Here, we present the whole genome sequence of Bt S2160-1, a potential alternative to the mosquitocidal model strain, Bti. One chromosome genome and four mega-plasmids were contained in Bt S2160-1, and 13 predicted genes encoding predicted insecticidal crystal proteins were identified clustered on one plasmid pS2160-1p2 containing two pathogenic islands (PAIs) designed as PAI-1 (Cry54Ba, Cry30Ea4, Cry69Aa-like, Cry50Ba2-like, Cry4Ca1-like, Cry30Ga2, Cry71Aa-like, Cry72Aa-like, Cry70Aa-like, Cyt1Da2-like and Vpb4C1-like) and PAI-2 (Cyt1Aa-like, and Tpp80Aa1-like). The clusters appear to represent mosquitocidal toxin islands similar to pathogenicity islands. Transcription/translation of 10 of the 13 predicted genes was confirmed by whole-proteome analysis using LTQ-Orbitrap LC-MS/MS. In summary, the present study identified the existence of a mosquitocidal toxin island in Bacillus thuringiensis, and provides important genomic information for understanding the insecticidal mechanism of B. thuringiensis.

The Aedes aegypti cadherin-like protein (Aae-Cad) and the membrane-bound alkaline phosphatase (Aae-mALP) are membrane proteins identified as putative receptors for the larvicidal Cry toxins produced by Bacillus thuringiensis subsp. israelensis bacteria. Cry toxins are the most used toxins in the control of different agricultural pest and mosquitos. Despite the relevance of Aae-Cad and Aae-mALP as possible toxin-receptors in mosquitoes, previous efforts to establish a clear functional connection among them and Cry toxins activity have been relatively limited. In this study, we used CRISPR-Cas9 to generate knockout (KO) mutations of Aae-Cad and Aae-mALP. The Aae-mALP KO was successfully generated, in contrast to the Aae-Cad KO which was obtained only in females. The female-linked genotype was due to the proximity of aae-cad gene to the sex-determining loci (M:m). Both A. aegypti KO mutant populations were viable and their insect-development was not affected, although a tendency on lower egg hatching rate was observed. Bioassays were performed to assess the effects of these KO mutations on the susceptibility of A. aegypti to Cry toxins, showing that the Aae-Cad female KO or Aae-mALP KO mutations did not significantly alter the susceptibility of A. aegypti larvae to the mosquitocidal Cry toxins, including Cry11Aa, Cry11Ba, Cry4Ba, and Cry4Aa. These findings suggest that besides the potential participation of Aae-Cad and Aae-mALP as Cry toxin receptors in A. aegypti, additional midgut membrane proteins are involved in the mode of action of these insecticidal toxins.

Prolyl-tRNA synthetases (ProRSs) are unique among aminoacyl-tRNA synthetases (aaRSs) in having two distinct structural architectures across different organisms: prokaryote-like (P-type) and eukaryote/archaeon-like (E-type). Interestingly, Bacillus thuringiensis harbors both types, with P-type (BtProRS1) and E-type ProRS (BtProRS2) coexisting. Despite their differences, both enzymes are constitutively expressed and functional in vivo. Similar to BtProRS1, BtProRS2 selectively charges the P-type tRNAPro and displays higher halofuginone tolerance than canonical E-type ProRS. However, these two isozymes recognize the primary identity elements of the P-type tRNAPro-G72 and A73 in the acceptor stem-through distinct mechanisms. Moreover, BtProRS2 exhibits significantly higher tolerance to stresses (such as heat, hydrogen peroxide, and dithiothreitol) than BtProRS1 does. This study underscores how an E-type ProRS adapts to a P-type tRNAPro and how it may contribute to the bacterium\'s survival under stress conditions.

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.

