Characterization of pesticidal crystal toxin protein Cry11Aa from Bacillus thuringiensis serovar israelensis VCRC-B646 for mosquito control.

Characterization of pesticidal crystal toxin protein Cry11Aa from Bacillus thuringiensis serovar israelensis VCRC-B646 for mosquito control. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characterization of pesticidal crystal toxin protein Cry11Aa from Bacillus thuringiensis serovar israelensis VCRC-B646 for mosquito control. Abhisubesh V, Sahadiya Mandodan, Jibi Lukose, Aneha Rajan, Kakhuangailiu Gangmei, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6163570/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Sep, 2025 Read the published version in Biotechnology Letters → Version 1 posted 5 You are reading this latest preprint version Abstract Effective mosquito control is essential for reducing the transmission of vector-borne diseases. This study focuses on the comprehensive characterization of mosquitocidal toxins produced by Bacillus thuringiensis serovar israelensis (Bti) VCRC B646 and the associated insecticidal genes. The bacterium was cultured, and the spore-crystal complex was purified to identify the mosquitocidal proteins. The isolate produced mosquitocidal toxins were effective against Aedes aegypti , Anopheles stephensi , and Culex quinquefasciatus , Toxicity bioassays indicated lethal concentrations (LC 50 and LC 90 ) for Aedes aegypti (0.0022 mg/L and 0.004 mg/L), and Culex quinquefasciatus (0.0025 mg/L and 0.0044 mg/L). SDS-PAGE and LC-MS analysis revealed that Cry11Aa5 (Pesticidal Crystal Protein) is the predominant toxin produced by this strain. PCR amplification confirmed the presence of genes encoding various insecticidal proteins, including Cry and Cyt toxins. Phylogenetic analysis was performed to assess the genetic relatedness and toxin profiles of the bacterial isolate. This detailed characterization of Bti VCRC B646 highlights its potential as a promising biopesticide candidate for mosquito control, contributing to the development of sustainable and eco-friendly strategies for vector management. Bacillus thuringiensis serovar israelensis Cry Cyt Mosquito species Toxin protein Phylogenetic analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Please have a look at courier new font provided for text in article. Mosquitoes are vectoring various pathogens such as protozoa, helminths, and arboviruses, which are responsible for numerous destructive diseases like dengue, Zika, malaria, chikungunya, and filariasis. These vector-borne diseases (VBD) pose significant challenges to public health in tropical and subtropical countries, further magnified by climate change, globalization, urbanization, and the development of resistance (Dhanasekaran and Thangaraj, 2014 ). The severity of the VBDs across globe, lack of medication and effective vaccines make the control methods prominent. The global severity of VBDs, combined with the lack of effective treatments and vaccines, underscores the importance of mosquito control methods targeting different stages of their life cycle, particularly the vulnerable immature stages, which prevent future infestations and ensure effective population control. Synthetic insecticides like organophosphates, which affect the nervous system, are commonly used against mosquitoes; however, the development of insecticide resistance and potential harm to beneficial organisms have driven the search for eco-friendly alternatives. Bacillus thuringiensis (Bt) is a ubiquitous environmental bacterium that has been extensively used as a biopesticide against mosquito vectors due to its metabolites, which are specifically toxic at the larval stage (Mansour et al., 2023 ). It is a Gram-positive, spore-forming bacterium known for producing parasporal crystals composed of multiple toxic proteins during sporulation and exhibiting a broad spectrum of toxicity against various insect orders, including Diptera, Lepidoptera, and Coleoptera, as well as other invertebrates (Schnepf et al., 1998 ). The subspecies Bacillus thuringiensis serovar israelensis ( Bti ) produces delta-endotoxins that are specific to a narrow range of mosquito larvae (Osman et al., 2015 ). Major endotoxins both Crystal ( Cry ) and Cytolytic ( Cyt ) toxins are non-homologous in origin and mode of action and belongs to distinct protein families. Cry toxins, exhibit a broader insecticidal potential targeting multiple orders, including Diptera, Lepidoptera, and Coleoptera, while Cy t toxins primarily target Diptera. Both are synthesized as protoxins and are highly specific, binding to receptors in the midgut of susceptible insects. Their action as pore-forming toxins (PFTs) occurs under the alkaline conditions of the midgut, upon conversion to active toxins leading to alterations in cell permeability, cell lysis, and ultimately the death of the larvae. The complex interaction of these toxins with the host's immune system and gut cells and the synergetic effect of Cry and Cyt toxin underscores their effectiveness in vector control (Belousova et al., 2021 ; Palma et al., 2014 ; Poopathi et al., 2014 ). The toxicity of Bti varies among strains and is species-dependent, with the combination of toxins determining each strain's effectiveness against different mosquito vectors crucial for mosquito control programs. Therefore, it is essential to characterize the major toxic protein responsible for each indigenous Bti strains and understand the genomic toxin profile This study focuses on the molecular characterization of toxic genes in the highly toxic indigenous Bti strain VCRC B646, isolated from the soils of Tamil Nadu, India. It aims to identify and characterize the toxic genes, assess their phylogenetic relatedness to reference strains, and purify the major toxic proteins responsible for the strain's heightened toxicity. Materials and methods Bacterial strains and growth conditions The Bacillus thuringiensis var. israelensis ( Bti ) strain VCRC B646, isolated from the soils of Kanchipuram, India, was cultured in Nutrient Yeast Salt Medium (NYSM) consisting of glucose (5 g/L), peptone (5 g/L), sodium chloride (5 g/L), HM peptone (3 g/L), yeast extract (5 g/L) and salt solution (g/L: MgCl 2 2.03, CaCl 2 1.0 and MnCl 2 , 0.1). The bacterial culture was incubated until complete sporulation and the formation of parasporal crystalline inclusions were observed (Manikandan et al., 2016 ). Microscopic examination with staining confirmed the presence of crystalline inclusions. The cellular mass was subsequently harvested by centrifugation at 8000 rpm for 15 minutes (Vijayakumar et al., 2024 ). Extraction of bacterial genomic DNA Bacterial genomic DNA was extracted from a pure individual colony, which was cultured overnight in nutrient broth at 37°C with shake culture. After harvesting the bacterial cells by centrifugation, the Gen-Elute bacterial genomic DNA kit (Sigma-Aldrich) was used for DNA extraction following the manufacturer's protocol. This included cell lysis, DNA binding to a silica membrane, washing to remove contaminants, and eluting the purified DNA. The extracted genomic DNA was visualized using 1.2% agarose gel electrophoresis and quantified spectrophotometrically at absorbance ratio at 260 nm/280 nm ( Bora et al ., 2024). PCR Amplification of Cry and C yt gene Bt specific toxin genes cry and cyt genes amplification for the extracted DNA of Bti VCRC B646 was accomplished with using an automated thermal cycler (Bio-Rad, USA). Cry gene and Cyt gene specific oligonucleotide primers ( Cry4A , Cry4B , Cry11 , Cry 10, Cyt 2) were used for amplification with varied cyclic conditions, the expected size and the sequences were described in (Table 1). Table.1: Characteristics of Primers for Cry and Cyt Toxin Genes Primer Oligonucleotide Primer (5 1 − 3 1 ) Melting temperature Optical Density Cry 4A (F) Cry 4A (R) TCA AAG ATC ATT TCA AAA TTA CAT G 56 9.8 CGG CTT GAT CTA TGT CAT AAT CTG T 63 10.2 Cry 4B (F) Cry 4B (R) CGT TTT CAA GAC CTA ATA ATA TAA TAC C 61 8.5 CGG CTT GAT CTA TGT CAT AAT CTG T 63 10.6 Cry 10 (F) Cry 10 (R) TCA ATG CTC CAT CCA ATG 52 9.0 CTT GTA TAG GCC TTC CTC CG 60 9.8 Cry 11 (F) Cry 11 (R) CGC TTA CAG GAT GGA TAG G 57 9.1 GCT GAA ACG GCA CGA ATA TAA TA 59 8.9 Cyt 2 (F) Cyt 2 (R) ATT ACA AAT TGC AAA TGG TAT TCC 57 9.3 TTT CAA CAT CCA CAG TAA TTT CAA ATG C 63 10.9 The total reaction mixtures consist a final volume of 25µl; (12.5 µl of master mix (dNTPs, polymerase, and buffers), 1µl (10ng) of forward and reverse primers, 50 ng Template DNA of VCRC B646, 9.5µl of Nuclease free water). The amplification involved an initial denaturation step, followed by 35 cycles consisting of denaturation, annealing, and extension steps, with a final extension phase (Table 2). The amplified PCR product was separated through 1.2% agarose gel in a 1X Tris-Acetate-EDTA (TAE) buffer system and visualized under a UV transilluminator using ethidium bromide (EtBr) as a fluorescent tag against a 100-bp molecular weight marker (HiMedia). Table.2: PCR conditions for each of the Cry and Cyt toxin genes Gene Initial denaturation Cycles Final Extension Denaturation Annealing Extension Cry 4A 3 min at 95°C 1 min at 95°C 1 min at 58°C 1 min at 72°C 5 min at 72°C Cry 4B 3 min at 95°C 1 min at 95°C 1 min at 58°C 1 min at 72°C 5 min at 72°C Cry 11 5 min at 95°C 30 sec at 95°C 30 sec at 57°C 30 sec at 72°C 1 min at 72°C Cry 10 4 min at 95°C 30 sec at 95°C 30 sec at 56°C 30 sec at 72°C 2 min at 72°C Cyt 2 5 min at 95°C 30 sec at 95°C 30 sec at 61°C 30 sec at 72°C 5 min at 72°C Purification and sequence analysis of Amplified Cry and Cyt genes Through spin column chromatography the amplified Cry and Cyt genes were recovered from the unwanted impurities (dNTPs, Salts, and excess primers). Qiaquick PCR purification kit (Qiagen, USA) were used as per the instructions of the manufacturer and Purified DNAs were quantified using spectrophotometry. Sequence analysis was performed in both forward and reverse direction separately as per the conditions prescribed in Mandodan et al., 2024 in an automated Thermal Cycler (Bio-Rad, USA). The DNA sequencing reaction mixture purified from the impurities and dye through size-exclusion chromatography with a pre-filled gel filtration matrix; NucleoSEQ columns (Macherey-Nagel, Germany) as per the manufacturer’s protocol to improve the accuracy of DNA sequences. Thus, purified DNA was analyzed using the Applied Biosystems 3130XL Genetic Analyzer by the Sanger technique, for high-throughput DNA sequencing and fragment analysis at ICMR VCRC, Puducherry, India. The chromatogram generated were edited for consensus sequence and related sequences with maximum percentage similarity were obtained through NCBI BLAST. The nucleotide sequence of all the genes studied were submitted to Gene Bank database and obtained NCBI accession number. Phylogenetic relatedness of Cry and Cyt genes The closest sequences with highest percentage similarity were obtained through neighbour-joining method. By employing the Kimura 2-parameter method, gaps and missing data were removed, and using a consensus sequence generated for multiple sequence alignment subsequent phylogenetic tree construction using the MEGA 11 software. Spore-Crystal production and purification The Bti strain VCRC B646 was