied, mainly for nontarget organisms. We evaluated, in Swiss mice, the hematotoxicity and genotoxicity of the genetically modified strains of Bt spore crystals Cry1Aa, 1Ab, 1Ac, or 2Aa at 27 mg/kg, and Cry1Aa, 1Ab and 2Aa also at 136 and 270 mg/kg, administered with a single intraperitoneal injection 24 h before euthanasia. Controls received filtered water or cyclophosphamide. Blood samples collected by cardiac puncture were used to perform hemogram, and bone marrow was extracted for the micronucleus test. Bt spore crystals presented toxicity for lymphocytes when in higher doses, which varied according to the type of spore crystal studied, besides promoting cytotoxic and genotoxic effects for the erythroid lineage of bone marrow, mainly at highest doses. Although the profile of such adverse side effects can be related to their high level of exposure, which is not commonly found in the environment, results indicated that these Bt spore crystals were not harmless to mice. This suggests that a more specific approach should be taken to increase knowledge about their toxicological properties and to establish the toxicological risks to nontarget organisms. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 970–978, 2016.     

Aedes aegypti Mos20 Cells Internalizes Cry Toxins by Endocytosis,and Actin Has a Role in the Defense against Cry11Aa Toxin

Bacillus thuringiensis (Bt) Cry toxins are used to control Aedes aegypti, an important vector of dengue fever and yellow fever. Bt Cry toxin forms pores in the gut cells, provoking larvae death by osmotic shock. Little is known, however, about the endocytic and/or degradative cell processes that may counteract the toxin action at low doses. The purpose of this work is to describe the mechanisms of internalization and detoxification of Cry toxins, at low doses, into Mos20 cells from A. aegypti, following endocytotic and cytoskeletal markers or specific chemical inhibitors. Here, we show that both clathrin-dependent and clathrin-independent endocytosis are involved in the internalization into Mos20 cells of Cry11Aa, a toxin specific for Dipteran, and Cry1Ab, a toxin specific for Lepidoptera. Cry11Aa and Cry1Ab are not directed to secretory lysosomes. Instead, Mos20 cells use the Rab5 and Rab11 pathways as a common mechanism, most probably for the expulsion of Cry11Aa and Cry1Ab toxins. In conclusion, we propose that endocytosis is a mechanism induced by Cry toxins independently of specificity, probably as part of a basal immune response. We found, however, that actin is necessary for defense-specific response to Cry11Aa, because actin-silenced Mos20 cells become more sensitive to the toxic action of Cry11A toxin. Cry toxin internalization analysis in insect cell lines may contribute to a better understanding to Cry resistance in mosquitoes.     

Resistance Allele Frequency to Cry1Ab and Vip3Aa20 in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in Louisiana and Three Other Southeastern U.S. States

The corn earworm/bollworm, Helicoverpa zea (Boddie), is a pest species that is targeted by both Bacillus thuringiensis (Bt) maize and cotton in the United States. Cry1Ab and Vip3Aa20 are two common Bt toxins that are expressed in transgenic maize. The objective of this study was to determine the resistance allele frequency (RAF) to Cry1Ab and Vip3Aa20 in H. zea populations that were collected during 2018 and 2019 from four southeastern U.S. states: Louisiana, Mississippi, Georgia, and South Carolina. By using a group-mating approach, 104 F₂ iso-lines of H. zea were established from field collections with most iso-lines (85) from Louisiana. These F₂ iso-lines were screened for resistance alleles to Cry1Ab and Vip3Aa20, respectively. There was no correlation in larval survivorship between Cry1Ab and Vip3Aa20 when the iso-lines were exposed to these two toxins. RAF to Cry1Ab maize was high (0.256) and the RAFs were similar between Louisiana and the other three states and between the two sampling years. In contrast, no functional major resistance allele (RA) that allowed resistant insects to survive on Vip3Aa20 maize was detected and the expected RAF of major RAs with 95% probability was estimated to 0 to 0.0073. However, functional minor RAs to Vip3Aa20 maize were not uncommon; the estimated RAF for minor alleles was 0.028. The results provide further evidence that field resistance to Cry1Ab maize in H. zea has widely occurred, while major RAs to Vip3Aa20 maize are uncommon in the southeastern U.S. region. Information that was generated from this study should be useful in resistance monitoring and refinement of resistance management strategies to preserve Vip3A susceptibility in H. zea.     