BACKGROUND: As part of a publicly funded initiative to develop genetically engineered Brassicas (cabbage, cauliflower, and canola) expressing Bacillus thuringiensis Crystal (Cry)-encoded insecticidal (Bt) toxin for Indian and Australian farmers, we designed several constructs that drive high-level expression of modified Cry1B and Cry1C genes (referred to as Cry1BM and Cry1CM; with M indicating modified). The two main motivations for modifying the DNA sequences of these genes were to minimise any licensing cost associated with the commercial cultivation of transgenic crop plants expressing CryM genes, and to remove or alter sequences that might adversely affect their activity in plants.
RESULTS: To assess the insecticidal efficacy of the Cry1BM/Cry1CM genes, constructs were introduced into the model Brassica Arabidopsis thaliana in which Cry1BM/Cry1CM expression was directed from either single (S4/S7) or double (S4S4/S7S7) subterranean clover stunt virus (SCSV) promoters. The resulting transgenic plants displayed a high-level of Cry1BM/Cry1CM expression. Protein accumulation for Cry1CM ranged from 5.18 to 176.88 µg Cry1CM/g dry weight of leaves. Contrary to previous work on stunt promoters, we found no correlation between the use of either single or double stunt promoters and the expression levels of Cry1BM/Cry1CM genes, with a similar range of Cry1CM transcript abundance and protein content observed from both constructs. First instar Diamondback moth (Plutella xylostella) larvae fed on transgenic Arabidopsis leaves expressing the Cry1BM/Cry1CM genes showed 100% mortality, with a mean leaf damage score on a scale of zero to five of 0.125 for transgenic leaves and 4.2 for wild-type leaves.
CONCLUSIONS: Our work indicates that the modified Cry1 genes are suitable for the development of insect resistant GM crops. Except for the PAT gene in the USA, our assessment of the intellectual property landscape of components presents within the constructs described here suggest that they can be used without the need for further licensing. This has the capacity to significantly reduce the cost of developing and using these Cry1M genes in GM crop plants in the future.

A riboswitch generally regulates the expression of its downstream genes through conformational change in its expression platform (EP) upon ligand binding. The cyclic diguanosine monophosphate (c-di-GMP) class I riboswitch Bc1 is widespread and conserved among Bacillus cereus group species. In this study, we revealed that Bc1 has a long EP with two typical ρ-independent terminator sequences 28 bp apart. The upstream terminator T1 is dominant in vitro, while downstream terminator T2 is more efficient in vivo. Through mutation analysis, we elucidated that Bc1 exerts a rare and incoherent \"transcription-translation\" dual regulation with T2 playing a crucial role. However, we found that Bc1 did not respond to c-di-GMP under in vitro transcription conditions, and the expressions of downstream genes did not change with fluctuation in intracellular c-di-GMP concentration. To explore this puzzle, we conducted SHAPE-MaP and confirmed the interaction of Bc1 with c-di-GMP. This shows that as c-di-GMP concentration increases, T1 unfolds but T2 remains almost intact and functional. The presence of T2 masks the effect of T1 unwinding, resulting in no response of Bc1 to c-di-GMP. The high Shannon entropy values of EP region imply the potential alternative structures of Bc1. We also found that zinc uptake regulator can specifically bind to the dual terminator coding sequence and slightly trigger the response of Bc1 to c-di-GMP. This work will shed light on the dual-regulation riboswitch and enrich our understanding of the RNA world.IMPORTANCEIn nature, riboswitches are involved in a variety of metabolic regulation, most of which preferentially regulate transcription termination or translation initiation of downstream genes in specific ways. Alternatively, the same or different riboswitches can exist in tandem to enhance regulatory effects or respond to multiple ligands. However, many putative conserved riboswitches have not yet been experimentally validated. Here, we found that the c-di-GMP riboswitch Bc1 with a long EP could form a dual terminator and exhibit non-canonical and incoherent \"transcription-translation\" dual regulation. Besides, zinc uptake regulator specifically bound to the coding sequence of the Bc1 EP and slightly mediated the action of Bc1. The application of SHAPE-MaP to the dual regulation mechanism of Bc1 may establish the foundation for future studies of such complex untranslated regions in other bacterial genomes.

