Cyt Toxins Research Articles

20-kDa accessory protein (P20) from Bacillus thuringiensis subsp. israelensis ISPC-12: Purification, characterization, solution scattering and structural analysis

The 20-kDa accessory protein (P20) from Bacillus thuringiensis subsp. israelensis (Bti) has been identified as an essential molecular chaperone in the enhancement of Cry11Aa and Cyt1Aa toxins production and their bio-crystallization. Additionally, P20 plays a vital role in suppressing the toxic effect of Cyt toxin on the host bacterium and also enhances insecticidal activity of Cry1Ac protein. Thus, the function of P20 is more specific than that of the chaperones. However, P20 is poorly investigated and insufficiently characterized. In the present study, we recombinantly expressed p20 from local isolate Bti ISPC-12 in heterologous bacterium E. coli and P20 protein was purified to homogeneity. Detailed biochemical and biophysical characterization provides crucial insights about in-vitro behavior as well as spatial conformations of P20 protein. Further, structural modelling and analysis provides insights into three-dimensional organization of the protein and shows that P20 is a non-toxic member of cytolytic (Cyt) toxin family similar to Cyt1Ca, with presence of conserved cytolysin fold. Additionally, solution scattering reveals that P20 is present as a dimer in the solution and probable dimeric assembly of P20 is presented. The findings reported here reveal engaging facts about P20 thereby advancing our understanding about this protein, which will expedite future studies.

Bacillus thuringiensis toxin Cyt2Aa forms filamentous oligomers when exposed to lipid membranes or detergents

The bacterium Bacillus thuringiensis (Bt) produces insecticidal proteins during the sporulation phase. These proteins are located in parasporal crystals consisting of two delta-endotoxin classes, crystal (Cry) and cytolytic (Cyt) toxins. In vitro, Cyt toxins show cytolytic activity against bacterial and a variety of insect and mammalian cells. They bind to cell membranes with unsaturated phospholipids and sphingomyelin. Although Bt and its parasporal crystals containing both Cry and Cyt toxins have been successfully used as bioinsecticides, the molecular mechanism of action of Cyt toxins is not yet fully understood. To address this, we exposed Cyt2Aa to lipid membranes and visualized membrane disruption process using cryo-electron microscopy. We observed two types of Cyt2Aa oligomers. First, Cyt2Aa forms smaller curved oligomers on the membrane surface that become linear over time, and detach when the membrane ruptures. Similar linear filamentous oligomers were also formed by Cyt2Aa in the presence of detergents without prior exposure to lipid membranes, which exhibited attenuated cytolytic activity. Furthermore, our data suggest that Cyt2Aa adopts different conformations between its monomeric and oligomeric forms. Overall, our results provide new evidence for a detergent-like mechanism of action of Cyt2Aa rather than the pore-forming model of target membrane disruption of this important class of insecticidal proteins.

Cyt1Aa toxin gene frequency in Bacillus thuringiensis isolates and its relation with pathogenicity for vector mosquitoes

Bacillus thuringiensis produces several virulence factors, the main ones being the Cry and Cyt toxins, present in the parasporal body produced during sporulation. The Cyt toxins have mechanisms specific for mosquitoes and Cyt1Aa, the most studied cytolytic toxin, is effective for mosquito control by acting in synergism with Cry toxins. The goal of the present work was to study the frequency of the codifying gene for Cyt1Aa in B. thuringiensis native isolates acquired from samples of soil, insect and water, as well as to verify any possible genetic polymorphism. 1,448 B thuringiensis strains were used for DNA extraction and PCR technique, all with the use of a primer that amplifies a fragment of 300 pairs of the cyt1Aa gene. The strains that showed amplification in the PCR reaction were sequenced and compared to each other and to the sequences available at Genbank. 32 (2.3%) strains of B. thuringiensis showed positive amplification for the cyt1Aa gene. The highest frequency of isolates with cyt1Aa gene was acquired from samples coming from the Cerrado biome, both isolates from soil and from insects, equally with 3.4%. The cyt1Aa gene sequencing highlighted that, for that 300 bp region, the gene is conserved and there is no single-base polymorphism.

