Cry Proteins Research Articles

Cry Toxins Use Multiple ATP-Binding Cassette Transporter Subfamily C Members as Low-Efficiency Receptors in Bombyx mori.

Recent studies have suggested that ABC transporters are the main receptors of Cry toxins. However, the receptors of many Cry toxins have not been identified. In this study, we used a heterologous cell expression system to identify Bombyx mori ABC transporter subfamily C members (BmABCCs) that function as receptors for five Cry toxins active in Lepidopteran insects: Cry1Aa, Cry1Ca, Cry1Da, Cry8Ca, and Cry9Aa. All five Cry toxins can use multiple ABCCs as low-efficiency receptors, which induce cytotoxicity only at high concentrations. Surface plasmon resonance analysis revealed that the KD values between the toxins and BmABCC1 and BmABCC4 were 10-5 to 10-9 M, suggesting binding affinities 8- to 10,000-fold lower than those between Cry1Aa and BmABCC2, which are susceptibility-determining receptors for Cry1Aa. Bioassays in BmABCC-knockout silkworm strains showed that these low-efficiency receptors are not involved in sensitivity to Cry toxins. The findings suggest that each family of Cry toxins uses multiple BmABCCs as low-efficiency receptors in the insect midgut based on the promiscuous binding of their receptor-binding regions. Each Cry toxin seems to have evolved to utilize one or several ABC transporters as susceptibility-determining receptors.

Diverse genetic basis of Vip3Aa resistance in five independent field-derived strains of Helicoverpa zea in the US.

Practical resistance of Helicoverpa zea to Cry proteins has become widespread in the US, making Vip3Aa the only effective Bacillus thuringiensis (Bt) protein for controlling this pest. Understanding the genetic basis of Vip3Aa resistance in H. zea is essential in sustaining the long-term efficacy of Vip3Aa. The objectives of this study were to characterize the inheritance of Vip3Aa resistance in four distinct field-derived H. zea strains (M1-RR, AC4-RR, R2-RR and R15-RR), and to test for shared genetic basis among these strains and a previously characterized Texas resistant strain (LT#70-RR). Maternal effects and sex linkage were absent, and the effective dominance level (DML) was 0.0 across Vip3Aa39 concentrations ranging from 1.0 to 31.6 μg cm-2, in all H. zea resistant strains. Mendelian monogenic model tests indicated that Vip3Aa resistance in each of the four strains was controlled by a single gene. However, interstrain complementation tests indicated that three distinct genetic loci are involved in Vip3Aa resistance in the five resistant H. zea strains: one shared by M1-RR and LT#70-RR; another shared by R2-RR and R15-RR; and a distinct one for AC4-RR. Results of this study indicate that Vip3Aa resistance in all H. zea strains was controlled by a single, recessive and autosomal gene. However, there were three distinct genetic loci associated with Vip3Aa resistance in the five resistant H. zea strains. The information generated from this study is valuable for exploring mechanisms of Vip3Aa resistance, monitoring the evolution of Vip3Aa resistance, and devising effective strategies for managing Vip3Aa resistance in H. zea. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Bacillus thuringiensis translocation in citrus scion/rootstock combinations and binding of cry toxins with the Asian citrus psyllid gut receptors

Bacillus thuringiensis (Bt) can be effective in controlling insect pests either in spray products or by their toxins expressed in transgenic crops. The discovery of Bt as endophytic in citrus opened new perspectives for the control of Diaphorina citri Kuwayama, the vector of the bacteria Candidatus Liberibacter spp. associated with Huanglongbing. In this study, the endophytic translocation of Bt in citrus seedlings (small and large plants) and in different scion-rootstock combinations were investigated. We also assessed the lethal concentration of D. citri nymphs caused by systemic Bt in citrus and, moreover, the binding of Cry toxins with the Asian citrus psyllid (ACP) gut receptors. Endophytic translocation of Bt in citrus seedlings and nursery trees was confirmed by colony isolation, PCR detection, and Btk-GFP microscopy analyses, which demonstrated that, after Bt inoculation into the substrate, the bacteria enter through lateral root emergence sites and are localized in stem xylem vessel elements. Mean lethal concentrations for D. citri third instar nymphs were 4.92 × 104 and 2.19 × 104 spores mL−1 for S1450 and S1302 strains, respectively. Nymphal mortality induced by the bacteria was higher in citrus seedlings than in citrus nursery trees. All Cry-toxins tested were able to bind to D. citri brush border membrane vesicles, suggesting the potential pathogenicity of Bt strains against psyllid. This study clearly demonstrates the penetration and Bt translocation in the xylem of small and large citrus plants and the potential of the bacteria to cause ACP nymphs mortality that fed on shoots from Bt-treated citrus.