BACKGROUND: The recent reduction in malaria burden in Côte d\'Ivoire is largely attributable to the use of long-lasting insecticidal nets (LLINs). However, this progress is threatened by insecticide resistance and behavioral changes in Anopheles gambiae sensu lato (s.l.) populations and residual malaria transmission, and complementary tools are required. Thus, this study aimed to assess the efficacy of the combined use of LLINs and Bacillus thuringiensis israelensis (Bti), in comparison with LLINs.
METHODS: This study was conducted in the health district of Korhogo, northern Côte d\'Ivoire, within two study arms (LLIN + Bti arm and LLIN-only arm) from March 2019 to February 2020. In the LLIN + Bti arm, Anopheles larval habitats were treated every fortnight with Bti in addition to the use of LLINs. Mosquito larvae and adults were sampled and identified morphologically to genus and species using standard methods. The members of the An. gambiae complex were determined using a polymerase chain reaction technique. Plasmodium infection in An. gambiae s.l. and malaria incidence in local people was also assessed.
RESULTS: Overall, Anopheles spp. larval density was lower in the LLIN + Bti arm 0.61 [95% CI 0.41-0.81] larva/dip (l/dip) compared with the LLIN-only arm 3.97 [95% CI 3.56-4.38] l/dip (RR = 6.50; 95% CI 5.81-7.29; P < 0.001). The overall biting rate of An. gambiae s.l. was 0.59 [95% CI 0.43-0.75] biting/person/night in the LLIN + Bti arm against 2.97 [95% CI 2.02-3.93] biting/person/night in LLIN-only arm (P < 0.001). Anopheles gambiae s.l. was predominantly identified as An. gambiae sensu stricto (s.s.) (95.1%, n = 293), followed by Anopheles coluzzii (4.9%; n = 15). The human-blood index was 80.5% (n = 389) in study area. EIR was 1.36 infected bites/person/year (ib/p/y) in the LLIN + Bti arm against 47.71 ib/p/y in the LLIN-only arm. Malaria incidence dramatically declined from 291.8‰ (n = 765) to 111.4‰ (n = 292) in LLIN + Bti arm (P < 0.001).
CONCLUSIONS: The combined use of LLINs with Bti significantly reduced the incidence of malaria. The LLINs and Bti duo could be a promising integrated approach for effective vector control of An. gambiae for elimination of malaria.

The entomopathogenic fungus Beauveria bassiana provides an eco-friendly substitute to chemical insecticides for mosquito control. Nevertheless, its widespread application has been hindered by its comparatively slow efficacy in eliminating mosquitoes. To augment the potency of B. bassiana against Aedes mosquitoes, a novel recombinant strain, Bb-Cyt1Aa, was developed by incorporating the Bacillus thuringiensis toxin gene Cyt1Aa into B. bassiana. The virulence of Bb-Cyt1Aa was evaluated against Aedes aegypti and Aedes albopictus using insect bioassays. Compared to the wild-type (WT) strain, the median lethal time (LT50) for A. aegypti larvae infected with Bb-Cyt1Aa decreased by 33.3% at a concentration of 1 × 108 conidia/mL and by 22.2% at 1 × 107 conidia/mL. The LT50 for A. aegypti adults infected with Bb-Cyt1Aa through conidia ingestion was reduced by 37.5% at 1 × 108 conidia/mL and by 33.3% at 1 × 107 conidia/mL. Likewise, the LT50 for A. aegypti adults infected with Bb-Cyt1Aa through cuticle contact decreased by 33.3% and 30.8% at the same concentrations, respectively. Furthermore, the Bb-Cyt1Aa strain also demonstrated increased toxicity against both larval and adult A. albopictus, when compared to the WT strain. In conclusion, our study demonstrated that the expression of B. thuringiensis toxin Cyt1Aa in B. bassiana enhanced its virulence against Aedes mosquitoes. This suggests that B. bassiana expressing Cyt1Aa has potential value for use in mosquito control.
OBJECTIVE: Beauveria bassiana is a naturally occurring fungus that can be utilized as a bioinsecticide against mosquitoes. Cyt1Aa is a delta-endotoxin protein produced by Bacillus thuringiensis that exhibits specific and potent insecticidal activity against mosquitoes. In our study, the expression of this toxin Cyt1Aa in B. bassiana enhances the virulence of B. bassiana against Aedes aegypti and Aedes albopictus, thereby increasing their effectiveness in killing mosquitoes. This novel strain can be used alongside chemical insecticides to reduce dependence on harmful chemicals, thereby minimizing negative impacts on the environment and human health. Additionally, the potential resistance of B. bassiana against mosquitoes in the future could be overcome by acquiring novel combinations of exogenous toxin genes. The presence of B. bassiana that expresses Cyt1Aa is of significant importance in mosquito control as it enhances genetic diversity, creates novel virulent strains, and contributes to the development of safer and more sustainable methods of mosquito control.