Characterization of a novel Bacillus thuringiensis toxin active against Aedes aegypti larvae

Some Bacillus thuringiensis (Bt) strains produce dipteran-active toxins and can control larval mosquitoes. We identified a novel mosquitocidal toxin named Xpp81Aa1 with the thioredoxin domain from Bt strain HSY204. This toxin has very little sequence similarity to the three-domain Cry toxin and Cyt toxins and has significant toxicity to Aedes aegypti larvae. A safety assessment indicated that the Xpp81Aa1 toxin has no cytocidal activity against red blood cells and did not induce allergic reactions. The Xpp81Aa1 toxin exhibited a synergistic effect in combination with Cry2Aa and Cry4Aa protein toxins. Thus, the Xpp81Aa1 toxin could be a good candidate for mosquito control applications to reduce the mosquito-borne disease.

Viability and reconstitution of delta-endotoxins from Bacillus thuringiensis var. israelensis extracts after forty\u2009years of storage against Aedes aegypti (Diptera: Culicidae)

BackgroundBacillus thuringiensis subsp. israelensis (Bti) produces insecticidal endotoxins known as Cry and Cyt. Its efficiency and specificity make it the most widely used substance as a biopesticide for controlling disease from vector insects, such as mosquitoes, responsible for important human diseases such as malaria, filariasis, dengue, and yellow fevers. To date, it is proven difficult to develop a commercial product that has more than 2 years of shelf life, and there is little information on the viability of these commercial proteins under prolonged storage conditions.ResultsThis study aimed to evaluate biological activity of reconstituted Bti endotoxins after 40 years of storage against the mosquito Aedes aegypti larvae. Five concentrations of Bti extracts were used for bioassays against 3rd and 4th instars of A. aegypti larvae. All reconstituted endotoxins from stored extracts showed a potency increase. The strain HD-500 from extract 3260 was the most effective insecticide (LC50 = 0.0014 mg/l), followed by 3756 (LC50 = 0.0037 mg/l). These strains were particularly notable, increasing their larvicidal potency one hundredfold and one thousandfold, respectively. Protein profiles in polyacrylamide gels revealed a greater presence of Cyt toxins compared to the stored Bti extracts, which maintained their activity at high concentrations.ConclusionThe reconstituted Bti strains presented a great biological activity against A. aegypti larvae, specially extract 3260 (median lethal concentration (LC50) value = 0.0014 mg/l). This considerable larvicidal activity after 40 years under storage was an encouraging signal for the development of future formulation strategies regarding their useful life. The stability of extracts of stored endotoxins produced by Bti decreased significantly, particularly Cyt1A protein, which is responsible for their synergistic activity.

TOXiTAXi: a web resource for toxicity of Bacillus thuringiensis protein compositions towards species of various taxonomic groups

Bioinsecticides consisting of different sets of Bacillus thuringiensis (Bt) Cry, Cyt and Vip toxins are broadly used in pest control. Possible interactions (synergistic, additive or antagonistic) between these proteins can not only influence the overall efficacy of certain Bt-based bioinsecticide, but also raise questions regarding environmental safety. Here, we assemble, summarize and analyze the outcomes of experiments published over 30 years, investigating combinatorial effects among Bt Cry, Cyt and Vip toxins. We collected the results on 118 various two-to-five-component combinations that have been bioassayed against 38 invertebrate species. Synergism, additive effect and antagonism was indicated in 54%, 32% and 14% of experiments, respectively. Synergism was noted most frequently for Cry/Cyt combinations, followed by Cyt/Vip and Cry/Cry. In Cry/Vip combinations, antagonism is more frequent and higher in magnitude compared to other categories. Despite a significant number of tested Bt toxin combinations, most of them have been bioassayed only against one pest species. To aid the research on Bt pesticidal protein activity, we present TOXiTAXi (http://www.combio.pl/toxitaxi/), a universal database and a dedicated web tool to conveniently gather and analyze the existing and future bioassay results on biocidal activity of toxins against various taxonomic groups.

