Granulocyte dynamics: a key player in the immune priming effects of crickets.

This study investigates immune priming effects associated with granulocytes in crickets through a comprehensive analysis. Kaplan-Meier survival analysis reveals a significant contrast in survival rates, with the heat-killed Bacillus thuringiensis (Bt)-primed group exhibiting an impressive ~80% survival rate compared to the PBS buffer-primed group with only ~10% survival 60 hours post live Bt infection. Hemocyte analysis underscores elevated hemocyte counts, particularly in granulocytes of the killed Bt-primed group, suggesting a correlation between the heat-killed Bt priming and heightened immune activation. Microscopy techniques further explore granulocyte morphology, unveiling distinctive immune responses in the killed Bt-primed group characterized by prolonged immune activation, heightened granulocyte activity, phagocytosis, and extracellular trap formation, contributing to enhanced survival rates. In particular, after 24 hours of injecting live Bt, most granulocytes in the PBS buffer-primed group exhibited extracellular DNA trap cell death (ETosis), while in the killed Bt-primed group, the majority of granulocytes were observed to maintain highly activated extracellular traps, sustaining the immune response. Gene expression analysis supports these findings, revealing differential regulation of immune-related genes such as antibacterial humoral response, detection of bacterial lipopeptides, and cellular response to bacteria lipopeptides. Additionally, the heat-killed Bt-primed group, the heat-killed E. coli-primed group, and the PBS-primed group were re-injected with live Bt 2 and 9 days post priming. Two days later, only the PBS-primed group displayed low survival rates. After injecting live Bt 9 days later, the heat-killed E. coli-primed group surprisingly showed a similarly low survival rate, while the heat-killed Bt-primed group exhibited a high survival rate of ~60% after 60 hours, with actively moving and healthy crickets. In conclusion, this research provides valuable insights into both short-term and long-term immune priming effects in crickets, contributing to our understanding of invertebrate immunity with potential applications in public health.

Immune Inhibitor A Metalloproteases Contribute to Virulence in Bacillus Endophthalmitis.

Endophthalmitis is a devastating infection that can cause blindness. Over half of Bacillus endophthalmitis cases result in significant loss of useful vision. Bacillus produces many virulence factors that may contribute to retinal damage and robust inflammation. We analyzed Bacillus immune inhibitor A (InhA) metalloproteases in the context of this disease, hypothesizing that InhAs contribute to Bacillus intraocular virulence and inflammation. We analyzed phenotypes and infectivity of wild-type (WT), InhA1-deficient (ΔinhA1), InhA2-deficient (ΔinhA2), or InhA1, A2, and A3-deficient (ΔinhA1-3) Bacillus thuringiensis. In vitro analysis of growth, proteolysis, and cytotoxicity were compared. WT and InhA mutants were similarly cytotoxic to retinal cells. The ΔinhA1 and ΔinhA2 mutants entered log-phase growth earlier than WT B. thuringiensis. Proteolysis by the ΔinhA1-3 mutant was decreased, but this strain grew similar to WT in vitro. Experimental endophthalmitis was initiated by intravitreally infecting C57BL/6J mice with 200 CFU of WT B. thuringiensis or InhA mutants. Eyes were analyzed for intraocular Bacillus and myeloperoxidase concentrations, retinal function loss, and gross histological changes. Eyes infected with the ΔinhA1 or ΔinhA2 mutant strains contained greater numbers of bacteria than eyes infected with WT throughout the infection course. Eyes infected with single mutants had inflammation and retinal function loss similar to eyes infected with the WT strain. Eyes infected with the ΔinhA1-3 mutant cleared the infection. Quantitative real-time PCR (qRT-PCR) results suggested that there may be compensatory expression of the other InhAs in the single InhA mutant. These results indicate that together, the InhA metalloproteases contribute to the severity of infection and inflammation in Bacillus endophthalmitis.

Differential capability of Bacillus thuringiensis Cry1Ac protoxin and toxin to induce in vivo activation of dendritic cells and B lymphocytes.