Proteotranscriptomic analyses of the midgut and Malpighian tubules after a sublethal concentration of Cry1Ab exposure on Spodoptera litura.

Cry1Ab has emerged as a bio-insecticide to control Spodoptera litura (Lepidoptera: Noctuidae). However, the sublethal effects of Cry1Ab on the physiological changes and molecular level of S. litura have not been well documented. Our aims in this study were to assess the sublethal effect of Cry1Ab on S. litura, including midgut and Malpighian tubules as targets. After sublethal Cry1Ab exposure, distinct histological alterations were mainly observed in the midgut. Furthermore, the results of comparative RNA sequencing and tandem mass tag-based proteomics showed that, in the midgut, most differential expression genes (DEGs) were up-regulated and significantly enriched in the serine protease activity pathway, and up-regulated differential expression proteins (DEPs) were mainly associated with the oxidative phosphorylation pathway, whereas the down-regulated involved in the ribosome pathways. In the Malpighian tubules, DEGs and DEPs were significantly enriched in the ribosome pathway. We proposed that ribosome may act as a universal target in energy metabolism with other pathways via the results of protein-protein interaction analysis. Further, by verification of the mRNA expression of some Cry protein receptor and detoxification genes after Cry1Ab treatment, it was suggested that the ribosomal proteins (RPs) possibly participate in influencing the Bt-resistance of S. litura larvae under sublethal Cry1Ab exposure. Under sublethal Cry1Ab exposure, the midgut of S. litura was damaged, and the proteotranscriptomic analysis elucidated that Cry1Ab disrupted the energy homeostasis of larvae. Furthermore, we emphasized the potential role of ribosomes in sublethal Cry1Ab exposure. © 2024 Society of Chemical Industry.

MiR-2b-3p Downregulated PxTrypsin-9 Expression in the Larval Midgut to Decrease Cry1Ac Susceptibility of the Diamondback Moth, Plutella xylostella (L.).

Crystal (Cry) toxins, produced by Bacillus thuringiensis, are widely used as effective biological pesticides in agricultural production. However, insects always quickly evolve adaptations against Cry toxins within a few generations. In this study, we focused on the Cry1Ac protoxin activated by protease. Our results identified PxTrypsin-9 as a trypsin gene that plays a key role in Cry1Ac virulence in Plutella xylostella larvae. In addition, P. xylostella miR-2b-3p, a member of the micoRNA-2 (miR-2) family, was significantly upregulated by Cry1Ac protoxin and targeted to PxTrypsin-9 downregulated its expression. The mRNA level of PxTrypsin-9, regulated by miR-2b-3p, revealed an increased tolerance of P. xylostella larvae to Cry1Ac at the post-transcriptional level. Considering that miR-2b and trypsin genes are widely distributed in various pest species, our study provides the basis for further investigation of the roles of miRNAs in the regulation of the resistance to Cry1Ac and other insecticides.

Blue light strongly promotes de-etiolation over green, moderate over red, but have limited action over far-red lights in lettuce plants

The role of photoreceptors (phytochromes and cryptochromes) was evaluated in lettuce regarding (de)etiolation processes. The manipulation of light quality and intensity can modulate their activity and response and, consequently, promote morphophysiological changes in plants. To this end, lettuce plants were grown under controlled conditions at two photosynthetically active radiation levels (300 and 600 µmol m−2 s−1) and at different qualities, including white, blue, green, red and far-red light, as well as combinations, totaling 18 treatments. Biomass gain, internode length, light interception, pigment content, morpho-anatomical changes in the leaves and ultrastructural changes in the chloroplast and cell wall were evaluated. It was verified that in green, there was the highest degree of etiolation; however, the supplementation of blue promotes a strong de-etiolation process. In pure red (R), the plants were slightly less etiolated, but with limited action of blue in stimulating de-etiolation, and in R plus far-red (FR) (R:FR ratio was 0.33 in R + FR treatment), there was a strong degree of etiolation and weak action of blue on those set of treatments. In general, the plants submitted to the highest intensities of blue light, alone or in combination, approach the control phenotype (white light at total 300 µmol m−2 s−1; 18 % blue = 54 µmol m−2 s−1). The morpho-anatomical phenotype supports the hypothesis that blue light is capable of inhibiting or reducing etiolation-related responses, but the effects are dependent not only on the quality alone but also on the light intensity. Based on the known light-dependent response of phytochromes and cryptochromes, we suggest a weak interference of PHYB on CRY and a strong negative action of PHYA on CRY proteins.