Functional significance of membrane associated proteolysis in the toxicity of Bacillus thuringiensis Cry3Aa toxin against Colorado potato beetle

Bacillus thuringiensis Cry toxins are widely used as biocontrol agents in bioinsecticides and transgenic plants. In the three domain-Cry toxins, domain II has been identified as an important determinant of their highly specific activity against insects. In this work, we assessed the role in membrane associated proteolysis and toxicity in Colorado potato beetle (CPB) of a previously reported ADAM recognition motif present in Cry3Aa toxin domain II. We used site-directed mutagenesis to modify the Bacillus thuringiensis cry3A gene in amino acid residues 344, 346, 347, 351 and 353 of the ADAM recognition motif in Cry3Aa toxin. Cry3Aa toxin mutants displayed decreased toxicity when compared to the wild type toxin and impaired ability to compete CPB brush border membrane associated cleavage of an ADAM fluorogenic substrate. Although the proteolytic profile of Cry3Aa toxin mutants generated by brush border membrane associated proteases was similar to that of Cry3Aa toxin, the metalloprotease inhibitor 1,10-phenanthroline was less efficient on the proteolysis of mutants than on that of the wild type toxin. The relevance of the Cry3Aa-ADAM interaction through the predicted recognition sequence was further confirmed by analyzing the effect of membrane integrity disturbance on Cry3Aa toxin membrane associated proteolysis and CPB larvae toxicity. Data support that Cry3Aa proteolysis, as a result of the interaction with ADAM through the Cry3Aa recognition motif, is essential for Cry3Aa toxic action in CPB. Detailed knowledge of Cry3Aa interaction with CPB midgut membrane should facilitate the development of more effective Bt based products against this devastating pest and other Coleoptera.     

Toxicological Evaluation of a Potential Immunosensitizer for Use as a Mucosal Adjuvant—Bacillus thuringiensis Cry1Ac Spore-Crystals: A Possible Inverse Agonist that Deserves Further Investigation

In addition to their applicability as biopesticides, Bacillus thuringiensis (Bt) Cry1Ac spore-crystals are being researched in the immunology field for their potential as adjuvants in mucosal and parenteral immunizations. We aimed to investigate the hematotoxicity and genotoxicity of Bt spore-crystals genetically modified to express Cry1Ac individually, administered orally (p.o.) or with a single intraperitoneal (i.p.) injection 24 h before euthanasia, to simulate the routes of mucosal and parenteral immunizations in Swiss mice. Blood samples were used to perform hemogram, and bone marrow was used for the micronucleus test. Cry1Ac presented cytotoxic effects on erythroid lineage in both routes, being more severe in the i.p. route, which also showed genotoxic effects. The greater severity noted in this route, mainly at 6.75 mg/kg, as well as the intermediate effects at 13.5 mg/kg, and the very low hematotoxicity at 27 mg/kg, suggested a possible inverse agonism. The higher immunogenicity for the p.o. route, particularly at 27 mg/kg, suggested that at this dose, Cry 1Ac could potentially be used as a mucosal adjuvant (but not in parenteral immunizations, due to the genotoxic effects observed). This potential should be investigated further, including making an evaluation of the proposed inverse agonism and carrying out cytokine profiling.     