The insecticidal Bacillus thuringiensis protein Cry1Ac is produced as a protoxin and becomes activated to a toxin when ingested by larvae. Both proteins are immunogenic and able to activate macrophages. The proposed mechanism of immunostimulation by Cry1Ac protoxin has been related to its capacity to activate antigen-presenting cells (APC), but its ability to activate dendritic cells (DC) has not been explored. Here we evaluated, in the popliteal lymph nodes (PLN), spleen and peritoneum, the activation of DC CD11c+ MHC-II+ following injection with single doses (50 µg) of Cry1Ac toxin or protoxin via the intradermal (i.d.) and intraperitoneal (i.p.) routes in C57BL/6 mice. In vivo stimulation with both Cry1Ac proteins induced activation of DC via upregulation of CD86, primarily in PLN 24 h after i. d. injection. Moreover, this activation was detected in DC, displaying CD103+, a typical marker of migratory DC, while upregulation of CD80 was uniquely induced by toxin. Tracking experiments showed that Cy5-labeled Cry1Ac proteins could rapidly reach the PLN and localize near DC, but some label remained in the footpad. When the capacity of Cry1Ac-activated DC to induce antigen presentation was examined, significant proliferation of naïve T lymphocytes was induced exclusively by the protoxin. The protoxin elicited a Th17-biased cytokine profile. Moreover, only the Cry1Ac toxin induced a pronounced proliferation of B cells from both untreated and Cry1Ac-injected mice, suggesting that it acts as a polyclonal activator. In conclusion, Cry1Ac protoxin and toxin show a distinctive capacity to activate APCs.

Molecular characterization and expression patterns of heat shock proteins in Spodoptera littoralis, heat shock or immune response?

The Egyptian cotton leaf worm, Spodoptera littoralis (Boisd.), is a major agricultural lepidopterous pest causing extensive damage in a variety of crops including vegetable, cotton, fodder, and fiber crops. Heat shock protein (HSP) family members play important roles in protecting insects against environmental stressors. In this study, we characterized three putative heat shock proteins (SpliHsp70, SpliHsp90, and SpliHSF) from S. littoralis and analyzed their expression levels in response to heat, cold, ultraviolet irradiation, Bacillus thuringiensis, and Spodoptera littoralis nucleopolyhedrovirus treatments. Significant upregulation of SpliHsp70 was observed in female pupae, while the highest expression levels of SpliHsp90 and SpliHSF were found in female adults. Heat shock triggered increases in SpliHsp levels compared to cold treatment. SpliHsp90 exhibited the highest expression levels during the first 30 min of UV treatment. Both bacterial and viral pathogenic agents effected the regulation of Hsps in S. littoralis. These findings suggest that SpliHsp genes might play significant roles in the response to biotic and abiotic stress, as well as in the regulation of developmental stages.

The Tripartite Interaction of Host Immunity-Bacillus thuringiensis Infection-Gut Microbiota.

Bacillus thuringiensis (Bt) is an important cosmopolitan bacterial entomopathogen, which produces various protein toxins that have been expressed in transgenic crops. The evolved molecular interaction between the insect immune system and gut microbiota is changed during the Bt infection process. The host immune response, such as the expression of induced antimicrobial peptides (AMPs), the melanization response, and the production of reactive oxygen species (ROS), varies with different doses of Bt infection. Moreover, B. thuringiensis infection changes the abundance and structural composition of the intestinal bacteria community. The activated immune response, together with dysbiosis of the gut microbiota, also has an important effect on Bt pathogenicity and insect resistance to Bt. In this review, we attempt to clarify this tripartite interaction of host immunity, Bt infection, and gut microbiota, especially the important role of key immune regulators and symbiotic bacteria in the Bt killing activity. Increasing the effectiveness of biocontrol agents by interfering with insect resistance and controlling symbiotic bacteria can be important steps for the successful application of microbial biopesticides.

Comparative genomic analysis and mosquito larvicidal activity of four Bacillus thuringiensis serovar israelensis strains.

