The Resistance of Bacillus Spores: Implications for the Strain-Specific Response to High-Performance Disinfectants.

Bacterial spores in materials and equipment pose significant biosecurity risks, making effective disinfection crucial. This study evaluated Ortho-phthalaldehyde (OPA) and a quaternary ammonia-glutaraldehyde solution (AG) for inactivating spores of Bacillus thuringiensis (BT), B. cereus (BC), and two strains of B. velezensis (BV1 and BV2). Spores of BV1 and BT were treated with 22.5 mg/m3 OPA by dry fumigation or 1 mg/mL AG by spray for 20 min, according to the manufacturer's recommendation. As no sporicidal effect was observed, OPA was tested at 112.5 mg/m3 for 40 min, showing effectiveness for BT but not for BV1. Minimum bactericidal concentration (MBC) tests revealed higher MBC values for glutaraldehyde, prompting an overnight test with 112.5 mg/m3 OPA by dry fumigation and 50 mg/mL AG by spray, using formaldehyde as a control. AG reduced all Bacillus strains, but with limited sporicidal effect. OPA was sporicidal for BT and BV1 but not for BC and BV2, indicating a strain-dependent effect. Formaldehyde performed better overall but did not completely inactivate BV2 spores. Our findings suggest that OPA and AG have potential as formaldehyde replacements in wet disinfection procedures.

Potential risk of biofilm-forming Bacillus cereus group in fresh-cut lettuce production chain.

Bacillus cereus and Bacillus thuringiensis, which belong to the B. cereus group, are widely distributed in nature and can cause food poisoning symptoms. In this study, we collected 131 isolates belonging to the B. cereus group, comprising 124B. cereus and seven B. thuringiensis isolates, from fresh-cut lettuce production chain and investigated their potential risk by analyzing genotypic (enterotoxin and emetic toxin gene profiles) and phenotypic (antibiotic susceptibility, sporulation, and biofilm formation) characteristics. Enterotoxin genes were present only in B. cereus, whereas the emetic toxin gene was not detected in any of the B. cereus isolates. All isolates were susceptible to vancomycin, which is a last resort for treating B. cereus group infection symptoms, but generally resistant to ß-lactam antimicrobials, and had the ability to form spores (at an average sporulation rate of 24.6 %) and biofilms at 30 °C. Isolates that formed strong biofilms at 30 °C had a superior possibility of forming a dense biofilm by proliferating at 10 °C compared to other isolates. Additionally, confocal laser scanning microscopy (CLSM) images revealed a notable presence of spores within the submerged biofilm formed at 10 °C, and the strengthened attachment of biofilm inner cells to the substrate was further revealed through biofilm structure parameters analysis. Collectively, our study revealed the prevalence and contamination levels of B. cereus and B. thuringiensis at fresh-cut lettuce production chain and investigated their genotypic and phenotypic characteristics, aiming to provide valuable insights for the development of potential risk management strategies to ensure food safety, especially along the cold chain.

Inoculation with Bacillus thuringiensis reduces uptake and translocation of Pb/Cd in soil-wheat system: A life cycle study.