Acute and repeated dose (28 day) mouse oral toxicology studies with Cry34Ab1 and Cry35Ab1 Bt proteins used in coleopteran resistant DAS-59122-7 corn

Expression of the Cry34Ab1 and Cry35Ab1 proteins from Bacillus thuringiensis (Bt) Berliner strain PS149B1 in genetically modified maize (event DAS-59122-7) protects the crop from damage due to feeding by Diabrotica larvae including the western corn rootworm (Diabrotica virgifera virgifera). As part of the safety assessment of this maize, mammalian toxicology studies were conducted with heterologously produced Cry34Ab1 and Cry35Ab1 proteins. No evidence of acute toxicity was observed in mice following oral exposure to either the Cry34Ab1 or Cry35Ab1 proteins individually (2700 and 1850 mg/kg, respectively) or concomitantly (482 and 1520 mg/kg, respectively; 1:1 molar ratio). Similarly, no adverse effects were observed in mice in a repeated dose (28 day) dietary toxicity study that incorporated these proteins into diets at concentrations corresponding up to 1000-fold greater than the highest estimate of human exposure based on the concentrations of these proteins expressed in 59122 maize grain. These studies demonstrate that the Cry34Ab1 and Cry35Ab1 proteins do not represent a risk to human health and support previous studies indicating that 59122 maize grain is as safe and wholesome as non-GM maize grain.     

Independent and Synergistic Effects of Knocking out Two ABC Transporter Genes on Resistance to Bacillus thuringiensis Toxins Cry1Ac and Cry1Fa in Diamondback Moth

Insecticidal proteins from Bacillus thuringiensis (Bt) are used widely in sprays and transgenic crops to control insect pests. However, evolution of resistance by pests can reduce the efficacy of Bt toxins. Here we analyzed resistance to Bt toxins Cry1Ac and Cry1Fa in the diamondback moth (Plutella xylostella), one of the world’s most destructive pests of vegetable crops. We used CRISPR/Cas9 gene editing to create strains with knockouts of the ATP-binding cassette (ABC) transporter genes PxABCC2, PxABCC3, or both. Bioassay results show that knocking out either gene alone caused at most 2.9-fold resistance but knocking out both caused >10,320-fold resistance to Cry1Ac and 380-fold resistance to Cry1Fa. Cry1Ac resistance in the double knockout strain was recessive and genetically linked with the PxABCC2/PxABCC3 loci. The results provide insight into the mechanism of cross-resistance to Cry1Fa in diamondback moth. They also confirm previous work with this pest showing that mutations disrupting both genes cause higher resistance to Cry1Ac than mutations affecting either PxABCC2 or PxABCC3 alone. Together with previous work, the results here highlight the value of using single and multiple gene knockouts to better understand the independent and synergistic effects of putative Bt toxin receptors on resistance to Bt toxins.     

Alteration of a Cry1A Shared Binding Site in a Cry1Ab-Selected Colony of Ostrinia furnacalis

The Asian corn borer, Ostrinia furnacalis (Guenée, 1854), is a highly damaging pest in Asia and the Pacific islands, and larvae feed mainly from corn crops. To determine the suitability of Bt-corn technology for the future control of this pest, understanding the potential to develop resistance to Cry1Ab and the basis of cross-resistance to other Cry1 proteins is of great interest. Here, we have explored the binding of Cry1A proteins to brush border membrane vesicles from two O. furnacalis colonies, one susceptible (ACB-BtS) and one laboratory-selected with Cry1Ab (ACB-AbR). The insects developed resistance to Cry1Ab and showed cross-resistance to Cry1Aa, Cry1Ac, and Cry1F. Binding assays with radiolabeled Cry1Ab and brush border membrane vesicles from susceptible insects showed that Cry1A proteins shared binding sites, though the results were not conclusive for Cry1F. The results were confirmed using radiolabeled Cry1Aa. The resistant insects showed a reduction of the specific binding of both Cry1Ab and Cry1Aa, suggesting that part of the binding sites were lost or altered. Competition binding assays showed full competition between Cry1Ab and Cry1Aa proteins in the susceptible colony but only partial competition in resistant insects, confirming the alteration of some, but not all, binding sites for these two proteins. The binding site model for Cry1A proteins in O. furnacalis is in agreement with the occurrence of multiple membrane receptors for these proteins.     