Bacillus subtilis as a host for mosquitocidal toxins production.

Summary Aedes albopictus transmits several arboviral infections. In the absence of vaccines, control of mosquito populations is the only strategy to prevent vector‐borne diseases. As part of the search for novel, biological and environmentally friendly strategies for vector control, the isolation of new bacterial species with mosquitocidal activity represents a promising approach. However, new bacterial isolates may be difficult to grow and genetically manipulate. To overcome these limits, here we set up a system allowing the expression of mosquitocidal bacterial toxins in the well‐known genetic background of Bacillus subtilis. As a proof of this concept, the ability of B. subtilis to express individual or combinations of toxins of Bacillus thuringiensis israelensis (Bti) was studied. Different expression systems in which toxin gene expression was driven by IPTG‐inducible, auto‐inducible or toxin gene‐specific promoters were developed. The larvicidal activity of the resulting B. subtilis strains against second‐instar Ae. albopictus larvae allowed studying the activity of individual toxins or the synergistic interaction among Cry and Cyt toxins. The expression systems here presented lay the foundation for a better improved system to be used in the future to characterize the larvicidal activity of toxin genes from new environmental isolates.

The Cyt1Aa toxin from Bacillus thuringiensis inserts into target membranes via different mechanisms in insects, red blood cells, and lipid liposomes

Bacillus thuringiensis subsp. israelensis produces crystal inclusions composed of three-domain Cry proteins and cytolytic Cyt toxins, which are toxic to different mosquito larvae. A key component is the Cyt toxin, which synergizes the activity of the other Cry toxins, thereby resulting in high toxicity. The precise mechanism of action of Cyt toxins is still debated, and two models have been proposed: the pore formation model and the detergent effect. Here, we performed a systematic structural characterization of the Cyt toxin interaction with different membranes, including in Aedes aegypti larval brush border membrane vesicles, small unilamellar vesicle liposomes, and rabbit erythrocytes. We examined Cyt1Aa insertion into these membranes by analyzing fluorescence quenching in solution and in the membrane-bound state. For this purpose, we constructed several Cyt1Aa variants having substitutions with a single cysteine residue in different secondary structures, enabling Cys labeling with Alexa Fluor 488 for quenching analysis using I-soluble quencher in solution and in the membrane-bound state. We identified the Cyt1Aa residues exposed to the solvent upon membrane insertion, predicting a possible topology of the membrane-inserted toxin in the different membranes. Moreover, toxicity assays with these variants revealed that Cyt1Aa exerts its insecticidal activity and hemolysis through different mechanisms. We found that Cyt1Aa exhibits variable interactions with each membrane system, with deeper insertion into mosquito larva membranes, supporting the pore formation model, whereas in the case of erythrocytes and small unilamellar vesicles, Cyt1Aa's insertion was more superficial, supporting the notion that a detergent effect underlies its hemolytic activity.

The Cytocidal Spectrum of Bacillus thuringiensis Toxins: From Insects to Human Cancer Cells.