Induced CTL-S15 gene expression by Bacillus thuringiensis declines susceptibility in Spodoptera exigua

It has been reported that C-type lectins (CTLs), which are pattern recognition receptors of the insect innate immunity response, may compete with Cry toxin for the receptor alkaline phosphatase to decrease its toxicity in insects. However, to date, which CTLs affect larval susceptibility to Bt in Spodoptera exigua is not clear. In this study, 33 CTL genes were identified from S. exigua. Based on the number of carbohydrate-recognition domains (CRDs) and the domain architectures, they were classified into three groups: (1) nineteen CTL-S (single-CRD), (2) eight immulectin (dual-CRD) and (3) six CTL-X (CRD with other domains). RT-qPCR analysis revealed that expression levels of SeCTL-S15, IML-4 and CTL-X6 were upregulated after challenge with Bt and Cry1Ab. Tissue and developmental stage expression analysis showed that only SeCTL-S15 was mainly expressed in the midgut and larva, respectively. Knockdown of SeCTL-S15 significantly increased Bt susceptibility, as indicated by reduced survival and larval weight. These results suggest that CTL-S15 might play a vital role in the low susceptibility of larvae to Bt in S. exigua. Our results provide new insights into CTL function in insects.

Peritrophins are involved in the defense against Bacillus thuringiensis and nucleopolyhedrovirus formulations in Spodoptera littoralis (Lepidoptera: Noctuidae)

The peritrophic matrix (or peritrophic membrane, PM) is present in most insects where it acts as a barrier to mechanical insults and pathogens, as well as a facilitator of digestive processes. The PM is formed by the binding of structural PM proteins, referred to as peritrophins, to chitin fibrils and spans the entire midgut in lepidopterans. To investigate the role of peritrophins in a highly polyphagous lepidopteran pest, namely the cotton leafworm (Spodoptera littoralis), we generated Insect Intestinal Mucin (IIM−) and non-mucin Peritrophin (PER−) mutant strains via CRISPR/Cas9 mutagenesis. Both strains exhibited deformed PMs and retarded developmental rates. Bioassays conducted with Bacillus thuringiensis (Bt) and nucleopolyhedrovirus (SpliNPV) formulations showed that both the IIM− and PER− mutant larvae were more susceptible to these bioinsecticides compared to the wild-type (WT) larvae with intact PM. Interestingly, the provision of chitin-binding agent Calcofluor (CF) in the diet lowered the toxicity of Bt formulations in both WT and IIM− larvae and the protective effect of CF was significantly lower in PER− larvae. This suggested that the interaction of CF with PER is responsible for Bt resistance mediated by CF. In contrast, the provision of CF caused increased susceptibility to SpliNPV in both mutants and WT larvae. The study showed the importance of peritrophins in the defense against pathogens in S. littoralis and revealed novel insights into CF-mediated resistance to Cry toxin.

Bacillus thuringiensis Cry9Aa Insecticidal Protein Domain I Helices α3 and α4 Are Two Core Regions Involved in Oligomerization and Toxicity.

Bacillus thuringiensis Cry9 proteins show high insecticidal activity against different lepidopteran pests. Cry9 could be a valuable alternative to Cry1 proteins because it showed a synergistic effect with no cross-resistance. However, the pore-formation region of the Cry9 proteins is still unclear. In this study, nine mutations of certain Cry9Aa helices α3 and α4 residues resulted in a complete loss of insecticidal activity against the rice pest Chilo suppressalis; however, the protein stability and receptor binding ability of these mutants were not affected. Among these mutants, Cry9Aa-D121R, Cry9Aa-D125R, Cry9Aa-D163R, Cry9Aa-E165R, and Cry9Aa-D167R are unable to form oligomers in vitro, while the oligomers formed by Cry9Aa-R156D, Cry9Aa-R158D, and Cry9Aa-R160D are unstable and failed to insert into the membrane. These data confirmed that helices α3 and α4 of Cry9Aa are involved in oligomerization, membrane insertion, and toxicity. The knowledge of Cry9 pore-forming action may promote its application as an alternative to Cry1 insecticidal proteins.

High-throughput screening for novel Bacillus thuringiensis insecticidal proteins revealed evidence that the bacterium exchanges Domain III to enhance its insecticidal activity.