Microbial inoculation is an important strategy to reduce the supply of heavy metals (HMs) in soil-crop systems. However, the mechanisms of microbial inoculation for the availability of HMs in soil and their accumulation/transfer in crops remain unclear. Here, the inhibitory effect of inoculation with Bacillus thuringiensis on the migration and accumulation of Pb/Cd in the soil-wheat system during the whole growth period was investigated by pot experiments. The results showed that inoculation with Bacillus thuringiensis increased soil pH and available nutrients (including carbon, nitrogen, and phosphorus), and enhanced the activities of nutrient-acquiring enzymes. Dominance analysis showed that dissolved organic matter (DOM) is the key factor affecting the availability of HMs. The content of colored spectral clusters and humification characteristics of DOM were significantly improved by inoculation, which is conducive to reducing the availability of Pb/Cd, especially during the flowering stage, the decrease was 12.8 %. Inoculation decreased Pb/Cd accumulation in the shoot and the transfer from root to shoot, with the greatest decreases at the jointing and seedling stages (27.0-34.1 % and 6.9-11.8 %), respectively. At the maturity stage, inoculation reduced the Pb/Cd accumulation in grain (12.9-14.7 %) and human health risk (4.1-13.2 %). The results of Pearson correlation analysis showed that the availability of Pb/Cd was positively correlated with the humification of DOM. Least square path model analysis showed that Bacillus thuringiensis could significantly reduce Pb/Cd accumulation in the grain and human health risks by regulating DOM spectral characteristics, the availability of HMs in soil and metals accumulation/transport in wheat at different growth stages. This study revealed the inhibition mechanism of Bacillus thuringiensis on migration of Pb/Cd in a soil-wheat system from a viewpoint of a full life cycle, which offers a valuable reference for the in-situ remediation of HM-contaminated soil and the safe production of food crops in field.

Biofilm-forming Ability of Bacillus thuringiensis Strains from Biopesticides on Polystyrene and their Attachment on Spinach.

Bacillus thuringiensis-based commercial products as a biopesticide have been used for more than 60 years in agriculture. However, as one of the species in B. cereus group, B. thuringiensis has been considered as an emerging hazard with the potential to cause food toxico-infections. The present study aimed to evaluate the biofilm-forming ability of B. thuringiensis biopesticide strains and their attachment on spinach, compared to foodborne B. cereus strains. Biofilm formations of tested strains were found to be strain-specific and affected by the nutrient conditions more than the incubation time. Nutrient starvation conditions generally reduced the biofilm formation of tested B. thuringiensis and B. cereus strains, particularly B. thuringiensis ABTS-1857 strain was found as the nonbiofilm former in starvation conditions. It is worth mentioning that B. thuringiensis SA-11 strain showed stronger biofilm-forming ability with more air-liquid interface biofilm than the other two B. thuringiensis biopesticide strains, but no such higher attachment of B. thuringiensis SA-11 to spinach was observed. These results indicate that B. thuringiensis SA-11 strain can enter the food processing lines by the attachment on spinach leaves, and it has the potential to form biofilms throughout the processing lines or the production environment when sufficient nutrients are available. However, more biofilm tests of B. thuringiensis biopesticide strains in the vegetable production chain should be performed. The dry formulation of commercial B. thuringiensis biopesticides enhanced their adhesion on spinach leaves, whereas the strength of adhesion was not improved by the formulation. In addition, 1-2 log reductions of spores after the intensive washing of spinach leaves in the lab were detected. However, the log reduction due to the actual washing done by the food processing companies in large-volume washing baths or by consumers at home would be limited and less than this lab simulation.

Differential proteome profiling of bacterial culture supernatants reveals candidates for the induction of oral immune priming in the red flour beetle.

Most organisms are host to symbionts and pathogens, which led to the evolution of immune strategies to prevent harm. Whilst the immune defences of vertebrates are classically divided into innate and adaptive, insects lack specialized cells involved in adaptive immunity, but have been shown to exhibit immune priming: the enhanced survival upon infection after a first exposure to the same pathogen or pathogen-derived components. An important piece of the puzzle are the pathogen-associated molecules that induce these immune priming responses. Here, we make use of the model system consisting of the red flour beetle (Tribolium castaneum) and its bacterial pathogen Bacillus thuringiensis, to compare the proteomes of culture supernatants of two closely related B. thuringiensis strains that either induce priming via the oral route, or not. Among the proteins that might be immunostimulatory to T. castaneum, we identify the Cry3Aa toxin, an important plasmid-encoded virulence factor of B. thuringiensis. In further priming-infection assays we test the relevance of Cry-carrying plasmids for immune priming. Our findings provide valuable insights for future studies to perform experiments on the mechanisms and evolution of immune priming.

Alteration of diet microbiota limits the experimentally evolved immune priming response in flour beetles, but not pathogen resistance.