Expression, purification and characterization of the Cry2Aa14 toxin from Bacillus thuringiensis subsp. kenyae

An indigenous strain HD-550 of Bacillus thuringiensis subsp. kenyae was found to be toxic to lepidopteran as well as dipteran insects. The cry2Aa gene (classified as cry2Aa14) from this isolate was cloned and expressed in Escherichia coli. Only a little amount of the expressed Cry2Aa14 protein was observed in soluble fraction under normal induction condition. The inclusions were non-toxic to test insects, whereas solubilized Cry2Aa14 was highly toxic to lepidopteran and dipteran insects. Cry2Aa14 protein was expressed as thioredoxin (trx) fusion protein for improving the yield of active protein. An enhancement of nearly 15% was observed in the yield of active Cry2Aa14. The TrxA-Cry2Aa14 protein purified from the solubilized fraction also showed toxicity profile similar to the wild-type protein. The LC₅₀ values of Cry2Aa14 and TrxA-Cry2Aa14 protein against Spodoptera litura was 694 and 696 ng/cm², respectively, while for Culex quinquefasciatus the LC₅₀ values were 894 and 902 ng/ml, respectively. The broad spectrum toxicity of the Cry2Aa14 thus indicates that this protein could be an important component in integrated pest management. Further, the trx tag clearly led to higher yield, which facilitates protein purification for biophysical and biochemical characterization.     

Cadherin Protein Is Involved in the Action of Bacillus thuringiensis Cry1Ac Toxin in Ostrinia furnacalis

Transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal proteins have been extensively planted for insect pest control, but the evolution of Bt resistance in target pests threatens the sustainability of this approach. Mutations of cadherin in the midgut brush border membrane was associated with Cry1Ac resistance in several lepidoptera species, including the Asian corn borer, Ostrinia furnacalis, a major pest of maize in Asian–Western Pacific countries. However, the causality of O. furnacalis cadherin (OfCad) with Cry1Ac resistance remains to be clarified. In this study, in vitro and in vivo approaches were employed to examine the involvement of OfCad in mediating Cry1Ac toxicity. Sf9 cells transfected with OfCad showed significant immunofluorescent binding with Cry1Ac toxin and exhibited a concentration-dependent mortality effect when exposed to Cry1Ac. The OfCad knockout strain OfCad-KO, bearing homozygous 15.4 kb deletion of the OfCad gene generated by CRISPR/Cas9 mutagenesis, exhibited moderate-level resistance to Cry1Ac (14-fold) and low-level resistance to Cry1Aa (4.6-fold), but no significant changes in susceptibility to Cry1Ab and Cry1Fa, compared with the original NJ-S strain. The Cry1Ac resistance phenotype was inherited as autosomal, recessive mode, and significantly linked with the OfCad knockout in the OfCad-KO strain. These results demonstrate that the OfCad protein is a functional receptor for Cry1Ac, and disruption of OfCad confers a moderate Cry1Ac resistance in O. furnacalis. This study provides new insights into the mode of action of the Cry1Ac toxin and useful information for designing resistance monitoring and management strategies for O. furnacalis.     

Recombinant Expression of ABCC2 Variants Confirms the Importance of Mutations in Extracellular Loop 4 for Cry1F Resistance in Fall Armyworm

Fall armyworm (FAW), Spodoptera frugiperda, is a highly destructive and invasive global noctuid pest. Its control is based on insecticide applications and Bacillus thuringiensis (Bt) insecticidal Cry toxins expressed in transgenic crops, such as Cry1F in Bt corn. Continuous selection pressure has resulted in populations that are resistant to Bt corn, particularly in Brazil. FAW resistance to Cry1F was recently shown to be conferred by mutations of ATP-binding cassette transporter C2 (ABCC2), but several mutations, particularly indels in extracellular loop 4 (ECL4), are not yet functionally validated. We addressed this knowledge gap by baculovirus-free insect cell expression of ABCC2 variants (and ABCC3) by electroporation technology and tested their response to Cry1F, Cry1A.105 and Cry1Ab. We employed a SYTOX^TM orange cell viability test measuring ABCC2-mediated Bt toxin pore formation. In total, we tested seven different FAW ABCC2 variants mutated in ECL4, two mutants modified in nucleotide binding domain (NBD) 2, including a deletion mutant lacking NBD2, and S. frugiperda ABCC3. All tested ECL4 mutations conferred high resistance to Cry1F, but much less to Cry1A.105 and Cry1Ab, whereas mutations in NBD2 hardly affected Bt toxin activity. Our study confirms the importance of indels in ECL4 for Cry1F resistance in S. frugiperda ABCC2.     