cultured in NYSM medium until spore-crystal formation (Kunnikuruvan et al. , 2014). The spore-crystal mixture was collected through centrifugation and purification of crystal protein from the complex carried out as per mounsef et al., 2014 . Briefly, Spore-Crystal biomass suspended first in 1M sodium chloride solution containing triton X-100 (0.01%) and washed twice. Then resuspended in saline solution (9 g of NaCl /1L water) to enhance the hydrophobic interactions. Organic solvent Hexane, was used for next wash in a proportion equal to or less than 10% to minimize the risk of crystal alteration during washing and repeated hexane wash 2–3 times. Thus, washed product obtained was washed 2–4 times with ice-cold water at 6000 rpm to obtain the final purified crystal (Mounsef et al., 2014 ). Protein characterization using ESI-nano LC-MS/MS The nano ACQUITY UPLC® chromatographic system (Waters, Manchester, UK) at Mass spectrometry & Proteomics Core facility of Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala was assessed for protein profiling. Separated protein bands were excised from the acrylamide gel and vortexed in MilliQ water prior to destaining (100 mM ammonium bicarbonate/ acetonitrile (1:1, v/v)) to remove excess dye. DTT (dithiothreitol) was used as a reducing agent, followed by alkylation with iodoacetamide (IAA). Gel pieces were dehydrated and proteins were digested into smaller peptides using trypsin buffer (13 ng /µl trypsin in 50 mM ammonium bicarbonate). The extracted peptides reconstituted in 3% acetonitrile and injected (3 µl) onto a Symmetry® trap column (180 µm x 20 mm, C18, 5 µm, Waters) and separated on an analytical column (75 µm X 200 mm, HSS T3 C18, 1.8 µm, Waters). A binary gradient of 0.1% formic acid /Water and 0.1% formic acid/Acetonitrile was used for the separation (flow rate;300 nL/min) and the column temperature was maintained at 35°C was maintained. The autosampler temperature was kept at 4ºC to preserve sample integrity (Wojtkiewicz et al., 2014 ) Mass spectrometry (MS) Mass spectrometry (MS) was performed using the Synapt G2 High-Definition Mass Spectrometry (HDMS) System, calibrated with sodium iodide and set to positive Electrospray Ionization (ESI) mode. Leucine encephalin (m/z = 556.2766) was utilized for online mass correction to ensure accuracy. The nano ESI capillary voltage was set to 3.4 kV to generate a fine spray of charged droplets, while the sample cone voltage was adjusted to 40 V to facilitate the transfer of ions from the ionization source to the mass spectrometer. The extraction cone voltage was maintained at 4 V to ensure efficient ion extraction into the vacuum system without significant fragmentation. In the ion mobility spectrometry (IMS) section, a nitrogen gas flow rate of 90 mL/min was used, with the T-Wave pulse height set to 40 V and the velocity adjusted to 800 m/s. The IMS voltage settings were configured to 8 V and 20 V to enhance ion mobility and separation. During data acquisition, the system operated in resolution mode, utilizing a continuum format to capture comprehensive spectral data, with collision energy set between 20 eV and 45 eV for effective ion fragmentation. Data analysis The resulting data was analyzed using Progenesis QI for Proteomics v4.2 software, focusing on the organism Bacillus thuringiensis to interpret mass spectra and identify compounds based on their mass-to-charge ratios (m/z). Each parameter is carefully chosen to balance the need for sensitivity and specificity in protein identification. The database source utilized for protein identification was Universal Protein Resource (UniProt). Toxicity Bioassay and analysis The purified crystal mixture was subjected to larvicidal activity against late third instar larvae of Cx. qunquefasciatus , and Ae. aegypti following protocol described by WHO and previously (Manikandan et al. , 2023; Mandodan et al., 2024 ; Vijayakumar et al., 2024 ). The 50% and 90% lethal concentration was calculated through probit analysis using SPSS (Gangmei et al. , 2024). Results Crystal Morphology of Bti isolate VCRC B646 In comparison with toxicity of standard strains through preliminary screening assays Bti VCRC B646 strain was selected due to higher toxicity. The spore crystal complex observed in 72 hour grown culture of Bti VCRC B646. The bacteria in rod shape were confirmed through microscopy and scanning electron microscopy showed the crystal morphology as irregular shape and spore size (Fig. 1 ). Spores were found in oval shape and stained with malachite green and formed at terminal end of vegetative cell. Spores and crystals found as a cluster under microscope (Fig. 2 ). Amplification of Cry and Cyt genes and phylogeny PCR product analysis revealed that the new larvicidal isolate Bti VCRC B646 contained all six genes studied in the total DNA. A 459-base pair (bp) fragment was obtained from PCR amplification of the Cry4A gene from the total DNA of Bti VCRC B646 (Fig. 3 ). There is a 99% identity to the reported Cry4A toxin gene, confirming the toxin sequence within the particular gene family. Similarly, other toxin genes were also confirmed with sequences similar to their corresponding families, and GenBank accession numbers were received as follows: Cry 4A(459): PP898123, Cry4B (321 bp): PP898125, Cyt2 (356 bp): PP898127, Cry11 (343 bp): PP898124, and Cry10 (348 bp): PP898126 (Fig. 3 ). Additionally, to investigate the the evolutionary relatedness of VCRC B646 strain with other previously sequenced Bt strains, constructed a phylogenomic tree based on the consensus sequences generated for all toxin genes ( Cry4A, Cry4B, Cry10, Cry11 , and Cyt2 ) of bacterial strain VCRC B646 to Bacillus thuringiensis var israelensis was confirmed through phylogenetic analysis. The maximum likelihood model with 1000 bootstrap replicates was used to construct the phylogenetic tree (MEGA software) for all amplified toxin genes: Cry4A (Fig. 4 ), Cry4B (Fig. 5 ), Cry10 (Fig. 6), Cry11 (Fig. 7 ), and Cyt2 (Fig. 8 ). Proteomic characterization of toxic protein The crystal toxins purified from spore-crystal mixture (dry biomass) irregular crystals were visualized in microscope (100X) with a yield of (10 mg/L) (Fig. 9 ). The freeze-dried protein separated through SDS-PAGE. The protein profile of Bti VCRC B646 -purified crystal toxins in an SDS-PAGE showed the main proteins with molecular weights of apparently 72, 35, 32, and 22 kDa in size (Fig. 10 ). SDS-PAGE pattern of solubilized proteins from crystal-spore mixture of strain HD1. LC-MS analysis confirmed the toxic protein band and identified as Cry11Aa5. LC- MS analysis Peptides of the all 4 main protein bands (A, B, C and D) were analyzed through LC-MS. Four protein bands were identified as Cry11Aa, also known as "Pesticidal crystal protein" based on the identified peptides and total score. Amino acid sequences of peptides were determined. Additionally, similarity indices were calculated for each band relative to the reference pesticidal crystal protein sp|P21256|: the first band showed a similarity index of 62.4%, the second band 67.76%, the third band 62.5%, and the fourth band 62.7%. These findings underscore a substantial resemblance between the identified Cry11Aa protein bands and the reference pesticidal crystal protein sp|P21256|, highlighting their functional similarity and potential efficacy in mosquito control strategies. An overestimation of Cry11Aa observed. The multiple sequence alignment of amino acid of all bands in comparison with Full length of Pesticidal crystal protein Cry11Aa sp|P21256| . Toxicity bioassay with Cry 11Aa Bioassays performed against the third instar larvae of Culex and Aedes with purified Crystals and the mortality found in response to different doses as linear (Table. 3). Table 3 Toxicity values (LC 50 and LC 90 ) of Bti VCRC B646 Crystal against mosquito vectors. Bacterial strain Mosquito species LC 50 (µg/ml) * (LCL –UCL) LC 90 (µg/ml) * (LCL –UCL) Intercept Slope χ 2 (df) Bti (VCRC –B646) Culex quinquefasciatus 0.0025 (0.0023–0.0026) 0.0044 (0.0042–0.0047) -1.665 0.016 15.089 Aedes aegypti 0.0022 (0.0020–0.0024) 0.0040 (0.0038–0.0043) − 1.723 0.018 26.250 DISCUSSION Bacillus thuringiensis israelensis (Bti) has emerged as a safe alternative to synthetic insecticides for mosquito control since its discovery in the 1970s (Gangmei et al ., 2024, Lukose et al ., 2024). The specificity and toxicity of Bti to various mosquito vectors are closely linked to the crystalline inclusion bodies it produces, particularly the Cry and Cyt proteins. The diversity of these genes varies among Bti strains, with each protein demonstrating distinct specificity against related mosquito species. Variations in the single amino acid sequence of Cry toxins lead to differences in toxicity levels among insect pests. Therefore, there is an ongoing need for novel Cry and Cyt toxin genes with sequence variations from indigenous strains for improved resistance management. This study focuses on the soil isolate Bti VCRC B646, selected for its high toxicity against mosquito larvae. There are several reports of Bti strains exhibiting larvicidal activity whereas the strain VCRC B646 exhibits an enhanced toxicity against A.aegypti and C.quinquefasciatus . The specific toxicity of Bti to dipteran insects is correlated to its combination of Cry and Cyt proteins (Ma et al., 2023 ). Research indicates that the combinations of Cry 4Aa, Cry 10, Cry 11, and Cy t proteins are particularly potent against mosquitoes (Ma et al., 2023 ). Notably, the strain Bti VCRC B646 contains all these effective toxin gene combinations, confirmed through specific gene amplification techniques Through PCR amplification, the presence of toxin genes belonging to the Cry and Cyt families in Bti VCRC B646 was confirmed. Additionally, the phylogenetic relatedness of this strain was assessed by sequencing all seven toxin genes present, revealing a 90% similarity to other known Bti strains. The specific toxicity of Bacillus thuringiensis ( Bt ) to dipteran insects, particularly mosquitoes, is a critical area of research in biological pest control. There is a multifaceted relationship between the genentic composition of Cry and Cyt genes in Bti and their toxicity to the susceptible insects which contributes to the varying level of toxicity among various isolates (Das et al., 2021 ). The environment of insect gut and synergetic effect contribute to the overall toxicity profile of certain isolates. Bioassays conducted with a purified crystal mixture containing the cry11 toxin have demonstrated remarkable toxicity against Aedes aegypti and Culex quinquefasciatus , showing higher efficacy compared to reference strains, highlighting its potential as a superior biocontrol agent. The specific toxicity of Bti to dipteran insects is closely linked to the combination of Cry and Cyt proteins in its genetic makeup. Research has shown that the combinations of Cry4Aa, Cry10, Cry11 , and Cyt proteins exhibit significant potency against mosquitoes (Ma et al., 2023 ). Interestingly, the strain Bti VCRC B646 contains all these effective combinations of toxin genes, confirmed through specific gene amplification techniques. Cry 11Aa is the most toxic δ-endotoxin produced by Bacillus thuringiensis israelensis ( Bti ) against mosquito larvae, particularly Aedes and Culex species. Purification of this toxin from the spore-crystal preparation of Bti VCRC revealed a 72 kDa polypeptide, identified as the pesticidal crystal protein Cry 11Aa. Bioassays of the purified toxin demonstrated high larvicidal efficacy, with the highest toxicity observed against the dengue vector Aedes aegypti and followed by the filarial vector Culex quinquefasciatus. The sporulation phase of the B. thuringiensis strains and the existence of Cry genes could vary due to the heterogeneity in environmental condition. The different Cry gene subgroups encode significantly different crystal proteins due to variations in the nucleotide sequences which results in varied insecticidal effect on insect larvae of different orders. Additionally, the synergistic interactions among these toxin proteins further enhance the potency of Bti for insecticidal activity, making it a highly efficient and environmentally friendly option for mosquito control programs (Arsov et al., 2023 ). This research contributes to the understanding of the genetic basis for the heightened toxicity observed in Bti VCRC B646 and highlights its potential as an effective biopesticide for mosquito control. The broad target range of Bti is attributed to the presence of different mosquitocidal proteins produced during sporulation and their combination in action, with the main ones being Cry11Aa, Cry4Ba, Cry4Aa , and Cyt1Aa. The Bti parasporal body is significantly different from the typical Bt bipyramidal crystal, which shows toxicity to lepidopteran larvae. The parasporal body formed of 4 main toxic proteins ( Cry11A (72 kDa), Cyt1A (27.3 kDa), Cry4B (134 kDa) and Cry4A (128 kDa) organized into three distinct inclusion types, forming a parasporal body (spherical) enclosed by a lamellar envelope. On the basis of amino acid sequence as well as toxicological properties Cyt1A protein is different significantly from the Cry proteins. In laboratory conditions, it exhibits notable cytolytic effects on various cells from both invertebrates and vertebrates, with a particular affinity for unsaturated fatty acids found in the lipid component of cell membranes. Bti produces several potent toxic proteins, such as, Cry11Aa, Cry4Aa, Cry4Ba , and Cyt1Aa , which act synergistically to target mosquito larvae by binding to specific receptors in the midgut brush border membrane, leading to cellular destruction and larval mortality. This specificity contributes to the lack of resistance among mosquito populations and ensures safety for non-target organisms (Poopathi et al., 2013 ; Valtierra et al. , 2020). The mosquitocidal activity of Bti VCRC B646 was also attributed to the effect of a specific polypeptide chain (72 kDa). The yield of Bti VCRC B646 crystal is 37 mg per liter. Through LC-MS analysis, the toxic protein has been identified and characterized as "Pesticidal crystal protein Cry11Aa". The amplification results revealed that the PCR amplification of specific regions within the Cry and Cy t genes, with fragment sizes of 459 bp for Cry4A , 321 bp for Cry4B , 477 bp for Cyt1 , 356 bp for Cyt2 , 343 bp for Cry11 , and 348 bp for Cry10 , indicating of target sequences. As Cry and Cyt genes, resulting from Bacillus thuringiensis israelensis ( Bti ), encode proteins with potential larvicidal activity against mosquito larvae. The construction of a phylogenetic tree using a neighbor joining model with 1000 bootstrap replicates, and the utilization of the Kimura 2-parameter model for sequence analysis, allowed for a comprehensive examination of the genetic relatedness of the bacterial isolate. Furthermore, the confirmation of Bacillus thuringiensis israelensis ( Bti ) VCRC B646 identity, along with the presence of specific Cry and Cyt toxin genes using targeted gene primers, provides valuable insights into the toxin profiles of the bacterial isolate. These proteins, including, Cry4Aa, Cry4Ba, Cry11Aa, Cry10Aa, Cyt1Aa , and Cyt2Aa , constitute the major toxic arsenal of Bti with broad range of target insects (de Souza et al., 1999 ). Additionally, the synergistic interactions among these toxin proteins further enhance the potency of Bti for insecticidal activity, making it a highly efficient and environmentally friendly option for mosquito control programs. Resistance to insecticidal proteins, such as Cry11Aa , has been reported, but there are currently no documented cases of resistance among field populations to Bacillus thuringiensis israelensis ( Bti ). To address potential resistance development, innovative strategies like recombinant technology are crucial for enhancing the efficacy of pesticidal proteins. Modification of proteins through recombinant techniques can optimize their effectiveness against target pests. For instance, Cry11Aa recombinant variants have shown activity against larval stages of Culex and Aedes mosquitoes. Detailed biophysical analyses have revealed insights into the stability and in-vitro behavior of recombinant Cry11Aa . Molecular dynamics simulations have further contributed to understanding Cry11Aa 's behavior and proteolytic processing, aiding in the strategic utilization of Bti for mosquito and vector management (Kinkar et al., 2023 ). In recent study, with Bti Cry11Aa toxins, a delivery system which is efficient stable was developed to control mosquito larvae. They applied a coating of Mg (OH)2 nanoparticles (MHNPs) to Cry11Aa proteins and subsequently evaluated the impact of MHNPs on the bioactivity and ultraviolet protection capabilities of the Cry11Aa proteins. Through this Cry protein insectical activity enhanced and protected from toxin degradation (reduction from 64.29–16.67%).) through MHNPs. Additionally, MHNPs enhanced the proteolysis of Cry11Aa in the midgut and intensified the damage inflicted by the Cry protein on gut epithelial cells, thereby augmenting its insecticidal efficacy against Culex quinquefasciatus . This indicates that MHNPs, acting as an excellent nano-carrier, have the potential to significantly enhance the insecticidal bioactivity and anti-ultraviolet capacity of Cry11Aa (Pan et al., 2017 ). According to Wang et al., ( 2021 ), Cry11Aa and Cry4Ba of Bti revealed highly toxicity to to Ae. aegypti larvae. The important membrane bound receptor for Cry toxin noted among Diptera, Lepidoptera, Coleoptera and other insects were glycosylphosphatidylinositol (GPI)-anchored APN. Two GPI-anchored APN isoforms in Ae. aegypti (AeAPN1 and AeAPN2), which bind Cry4Ba and Cry11Aa , were individually disabled using CRISPR/Cas9 mutagenesis. A double-mutant homozygous strain lacking both AeAPN1 and AeAPN2 was produced via : reverse genetics. Binding affinity of protoxins Cry11Aa and Cry4Ba to midgut brush border membrane vesicles was similar in in ELISA assays from these APN-knockouts to that of the wild. Toxicity results of bioassays indicated that neither single knockout of AeAPN1 nor AeAPN2, nor disruption of both AeAPN1 and AeAPN2 simultaneously, led to changes significantly in the Ae. aegypti larvae susceptibility to Cry11Aa and Cry4Ba . In conclusion, the use of Bacillus thuringiensis israelensis ( Bti ) VCRC B646 as biological larvicides represents a promising strategy for mosquito control, with a particular focus on their eco-friendly nature and effectiveness against a broad spectrum of mosquito species. Its enhanced toxicity against Aedes aegypti and Culex quinquefasciatus is attributed to the presence of multiple Cry and Cyt toxin genes, particularly Cry 10, Cry 11Aa, Cry 4Aa, Cry 4Ba, and Cyt 1Aa, which act synergistically to target mosquito larvae. Molecular characterization confirmed the presence of these toxin genes, with phylogenetic analysis revealing a close genetic relationship with other potent Bti strains. Statements and Declarations Acknowledgments The first author acknowledges the PhD supervisor (Dr. S. Poopathi). Authors acknowledges the Director, ICMR-Vector Control Research Centre, Puducherry for providing facilities. Ethical approval: Compliance with ethical standards. This study does not require ethical approval because it is a microbiological study. Conflict of interest: The authors declare no conflict of interests. Competing interests: The authors declare no competing interests. Funding: The authors declare no funding received. Data availability : The obtained sequence data to support the study was submitted to the NCBI GenBank and received the following accession numbers mentioned in manuscript, PP898123, PP898125, PP898127, PP898124, PP898126. Author contributions statement: A.V., Writing-Original Draft, experiments, Investigation, J.L., S.M., Formal Analysis, Validation, Data Curation K.G., Data Curation, Validation, A.K., B.B., Visualization, Validation, H.P., Methodology, Validation, M.A., Validation, Methodology, Formal Analysis, Review and Editing, P. S. Supervision and concept and administration References Dhanasekaran, D., and Thangaraj, R. (2014). Microbial secondary metabolites are an alternative approaches against insect vector to prevent zoonotic diseases. Asian Pacific Journal of Tropical Disease, 4(4), 253-261. Mansour, T., Radwan, W. H., Mansour, M., Gomaa, M., Farouk, F., Shepl, M., and Abu-Hussien, S. H. (2023). Larvicidal potential, toxicological assessment, and molecular docking studies of four Egyptian bacterial strains against Culex pipiens L(Diptera: Culicidae). Scientific Reports, 13(1), 17230. Schnepf E., Crickmore N., Van Rie J., Lereclus D., Baum J., Feitelson J., and Dean D. (1998). 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Manikandan, R., Muthukumar, C., Ramalakshmi, A., Balasubramani, V., and Udayasuriyan, V. (2016). Screening of new isolates of Bacillus thuringiensis for cry1 genes and testing of toxicity against Dichocrocis punctiferalis (Family: Pyralidae, Order: Lepidoptera). Microbiology , 85 , 191-197. Vijayakumar, A., Mandodan, S., Aneha, K., Gangmei, K., Padmanaban, H., Bora, B., Lukose, J., Mathivanan, A., Irudayaraj, G., and Subbiah, P. (2024). A New Aqueous Formulation from Indigenously Isolated Bacillus thuringiensis israelensis VCRC B646 for Mosquito Control. Indian Journal of Entomology . Ref. No. e24876. Bora, B., Lukose, J., Gangmei, K., Sivaprakasam, M., Vijayakumar, A., Mandodan, S., ... & Subbiah, P. (2024). A First Report on the isolation and characterization of a highly potential indigenous Mosquitocidal Bacterium (Bacillus thuringiensis subspecies Israelensis VCRC B647) from Red Soil, India. Indian Journal of Public Health, 68(1), 3-8. Manikandan, S., Aneha, K., Bora, B., Abhisubesh, V., Hemaladkshmi, P., Gangmei, K., ... & Poopathi, S. (2024). Identification and characterization of mosquitocidal toxins from Bacillus cereus VCRC-641., S., Aneha, K., Bora, B., Abhisubesh, V., Hemaladkshmi, P., Gangmei, K., & Poopathi, S. (2024). Identification and characterization of mosquitocidal toxins from Bacillus cereus VCRC-641. Mandodan, S., Gangmei, K., Vijayakumar, A., Kunnikuruvan, A., Lukose, J., Padmanaban, H., Bore, B., Ashokkumar, M Irudayaraj, G, and Subbiah, P. (2024). Molecular identification and GC-MS analysis of a newly isolated novel bacterium (Lysinibacillus sp. VCRC B655) for mosquito control. Molecular Biology Reports, 51(1), 800. Kunnikuruvan, A., Vijayakumar, A., Sivaprakasam, M. et