Bacillus thuringiensis (Bt) is a ubiquitous bacterium in soils, insect cadavers, phylloplane, water, and stored grain, that produces several proteins, each one toxic to different biological targets such as insects, nematodes, mites, protozoa, and mammalian cells. Most Bt toxins identify their particular target through the recognition of specific cell membrane receptors. Cry proteins are the best-known toxins from Bt and a great amount of research has been published. Cry are cytotoxic to insect larvae that affect important crops recognizing specific cell membrane receptors such as cadherin, aminopeptidase-N, and alkaline phosphatase. Furthermore, some Cry toxins such as Cry4A, Cry4B, and Cry11A act synergistically with Cyt toxins against dipteran larvae vectors of human disease. Research developed with Cry proteins revealed that these toxins also could kill human cancer cells through the interaction with specific receptors. Parasporins are a small group of patented toxins that may or may not have insecticidal activity. These proteins could kill a wide variety of mammalian cancer cells by recognizing specific membrane receptors, just like Cry toxins do. Surface layer proteins (SLP), unlike the other proteins produced by Bt, are also produced by most bacteria and archaebacteria. It was recently demonstrated that SLP produced by Bt could interact with membrane receptors of insect and human cancer cells to kill them. Cyt toxins have a structure that is mostly unrelated to Cry toxins; thereby, other mechanisms of action have been reported to them. These toxins affect mainly mosquitoes that are vectors of human diseases like Anopheles spp (malaria), Aedes spp (dengue, zika, and chikungunya), and Culex spp (Nile fever and Rift Valley fever), respectively. In addition to the Cry, Cyt, and parasporins toxins produced during spore formation as inclusion bodies, Bt strains also produce Vip (Vegetative insecticidal toxins) and Sip (Secreted insecticidal proteins) toxins with insecticidal activity during their vegetative growth phase.

Characterization of the Novel Insecticidal Crystal Protein Cyt3Aa1 from <i>Bacillus thuringiensis</i> TD516

Bacillus thuringiensis (Bt) produces two families of insecticidal crystal proteins, i.e., crystalline (Cry) and cytolytic (Cyt) toxins. Cyt3Aa1, the newest Cyt family member, is produced by Bt TD516. Bioassay results have shown that Cyt3Aa1 has weak hemolytic activity against human red blood cells and is not toxic to A. aegypti larvae, but causing a teratogenic effect. The three-dimensional structure of Cyt3Aa1 has a typical cytolysin fold containing a β-sheet held by two surrounding α-helical layers, resembling the previously reported Cyt1Aa and Cyt2Aa structures, which indicated that Cyt3Aa1 might be a membrane-perforation toxin and could induce synergism with Cry protein. This study provides a new source of insecticidal crystal proteins, and presents a foundation for understanding the biological characterization of it, which will aid in the development of strategies to cope with the potential problem of insect resistance.

Genome of Bacillus sp. strain QHF158 provides insights into its parasporal inclusions encoded by the S-layer gene.

Bacillus sp. strain QHF158, a Gram-positive, spore-forming and parasporal crystal-secreting bacterium, was isolated from soil of Limushan National Forest Park in China. Here we present the significant feature of parasporal inclusions of this organism, together with the draft genome sequence and annotation. Phylogenetic analysis suggested that strain QHF158 is possibly a novel species, most closely related to Bacillus mycoides. Genome annotation results revealed that strain QHF158 did not contain any typical Cry or Cyt toxin coding gene. Furthermore, the mass spectrometry analyses demonstrated that the parasporal crystalline inclusions were encoded by the orf_05273 gene, with 95% similarity to the S-layer protein (SLP) EA1 of B. mycoides, which indicated that the parasporal crystal from Bacillus sp. strain QHF158 was mainly formed by SLP, instead of the typical Cry or Cyt toxin proteins.

Genome sequencing of Bacillus thuringiensis isolate T414 toxic to pink bollworm (Pectinophora gossypiella Saunders) and its insecticidal genes

Bacillus thuringiensis is a spore-forming bacterium that is pathogenic towards a range of insect and nematode species and had been widely used as a biopesticide. In this study, we present the morphological, molecular and genetic characteristics of an indigenous Bt isolate T414 which displayed an effective toxicity against Pectinophora gossypiella. Scanning electron microscopy revealed the presence of bipyramidal, spherical and cubic shaped protein crystals in its spore-crystal suspension. SDS-PAGE analysis of its spore-crystal mixture showed the presence of two major protein bands viz.130 and 65 kDa. Whole genome sequencing with MiSeq divulged that it contains a chromosome and many plasmids. The assembled genome finally contained 6493494bp. Automated annotation of this genome draft predicted 6877 coding sequences and 152 RNAs (rRNAs, tRNAs and ncRNAs). NCBI blast analysis showed that assembled genome was distributed in a chromosome and 15 different types of plasmids. Further analysis of draft sequence revealed it harbors parasporal crystal protein genes (cry1Aa, cry1Ab, cry1Ac, cry1IAa, cry2Aa, cry2Ab and cyt1), vegetative insecticidal protein gene (vip3Aa), all plasmid borne and various additional virulence factors such as chitinases, proteases, bacteriocins and hemolysins. From the analysis it is evident that all the Cry, Cyt or Vip toxins are plasmid borne and are present on two types of plasmids named as p414A and p414E in the present study. A cry2A type gene was cloned and sequenced. It was named as cry2Aa21 by Bt nomenclature committee.