Approximately 3000 Bacillus thuringiensis (Bt) isolates were screened to discover novel three-domain (3D) Cry proteins active against Helicoverpa zea (corn earworm). From 400 active isolates found during the primary screening, Cry1Ac and Cry2A, which are known to be active against H. zea, were removed using multiplex-primer PCR and high-throughput column chromatography. This process reduced the number of active cultures to 48. DNA segments encoding Domain III of these 48 cultures were amplified by PCR and sequenced. Sequencing revealed two novel Cry1B-type Domain IIIs. Further sequencing of the flanking regions of these domains revealed that one was part of Cry1Bj (GenBank: KT952325). However, the other Domain III lacked Domains I and II. Instead, this Domain III was associated with two open reading frames, ORF1 and ORF2. ORF1 was identified as an ATP-binding protein, and ORF2 as an ATPase, suggesting that Bt exchanges Domain III among homologous Cry proteins.

Metadichol®-induced expression of circadian clock transcription factors in human fibroblasts

Circadian clock genes regulate many physiological processes, including sleep, metabolism, inflammation, and cancer. Disruptions in these cycles are associated with heightened vulnerability to cancer, psychiatric diseases, and neurological problems. Circadian clock genes play a vital role in the growth and progression of cancer. Manipulating the circadian clock mechanism can eliminate cancer cells, augment the effectiveness of chemotherapy, eliminate senescent cells, and reinstate regular circadian cycles. The circadian clock genes that are crucial for regulating the daily physiological processes of mammals include Cry1, Bmal1, Per1, Ppargc1a, and Clock. Currently, there is a lack of information regarding small molecules that can effectively control and activate all five transcription factors. By subjecting fibroblasts to treatment with Metadichol, we can induce the expression of all the genes, as evidenced by the results obtained from quantitative real-time polymerase chain reaction (qRT‒PCR). The administration of metadichol significantly enhanced the expression of the Cry1, Clock, and Ppargc1a genes in human fibroblasts, resulting in a four- to fivefold increase in expression. Metadichol successfully maintained the expression of the Per1 and Bmal1 genes at a dose of 100 ng. Clock and Bmal1 heterodimers initiate the activation of Cry1, Per1, and other clock genes via a feedback loop. The Cry1 and Per1 proteins inhibit the function of Clock-Bmal1. Ppargc1a regulates the expression of Clock and metabolic genes. Metadichol did not result in elevated levels of Per1 and Bmal1 expression, suggesting that it does not directly affect these genes. Metadichol is a nontoxic molecule, and its effects on circadian rhythm can have significant implications for human health to alleviate the progression of numerous ailments and diseases

Exposure to Cry1 Toxins Increases Long Flight Tendency in Susceptible but Not in Cry1F-Resistant Female Spodoptera frugiperda (Lepidoptera: Noctuidae).

The fall armyworm (JE Smith) (Spodoptera frugiperda) is a polyphagous pest targeted by selected Cry and Vip3A insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) that are produced in transgenic Bt corn and cotton. Available evidence suggests that sublethal larval exposure to Cry1Ac increases flight activity in adult Spodoptera spp. However, it is not known whether this effect is also observed in survivors from generally lethal exposure to Cry1Ac. Moreover, while multiple cases of field-evolved resistance to Bt proteins have been described in the native range of S. frugiperda, the effect of resistance on flight behavior has not been examined. Long-distance migratory flight capacity of S. frugiperda is of concern given its ongoing global spread and the possibility that migrants may be carrying resistance alleles against pesticides and Bt crops. In this study, we used rotational flight mills to test the effects of generally lethal exposure to Cry1Ac in susceptible and sublethal exposure in Cry1F-resistant S. frugiperda strains. The results detected altered pupal weight after larval feeding on diet containing Cry proteins, which only translated in significantly increased tendency for longer flights in female moths from the susceptible strain. This information has relevant implications when considering current models and assumptions for resistance management of Bt crops.