Bacillus thuringiensis serovar israelensis (Bti) is used to control insect vectors of human and animal diseases. In the present study, the toxicity of four strains of Bti, named T0124, T0131, T0137, and T0139, toward Aedes aegypti and Culex quinquefasciatus larvae was analyzed. The T0131 strain showed the highest larvicidal activity against A. aegypti (LC50 = 0.015 µg/ml) and C. quinquefasciatus larvae (LC50 = 0.035 µg/ml) when compared to the other strains. Furthermore, the genomic sequences of the four strains were obtained and compared. These Bti strains had chromosomes sizes of approximately 5.4 Mb with GC contents of ~35% and 5472-5477 putative coding regions. Three small plasmids (5.4, 6.8, and 7.6 kb) and three large plasmids (127, 235, and 359 kb) were found in the extrachromosomal content of all four strains. The SNP-based phylogeny revealed close relationship among isolates from this study and other Bti isolates, and SNPs analysis of the plasmids 127 kb did not reveal any mutations in δ-endotoxins genes. This newly acquired sequence data for these Bti strains may be useful in the search for novel insecticidal toxins to improve existing ones or develop new strategies for the biological control of important insect vectors of human and animal diseases.

Regulator ThnR and the ThnDE ABC transporter proteins confer autoimmunity to thurincin H in Bacillus thuringiensis.

The structural gene that encodes thurincin H, a bacteriocin produced by Bacillus thuringiensis, is harboured in a genetic cluster (thnP, E, D, R, A1, A2, A3, B, T, I) that controls its synthesis, modification, secretion and autoimmunity. The specific genes in the cassette that confer immunity in B. thuringiensis to thurincin H are unknown. To identify these immunity determinants, we generated constructs that were used to transform a natural thurincin H-sensitive B. thuringiensis strain (i.e. Btk 404), and resistance or susceptibility to the bacteriocin in resultant recombinants was evaluated. When Btk 404/pHT3101-ThnARDEP and Btk 404/pHT3101-ThnABTI were exposed to thurincin H, immunity was demonstrated by the former only, indicating that ThnI does not play a role in resistance to the bacteriocin as previously proposed. Furthermore, we generated different sub-cassettes under the control of divergent promoters pThnR and pThur of the thurincin H locus, and pChi, and using the green fluorescent protein gene as reporter, which demonstrated that all promoters were recognised by ThnR, except pChi. We show for the first time that the small operon composed of thnR, thnD and thnE is required for immunity of B. thuringiensis to thurincin H, and thnI is not necessary for this response.

Trans-generational immune priming in the mealworm beetle protects eggs through pathogen-dependent mechanisms imposing no immediate fitness cost for the offspring.

Immune-challenged mothers can improve their offspring immunity through trans-generational immune priming (TGIP). In insects, TGIP endows the offspring with lifetime immunity, including the eggs, which are likely exposed soon after maternal infection. Egg protection may rely on the transfer of maternal immune effectors to the egg or/and the induction of egg immune genes. These respective mechanisms are assumed to have early-life fitness costs of different magnitude for the offspring. We provide evidence in the mealworm beetle Tenebrio molitor that enhanced egg immunity following a maternal immune challenge is achieved by both of these mechanisms but in a pathogen-dependent manner. While previously found having late-life fitness costs for the offspring, TGIP here improved egg hatching success and early larval survival, in addition of improving offspring immunity. These results suggest that early-life of primed offspring is critical in the optimization of life history trajectory of this insect under trans-generational pathogenic threats.

A dietary carotenoid reduces immunopathology and enhances longevity through an immune depressive effect in an insect model.

Immunopathology corresponds to self-damage of the inflammatory response, resulting from oxidizing molecules produced when the immune system is activated. Immunopathology often contributes to age-related diseases and is believed to accelerate ageing. Prevention of immunopathology relies on endogenous antioxidant enzymes and the consumption of dietary antioxidants, including carotenoids such as astaxanthin. Astaxanthin currently raises considerable interest as a powerful antioxidant and for its potential in alleviating age-related diseases. Current in vitro and short-term in vivo studies provide promising results about immune-stimulating and antioxidant properties of astaxanthin. However, to what extent dietary supplementation with astaxanthin can prevent long-term adverse effects of immunopathology on longevity is unknown so far. Here, using the mealworm beetle, Tenebrio molitor, as biological model we tested the effect of lifetime dietary supplementation with astaxanthin on longevity when exposed to early life inflammation. While supplementation with astaxanthin was found to lessen immunopathology cost on larval survival and insect longevity, it was also found to reduce immunity, growth rate and the survival of non immune-challenged larvae. This study therefore reveals that astaxanthin prevents immunopathology through an immune depressive effect and can have adverse consequences on growth.