Effect of Chlorine and Temperature on Larvicidal Activity of Cuban Bacillus thuringiensis Isolates

The efficacy of biolarvicides may be influenced by species of mosquito, larval age and density, temperature, water quality, bacterial formulation, and others. The aim of this study was to evaluate the influence of temperature and chlorine on larvicidal activity of Bacillus thuringiensis Cuban isolates against Aedes aegypti. The influence of temperature (25, 30, 35 °C) and chlorine (2.25mg/L) on the larvicidal activity of eleven B. thuringiensis Cuban isolates (collected between 2007 and 2009) were tested under laboratory conditions following WHO protocols. Bioassay data were analyzed by Probit program. The effect of chlorine and temperature (25, 30, 35 and 40 °C) on the Cry and Cyt proteins of these isolates was determined by SDS-PAGE polyacrylamide gel electrophoresis. The pathogenicity of the isolates U81, X48 was affected at 35 °C. However, A21, A51, L910, and R89 isolates increase their entomopathogen activity at 35 °C. No differences were observed in toxicity of M29, R84, R85 and R87 isolates at different temperatures. The Cry 4, Cry 10 and Cry 11 proteins were reduced in A21, X48, R85 isolates at 35 and 40 °C. The Cyt proteins were reduced at 35 and 40 °C in A21, X48, R85, and A51 isolates. In L910 and R84 isolates, the Cyt toxin was degraded only at 40 °C. In chlorinated water, the lethal concentrations 50 and 90 in A21, A51, M29, R84, U81, and X48 isolates were increase. A21, A51, L910, R85, and X48 isolates have a strong larvicidal activity for the treatment of Ae. aegypti breeding's sites exposed to high temperature and chlorine.

Structural characterization and heterologous expression of a new cyt gene cloned from Bacillus thuringiensis.

Bacillus thuringiensis (Bt) strains produce Cry (crystal) and Cyt (cytolytic) proteins belonging to the group of bacterial toxins known as pore-forming toxins (PFTs), which interact with midgut cells of target insects to create pores, disruption of ion homeostasis and eventual death. PFTs have synergistic insecticidal activities and have been used as biopesticides against agriculturally important insects. Identification of new Cyt proteins is important because of their specific toxicity towards hemipteran pests, against which the Cry proteins are not effective. We have structurally characterized a cyt (cyt1007) gene from an Indian Bt isolate SK-1007. The presence of a "Bacillus thuringiensis toxin" domain and maximum identity of 36% with Cyt2Ca in the deduced amino acid sequence indicated Cyt1007 protein to be a new member of Cyt family. Three dimensional (3D) modeling (PMDB ID: PM0081490) revealed that it adopts a typical ferredoxin-like fold, and is composed of a single domain of α/β architecture, in which a single β sheet is surrounded by two α helical layers. The putative lipid binding site and probable mode of action of Cyt1007 protein were predicted through comparative analysis with other Cyt toxins and their distant homologs Evf (Erwinia virulence factor) and VVA2 (Volvatoxin A2). Heterologous expression of cyt1007 gene as a 25kDa protein in Escherichia coli was achieved at high levels in both soluble and insoluble fractions. Affinity chromatography-based purification yielded 83.6% purified Cyt1007 protein, which can be used for downstream applications for the investigation of its toxicity. Graphical abstract Steps in the structural characterization and heterologous expression of a new cyt gene cloned from Bacillus thuringiensis.