al. Enhanced Mosquito Larvicidal Efficacy and Dehairing Properties of Bacillus thuringiensis Serovar israelensis Strain VCRC-B649 Isolated from Malabar Coast, India. Curr Microbiol 82, 93 (2025). https://doi.org/10.1007/s00284-025-04070-y. Mounsef, J. R., Salameh, D., kallassy Awad, M., Chamy, L., Brandam, C., & Lteif, R. (2014). A simple method for the separation of Bacillus thuringiensis spores and crystals. Journal of Microbiological Methods , 107 , 147-149. Wojtkiewicz, M., Barnett, K., and Ciborowski, P. (2014). Protein Identification by Mass Spectrometry: Proteomics. Current Laboratory Methods in Neuroscience Research , 399-409. Gangmei, K., Lukose, J., Vijayakumar, A., Padmanaban, H., Mandodan, S., Bora, B., Sivaprakasam, M., Kunnikuruvan, A., Ashokkumar, M., Balakrishnan, V. and Irudayaraj, G., & Subbiah, P. (2025). Isolation of Bacillus thuringiensis serovar israelensis VCRC-B650 from Indian clay soil with enhanced mosquitocidal activity and dehairing property. BioControl , 70 (1), 79-93. Lukose, J., Gangmei, K., Bora, B., Hemaladkshmi, P., Abhisubesh, V., Mandodan, S., ... & Poopathi, S. (2024). Bacillus thuringiensis israelensis VCRC B650 culture filtrate useful for mosquito oviposition attractant and larvicidal action. Entomon, 49(3), 425-434. Ma, X., Hu, J., Ding, C. et al. New native Bacillus thuringiensis strains induce high insecticidal action against Culex pipiens pallens larvae and adults. BMC Microbiol 23 , 100 (2023). https://doi.org/10.1186/s12866-023-02842-9 . Das, S. K., Pradhan, S. K., Samal, K. C., & Singh, N. R. (2021). Structural, functional, and evolutionary analysis of Cry toxins of Bacillus thuringiensis: an in silico study. Egyptian Journal of Biological Pest Control, 31, 1-14. Arsov, A., Gerginova, M., Paunova-Krasteva, T., Petrov, K., & Petrova, P. (2023). Multiple cry genes in Bacillus thuringiensis strain BTG suggest a broad-spectrum insecticidal activity. International Journal of Molecular Sciences , 24 (13), 11137. Poopathi, S., Mani, C., Vignesh, V., Praba, V.L., and Thirugnanasambantham, K. (2013) Genotypic diversity of mosquitocidal bacteria ( Bacillus sphaericus , B. thuringiensis , and B. cereus ) newly isolated from natural sources. Applied Biochemistry and Biotechnology , 171(8): 2233–2246. Valtierra-de-Luis, D., Villanueva, M., Berry, C., and Caballero, P. (2020). Potential for Bacillus thuringiensis and other bacterial toxins as biological control agents to combat dipteran pests of medical and agronomic importance. Toxins , 12(12): 773. De Souza, M. P., Huang, C. P. A., Chee, N. and Terry, N. (1999). Rhizosphere bacteria enhance the accumulation of selenium and mercury in wetland plants. Planta, 209, 259-263. Kinkar, O. U., Prashar, A., Yadav, B., Kumar, A., Hadapad, A. B., Hire, R. S., and Makde, R. D. (2023). Purification, characterization and proteolytic processing of mosquito larvicidal protein Cry11Aa from Bacillus thuringensis subsp. isralensis ISPC-12. International Journal of Biological Macromolecules , 124979. Pan, X., Xu, Z., Li, L., Shao, E., Chen, S., Huang, T., and Guan, X. (2017). Adsorption of insecticidal crystal protein Cry11Aa onto nano-Mg (OH) 2: effects on bioactivity and anti-ultraviolet ability. Journal of Agricultural and Food Chemistry , 65(43), 9428-9434. Wang, J., Yang, X., He, H., Chen, J., Liu, Y., Huang, W., and Wu, S. (2021). Knockout of two Cry-binding aminopeptidase N isoforms does not change susceptibility of aedes aegypti larvae to Bacillus thuringiensis subsp. israelensis Cry4Ba and Cry11Aa toxins. Insects , 12(3): 223. Cite Share Download PDF Status: Published Journal Publication published 10 Sep, 2025 Read the published version in Biotechnology Letters → Version 1 posted Reviewers agreed at journal 21 Mar, 2025 Reviewers invited by journal 20 Mar, 2025 Editor assigned by journal 20 Mar, 2025 First submitted to journal 17 Mar, 2025 Editorial decision: Minor revisions 10 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6163570","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":431604707,"identity":"0cb04a37-b760-496d-beec-7653615cf1c0","order_by":0,"name":"Abhisubesh V","email":"","orcid":"","institution":"Vector Control Research Centre","correspondingAuthor":false,"prefix":"","firstName":"Abhisubesh","middleName":"","lastName":"V","suffix":""},{"id":431604708,"identity":"b0fbb048-347a-4780-a8ce-0f66cbade3fd","order_by":1,"name":"Sahadiya Mandodan","email":"","orcid":"","institution":"Vector Control Research 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tree\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6163570/v1/690985d83535f4e171bd55c2.png"},{"id":79095844,"identity":"7f0a6c38-d1fb-497b-bbeb-b951fd89c048","added_by":"auto","created_at":"2025-03-24 10:55:37","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":106645,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eCyt\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e 2 Phylogenetic tree\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6163570/v1/d12db7529291b25e5d7c4032.png"},{"id":79095850,"identity":"83145d97-ca13-4449-919e-cc55420604f0","added_by":"auto","created_at":"2025-03-24 10:55:37","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":263244,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpores and crystal separation\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-6163570/v1/311860316bc4ed0697201b6a.png"},{"id":79096149,"identity":"62e61f3d-71c3-4c3e-b66b-608f70accde6","added_by":"auto","created_at":"2025-03-24 11:03:37","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":55572,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSDS page of purified crystal\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-6163570/v1/8e21dfce9b234af95ec324e9.png"},{"id":79095858,"identity":"7f720db5-58ec-4055-977d-13e1608d8899","added_by":"auto","created_at":"2025-03-24 10:55:38","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":688763,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of deducted Amino acid sequences of Band A, Band B, Band C and Band D from \u003cem\u003eBti\u003c/em\u003e VCRC B646 and reference sequence of full-length Cry 11Aa pesticidal crystal protein of sp|P21256|.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-6163570/v1/f978fb54e34ca3eba378bb30.png"},{"id":91359069,"identity":"4005f632-6116-4677-9761-f8ce94fb9dd0","added_by":"auto","created_at":"2025-09-15 16:04:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2977907,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6163570/v1/58803380-583d-47a1-a097-b37a00c9659c.pdf"}],"financialInterests":"","formattedTitle":"Characterization of pesticidal crystal toxin protein Cry11Aa from Bacillus thuringiensis serovar israelensis VCRC-B646 for mosquito control.","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cspan fontcategory=\"NonProportional\" class=\"\" name=\"Emphasis\"\u003ePlease have a look at courier new font provided for text in article.\u003c/span\u003e\u003c/p\u003e \u003cp\u003eMosquitoes are vectoring various pathogens such as protozoa, helminths, and arboviruses, which are responsible for numerous destructive diseases like dengue, Zika, malaria, chikungunya, and filariasis. These vector-borne diseases (VBD) pose significant challenges to public health in tropical and subtropical countries, further magnified by climate change, globalization, urbanization, and the development of resistance (Dhanasekaran and Thangaraj, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The severity of the VBDs across globe, lack of medication and effective vaccines make the control methods prominent. The global severity of VBDs, combined with the lack of effective treatments and vaccines, underscores the importance of mosquito control methods targeting different stages of their life cycle, particularly the vulnerable immature stages, which prevent future infestations and ensure effective population control. Synthetic insecticides like organophosphates, which affect the nervous system, are commonly used against mosquitoes; however, the development of insecticide resistance and potential harm to beneficial organisms have driven the search for eco-friendly alternatives.\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacillus thuringiensis (Bt)\u003c/em\u003e is a ubiquitous environmental bacterium that has been extensively used as a biopesticide against mosquito vectors due to its metabolites, which are specifically toxic at the larval stage (Mansour et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It is a Gram-positive, spore-forming bacterium known for producing parasporal crystals composed of multiple toxic proteins during sporulation and exhibiting a broad spectrum of toxicity against various insect orders, including Diptera, Lepidoptera, and Coleoptera, as well as other invertebrates (Schnepf et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1998\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe subspecies \u003cem\u003eBacillus thuringiensis\u003c/em\u003e serovar \u003cem\u003eisraelensis\u003c/em\u003e (\u003cem\u003eBti\u003c/em\u003e) produces delta-endotoxins that are specific to a narrow range of mosquito larvae (Osman et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Major endotoxins both Crystal (\u003cem\u003eCry\u003c/em\u003e) and Cytolytic (\u003cem\u003eCyt\u003c/em\u003e) toxins are non-homologous in origin and mode of action and belongs to distinct protein families. \u003cem\u003eCry\u003c/em\u003e toxins, exhibit a broader insecticidal potential targeting multiple orders, including Diptera, Lepidoptera, and Coleoptera, while \u003cem\u003eCy\u003c/em\u003et toxins primarily target Diptera. Both are synthesized as protoxins and are highly specific, binding to receptors in the midgut of susceptible insects. Their action as pore-forming toxins (PFTs) occurs under the alkaline conditions of the midgut, upon conversion to active toxins leading to alterations in cell permeability, cell lysis, and ultimately the death of the larvae. The complex interaction of these toxins with the host's immune system and gut cells and the synergetic effect of \u003cem\u003eCry\u003c/em\u003e and \u003cem\u003eCyt\u003c/em\u003e toxin underscores their effectiveness in vector control (Belousova et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Palma et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Poopathi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe toxicity of \u003cem\u003eBti\u003c/em\u003e varies among strains and is species-dependent, with the combination of toxins determining each strain's effectiveness against different mosquito vectors crucial for mosquito control programs. Therefore, it is essential to characterize the major toxic protein responsible for each indigenous \u003cem\u003eBti\u003c/em\u003e strains and understand the genomic toxin profile This study focuses on the molecular characterization of toxic genes in the highly toxic indigenous \u003cem\u003eBti\u003c/em\u003e strain VCRC B646, isolated from the soils of Tamil Nadu, India. It aims to identify and characterize the toxic genes, assess their phylogenetic relatedness to reference strains, and purify the major toxic proteins responsible for the strain's heightened toxicity.