Structural Insights into Bacillus thuringiensis Cry,Cyt and Parasporin Toxins

Since the first X-ray structure of Cry3Aa was revealed in 1991, numerous structures of B. thuringiensis toxins have been determined and published. In recent years, functional studies on the mode of action and resistance mechanism have been proposed, which notably promoted the developments of biological insecticides and insect-resistant transgenic crops. With the exploration of known pore-forming toxins (PFTs) structures, similarities between PFTs and B. thuringiensis toxins have provided great insights into receptor binding interactions and conformational changes from water-soluble to membrane pore-forming state of B. thuringiensis toxins. This review mainly focuses on the latest discoveries of the toxin working mechanism, with the emphasis on structural related progress. Based on the structural features, B. thuringiensis Cry, Cyt and parasporin toxins could be divided into three categories: three-domain type α-PFTs, Cyt toxin type β-PFTs and aerolysin type β-PFTs. Structures from each group are elucidated and discussed in relation to the latest data, respectively.     

An overview of the safety and biological effects of Bacillus thuringiensis Cry toxins in mammals

Crystal proteins (Cry) produced during the growth and sporulation phases of Bacillus thuringiensis (Bt) bacterium are known as delta endotoxins. These toxins are being used worldwide as bioinsecticides to control pests in agriculture, and some Cry toxins are used against mosquitoes to control vector transmission. This review summarizes the relevant information currently available regarding the biosafety and biological effects that Bt and its insecticidal Cry proteins elicit in mammals. This work was performed because of concerns regarding the possible health impact of Cry toxins on vertebrates, particularly because Bt toxins might be associated with immune‐activating or allergic responses. The controversial data published to date are discussed in this review considering earlier toxicological studies of B. thuringiensis, spores, toxins and Bt crops. We discussed the experimental studies performed in humans, mice, rats and sheep as well as in diverse mammalian cell lines. Although the term ’toxic‘ is not appropriate for defining the effects these toxins have on mammals, they cannot be considered innocuous, as they have some physiological effects that may become pathological; thus, trials that are more comprehensive are necessary to determine their effects on mammals because knowledge in this field remains limited. Copyright © 2015 John Wiley & Sons, Ltd.     

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

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

Baseline Susceptibility and Resistance Allele Frequency in Ostrinia furnacalis in Relation to Cry1Ab Toxins in China

Asian corn borer (ACB), Ostrinia furnacalis (Guenée) is a destructive pest of corn and major target of transgenic corn expressing Bt (Bacillus thuringiensis) toxins in China. It is necessary to establish the baseline susceptibility of geographically distinct ACB populations to Cry1Ab protein and estimate the resistance alleles frequency. The median lethal concentration (LC₅₀) and LC₉₅ values of Bt toxin Cry1Ab for 25 geographically distinct populations collected in 2018–2019 ranged from 0.86 to 71.33, 18.58 to 5752.34 ng/cm², respectively. The median effective concentration (EC₅₀) and EC₉₅ values ranged from 0.03 to 10.40 ng/cm² and 3.75 to 172.86 ng/cm², respectively. We used the F₂ screening method for estimating the expected frequency of resistance alleles of the 13 ACB populations, to Bt corn (Bt11 × GA21) expressing the Cry1Ab toxin. The neonates could not survive on the leaves of transgenic maize Bt11 × GA21 with cry1Ab gene, the Cry1Ab resistance allele frequency was still low in each geographic population in the field, ranging from 0.0032–0.0048, indicating that the sensitivity of ACB to Cry1Ab was still at a high level, and there were no viable resistant individuals in the field at present. The susceptibility of 25 populations of ACB collected in China showed regional differences, although the Cry1Ab resistance allele frequency in these ACB populations is still at a low level. This provides essential knowledge for making the decision to commercialize Bt maize, and monitoring resistance development and evaluating resistance management strategies in the future in China.     