Host-associated microbiota play a fundamental role in the training and induction of different forms of immunity, including inducible as well as constitutive components. However, direct experiments analysing the relative importance of microbiota on diverse forms of evolved immune functions are missing. We addressed this gap by using experimentally evolved lines of Tribolium castaneum that either produced inducible immune memory-like responses (immune priming) or constitutively expressed basal resistance (without priming), as divergent counterstrategies against Bacillus thuringiensis infection. We altered the microbial communities present in the diet (i.e. wheat flour) of these evolved lines using UV irradiation and estimated the impact on the beetle's ability to mount a priming response versus basal resistance. Populations that had evolved immune priming lost the ability to mount a priming response upon alteration of diet microbiota. Microbiota manipulation also caused a drastic reduction in their reproductive output and post-infection longevity. In contrast, in pathogen-resistant beetles, microbiota manipulation did not affect post-infection survival or reproduction. The divergent evolution of immune responses across beetle lines was thus associated with divergent reliance on the microbiome. Whether the latter is a direct outcome of differential pathogen exposure during selection or reflects evolved immune functions remains unclear. We hope that our results will motivate further experiments to understand the mechanistic basis of these complex evolutionary associations between microbiota, host immune strategies and fitness outcomes.

Bacillus thuringiensis-based bioinsecticides affect predation of Euborellia annulipes on diamondback moth larvae.

Interactions between earwigs and entomopathogens, such as Bacillus thuringiensis (Bt), are still poorly understood. This study tested whether Bt-based bioinsecticides have any effect on the predation of Euborellia annulipes (Lucas) (Dermaptera: Anisolabididae) on Plutella xylostella (L.) (Lepidoptera: Plutellidae), one of the pests with the largest number of cases of use and resistance to Bt. Fourth instar larvae were Bt infected by feeding on collard green leaves treated with Dipel®WG and XenTari®WG at the manufacturer-recommended doses. We used one no-choice condition, in which the predator had access to uninfected or Bt-infected larvae separately, and four free-choice conditions: uninfected vs Dipel®-infected larvae, uninfected vs XenTari®-infected larvae, Dipel®-infected vs XenTari®-infected larvae, and uninfected vs Bt-infected larvae with both bioinsecticides. Uninfected larvae were less consumed than those infected by both Bt-bioinsecticides in the no-choice condition. There was a higher consumption of uninfected over Dipel®-infected larvae in the free-choice condition. Overall, uninfected larvae were preferred over both Bt-based bioinsecticides infected larvae. We also used six different prey densities. The ringlegged earwig's predation rate enhanced as the prey population density increased, but the functional response was not affected by Bt-infection, being type II. The predator invested a low amount of handling time on Bt-fed prey and increased the maximum predation rate. Bt-based bioinsecticides cause effects on E. annulipes predation by altering their feeding preference and some aspects of its predatory behavior. The results of our study provide an important background for understanding interactions between earwigs and Bt. In addition, they can be used for decision making during approaches to integrated P. xylostella management.

Bacteria-derived pesticidal proteins active against hemipteran pests.

Hemipteran pests are among the most important threats to agricultural production. Losses associated with these insects result from both feeding-associated damage and the transmission of plant pathogens by some species. Key among hemipteran pests of agricultural importance are stink bugs, whitefly, aphids and psyllids. While bacteria provide an excellent resource for identification of environmentally benign pesticidal proteins for use against pest insects, relatively few with activity against hemipteran species have been identified. In this comprehensive review including the patent literature, we describe physiological features unique to Hemiptera that may restrict the toxicity of bacterial pesticidal proteins, provide an overview of Hemiptera-active pesticidal proteins and associated structural classes, and summarize biotechnological strategies used for optimization of toxicity against target hemipteran species.

Dose-response analysis of Bacillus thuringiensis HD-1 cry- spore reduction on surfaces using formaldehyde with pre-germination.