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBacterial strains and growth conditions\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eBacillus thuringiensis\u003c/em\u003e var. \u003cem\u003eisraelensis\u003c/em\u003e (\u003cem\u003eBti\u003c/em\u003e) strain VCRC B646, isolated from the soils of Kanchipuram, India, was cultured in Nutrient Yeast Salt Medium (NYSM) consisting of glucose (5 g/L), peptone (5 g/L), sodium chloride (5 g/L), HM peptone (3 g/L), yeast extract (5 g/L) and salt solution (g/L: MgCl\u003csub\u003e2\u003c/sub\u003e 2.03, CaCl\u003csub\u003e2\u003c/sub\u003e 1.0 and MnCl\u003csub\u003e2\u003c/sub\u003e, 0.1). The bacterial culture was incubated until complete sporulation and the formation of parasporal crystalline inclusions were observed (Manikandan et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Microscopic examination with staining confirmed the presence of crystalline inclusions. The cellular mass was subsequently harvested by centrifugation at 8000 rpm for 15 minutes (Vijayakumar et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eExtraction of bacterial genomic DNA\u003c/h3\u003e\n\u003cp\u003eBacterial genomic DNA was extracted from a pure individual colony, which was cultured overnight in nutrient broth at 37\u0026deg;C with shake culture. After harvesting the bacterial cells by centrifugation, the Gen-Elute bacterial genomic DNA kit (Sigma-Aldrich) was used for DNA extraction following the manufacturer's protocol. This included cell lysis, DNA binding to a silica membrane, washing to remove contaminants, and eluting the purified DNA. The extracted genomic DNA was visualized using 1.2% agarose gel electrophoresis and quantified spectrophotometrically at absorbance ratio at 260 nm/280 nm ( Bora \u003cem\u003eet al\u003c/em\u003e., 2024).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePCR Amplification of\u003c/b\u003e \u003cb\u003eCry\u003c/b\u003e \u003cb\u003eand C\u003c/b\u003e\u003cb\u003eyt\u003c/b\u003e \u003cb\u003egene\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eBt\u003c/em\u003e specific toxin genes \u003cem\u003ecry\u003c/em\u003e and \u003cem\u003ecyt\u003c/em\u003e genes amplification for the extracted DNA of \u003cem\u003eBti\u003c/em\u003e VCRC B646 was accomplished with using an automated thermal cycler (Bio-Rad, USA). \u003cem\u003eCry\u003c/em\u003e gene and \u003cem\u003eCyt\u003c/em\u003e gene specific oligonucleotide primers (\u003cem\u003eCry4A\u003c/em\u003e, \u003cem\u003eCry4B\u003c/em\u003e, \u003cem\u003eCry11\u003c/em\u003e, \u003cem\u003eCry\u003c/em\u003e10, \u003cem\u003eCyt\u003c/em\u003e2) were used for amplification with varied cyclic conditions, the expected size and the sequences were described in (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable.1: Characteristics of Primers for\u003c/b\u003e \u003cb\u003eCry\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eCyt\u003c/b\u003e \u003cb\u003eToxin Genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOligonucleotide Primer (5\u003csup\u003e1\u003c/sup\u003e \u0026minus;\u0026thinsp;3\u003csup\u003e1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMelting temperature\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eOptical Density\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eCry 4A\u003c/em\u003e (F)\u003c/p\u003e \u003cp\u003e\u003cem\u003eCry 4A\u003c/em\u003e (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCA AAG ATC ATT TCA AAA TTA CAT G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCGG CTT GAT CTA TGT CAT AAT CTG T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eCry 4B\u003c/em\u003e (F)\u003c/p\u003e \u003cp\u003e\u003cem\u003eCry 4B\u003c/em\u003e (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCGT TTT CAA GAC CTA ATA ATA TAA TAC C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCGG CTT GAT CTA TGT CAT AAT CTG T\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eCry 10\u003c/em\u003e (F)\u003c/p\u003e \u003cp\u003e\u003cem\u003eCry 10\u003c/em\u003e (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCA ATG CTC CAT CCA ATG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTT GTA TAG GCC TTC CTC CG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eCry 11\u003c/em\u003e (F)\u003c/p\u003e \u003cp\u003e\u003cem\u003eCry 11\u003c/em\u003e (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCGC TTA CAG GAT GGA TAG G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCT GAA ACG GCA CGA ATA TAA TA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eCyt 2\u003c/em\u003e (F)\u003c/p\u003e \u003cp\u003e\u003cem\u003eCyt 2\u003c/em\u003e (R)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATT ACA AAT TGC AAA TGG TAT TCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTTT CAA CAT CCA CAG TAA TTT CAA ATG C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe total reaction mixtures consist a final volume of 25\u0026micro;l; (12.5 \u0026micro;l of master mix (dNTPs, polymerase, and buffers), 1\u0026micro;l (10ng) of forward and reverse primers, 50 ng Template DNA of VCRC B646, 9.5\u0026micro;l of Nuclease free water). The amplification involved an initial denaturation step, followed by 35 cycles consisting of denaturation, annealing, and extension steps, with a final extension phase (Table\u0026nbsp;2). The amplified PCR product was separated through 1.2% agarose gel in a 1X Tris-Acetate-EDTA (TAE) buffer system and visualized under a UV transilluminator using ethidium bromide (EtBr) as a fluorescent tag against a 100-bp molecular weight marker (HiMedia).\u003c/p\u003e \u003cp\u003e \u003cb\u003eTable.2: PCR conditions for each of the\u003c/b\u003e \u003cb\u003eCry\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eCyt\u003c/b\u003e \u003cb\u003etoxin genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eInitial denaturation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eCycles\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eFinal Extension\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDenaturation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnnealing\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExtension\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCry 4A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 min at 95\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 min at 95\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 min at 58\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 min at 72\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 min at 72\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCry 4B\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3 min at 95\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 min at 95\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 min at 58\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 min at 72\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 min at 72\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCry 11\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 min at 95\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 sec at 95\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30 sec at 57\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30 sec at 72\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1 min at 72\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCry 10\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 min at 95\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 sec at 95\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30 sec at 56\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30 sec at 72\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2 min at 72\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eCyt 2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 min at 95\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 sec at 95\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30 sec at 61\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30 sec at 72\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5 min at 72\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePurification and sequence analysis of Amplified\u003c/b\u003e \u003cb\u003eCry\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eCyt\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThrough spin column chromatography the amplified \u003cem\u003eCry\u003c/em\u003e and \u003cem\u003eCyt\u003c/em\u003e genes were recovered from the unwanted impurities (dNTPs, Salts, and excess primers). Qiaquick PCR purification kit (Qiagen, USA) were used as per the instructions of the manufacturer and Purified DNAs were quantified using spectrophotometry. Sequence analysis was performed in both forward and reverse direction separately as per the conditions prescribed in Mandodan et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e in an automated Thermal Cycler (Bio-Rad, USA). The DNA sequencing reaction mixture purified from the impurities and dye through size-exclusion chromatography with a pre-filled gel filtration matrix; NucleoSEQ columns (Macherey-Nagel, Germany) as per the manufacturer\u0026rsquo;s protocol to improve the accuracy of DNA sequences. Thus, purified DNA was analyzed using the Applied Biosystems 3130XL Genetic Analyzer by the Sanger technique, for high-throughput DNA sequencing and fragment analysis at ICMR VCRC, Puducherry, India. The chromatogram generated were edited for consensus sequence and related sequences with maximum percentage similarity were obtained through NCBI BLAST. The nucleotide sequence of all the genes studied were submitted to Gene Bank database and obtained NCBI accession number.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic relatedness of\u003c/b\u003e \u003cb\u003eCry\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eCyt\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe closest sequences with highest percentage similarity were obtained through neighbour-joining method. By employing the Kimura 2-parameter method, gaps and missing data were removed, and using a consensus sequence generated for multiple sequence alignment subsequent phylogenetic tree construction using the MEGA 11 software.\u003c/p\u003e\n\u003ch3\u003eSpore-Crystal production and purification\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eBti\u003c/em\u003e strain VCRC B646 was cultured in NYSM medium until spore-crystal formation (Kunnikuruvan \u003cem\u003eet al.