Construction of an Immunized Rabbit Phage Display Library for Selecting High Activity against Bacillus thuringiensis Cry1F Toxin Single-Chain Antibodies.

In the present study, a Cry1F-immunized rabbit phage display library (6.96 × 108 cfu/mL) was constructed for selecting high activity of anti-Cry1F toxin single-chain antibody (a single-chain variable fragment, scFv) by biopanning. A total of 16 positive monoclonal phage scFv's were obtained after 4 rounds of panning, which were identified by enzyme-linked immunosorbent assay (ELISA), polymerized chain reaction, and DNA sequencing. The most positive phage scFv (named RF4) was expressed in Escherichia coli HB2151, and a soluble protein of approximately 30 kDa was purified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An indirect competitive ELISA (IC-ELISA) was established on the basis of purified soluble RF4-scFv for Cry1F toxin. It indicated the 50% inhibition of the control (IC50) was 11.56 ng/mL and the detection limit (IC10) was 0.18 ng/mL and showed weak cross-reactivities for Cry1Ab (2.8%), Cry1Ac (1.3%), and Cry1B, Cry1C, Cry1Ie, and Cry2A (less than 0.1%). It was found that IC-ELISA detected Cry1F toxin spiked in rice, wheat, corn, and soil samples with good accuracy, stability, and repeatability. The recoveries were in the range of 80.2-99.6%, and the coefficients of variation were in the range of 2.5-10.0%. These results showed that IC-ELISA based on scFv from the immunized rabbit phage display library was promising for specific detection of Cry1F toxin in agroproducts and environmental samples.

Enhanced behavioral immune defenses in a C. elegans C-type lectin-like domain gene mutant.

C-type lectin-like domain (CTLD) proteins occupy crucial functions in the immune system of vertebrates, but their role in invertebrate immunity is much less understood. The nematode Caenorhabditis elegans possesses a highly diverse CTLD protein encoding (clec) gene repertoire. A role of C. elegans clec genes in pathogen defense is always assumed, yet experimental evidence for clec immune function is rare. To systematically test the potential function of clec genes in the C. elegans defense against pathogens, we screened 39 clec mutants for survival on the Gram-positive pathogen Bacillus thuringiensis (BT18247) and 37 clec mutants on the Gram-negative pathogen Pseudomonas aeruginosa (PA14). We found that clec mutants can exhibit either decreased or, unexpectedly, increased resistance to infection. Since we observed high escape behavior for some of the clec mutants on BT18247 during the initial screen, we then asked if increased pathogen avoidance behavior underlies the increased resistance of some clec mutants. We thus tested lawn leaving behavior of the resistant clec-29(ok3181), clec-34(ok2120), clec-151(ok2264), and C54G4.4(ok2110) mutant on BT18247. We found that C54G4.4(ok2110) mutant animals exhibit a particularly strong lawn leaving behavior, in addition to prolonged feeding cessation when exposed to BT18247. Together, our results indicate that clec genes mediate both resistance and susceptibility to infection. Further, behavioral analyses of the C54G4.4(ok2110) mutant implicate C54G4.4 in the regulation of pathogen avoidance behavior towards BT18247. We conclude that C. elegans clec genes may act both as positive and negative regulators of physiological as well as behavioral immune defense responses.

Simultaneous production of monoclonal antibodies against Bacillus thuringiensis (Bt) Cry1 toxins using a mixture immunization.

The detections of Cry1 toxins are mainly dependent on immunoassays based on specific monoclonal antibodies (mAb). In the present study, a mixture immunization with seven Cry1 toxins was administered. The results showed that five mAbs with different characteristics, especially one mAb named 5-E8 which could recognize all the seven Cry1 toxins were obtained. Based on the 5-E8 mAb, a double antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) which can specifically detect the seven Cry1 toxins without cross-reactivity to Cry2A and vip3 was developed with the limit of detection (LOD) and limit of quantification (LOQ) of 6.37-11.35 ng mL-1 and 17.36-24.48 ng mL-1, respectively. The recovery tests showed that the recoveries ranged from 78% to 110% within the quantitation range (LOQ-100 ng mL-1). The established DAS-ELISA can be a useful tool for monitoring the Cry1 toxins in agricultural products. Mixture immunization opens a new path for producing diverse mAbs simultaneously in a single immunization circle.