Small lentic water bodies are important emitters of methane (CH4) and carbon dioxide (CO2), but the processes regulating their dynamics and susceptibility to human-induced stressors are not fully understood. Bioturbation by chironomid larvae has been proposed as a potentially important factor controlling the dynamics of both gases in aquatic sediments. Chironomid abundance can be affected by the application of biocides for mosquito control, such as Bti (Bacillus thuringiensis var. israelensis). Previous research has attributed increases in CH4 and CO2 emissions after Bti application to reduced bioturbation by chironomids. In this study, we separately tested the effect of chironomid bioturbation and Bti addition on CH4 production and emission from natural sediments. In a set of 15 microcosms, we compared CH4 and CO2 emission and production rates with high and low densities of chironomid larvae at the bioturbating stage, and standard and five times (5x) standard Bti dose, with control sediments that contained neither chironomid larvae nor Bti. Regardless of larvae density, chironomid larvae did not affect CH4 nor CO2 emission and production of the sediment, although both rates were more variable in the treatments with organisms. 5xBti dosage, however, led to a more than three-fold increase in CH4 and CO2 production rates, likely stimulated by bioavailable dissolved carbon in the Bti excipient and priming effects. Our results suggest weak effects of bioturbating chironomid larvae on the CH4 and CO2 dynamics in aquatic ecosystems. Furthermore, our results point out towards potential functional implications of Bti for carbon cycling beyond those mediated by changes in the macroinvertebrate community.

Bacillus thuringiensis (Bt) secretes the nutritional insecticidal protein Vip3Aa11, which exhibits high toxicity against the fall armyworm (Spodoptera frugiperda). The Bt HD270 extracellular polysaccharide (EPS) enhances the toxicity of Vip3Aa11 protoxin against S. frugiperda by enhancing the attachment of brush border membrane vesicles (BBMVs). However, how EPS-HD270 interacts with Vip3Aa11 protoxin in vivo and the effect of EPS-HD270 on the toxicity of activated Vip3Aa11 toxin are not yet clear. Our results indicated that there is an interaction between mannose, a monosaccharide that composes EPS-HD270, and Vip3Aa11 protoxin, with a dissociation constant of Kd = 16.75 ± 0.95 mmol/L. When EPS-HD270 and Vip3Aa11 protoxin were simultaneously fed to third-instar larvae, laser confocal microscopy observations revealed the co-localization of the two compounds near the midgut wall, which aggravated the damage to BBMVs. EPS-HD270 did not have a synergistic insecticidal effect on the activated Vip3Aa11 protein against S. frugiperda. The activated Vip3Aa11 toxin demonstrated a significantly reduced binding capacity (548.73 ± 82.87 nmol/L) towards EPS-HD270 in comparison to the protoxin (34.96 ± 9.00 nmol/L). Furthermore, this activation diminished the affinity of EPS-HD270 for BBMVs. This study provides important evidence for further elucidating the synergistic insecticidal mechanism between extracellular polysaccharides and Vip3Aa11 protein both in vivo and in vitro.