\u003c/em\u003e, 2014). The spore-crystal mixture was collected through centrifugation and purification of crystal protein from the complex carried out as per mounsef et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e. Briefly, Spore-Crystal biomass suspended first in 1M sodium chloride solution containing triton X-100 (0.01%) and washed twice. Then resuspended in saline solution (9 g of NaCl /1L water) to enhance the hydrophobic interactions. Organic solvent Hexane, was used for next wash in a proportion equal to or less than 10% to minimize the risk of crystal alteration during washing and repeated hexane wash 2\u0026ndash;3 times. Thus, washed product obtained was washed 2\u0026ndash;4 times with ice-cold water at 6000 rpm to obtain the final purified crystal (Mounsef et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eProtein characterization using ESI-nano LC-MS/MS\u003c/h3\u003e\n\u003cp\u003eThe nano ACQUITY UPLC\u0026reg; chromatographic system (Waters, Manchester, UK) at Mass spectrometry \u0026amp; Proteomics Core facility of Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala was assessed for protein profiling. Separated protein bands were excised from the acrylamide gel and vortexed in MilliQ water prior to destaining (100 mM ammonium bicarbonate/ acetonitrile (1:1, v/v)) to remove excess dye. DTT (dithiothreitol) was used as a reducing agent, followed by alkylation with iodoacetamide (IAA). Gel pieces were dehydrated and proteins were digested into smaller peptides using trypsin buffer (13 ng /\u0026micro;l trypsin in 50 mM ammonium bicarbonate). The extracted peptides reconstituted in 3% acetonitrile and injected (3 \u0026micro;l) onto a Symmetry\u0026reg; trap column (180 \u0026micro;m x 20 mm, C18, 5 \u0026micro;m, Waters) and separated on an analytical column (75 \u0026micro;m X 200 mm, HSS T3 C18, 1.8 \u0026micro;m, Waters). A binary gradient of 0.1% formic acid /Water and 0.1% formic acid/Acetonitrile was used for the separation (flow rate;300 nL/min) and the column temperature was maintained at 35\u0026deg;C was maintained. The autosampler temperature was kept at 4\u0026ordm;C to preserve sample integrity (Wojtkiewicz et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2014\u003c/span\u003e)\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMass spectrometry (MS)\u003c/h2\u003e \u003cp\u003eMass spectrometry (MS) was performed using the Synapt G2 High-Definition Mass Spectrometry (HDMS) System, calibrated with sodium iodide and set to positive Electrospray Ionization (ESI) mode. Leucine encephalin (m/z\u0026thinsp;=\u0026thinsp;556.2766) was utilized for online mass correction to ensure accuracy. The nano ESI capillary voltage was set to 3.4 kV to generate a fine spray of charged droplets, while the sample cone voltage was adjusted to 40 V to facilitate the transfer of ions from the ionization source to the mass spectrometer.\u003c/p\u003e \u003cp\u003eThe extraction cone voltage was maintained at 4 V to ensure efficient ion extraction into the vacuum system without significant fragmentation. In the ion mobility spectrometry (IMS) section, a nitrogen gas flow rate of 90 mL/min was used, with the T-Wave pulse height set to 40 V and the velocity adjusted to 800 m/s. The IMS voltage settings were configured to 8 V and 20 V to enhance ion mobility and separation. During data acquisition, the system operated in resolution mode, utilizing a continuum format to capture comprehensive spectral data, with collision energy set between 20 eV and 45 eV for effective ion fragmentation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe resulting data was analyzed using Progenesis QI for Proteomics v4.2 software, focusing on the organism \u003cem\u003eBacillus thuringiensis\u003c/em\u003e to interpret mass spectra and identify compounds based on their mass-to-charge ratios (m/z). Each parameter is carefully chosen to balance the need for sensitivity and specificity in protein identification. The database source utilized for protein identification was Universal Protein Resource (UniProt).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eToxicity Bioassay and analysis\u003c/h3\u003e\n\u003cp\u003eThe purified crystal mixture was subjected to larvicidal activity against late third instar larvae of \u003cem\u003eCx. qunquefasciatus\u003c/em\u003e, and \u003cem\u003eAe. aegypti\u003c/em\u003e following protocol described by WHO and previously (Manikandan \u003cem\u003eet al.\u003c/em\u003e, 2023; Mandodan et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Vijayakumar et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The 50% and 90% lethal concentration was calculated through probit analysis using SPSS (Gangmei \u003cem\u003eet al.\u003c/em\u003e, 2024).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCrystal Morphology of\u003c/strong\u003e \u003cstrong\u003eBti\u003c/strong\u003e \u003cstrong\u003eisolate VCRC B646\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn comparison with toxicity of standard strains through preliminary screening assays \u003cem\u003eBti\u003c/em\u003e VCRC B646 strain was selected due to higher toxicity. The spore crystal complex observed in 72 hour grown culture of \u003cem\u003eBti\u003c/em\u003e VCRC B646. The bacteria in rod shape were confirmed through microscopy and scanning electron microscopy showed the crystal morphology as irregular shape and spore size (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Spores were found in oval shape and stained with malachite green and formed at terminal end of vegetative cell. Spores and crystals found as a cluster under microscope (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmplification of\u003c/strong\u003e \u003cstrong\u003eCry\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eCyt\u003c/strong\u003e \u003cstrong\u003egenes and phylogeny\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePCR product analysis revealed that the new larvicidal isolate \u003cem\u003eBti\u003c/em\u003e VCRC B646 contained all six genes studied in the total DNA. A 459-base pair (bp) fragment was obtained from PCR amplification of the \u003cem\u003eCry4A\u003c/em\u003e gene from the total DNA of \u003cem\u003eBti\u003c/em\u003e VCRC B646 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). There is a 99% identity to the reported \u003cem\u003eCry4A\u003c/em\u003e toxin gene, confirming the toxin sequence within the particular gene family. Similarly, other toxin genes were also confirmed with sequences similar to their corresponding families, and GenBank accession numbers were received as follows: \u003cem\u003eCry\u003c/em\u003e4A(459): PP898123, \u003cem\u003eCry4B\u003c/em\u003e (321 bp): PP898125, \u003cem\u003eCyt2\u003c/em\u003e (356 bp): PP898127, \u003cem\u003eCry11\u003c/em\u003e (343 bp): PP898124, and \u003cem\u003eCry10\u003c/em\u003e (348 bp): PP898126 (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eAdditionally, to investigate the the evolutionary relatedness of VCRC B646 strain with other previously sequenced \u003cem\u003eBt\u003c/em\u003e strains, constructed a phylogenomic tree based on the consensus sequences generated for all toxin genes (\u003cem\u003eCry4A, Cry4B, Cry10, Cry11\u003c/em\u003e, and \u003cem\u003eCyt2\u003c/em\u003e) of bacterial strain VCRC B646 to \u003cem\u003eBacillus thuringiensis\u003c/em\u003e var \u003cem\u003eisraelensis\u003c/em\u003e was confirmed through phylogenetic analysis. The maximum likelihood model with 1000 bootstrap replicates was used to construct the phylogenetic tree (MEGA software) for all amplified toxin genes: \u003cem\u003eCry4A\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), \u003cem\u003eCry4B\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e), \u003cem\u003eCry10\u003c/em\u003e (Fig. 6), \u003cem\u003eCry11\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e), and \u003cem\u003eCyt2\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eProteomic characterization of toxic protein\u003c/h2\u003e\n \u003cp\u003eThe crystal toxins purified from spore-crystal mixture (dry biomass) irregular crystals were visualized in microscope (100X) with a yield of (10 mg/L) (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e). The freeze-dried protein separated through SDS-PAGE. The protein profile of \u003cem\u003eBti\u003c/em\u003e VCRC B646 -purified crystal toxins in an SDS-PAGE showed the main proteins with molecular weights of apparently 72, 35, 32, and 22 kDa in size (Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eSDS-PAGE pattern of solubilized proteins from crystal-spore mixture of strain HD1.\u003c/p\u003e\n \u003cp\u003eLC-MS analysis confirmed the toxic protein band and identified as \u003cstrong\u003eCry11Aa5.\u003c/strong\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eLC- MS analysis\u003c/h2\u003e\n \u003cp\u003ePeptides of the all 4 main protein bands (A, B, C and D) were analyzed through LC-MS. Four protein bands were identified as Cry11Aa, also known as \u0026quot;Pesticidal crystal protein\u0026quot; based on the identified peptides and total score. Amino acid sequences of peptides were determined. Additionally, similarity indices were calculated for each band relative to the reference pesticidal crystal protein sp|P21256|: the first band showed a similarity index of 62.4%, the second band 67.76%, the third band 62.5%, and the fourth band 62.7%. These findings underscore a substantial resemblance between the identified Cry11Aa protein bands and the reference pesticidal crystal protein sp|P21256|, highlighting their functional similarity and potential efficacy in mosquito control strategies. An overestimation of Cry11Aa observed. The multiple sequence alignment of amino acid of all bands in comparison with Full length of Pesticidal crystal protein Cry11Aa sp|P21256| .\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eToxicity bioassay with Cry 11Aa\u003c/h2\u003e\n \u003cp\u003eBioassays performed against the third instar larvae of \u003cem\u003eCulex and Aedes\u003c/em\u003e with purified Crystals and the mortality found in response to different doses as linear (Table. 3).\u0026nbsp;\u003c/p\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eToxicity values (LC\u003csub\u003e50\u003c/sub\u003e and LC\u003csub\u003e90\u003c/sub\u003e) of \u003cem\u003eBti\u003c/em\u003e VCRC B646 Crystal against mosquito vectors.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBacterial strain\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMosquito species\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/ml)\u003c/p\u003e\n \u003cp\u003e* (LCL \u0026ndash;UCL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLC\u003csub\u003e90\u003c/sub\u003e (\u0026micro;g/ml)\u003c/p\u003e\n \u003cp\u003e* (LCL \u0026ndash;UCL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIntercept\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSlope\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u0026chi;\u003csup\u003e2\u003c/sup\u003e(df)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eBti (VCRC \u0026ndash;B646)\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eCulex quinquefasciatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0025\u003c/p\u003e\n \u003cp\u003e(0.0023\u0026ndash;0.0026)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0044\u003c/p\u003e\n \u003cp\u003e(0.0042\u0026ndash;0.0047)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e-1.665\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.089\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAedes aegypti\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0022\u003c/p\u003e\n \u003cp\u003e(0.0020\u0026ndash;0.0024)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0040\u003c/p\u003e\n \u003cp\u003e(0.0038\u0026ndash;0.0043)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e\u0026minus;\u0026thinsp;1.723\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.250\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e \u003cem\u003eBacillus thuringiensis israelensis (Bti)\u003c/em\u003e has emerged as a safe alternative to synthetic insecticides for mosquito control since its discovery in the 1970s (Gangmei \u003cem\u003eet al\u003c/em\u003e., 2024, Lukose \u003cem\u003eet al\u003c/em\u003e., 2024). The specificity and toxicity of \u003cem\u003eBti\u003c/em\u003e to various mosquito vectors are closely linked to the crystalline inclusion bodies it produces, particularly the \u003cem\u003eCry\u003c/em\u003e and \u003cem\u003eCyt\u003c/em\u003e proteins. The diversity of these genes varies among \u003cem\u003eBti\u003c/em\u003e strains, with each protein demonstrating distinct specificity against related mosquito species. Variations in the single amino acid sequence of \u003cem\u003eCry\u003c/em\u003e toxins lead to differences in toxicity levels among insect pests. Therefore, there is an ongoing need for novel \u003cem\u003eCry\u003c/em\u003e and \u003cem\u003eCyt\u003c/em\u003e toxin genes with sequence variations from indigenous strains for improved resistance management. This study focuses on the soil isolate \u003cem\u003eBti\u003c/em\u003e VCRC B646, selected for its high toxicity against mosquito larvae.