AIM: To establish a basis for rapid remediation of large areas contaminated with Bacillus anthracis spores. METHODS AND RESULTS: Representative surfaces of wood, steel and cement were coated by nebulization with B. thuringiensis HD-1 cry- (a simulant for B. anthracis) at 5.9 ± 0.2, 6.3 ± 0.2 and 5.8 ± 0.2 log10 CFU per cm2 , respectively. These were sprayed with formaldehyde, either with or without pre-germination. Low volume (equivalent to ≤2500 L ha-1 ) applications of formaldehyde at 30 g l-1 to steel or cement surfaces resulted in ≥4 or ≤2 log10 CFU per cm2 reductions respectively, after 2 h exposure. Pre-germinating spores (500 mmol l-1 l-alanine and 25 mmol l-1 inosine, pH 7) followed by formaldehyde application showed higher levels of spore inactivation than formaldehyde alone with gains of up to 3.4 log10 CFU per cm2 for a given dose. No loss in B. thuringiensis cry- viability was measured after the 2 h germination period, however, a pre-heat shock log10 reduction was seen for B. anthracis strains: LSU149 (1.7 log10), Vollum and LSU465 (both 0.9 log10), LSU442 (0.2 log10), Sterne (0.8 log10) and Ames (0.6 log10). CONCLUSIONS: A methodology was developed to produce representative spore contamination of surfaces along with a laboratory-based technique to measure the efficacy of decontamination. Dose-response analysis was used to optimize decontamination. Pre-germinating spores was found to increase effectiveness of decontamination but requires careful consideration of total volume used (germinant and decontaminant) by surface type. SIGNIFICANCE AND IMPACT OF THE STUDY: To be practically achievable, decontamination of a wide area contaminated with B. anthracis spores must be effective, timely and minimize the amount of materials required. This study uses systematic dose-response methodology to demonstrate that such an approach is feasible.

Cd exposure-triggered susceptibility to Bacillus thuringiensis in Lymantria dispar involves in gut microbiota dysbiosis and hemolymph metabolic disorder.

The immunotoxicity induced by heavy metals on herbivorous insects reflect the alterations of the susceptibility to entomopathogenic agents in herbivorous insects exposed to heavy metal. In the present study, the susceptibility of gypsy moth larvae to Bacillus thuringiensis under Cd treatment at low and high dosages was investigated, and the gut microbiome-hemolymph metabolome responses that affected larval disease susceptibility caused by Cd exposure were examined. Our results showed that mortality of gypsy moth larvae caused by B. thuringiensis was significantly higher in larvae pre-exposed to Cd stress, and there was a synergistic effect between Cd pre-exposure and bacterial infection. Exposure to Cd significantly decreased the abundance of several probiotics (e.g., Serratia for the low Cd dosage and Weissella, Aeroonas, and Serratia for the high Cd dosage) and increased the abundances of several pathogenic bacteria (e.g., Stenotrophomonas, Gardnerella, and Cutibacterium for the low Cd dosage and Pluralibacter and Tsukamurella for the high Cd dosage) compared to the controls. Moreover, metabolomics analysis indicated that amino acid biosynthesis and metabolism were significantly perturbed in larval hemolymph under Cd exposure at both the low and high dosages. Correlation analysis demonstrated that several altered metabolites in larval hemolymph were significantly correlated with changes in the gut microbial community. The results demonstrate that prior exposure to Cd increases the susceptibility of gypsy moth larvae to B. thuringiensis in a synergistic fashion due to gut microbiota dysbiosis and hemolymph metabolic disorder, and thus microbial-based biological control may be the best pest control strategy in heavy metal-polluted areas.

A specific primed immune response in red palm weevil, Rhynchophorus ferrugineus, is mediated by hemocyte differentiation and phagocytosis.