Cry1A(b)16 toxin from Bacillus thuringiensis: Theoretical refinement of three-dimensional structure and prediction of peptides as molecular markers for detection of genetically modified organisms.

Transgenic maize produced by the insertion of the Cry transgene into its genome became the second most cultivated crop worldwide. Cry gene from Bacillus thuringiensis kurstaki expresses protein derivatives of crystalline endotoxins which confer insect resistance onto the maize crop. Mandatory labeling of processed food containing or made by genetically modified organisms is in force in many countries, so, it is very urgent to develop fast and practical methods for GMO identification, for example, biosensors. In the absence of an available empirical structure of Cry1A(b)16 protein, a theoretical model was effectively generated, in this work, by homology modeling and molecular dynamics simulations based on two available homologous protein structures. Molecular dynamics simulations were carried out to refine the selected model, and an analysis of its global structure was performed. The refined models of Cry1A(b)16 showed a standard fold and structural characteristics similar to those seen in Bacillus thuringiensis Cry1A(a) insecticidal toxin and Bacillus thuringiensis serovar kurstaki Cry1A(c) toxin. After in silico analysis of Cry1A(b)16, two immunoreactive candidate peptides were selected and specific polyclonal antibodies were produced resulting in antibody-peptide interaction. Biosensing devices are expected to be developed for detection of the Cry1A(b) protein as a marker of transgenic maize in food. Proteins 2017; 85:1248-1257. © 2017 Wiley Periodicals, Inc.

Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model.

Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab's amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10-8 M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL-1, respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5-100 ng mL-1), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products. Graphical Abstract Establishment of a DAS-ELISA for the specific detecting of Bacillus thuringiensis (Bt) Cry1Ab toxin.

Fulminant phlegmonitis of the esophagus, stomach, and duodenum due to Bacillus thuringiensis.

We report a case of phlegmonitis of the esophagus, stomach, and duodenum in patient in an immunocompromised state. Culture of gastric juice and blood yielded Bacillus thuringiensis. This case showed that even low-virulence bacilli can cause lethal gastrointestinal phlegmonous gastritis in conditions of immunodeficiency.

Intraperitoneal Immunization with Cry1Ac Protoxin from Bacillus thuringiensis Provokes Upregulation of Fc-Gamma-II/and Fc-Gamma-III Receptors Associated with IgG in the Intestinal Epithelium of Mice.

In humans, intestinal epithelial FcRn is expressed throughout life and mediates the bidirectional transport of IgG, but in mice, it is markedly expressed in neonatal intestine. In adults, its expression is only faintly upregulated after intestinal IgG induction such as that elicited by i.p. immunization with Cry1Ac protoxin (pCry1Ac) Bacillus thuringiensis. This led us to suggest that additional Fcγ receptors (Fcγ-R) may be participating in epithelial IgG uptake. So, first we determined whether CD16/32 [an epitope shared by Fcγ-RII (CD32) and Fcγ-RIII (CD16)] was expressed in the intestinal epithelia of mice. Using confocal microscopy and flow cytometry, we detected co-localization of IgG and CD16/32 in epithelial cells, whose frequency was increased by immunization with pCry1Ac. Western blot and cross-immunoprecipitation results with anti-CD16/32 and IgG antibodies in epithelial cell extracts suggested that epithelial cells bear both Fcγ-RII and Fcγ-RIII and contained IgG associated with Fcγ-RII/RIII. Using anti-CD32 and anti-CD16 antibodies, we confirmed by Western blot, confocal microscopy and flow cytometry that both Fcγ-RII and Fcγ-RIII were expressed and suggested that upregulation occurred upon immunization in intestinal epithelia. Finally, we examined the in vitro effect of anti-CD16/32, anti-CD16 and anti-CD32 antibodies on IgG uptake and transport by intestinal epithelial cells and found that it was partially reduced. Although further studies are still required, our results suggest that Fcγ-RII and Fcγ-RIII might participate in the uptake and/or transport of IgG through the intestinal epithelia of adult mice.

Overlapping and unique signatures in the proteomic and transcriptomic responses of the nematode Caenorhabditis elegans toward pathogenic Bacillus thuringiensis.