\u003c/p\u003e \u003cp\u003eThere are several reports of \u003cem\u003eBti\u003c/em\u003e strains exhibiting larvicidal activity whereas the strain VCRC B646 exhibits an enhanced toxicity against \u003cem\u003eA.aegypti\u003c/em\u003e and \u003cem\u003eC.quinquefasciatus\u003c/em\u003e. The specific toxicity of \u003cem\u003eBti\u003c/em\u003e to dipteran insects is correlated to its combination of Cry and \u003cem\u003eCyt\u003c/em\u003e proteins (Ma et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Research indicates that the combinations of \u003cem\u003eCry\u003c/em\u003e4Aa, \u003cem\u003eCry\u003c/em\u003e10, \u003cem\u003eCry\u003c/em\u003e11, and \u003cem\u003eCy\u003c/em\u003et proteins are particularly potent against mosquitoes (Ma et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, the strain \u003cem\u003eBti\u003c/em\u003e VCRC B646 contains all these effective toxin gene combinations, confirmed through specific gene amplification techniques\u003c/p\u003e \u003cp\u003eThrough PCR amplification, the presence of toxin genes belonging to the \u003cem\u003eCry\u003c/em\u003e and \u003cem\u003eCyt\u003c/em\u003e families in \u003cem\u003eBti\u003c/em\u003e VCRC B646 was confirmed. Additionally, the phylogenetic relatedness of this strain was assessed by sequencing all seven toxin genes present, revealing a 90% similarity to other known \u003cem\u003eBti\u003c/em\u003e strains.\u003c/p\u003e \u003cp\u003eThe specific toxicity of \u003cem\u003eBacillus thuringiensis\u003c/em\u003e (\u003cem\u003eBt\u003c/em\u003e) to dipteran insects, particularly mosquitoes, is a critical area of research in biological pest control. There is a multifaceted relationship between the genentic composition of \u003cem\u003eCry\u003c/em\u003e and \u003cem\u003eCyt\u003c/em\u003e genes in \u003cem\u003eBti\u003c/em\u003e and their toxicity to the susceptible insects which contributes to the varying level of toxicity among various isolates (Das et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The environment of insect gut and synergetic effect contribute to the overall toxicity profile of certain isolates.\u003c/p\u003e \u003cp\u003eBioassays conducted with a purified crystal mixture containing the \u003cem\u003ecry11\u003c/em\u003e toxin have demonstrated remarkable toxicity against \u003cem\u003eAedes aegypti\u003c/em\u003e and \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e, showing higher efficacy compared to reference strains, highlighting its potential as a superior biocontrol agent. The specific toxicity of \u003cem\u003eBti\u003c/em\u003e to dipteran insects is closely linked to the combination of \u003cem\u003eCry\u003c/em\u003e and \u003cem\u003eCyt\u003c/em\u003e proteins in its genetic makeup. Research has shown that the combinations of \u003cem\u003eCry4Aa, Cry10, Cry11\u003c/em\u003e, and \u003cem\u003eCyt\u003c/em\u003e proteins exhibit significant potency against mosquitoes (Ma et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Interestingly, the strain \u003cem\u003eBti\u003c/em\u003e VCRC B646 contains all these effective combinations of toxin genes, confirmed through specific gene amplification techniques.\u003c/p\u003e \u003cp\u003e \u003cem\u003eCry\u003c/em\u003e11Aa is the most toxic δ-endotoxin produced by \u003cem\u003eBacillus thuringiensis israelensis\u003c/em\u003e (\u003cem\u003eBti\u003c/em\u003e) against mosquito larvae, particularly \u003cem\u003eAedes\u003c/em\u003e and \u003cem\u003eCulex\u003c/em\u003e species. Purification of this toxin from the spore-crystal preparation of \u003cem\u003eBti\u003c/em\u003e VCRC revealed a 72 kDa polypeptide, identified as the pesticidal crystal protein \u003cem\u003eCry\u003c/em\u003e11Aa. Bioassays of the purified toxin demonstrated high larvicidal efficacy, with the highest toxicity observed against the dengue vector \u003cem\u003eAedes aegypti\u003c/em\u003e and followed by the filarial vector \u003cem\u003eCulex quinquefasciatus.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eThe sporulation phase of the \u003cem\u003eB. thuringiensis\u003c/em\u003e strains and the existence of \u003cem\u003eCry\u003c/em\u003e genes could vary due to the heterogeneity in environmental condition. The different \u003cem\u003eCry\u003c/em\u003e gene subgroups encode significantly different crystal proteins due to variations in the nucleotide sequences which results in varied insecticidal effect on insect larvae of different orders. Additionally, the synergistic interactions among these toxin proteins further enhance the potency of \u003cem\u003eBti\u003c/em\u003e for insecticidal activity, making it a highly efficient and environmentally friendly option for mosquito control programs (Arsov et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis research contributes to the understanding of the genetic basis for the heightened toxicity observed in \u003cem\u003eBti\u003c/em\u003e VCRC B646 and highlights its potential as an effective biopesticide for mosquito control. The broad target range of \u003cem\u003eBti\u003c/em\u003e is attributed to the presence of different mosquitocidal proteins produced during sporulation and their combination in action, with the main ones being \u003cem\u003eCry11Aa, Cry4Ba, Cry4Aa\u003c/em\u003e, and \u003cem\u003eCyt1Aa.\u003c/em\u003e The \u003cem\u003eBti\u003c/em\u003e parasporal body is significantly different from the typical \u003cem\u003eBt\u003c/em\u003e bipyramidal crystal, which shows toxicity to lepidopteran larvae. The parasporal body formed of 4 main toxic proteins (\u003cem\u003eCry11A\u003c/em\u003e (72 kDa), \u003cem\u003eCyt1A\u003c/em\u003e (27.3 kDa), \u003cem\u003eCry4B\u003c/em\u003e (134 kDa) and \u003cem\u003eCry4A\u003c/em\u003e (128 kDa) organized into three distinct inclusion types, forming a parasporal body (spherical) enclosed by a lamellar envelope.\u003c/p\u003e \u003cp\u003eOn the basis of amino acid sequence as well as toxicological properties \u003cem\u003eCyt1A\u003c/em\u003e protein is different significantly from the Cry proteins. In laboratory conditions, it exhibits notable cytolytic effects on various cells from both invertebrates and vertebrates, with a particular affinity for unsaturated fatty acids found in the lipid component of cell membranes. \u003cem\u003eBti\u003c/em\u003e produces several potent toxic proteins, such as, \u003cem\u003eCry11Aa, Cry4Aa, Cry4Ba\u003c/em\u003e, and \u003cem\u003eCyt1Aa\u003c/em\u003e, which act synergistically to target mosquito larvae by binding to specific receptors in the midgut brush border membrane, leading to cellular destruction and larval mortality. This specificity contributes to the lack of resistance among mosquito populations and ensures safety for non-target organisms (Poopathi et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Valtierra \u003cem\u003eet al.\u003c/em\u003e, 2020).\u003c/p\u003e \u003cp\u003eThe mosquitocidal activity of \u003cem\u003eBti\u003c/em\u003e VCRC B646 was also attributed to the effect of a specific polypeptide chain (72 kDa). The yield of \u003cem\u003eBti\u003c/em\u003e VCRC B646 crystal is 37 mg per liter. Through LC-MS analysis, the toxic protein has been identified and characterized as \"Pesticidal crystal protein Cry11Aa\". The amplification results revealed that the PCR amplification of specific regions within the \u003cem\u003eCry\u003c/em\u003e and \u003cem\u003eCy\u003c/em\u003et genes, with fragment sizes of 459 bp for \u003cem\u003eCry4A\u003c/em\u003e, 321 bp for \u003cem\u003eCry4B\u003c/em\u003e, 477 bp for \u003cem\u003eCyt1\u003c/em\u003e, 356 bp for \u003cem\u003eCyt2\u003c/em\u003e, 343 bp for \u003cem\u003eCry11\u003c/em\u003e, and 348 bp for \u003cem\u003eCry10\u003c/em\u003e, indicating of target sequences. As \u003cem\u003eCry\u003c/em\u003e and \u003cem\u003eCyt\u003c/em\u003e genes, resulting from \u003cem\u003eBacillus thuringiensis israelensis\u003c/em\u003e (\u003cem\u003eBti\u003c/em\u003e), encode proteins with potential larvicidal activity against mosquito larvae. The construction of a phylogenetic tree using a neighbor joining model with 1000 bootstrap replicates, and the utilization of the Kimura 2-parameter model for sequence analysis, allowed for a comprehensive examination of the genetic relatedness of the bacterial isolate. Furthermore, the confirmation of \u003cem\u003eBacillus thuringiensis israelensis\u003c/em\u003e (\u003cem\u003eBti\u003c/em\u003e) VCRC B646 identity, along with the presence of specific \u003cem\u003eCry\u003c/em\u003e and \u003cem\u003eCyt\u003c/em\u003e toxin genes using targeted gene primers, provides valuable insights into the toxin profiles of the bacterial isolate. These proteins, including, \u003cem\u003eCry4Aa, Cry4Ba, Cry11Aa, Cry10Aa, Cyt1Aa\u003c/em\u003e, and \u003cem\u003eCyt2Aa\u003c/em\u003e, constitute the major toxic arsenal of \u003cem\u003eBti\u003c/em\u003e with broad range of target insects (de Souza et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Additionally, the synergistic interactions among these toxin proteins further enhance the potency of \u003cem\u003eBti\u003c/em\u003e for insecticidal activity, making it a highly efficient and environmentally friendly option for mosquito control programs.