Red palm weevil, Rhynchophorus ferrugineus, is an invasive and destructive pest that causes serious damages to palm trees. Like other invertebrates, red palm weevil relies solely on its innate immune response to fight invading microbes; by definition, innate immunity lacks adaptive characteristics. However, we show here that priming the red palm weevil larvae with heat-killed Bacillus thuringiensis specifically increased survival of the larvae during a secondary lethal infection with live bacteria, and B. thuringiensis primed larvae also showed a higher clearance efficiency for this bacterium, which indicated that the red palm weevil larvae possessed a strong immune priming response. The degree of enhanced immune protection was positively correlated with hemocyte proliferation and the level of phagocytic ability of hemocytes. Moreover, the red palm weevil larvae primed by B. thuringiensis induced the continuous synthesis of serotonin in the hemolymph, which in turn enhanced the phagocytic ability and pathogen clearance ability of the host, representing an important mechanism for the red palm weevil to achieve priming protection. Our findings reveal a specific immune priming of the red palm weevil larvae mediated by the continuous secretion of serotonin, and provide new insights into the mechanisms of invertebrates immune priming.

The costs and benefits of basal infection resistance vs immune priming responses in an insect.

In insects, basal pathogen resistance and immune priming can evolve as mutually exclusive strategies, with distinct infection outcomes. However, the evolutionary drivers of such diverse immune functions remain poorly understood. Here, we addressed this key issue by systematically analyzing the differential fitness costs and benefits of priming vs resistance evolution in Tribolium beetle populations infected with Bacillus thuringiensis. Surprisingly, resistant beetles had increased post-infection reproduction and a longer lifespan under both starving as well as fed conditions, with no other measurable costs. In contrast, priming reduced offspring early survival, development rate and reproduction. Priming did improve post-infection survival of offspring, but this added trans-generational benefit of immune priming might not compensate for its pervasive costs. Resistance was thus consistently more beneficial. Overall, our work demonstrates the evolutionary change in trans-generational priming response, and provides a detailed comparison of the complex fitness consequences of evolved priming vs resistance.

ABCC2 is a functional receptor of Bacillus thuringiensis Cry1Ca in Spodoptera litura.

Spodoptera litura is a serious polyphagous pest in the whole world, which has developed resistance to most conventional insecticides and even some Bacillus thuringiensis (Bt) toxins. Cry1Ca has excellent insecticide activity against S. litura with potential application to control S. litura and delay the development of insect resistance. However, the mode of action of Cry1Ca in S. litura is poorly understood. Here, Cry1Ca-binding proteins were identified from S. litura by using pull down assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicated that aminopeptidase-N (APN), ATP binding cassette subfamily C member 2 (ABCC2), polycalin, actin and V-type proton ATPase subunit A may bind with Cry1Ca. Further study confirmed that ABCC2 fragment expressed in vitro can bind to Cry1Ca as demonstrated by Ligand blot and homologous competition experiments. The over-expression of endogenous SlABCC2 in Sf9 cells increased Cry1Ca cytotoxicity. Correspondingly, the vivo loss of function analyses by SlABCC2 small interfering RNAs (siRNAs) in S. litura larvae decreased the toxicity of Cry1Ca to larvae. Altogether, these results show that ABCC2 of S. litura is a functional receptor that is involved in the action mode of Cry1Ca.

Genome-wide analysis of V-ATPase genes in Plutella xylostella (L.) and the potential role of PxVHA-G1 in resistance to Bacillus thuringiensis Cry1Ac toxin.

The rapid development of insecticide resistance has hampered the use of Bacillus thuringiensis (Bt), a widely used bio-pesticide. Plutella xylostella (L.) is a globally distributed lepidopteran pest of cruciferous vegetables and has developed severe field resistance to the Bt toxin. Vacuolar H+-ATPases (VHA) are multi-subunit complexes and participate in multiple physiological processes. However, the characterization and functional studies of VHA genes are lacking in insects. This study performed a genome-wide analysis and identified 35 VHA gene family members divided into 15 subfamilies in P. xylostella. We cloned a V-ATPase subunit G gene, PxVHA-G1, in our previous midgut transcriptome profiles. Quantitative reverse transcriptase-polymerase chain reaction results showed that PxVHA-G1 was upregulated in the Cry1S1000-resistant strain than in the G88-susceptible strain, and its expression profile revealed that the midgut, Malpighian tubules, and larva stages generally showed high expression levels. RNAi-mediated knockdown of the PxVHA-G1 gene increased the susceptibility of P. xylostella (G88 and Cry1S1000) to Cry1Ac toxin. Our study is the first to explore the role of PxVHA-G1 on regulating Cry1Ac toxicity in P. xylostella, thus, providing new insights into the role of VHAs in the development of Cry1Ac resistance and sustainable development of pest management.