Influence of Cry1Ab protein on growth and development of a predatory spider, Pardosa pseudoannulata, from protective perspectives

The expression of Cry proteins in genetically modified rice varieties safeguards the crop from lepidopteran pests. These proteins have the potential to be transferred through the food chain to arthropods like planthoppers and predatory spiders, triggering defensive responses in these unintended organisms. Hence, we hypothesized that Cry protein might influence the growth and development of spiders by altering protective enzyme activities. The results showed that Cry1Ab protein could accumulate in tissues and subcellular organelles of Pardosa pseudoannulata from Nilaparvata lugens. Cry1Ab protein exposure prolonged the developmental duration in the 5th and 7th instar spiderlings but induced no alterations of other growth indicators, such as body length, median ocular area, and survival rate. In addition, Cry1Ab protein exerted no adverse impacts on several detoxifying enzymes (i.e., superoxide dismutase, catalase, glutathione peroxidase, and acetylcholine esterase) in muscle, midgut, ganglia, and hemolymph at subcellular components (i.e., microsome and cytoplasm). To further explore the effects of Cry1Ab protein on the spiderlings, we performed an integrated transcriptome analysis on spiderlings exposed to Cry1Ab protein. The results showed that Cry1Ab protein might prolong the development duration of P. pseudoannulata via the altered cuticle metabolism (e.g., chitin metabolic process and structural constituent of cuticle). In addition, the gene expression profile associated with detoxifying enzymes and three stress-responsive pathways (JAK/STAT, JNK/SAPK, and Hippo pathways) also displayed no significant alterations under Cry1Ab exposure. Collectively, this integrated analysis generates multidimensional insights to assess the effects of Cry1Ab protein on non-target spiders and demonstrates that Cry1Ab protein exerts no toxicity in P. pseudoannulata.

Random Mutagenesis to Enhance the Toxicity of Bacillus thuringiensis Cry Proteins Against Earias Vittella (F.)

Insecticidal proteins derived from Bacillus thuringiensis (Bt) are widely utilized in a variety of insect control applications,including sprays and transgenic crops.The development of resistance in pests, on the other hand, can lessen the effectiveness of Bttoxins. In this study, we made efforts to enhance the toxicity of two cry proteins Cry1Ac and Cry2Aa through random mutagenesis against cotton bollworm (Earias vitella), one of the most destructive cotton pests in Pakistan. Random mutagenesis is an important tool for elucidating protein structure-function relationships and for modifying proteins to enhance or change their characteristics. We focused on whole cry proteins for random mutagenesis througherror-prone PCR and constructed a recombinant library of cry proteins. Sequence analysis of eight mutants showed the mutations of 34 different nucleotides in Cry1Ac and Cry2Aa genes. All mutants were spared for toxicity bioassays against 2nd instar larvae of spotted bollworm. Cry1Ac mutant RM1AcM4 (D242E) and Cry2Aa mutants RM2AaM2 (T354A, T492R, F511L, G585E, D606Y) showed enhanced toxicity as compared to proteins without mutation. These two mutants comprise the mutations in domain-II of cry proteins important in specificity determining regions on midgut receptors in insect pests.

MINIREVIEW AND HYPOTHESIS: HOMOLOGY MODELLING OF SPODOPTERA LITURA (LEPIDOPTERA: NOCTUIDAE) AMINOPEPTIDASE N RECEPTOR

Pest insect aminopeptidase N (APN) is a receptor for Bacillus thuringiensis Cry1 proteins. A 108-kDa APN has been characterised in Spodoptera litura, here their APN three-dimensional structure was determined; it has 4 structural domains. Domain I is the region that recognizes Cry1 toxins, a loop of this section might be very important in this role. Probably, domain II has functions in Cry1 protein-APN interaction. Domain III has a sandwich topology and domain IV is a superhelix. The APN have conserved structures, with slightly mutations just like result of coevolution with Cry proteins.

Draft genome of neotropical Bacillus thuringiensis UFT038 and its potential against lepidopteran soybean pests.

Bacillus thuringiensis (Bt) is known for its Cry and Vip3A pesticidal proteins with high selectivity to target pests. Here, we assessed the potential of a novel neotropical Bt strain (UFT038) against six lepidopteran pests, including two Cry-resistant populations of fall armyworm, Spodoptera frugiperda. We also sequenced and analyzed the genome of Bt UFT038 to identify genes involved in insecticidal activities or encoding other virulence factors. In toxicological bioassays, Bt UFT038 killed and inhibited the neonate growth in a concentration-dependent manner.Bt UFT038and HD-1 were equally toxic againstS. cosmioides, S. frugiperda (S_Bt and R_Cry1 + 2Ab populations), Helicoverpa zea, and H. armigera. However, larval growth inhibition results indicated that Bt UFT038 was more toxic than HD-1 to S. cosmioides, while HD-1 was more active against Chrysodeixis includens. The draft genome of Bt UFT038 showed the cry1Aa8, cry1Ac11, cry1Ia44, cry2Aa9, cry2Ab35, and vip3Af5 genes. Besides this, genes encoding the virulence factors (inhA, plcA, piplC, sph, and chi1-2) and toxins (alo, cytK, hlyIII, hblA-D, and nheA-C) were also identified. Collectively, our findings reveal the potential of the Bt UFT038 strain as a source of insecticidal genes against lepidopteran pests, including S. cosmioides and S. frugiperda.