Susceptibilities of the Invasive Fall Armyworm (Spodoptera frugiperda) to the Insecticidal Proteins of Bt maize in China

To control the fall armyworm (FAW), Spodoptera frugiperda (Smith), a serious threat to maize production in China, the Chinese government has issued biosafety certificates for transgenic insect-resistant maize expressing Bt (Bacillus thuringiensis) toxins including Bt-Cry1Ab maize (crop event DBN9936), Bt-Vip3Aa maize (event DBN9501), Bt-(Cry1Ab+Vip3Aa) maize with superimposed traits (event DBN9936 × DBN9501) and Bt-(Cry1Ab+Vip3Aa) maize with superimposed traits (event Bt11 × MIR162), but the susceptibility baselines of geographically distinct FAW populations to these events, which form the basis for managing resistance development in the pest to these events, are not clear. We used the diet-incorporated bioassays method to detect the susceptibilities of the seven FAW populations collected from Yunnan, Henan and Hubei provinces in China in 2021 to the insecticidal proteins of the four Bt maize events. The result showed that the susceptibilities of different geographical populations to Bt insecticidal proteins were significantly different. In the seven populations, the range in median lethal concentrations (LC₅₀) of Cry1Ab expressed in DBN9936 was 0.87–2.63 μg/g, 0.14–0.30 μg/g for Vip3Aa expressed in DBN9501, 0.78–1.86 μg/g for Cry1Ab+Vip3Aa expressed in DBN9936 × DBN9501, and 0.36–1.42 μg/g for CryAb+Vip3Aa expressed in Bt11 × MIR162. The growth inhibition responses also showed that the susceptibilities varied with the different median growth inhibitory concentration (GIC₅₀) ranges (0.38–1.22, 0.08–0.28, 0.28–0.87, and 0.24–0.78 μg/g, respectively). The variations in the ranges of the susceptibility baselines of the geographical populations of fall armyworm in China to the insecticidal proteins expressed in the four events provide a scientific basis for monitoring FAW population resistance to Bt maize and managing the populations using different Bt maize events.     

Identification of Distinct Bacillus thuringiensis 4A4 Nematicidal Factors Using the Model Nematodes Pristionchus pacificus and Caenorhabditis elegans

Bacillus thuringiensis has been extensively used for the biological control of insect pests. Nematicidal B. thuringiensis strains have also been identified; however, virulence factors of such strains are poorly investigated. Here, we describe virulence factors of the nematicidal B. thuringiensis 4A4 strain, using the model nematodes Pristionchus pacificus and Caenorhabditis elegans. We show that B. thuringiensis 4A4 kills both nematodes via intestinal damage. Whole genome sequencing of B. thuringiensis 4A4 identified Cry21Ha, Cry1Ba, Vip1/Vip2 and β-exotoxin as potential nematicidal factors. Only Cry21Ha showed toxicity to C. elegans, while neither Cry nor Vip toxins were active against P. pacificus, when expressed in E. coli. Purified crystals also failed to intoxicate P. pacificus, while autoclaved spore-crystal mixture of B. thuringiensis 4A4 retained toxicity, suggesting that primary β-exotoxin is responsible for P. pacificus killing. In support of this, we found that a β-exotoxin-deficient variant of B. thuringiensis 4A4, generated by plasmid curing lost virulence to the nematodes. Thus, using two model nematodes we revealed virulence factors of the nematicidal strain B. thuringiensis 4A4 and showed the multifactorial nature of its virulence.