Bacillus thuringiensis Spores and Cry3A Toxins Act Synergistically to Expedite Colorado Potato Beetle Mortality.

The insect integument (exoskeleton) is an effective physiochemical barrier that limits disease-causing agents to a few portals of entry, including the gastrointestinal and reproductive tracts. The bacterial biopesticide Bacillus thuringiensis (Bt) enters the insect host via the mouth and must thwart gut-based defences to make its way into the body cavity (haemocoel) and establish infection. We sought to uncover the main antibacterial defences of the midgut and the pathophysiological features of Bt in a notable insect pest, the Colorado potato beetle Leptinotarsa decemlineata (CPB). Exposing the beetles to both Bt spores and their Cry3A toxins (crystalline δ-endotoxins) via oral inoculation led to higher mortality levels when compared to either spores or Cry3A toxins alone. Within 12 h post-exposure, Cry3A toxins caused a 1.5-fold increase in the levels of reactive oxygen species (ROS) and malondialdehyde (lipid peroxidation) within the midgut - key indicators of tissue damage. When Cry3A toxins are combined with spores, gross redox imbalance and 'oxidation stress' is apparent in beetle larvae. The insect detoxification system is activated when Bt spores and Cry3A toxins are administered alone or in combination to mitigate toxicosis, in addition to elevated mRNA levels of candidate defence genes (pattern-recognition receptor, stress-regulation, serine proteases, and prosaposin-like protein). The presence of bacterial spores and/or Cry3A toxins coincides with subtle changes in microbial community composition of the midgut, such as decreased Pseudomonas abundance at 48 h post inoculation. Both Bt spores and Cry3A toxins have negative impacts on larval health, and when combined, likely cause metabolic derangement, due to multiple tissue targets being compromised.

Role of Sesamia nonagrioides and Ostrinia nubilalis as Vectors of Fusarium spp. and Contribution of Corn Borer-Resistant Bt Maize to Mycotoxin Reduction.

Maize expressing Cry1Ab insecticidal toxin (Bt maize) is an effective method to control Sesamia nonagrioides and Ostrinia nubilalis, the most damaging corn borers of southern Europe. In this area, maize is prone to Fusarium infections, which can produce mycotoxins that pose a serious risk to human and animal health, causing significant economic losses in the agrifood industry. To investigate the influence of corn borer damage on the presence of Fusarium species and their mycotoxins, Bt maize ears and insect-damaged ears of non-Bt maize were collected from commercial fields in three Bt maize growing areas in Spain, and differences in contamination were assessed. Additionally, larvae of both borer species were collected to evaluate their role as vectors of these molds. Non-Bt maize ears showed significantly higher presence of F. verticillioides, F. proliferatum, and F. subglutinans than Bt maize ears. For the first time, Fusarium species have been isolated from larvae of the two species. The most frequently found mycotoxins in ears were fumonisins, with non-Bt ears being significantly more contaminated than those of Bt maize. High levels of fumonisins were shown to correlate with the occurrence of corn borers in the ear and the presence of F. verticillioides and F. proliferatum.

Tandem Mass Tag-Based Quantitative Proteomics and Virulence Phenotype of Hemolymph-Treated Bacillus thuringiensis kurstaki Cells Reveal New Insights on Bacterial Pathogenesis in Insects.