Lipid phase influences the binding of Bacillus thuringiensis Cyt2Aa2 toxin on model lipid membranes

Bacillus thuringiensis is a bacterium that produces many insecticidal proteins including cytolytic proteins or Cyt toxins. Although the Cyt toxin shows specific toxicity against Dipteran insect species, the toxin binds directly to the lipid membrane without a specific protein receptor requirement. In this work, we have investigated the interaction of Cyt2Aa2 toxin with lipid bilayers composed of different lipid phases. By means of atomic force microscopy (AFM), lipid phase separation was observed for 1:1 and 4:1 M mixtures of DPPC/POPC bilayers. The exposure of Cyt2Aa2 to these lipid bilayers revealed that the toxin selectively bound to Ld lipid bilayer (corresponding to POPC). In turn, it did not bind to the Lo and So phases (corresponding to DPPC). Interestingly, for the bilayer of 4:1 DPPC/POPC mixture, the binding of Cyt2Aa2 was localized at the lipid phase boundary instead of Ld domain as occurred for the 1:1 DPPC/POPC bilayer. In addition, quartz crystal microbalance with dissipation experiments confirmed AFM results. In particular, the measurements showed that amount of protein bound to 1:1 DPPC/POPC (with phase separation) was half of the binding quantified for the Ld phase lipid bilayer (pure POPC and 1:4 DPPC/POPC mixture). These results indicate that the lipid phase (lipid acyl chain) influences the Cyt2Aa2-lipid interaction.

Engineering Bacillus thuringiensis Cyt1Aa toxin specificity from dipteran to lepidopteran toxicity

The Cyt and Cry toxins are different pore-forming proteins produced by Bacillus thuringiensis bacteria, and used in insect-pests control. Cry-toxins have a complex mechanism involving interaction with several proteins in the insect gut such as aminopeptidase N (APN), alkaline phosphatase (ALP) and cadherin (CAD). It was shown that the loop regions of domain II of Cry toxins participate in receptor binding. Cyt-toxins are dipteran specific and interact with membrane lipids. We show that Cry1Ab domain II loop3 is involved in binding to APN, ALP and CAD receptors since point mutation Cry1Ab-G439D affected binding to these proteins. We hypothesized that construction of Cyt1A-hybrid proteins providing a binding site that recognizes gut proteins in lepidopteran larvae could result in improved Cyt1Aa toxin toward lepidopteran larvae. We constructed hybrid Cyt1Aa-loop3 proteins with increased binding interaction to Manduca sexta receptors and increased toxicity against two Lepidopteran pests, M. sexta and Plutella xylostella. The hybrid Cyt1Aa-loop3 proteins were severely affected in mosquitocidal activity and showed partial hemolytic activity but retained their capacity to synergize Cry11Aa toxicity against mosquitos. Our data show that insect specificity of Cyt1Aa toxin can be modified by introduction of loop regions from another non-related toxin with different insect specificity.

The parasporal crystals of Bacillus pumilus strain 15.1: a potential virulence factor?

Summary Bacillus pumilus strain 15.1 was previously found to cause larval mortality in the Med‐fly Ceratitis capitata and was shown to produce crystals in association with the spore. As parasporal crystals are well‐known as invertebrate‐active toxins in entomopathogenic bacteria such as Bacillus thuringiensis (Cry and Cyt toxins) and Lysinibacillus sphaericus (Bin and Cry toxins), the B. pumilus crystals were characterized. The crystals were composed of a 45 kDa protein that was identified as an oxalate decarboxylase by peptide mass fingerprinting, N‐terminal sequencing and by comparison with the genome sequence of strain 15.1. Synthesis of crystals by a plasmid‐cured derivative of strain 15.1 (produced using a novel curing strategy), demonstrated that the oxalate decarboxylase was encoded chromosomally. Crystals spontaneously solubilized when kept at low temperatures, and the protein produced was resistant to trypsin treatment. The insoluble crystals produced by B. pumilus 15.1 did not show significant toxicity when bioassayed against C. capitata larvae, but once the OxdD protein was solubilized, an increase of toxicity was observed. We also demonstrate that the OxdD present in the crystals has oxalate decarboxylate activity as the formation of formate was detected, which suggests a possible mechanism for B. pumilus 15.1 activity. To our knowledge, the characterization of the B. pumilus crystals as oxalate decarboxylase is the first report of the natural production of parasporal inclusions of an enzyme.

Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with aerolysin-type architecture

Cry46Ab is a Cry toxin derived from Bacillus thuringiensis TK-E6. Cry46Ab is not significantly homologous to other mosquitocidal Cry or Cyt toxins and is classified as an aerolysin-type pore-forming toxin based on structural similarity. In this study, the potency of Cry46Ab was assessed for its potential application to mosquito control. A synthetic Cry46Ab gene, cry46Ab-S1, was designed to produce recombinant Cry46Ab as a glutathione-S-transferase fusion in Escherichia coli. Recombinant Cry46Ab showed apparent toxicity to Culex pipiens larvae, with a 50% lethal dose of 1.02 μg/ml. In an artificial lipid bilayer, Cry46Ab activated by trypsin caused typical current transitions between open and closed states, suggesting it functions as a pore-forming toxin similar to other Cry and Cyt toxins. The single-channel conductance was 103.3 ± 4.1 pS in 150 mM KCl. Co-administration of recombinant Cry46Ab with other mosquitocidal Cry toxins, especially the combination of Cry4Aa and Cry46Ab, resulted in significant synergistic toxicity against C. pipiens larvae. Co-administration of multiple toxins exhibiting different modes of action is believed to prevent the onset of resistance in insects. Our data, taken in consideration with the differences in its structure, suggest that Cry46Ab could be useful in not only reducing resistance levels but also improving the insecticidal activity of Bt-based bio-insecticides.

Effects and mechanisms of Bacillus thuringiensis crystal toxins for mosquito larvae.

Bacillus thuringiensis is a Gram-positive aerobic bacterium that produces insecticidal crystalline inclusions during sporulation phases of the mother cell. The virulence factor, known as parasporal crystals, is composed of Cry and Cyt toxins. Most Cry toxins display a common 3-domain topology. Cry toxins exert intoxication through toxin activation, receptor binding and pore formation in a suitable larval gut environment. The mosquitocidal toxins of Bt subsp. israelensis (Bti) were found to be highly active against mosquito larvae and are widely used for vector control. Bt subsp. jegathesan is another strain which possesses high potency against broad range of mosquito larvae. The present review summarizes characterized receptors for Cry toxins in mosquito larvae, and will also discuss the diversity and effects of 3-D mosquitocidal Cry toxin and the ongoing research for Cry toxin mechanisms generated from investigations of lepidopteran and dipteran larvae.

Receptors are affected by selection with each Bacillus thuringiensis israelensis Cry toxin but not with the full Bti mixture in Aedes aegypti

Bacillus thuringiensis israelensis (Bti) toxins are increasingly used for mosquito control, but little is known about the precise mode of action of each of these toxins, and how they interact to kill mosquito larvae. By using RNA sequencing, we investigated change in gene transcription level and polymorphism variations associated with resistance to each Bti Cry toxin and to the full Bti toxin mixture in the dengue vector Aedes aegypti. The up-regulation of genes related to chitin metabolism in all selected strain suggests a generalist, non-toxin-specific response to Bti selection in Aedes aegypti. Changes in the transcription level and/or protein sequences of several putative Cry toxin receptors (APNs, ALPs, α-amylases, glucoside hydrolases, ABC transporters) were specific to each Cry toxin. Selective sweeps associated with Cry4Aa resistance were detected in 2 ALP and 1 APN genes. The lack of selection of toxin-specific receptors in the Bti-selected strain supports the hypothesis that Cyt toxin acts as a receptor for Cry toxins in mosquitoes.