\u003c/p\u003e \u003cp\u003eResistance to insecticidal proteins, such as \u003cem\u003eCry11Aa\u003c/em\u003e, has been reported, but there are currently no documented cases of resistance among field populations to \u003cem\u003eBacillus thuringiensis israelensis\u003c/em\u003e (\u003cem\u003eBti\u003c/em\u003e). To address potential resistance development, innovative strategies like recombinant technology are crucial for enhancing the efficacy of pesticidal proteins. Modification of proteins through recombinant techniques can optimize their effectiveness against target pests. For instance, \u003cem\u003eCry11Aa\u003c/em\u003e recombinant variants have shown activity against larval stages of \u003cem\u003eCulex\u003c/em\u003e and \u003cem\u003eAedes\u003c/em\u003e mosquitoes. Detailed biophysical analyses have revealed insights into the stability and in-vitro behavior of recombinant \u003cem\u003eCry11Aa\u003c/em\u003e. Molecular dynamics simulations have further contributed to understanding \u003cem\u003eCry11Aa\u003c/em\u003e's behavior and proteolytic processing, aiding in the strategic utilization of \u003cem\u003eBti\u003c/em\u003e for mosquito and vector management (Kinkar et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn recent study, with \u003cem\u003eBti Cry11Aa\u003c/em\u003e toxins, a delivery system which is efficient stable was developed to control mosquito larvae. They applied a coating of Mg (OH)2 nanoparticles (MHNPs) to \u003cem\u003eCry11Aa\u003c/em\u003e proteins and subsequently evaluated the impact of MHNPs on the bioactivity and ultraviolet protection capabilities of the \u003cem\u003eCry11Aa\u003c/em\u003e proteins. Through this \u003cem\u003eCry\u003c/em\u003e protein insectical activity enhanced and protected from toxin degradation (reduction from 64.29\u0026ndash;16.67%).) through MHNPs. Additionally, MHNPs enhanced the proteolysis of \u003cem\u003eCry11Aa\u003c/em\u003e in the midgut and intensified the damage inflicted by the \u003cem\u003eCry\u003c/em\u003e protein on gut epithelial cells, thereby augmenting its insecticidal efficacy against \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e. This indicates that MHNPs, acting as an excellent nano-carrier, have the potential to significantly enhance the insecticidal bioactivity and anti-ultraviolet capacity of \u003cem\u003eCry11Aa\u003c/em\u003e (Pan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAccording to Wang et al., (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), \u003cem\u003eCry11Aa\u003c/em\u003e and \u003cem\u003eCry4Ba\u003c/em\u003e of \u003cem\u003eBti\u003c/em\u003e revealed highly toxicity to to \u003cem\u003eAe. aegypti\u003c/em\u003e larvae. The important membrane bound receptor for \u003cem\u003eCry\u003c/em\u003e toxin noted among \u003cem\u003eDiptera, Lepidoptera, Coleoptera\u003c/em\u003e and other insects were glycosylphosphatidylinositol (GPI)-anchored APN. Two GPI-anchored APN isoforms in \u003cem\u003eAe. aegypti\u003c/em\u003e (AeAPN1 and AeAPN2), which bind \u003cem\u003eCry4Ba\u003c/em\u003e and \u003cem\u003eCry11Aa\u003c/em\u003e, were individually disabled using CRISPR/Cas9 mutagenesis. A double-mutant homozygous strain lacking both AeAPN1 and AeAPN2 was produced \u003cem\u003evia\u003c/em\u003e: reverse genetics. Binding affinity of protoxins \u003cem\u003eCry11Aa\u003c/em\u003e and \u003cem\u003eCry4Ba\u003c/em\u003e to midgut brush border membrane vesicles was similar in in ELISA assays from these APN-knockouts to that of the wild. Toxicity results of bioassays indicated that neither single knockout of AeAPN1 nor AeAPN2, nor disruption of both AeAPN1 and AeAPN2 simultaneously, led to changes significantly in the \u003cem\u003eAe. aegypti\u003c/em\u003e larvae susceptibility to \u003cem\u003eCry11Aa\u003c/em\u003e and \u003cem\u003eCry4Ba\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn conclusion, the use of \u003cem\u003eBacillus thuringiensis israelensis\u003c/em\u003e (\u003cem\u003eBti\u003c/em\u003e) VCRC B646 as biological larvicides represents a promising strategy for mosquito control, with a particular focus on their eco-friendly nature and effectiveness against a broad spectrum of mosquito species. Its enhanced toxicity against \u003cem\u003eAedes aegypti\u003c/em\u003e and \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e is attributed to the presence of multiple Cry and Cyt toxin genes, particularly \u003cem\u003eCry\u003c/em\u003e10, \u003cem\u003eCry\u003c/em\u003e11Aa, \u003cem\u003eCry\u003c/em\u003e4Aa, \u003cem\u003eCry\u003c/em\u003e4Ba, and \u003cem\u003eCyt\u003c/em\u003e1Aa, which act synergistically to target mosquito larvae. Molecular characterization confirmed the presence of these toxin genes, with phylogenetic analysis revealing a close genetic relationship with other potent Bti strains.\u003c/p\u003e"},{"header":"Statements and Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe first author acknowledges the PhD supervisor (Dr. S. Poopathi). Authors acknowledges the Director, ICMR-Vector Control Research Centre, Puducherry for providing facilities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u0026nbsp;\u003c/strong\u003eCompliance with ethical standards. This study does not require ethical approval because it is a microbiological study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors declare no funding received.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e: The obtained sequence data to support the study was submitted to the NCBI GenBank and received the following accession numbers mentioned in manuscript, PP898123, PP898125, PP898127, PP898124, PP898126.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statement:\u0026nbsp;\u003c/strong\u003eA.V., Writing-Original Draft, experiments, Investigation, J.L., S.M., Formal Analysis, Validation, Data Curation K.G., Data Curation, Validation, A.K., B.B., Visualization, Validation, H.P., Methodology, Validation, M.A., Validation, Methodology, Formal Analysis, Review and Editing, P. S. Supervision and concept and administration\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDhanasekaran, D., and Thangaraj, R. (2014). Microbial secondary metabolites are an alternative approaches against insect vector to prevent zoonotic diseases. Asian Pacific Journal of Tropical Disease, 4(4), 253-261.\u003c/li\u003e\n \u003cli\u003eMansour, T., Radwan, W. H., Mansour, M., Gomaa, M., Farouk, F., Shepl, M., and Abu-Hussien, S. H. (2023). Larvicidal potential, toxicological assessment, and molecular docking studies of four Egyptian bacterial strains against Culex pipiens L(Diptera: Culicidae). Scientific Reports, 13(1), 17230.\u003c/li\u003e\n \u003cli\u003eSchnepf E., Crickmore N., Van Rie J., Lereclus D., Baum J., Feitelson J., and Dean D. 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Potential for \u003cem\u003eBacillus thuringiensis\u003c/em\u003e and other bacterial toxins as biological control agents to combat dipteran pests of medical and agronomic importance. \u003cem\u003eToxins\u003c/em\u003e, 12(12): 773.\u003c/li\u003e\n \u003cli\u003eDe Souza, M. P., Huang, C. P. A., Chee, N. and Terry, N. (1999). Rhizosphere bacteria enhance the accumulation of selenium and mercury in wetland plants. Planta, 209, 259-263.\u003c/li\u003e\n \u003cli\u003eKinkar, O. U., Prashar, A., Yadav, B., Kumar, A., Hadapad, A. B., Hire, R. S., and Makde, R. D. (2023). Purification, characterization and proteolytic processing of mosquito larvicidal protein Cry11Aa from \u003cem\u003eBacillus thuringensis\u003c/em\u003e subsp. \u003cem\u003eisralensis\u003c/em\u003e ISPC-12. \u003cem\u003eInternational Journal of Biological Macromolecules\u003c/em\u003e, 124979.\u003c/li\u003e\n \u003cli\u003ePan, X., Xu, Z., Li, L., Shao, E., Chen, S., Huang, T., and Guan, X. (2017). Adsorption of insecticidal crystal protein \u003cem\u003eCry11Aa\u003c/em\u003e onto nano-Mg (OH) 2: effects on bioactivity and anti-ultraviolet ability. \u003cem\u003eJournal of Agricultural and Food Chemistry\u003c/em\u003e, 65(43), 9428-9434.\u003c/li\u003e\n \u003cli\u003eWang, J., Yang, X., He, H., Chen, J., Liu, Y., Huang, W., and Wu, S. (2021). Knockout of two Cry-binding aminopeptidase N isoforms does not change susceptibility of aedes aegypti larvae to \u003cem\u003eBacillus thuringiensis\u003c/em\u003e subsp. \u003cem\u003eisraelensis\u003c/em\u003e Cry4Ba and Cry11Aa toxins. \u003cem\u003eInsects\u003c/em\u003e, 12(3): 223.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bacillus thuringiensis serovar israelensis, Cry, Cyt, Mosquito species, Toxin protein, Phylogenetic analysis","lastPublishedDoi":"10.21203/rs.3.rs-6163570/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6163570/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEffective mosquito control is essential for reducing the transmission of vector-borne diseases. This study focuses on the comprehensive characterization of mosquitocidal toxins produced by \u003cem\u003eBacillus thuringiensis\u003c/em\u003e serovar \u003cem\u003eisraelensis (Bti)\u003c/em\u003e VCRC B646 and the associated insecticidal genes. The bacterium was cultured, and the spore-crystal complex was purified to identify the mosquitocidal proteins. The isolate produced mosquitocidal toxins were effective against \u003cem\u003eAedes aegypti\u003c/em\u003e, \u003cem\u003eAnopheles stephensi\u003c/em\u003e, and \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e, Toxicity bioassays indicated lethal concentrations (LC\u003csub\u003e50\u003c/sub\u003e and LC\u003csub\u003e90\u003c/sub\u003e) for \u003cem\u003eAedes aegypti\u003c/em\u003e (0.0022 mg/L and 0.004 mg/L), and \u003cem\u003eCulex quinquefasciatus\u003c/em\u003e (0.0025 mg/L and 0.0044 mg/L). SDS-PAGE and LC-MS analysis revealed that \u003cem\u003eCry11Aa5\u003c/em\u003e (Pesticidal Crystal Protein) is the predominant toxin produced by this strain. PCR amplification confirmed the presence of genes encoding various insecticidal proteins, including \u003cem\u003eCry\u003c/em\u003e and \u003cem\u003eCyt\u003c/em\u003e toxins. Phylogenetic analysis was performed to assess the genetic relatedness and toxin profiles of the bacterial isolate. This detailed characterization of \u003cem\u003eBti\u003c/em\u003e VCRC B646 highlights its potential as a promising biopesticide candidate for mosquito control, contributing to the development of sustainable and eco-friendly strategies for vector management.\u003c/p\u003e","manuscriptTitle":"Characterization of pesticidal crystal toxin protein Cry11Aa from Bacillus thuringiensis serovar israelensis VCRC-B646 for mosquito control.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 10:47:32","doi":"10.21203/rs.3.rs-6163570/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-03-21T09:00:21+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-20T13:10:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-20T12:02:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biotechnology Letters","date":"2025-03-18T02:40:28+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor revisions","date":"2025-03-10T09:03:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biotechnology-letters","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bile","sideBox":"Learn more about [Biotechnology Letters](https://www.springer.com/journal/10529)","snPcode":"10529","submissionUrl":"https://submission.nature.com/new-submission/10529/3","title":"Biotechnology Letters","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"929f2893-6ae5-4424-b6cf-0c494cad3341","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-15T16:02:07+00:00","versionOfRecord":{"articleIdentity":"rs-6163570","link":"https://doi.org/10.1007/s10529-025-03640-1","journal":{"identity":"biotechnology-letters","isVorOnly":false,"title":"Biotechnology Letters"},"publishedOn":"2025-09-10 15:57:25","publishedOnDateReadable":"September 10th, 2025"},"versionCreatedAt":"2025-03-24 10:47:32","video":"","vorDoi":"10.1007/s10529-025-03640-1","vorDoiUrl":"https://doi.org/10.1007/s10529-025-03640-1","workflowStages":[]},"version":"v1","identity":"rs-6163570","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6163570","identity":"rs-6163570","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}