The spore-forming bacterium Bacillus thuringiensis (Bt) of the Bacillus cereus group uses toxin-opened breaches at the insect midgut epithelium to infest the hemolymph, where it can rapidly propagate despite antimicrobial host defenses and induce host death by acute septicemia. The response of Bt to host hemolymph and the latter's role in bacterial pathogenesis is an area that needs clarification. Here, we report a proteomic analysis of the Bt kurstaki strain HD73 (Btk) hemolymph stimulon showing significant changes in 60 (34 up- and 26 downregulated) differentially accumulated proteins (DAPs). Gene ontology (GO) enrichment analysis revealed that DAPs were mainly related to glutamate metabolism, transketolase activity, and ATP-dependent transmembrane transport. KEGG analysis disclosed that DAPs were highly enriched in the biosynthesis of bacterial secondary metabolites, ansamycins. Interestingly, about 30% of all DAPs were in silico predicted as putative virulence factors. Further characterization of hemolymph effects on Btk showed enhanced autoaggregation in liquid cultures and biofilm formation in microtiter polystyrene plates. Hemolymph-exposed Btk cells were less immunogenic in mice, suggesting epitope masking of selected surface proteins. Bioassays with intrahemocoelically infected Bombyx mori larvae showed that hemolymph preexposure significantly increased Btk toxicity and reproduction within the insect (spore count per cadaver) at low inoculum doses, possibly due to 'virulence priming'. Collectively, our findings suggest that the Btk hemolymph stimulon could be partially responsible for bacterial survival and propagation within the hemolymph of infected insects, contributing to its remarkable success as an entomopathogen. All mass spectrometry data are available via ProteomeXchange with identifier PXD021830. IMPORTANCE After ingestion by a susceptible insect and damaging its midgut epithelium, the bacterium Bacillus thuringiensis (Bt) reaches the insect blood (hemolymph), where it propagates despite the host's antimicrobial defenses and induces insect death by acute septicemia. Although the hemolymph stage of the Bt toxic pathway is determinant for the infested insects' fate, the response of Bt to hemolymph and the latter's role in bacterial pathogenesis has been poorly explored. In this study, we identified the bacterial proteins differentially expressed by Bt after hemolymph exposure. We found that about 30% of hemolymph-regulated Bt proteins were potential virulence factors, including manganese superoxide dismutase, a described inhibitor of hemocyte respiratory burst. Additionally, contact with hemolymph enhanced Bt virulence phenotypes, such as cell aggregation and biofilm formation, altered bacterial immunogenicity, and increased Bt toxicity to intrahemocoelically injected insects.

ATP-binding cassette transporter subfamily C members 2, 3 and cadherin protein are susceptibility-determining factors in Bombyx mori for multiple Bacillus thuringiensis Cry1 toxins.

Field-evolved resistance of insect pests to Bacillus thuringiensis (Bt) toxins (Cry toxins) is a threat to the efficacy of Bt-based bio-insecticides and transgenic crops. Recent reports have suggested that ATP-binding cassette transporter subfamily C2 (ABCC2) and cadherin-like receptor play important roles in conferring susceptibility to Cry1 toxins. However, the receptors involved in Bt susceptibility in each insect remain unclear. To determine the receptors that are involved in the susceptibility of Bombyx mori to Cry1 toxins (1Ab, 1Ac and 1Fa), we conducted diet overlay bioassay using B. mori strains disrupted with one or two receptor (s) among BmABCC2, BmABCC3, and cadherin-like receptor (BtR175) generated by transcription activator-like effector nuclease (TALEN)-mediated gene editing. The single-knockout strains for BmABCC2 showed resistance to Cry1Ab and Cry1Ac, whereas only strains with double knockout of BmABCC2 and BmABCC3 exhibited high resistance to Cry1Fa. Progeny populations generated from the crossing of heterozygotes for BtR175 knockout allele included 25% theoretical homozygotes for the BtR175 knockout allele and they showed resistance to Cry1Ab and Cry1Ac. Then, through a cell swelling assay using Sf9 cells ectopically expressing the receptor, we analyzed the mechanisms underlying the different contributions of BmABCC2, BmABCC3, and BtR175 to larval susceptibility. The receptor activity of BmABCC2 for Cry1Ab and Cry1Ac was far higher than that of BmABCC3, and BtR175 synergistically enhanced the receptor activity of BmABCC2. This result well explained the important involvement of BmABCC2 and BtR175 in the larval susceptibility to Cry1A toxins. By contrast, the receptor activities of BmABCC2 and BmABCC3 for Cry1Fa were observed at a similar level and synergistic effect of BtR175 was small. This finding explains the equal importance of BmABCC2 and BmABCC3 and very small contribution of BtR175 on larval susceptibility to Cry1Fa. Thus, we demonstrated the different importance of BmABCC2, BmABCC3, and BtR175 to various Cry1 toxins as susceptibility-determining factors in B. mori larvae and the underlying basis for the observed differences. Furthermore, a weak correlation was indicated between the binding affinity and receptor activities of BmABCC2 and BmABCC3 to Cry1 toxins.