Pathogen infection can activate multiple signaling cascades that ultimately alter the abundance of molecules in cells. This change can be measured both at the transcript and protein level. Studies analyzing the immune response at both levels are, however, rare. Here, we compare transcriptome and proteome data generated after infection of the nematode and model organism Caenorhabditis elegans with the Gram-positive pathogen Bacillus thuringiensis. Our analysis revealed a high overlap between abundance changes of corresponding transcripts and gene products, especially for genes encoding C-type lectin domain-containing proteins, indicating their particular role in worm immunity. We additionally identified a unique signature at the proteome level, suggesting that the C. elegans response to infection is shaped by changes beyond transcription. Such effects appear to be influenced by AMP-activated protein kinases (AMPKs), which may thus represent previously unknown regulators of C. elegans immune defense.

Tribolium castaneum immune defense genes are differentially expressed in response to Bacillus thuringiensis toxins sharing common receptor molecules and exhibiting disparate toxicity.

In Tribolium castaneum larvae we have demonstrated by RNA interference knockdown that the Bacillus thuringiensis Cry3Ba toxin receptors Cadherin-like and Sodium solute symporter proteins are also functional receptors of the less active Cry3Aa toxin. Differences in susceptibility to B. thuringiensis infection might not only rely on toxin-receptor interaction but also on host defense mechanisms. We compared the expression of the immune related genes encoding Apolipophorin-III and two antimicrobial peptides, Defensin3 and Defensin2 after B. thuringiensis challenge. All three genes were up-regulated following Cry3Ba spore-crystal intoxication whereas only Defensins gene expression was induced upon Cry3Aa spore-crystal treatment, evidencing a possible association between host immune response and larval susceptibility to B. thuringiensis. We assessed the antimicrobial activity spectra of T. castaneum defensins peptide fragments and found that a peptide fragment of Defensin3 was effective against the human microbial pathogens, Escherichia coli, Staphylococcus aureus and Candida albicans, being S. aureus the most susceptible one.

Short communication: Naturally sensitive Bacillus thuringiensis EG10368 produces thurincin H and acquires immunity after heterologous expression of the one-step-amplified thurincin H gene cluster.

Heterologous expression of bacteriocin genetic determinants (or operons) has long been a research interest for the functional analysis of genes involved in bacteriocin biosynthesis, regulation, modification, and immunity. Previously, construction of genomic libraries of the bacteriocin producer strains was usually required to identify new bacteriocin operons, a method that is tedious and time consuming. For the first time, we directly amplified an 8.14-kb bioinformatically identified thurincin H gene cluster using a one-step PCR method with 100% accuracy. This amplified gene cluster was cloned into plasmid pHT315, resulting in plasmid pGW139, and subsequently transformed to Bacillus thuringiensis EG10368, a strain naturally sensitive to thurincin H. Heterologous expression of the gene cluster makes the sensitive B. thuringiensis EG10368 produce thurincin H at a higher level compared with the wild-type producer, B. thuringiensis SF361. Moreover, B. thuringiensis EG10368pGW139 acquired complete immunity to thurincin H. The results indicated that one-step PCR is a promising tool to accurately amplify long bacteriocin gene clusters used in bacteriocin functional analysis studies and it is an effective way to produce bacteriocins at a higher level, without the need to clone large chromosomal fragments.

Contributions of cellular and humoral immunity of Galleria mellonella larvae in defence against oral infection by Bacillus thuringiensis.

In this study the cellular and humoral immune reactions of the Greater wax moth Galleria mellonella have been investigated during bacterial infection caused by oral administration of Bacillus thuringiensis. Two different dose strengths were investigated to assess the contribution of immune parameters to induced Bt resistance. Low-dose (sublethal LC15) infection resulted in significantly elevated haemolymph phenoloxidase and lysozyme-like activity, enhanced phagocytic activity of haemocytes, and increased encapsulation responses in infected larvae at 48 and 72 h post infection. Higher doses of Bt (half-lethal LC50) also triggered significantly elevated haemolymph phenoloxidase and lysozyme-like activity, but decreased the coagulation index and activity of phenoloxidase in haemocytes of infected larvae. In both types of infection, the pool of circulating haemocytes became depleted. The importance of cellular and humoral immune reactions in induced insect resistance to intestinal bacterial infection Bt is herein discussed.