Comparative genome analysis of Bacillus thuringiensis strain HD521 and HS18-1.

Bacillus thuringiensis (Bt) is an important biological insecticide used to management of different agricultural pests by producing toxic parasporal crystals proteins. Strain HD521 has an antagonistic effect against Rhizoctonia solani AG1IA, the causal agent of rice sheath blight. This strain with three cry7 genes can the formation of bipyramidal parasporal crystals (BPCs). BPCs are used for insecticidal activities against Henosepilachna vigintioctomaculata larva (Coleoptera). Strain HS18-1 contains different types of BPCs encoding genes and has effective toxicity for Lepidoptera and Diptera insects. Here we report the whole genome sequencing and assembly of HD521 and HS18-1 strains and analyzed the genome constitution covering virulence factors, types of plasmid, insertion sequences, and prophage sequences. The results showed that the genome of strain HD521 contains a circular chromosome and six circular plasmids, encoding eight types of virulence protein factors [Immune Inhibitor A, Hemolytic Enterotoxin, S-layer protein, Phospholipase C, Zwittermicin A-resistance protein, Metalloprotease, Chitinase, and N-acyl homoserine lactonase (AiiA)], four families of insertion sequence, and comprises six pro-phage sequences. The genome of strain HS18-1 contains one circular chromosome and nine circular plasmids, encoding five types of virulence protein factors [Hemolytic Enterotoxin, S-layer protein, Phospholipase C, Chitinase, and N-acyl homoserine lactonase (AiiA)] and four families of insertion sequence, and comprises of three pro-phage sequences. The obtained results will contribute to deeply understand the B. thuringiensis strain HD521 and HS18-1 at the genomic level.

Polyurethane foam as an inert support using concentrated media improves quality and spore production from Bacillus thuringiensis.

Bacillus thuringiensis (Bt) (Bacillales:Bacillaceae) is a gram-positive bacterium that produces spores, several virulence factors and insecticidal toxins, making this microorganism the most used biopesticide worldwide. The use of inert supports such as polyurethane foam (PUF) in solid cultures has been a great alternative to produce various metabolites, including those produced by Bt. In this study we compared the yields, productivity and quality of the spores by two wild strains of Bt, (Y15 and EA3), grown in media with high substrate concentration in both culture systems: liquid and solid (PUF as solid inert support). Both strains showed 2.5- to 30-fold increases in spore production and productivity in solid culture, which showed an even greater increase when considering the spores retained in the PUF observed by scanning electron microscopy. Moreover, spore produced in solid culture showed up to sevenfold higher survival after a heat-shock treatment, relative to spores from liquid culture. The infectivity against larvae of Galleria mellonella (Lepidoptera:Pyralidae) improved also in spores from solid cultures. This comparison showed that the culture of Bt on solid support has clear advantages over liquid culture in terms of the production and quality of spores, and that those advantages can be attributed only to the culture system, as the same media composition was used in both systems.