Reduced toxin binding associated with resistance to Vip3Aa in the corn earworm (Helicoverpa zea).

Helicoverpa zea is a major crop pest in the United States that is managed with transgenic corn and cotton that produce insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt). However, H. zea has evolved widespread resistance to the Cry proteins produced in Bt corn and cotton, leaving Vip3Aa as the only plant-incorporated protectant in Bt crops that consistently provides excellent control of H. zea. The benefits provided by Bt crops will be substantially reduced if widespread Vip3Aa resistance develops in H. zea field populations. Therefore, it is important to identify resistance alleles and mechanisms that contribute to Vip3Aa resistance to ensure that informed resistance management strategies are implemented. This study is the first report of reduced binding of Vip3Aa to midgut receptors associated with resistance.

Channel Formation in Cry Toxins: An Alphafold-2 Perspective.

Bacillus thuringiensis (Bt) strains produce pore-forming toxins (PFTs) that attack insect pests. Information for pre-pore and pore structures of some of these Bt toxins is available. However, for the three-domain (I-III) crystal (Cry) toxins, the most used Bt toxins in pest control, this crucial information is still missing. In these Cry toxins, biochemical data have shown that 7-helix domain I is involved in insertion in membranes, oligomerization and formation of a channel lined mainly by helix α4, whereas helices α1 to α3 seem to have a dynamic role during insertion. In the case of Cry1Aa, toxic against Manduca sexta larvae, a tetrameric oligomer seems to precede membrane insertion. Given the experimental difficulty in the elucidation of the membrane insertion steps, we used Alphafold-2 (AF2) to shed light on possible oligomeric structural intermediates in the membrane insertion of this toxin. AF2 very accurately (<1 Å RMSD) predicted the crystal monomeric and trimeric structures of Cry1Aa and Cry4Ba. The prediction of a tetramer of Cry1Aa, but not Cry4Ba, produced an 'extended model' where domain I helices α3 and α2b form a continuous helix and where hydrophobic helices α1 and α2 cluster at the tip of the bundle. We hypothesize that this represents an intermediate that binds the membrane and precedes α4/α5 hairpin insertion, together with helices α6 and α7. Another Cry1Aa tetrameric model was predicted after deleting helices α1 to α3, where domain I produced a central cavity consistent with an ion channel, lined by polar and charged residues in helix α4. We propose that this second model corresponds to the 'membrane-inserted' structure. AF2 also predicted larger α4/α5 hairpin n-mers (14 ≤n ≤ 17) with high confidence, which formed even larger (~5 nm) pores. The plausibility of these models is discussed in the context of available experimental data and current paradigms.

Prospects of Indigenous Bacillus subtilis Strain LS9.1 as a Potential Biocontrol Agent against Aedes aegypti Larvae

Biocontrol is one way that can be developed to overcome public health and agricultural problems. Biocontrol agents could be a microorganism, for example, from the Bacillus group. This study aims to determine the larvicidal toxicity of the Bacillus subtilis strain LS9.1 against Aedes aegypti larvae. The mode of action of the isolate caused larval death, which was detected with parasporal inclusions and secondary metabolites. Larvicidal toxicity with the bioassay method. Parasporal inclusion proteins were detected with the SDS-PAGE method. The Cry toxin coding gene with the PCR method and cell ultrastructure with transmission and scanning electron microscopy (TEM and SEM). The action of secondary metabolites was detected with hemolytic activity test. The isolate high toxicity to A. aegypti larvae. Parasporal inclusion proteins with molecular weights ranging from 72-7 kDa and the gene encoding a protein toxin in size 300 bp. The TEM and SEM results did not Cry-IV toxin. The hemolytic activity test showed a positive reaction correlated with biosurfactant production. Based on this study, the death of larvae is probably caused by secondary metabolites produced by Bacillus subtilis strain LS9.1. Thus, this isolate has the prospect of being an environmentally friendly biocontrol agent to disease vectors and insect pests.

Establishment of novel receptor-antibody sandwich assays to broadly detect Bacillus thuringiensis Cry1 and Cry2 toxins

Bacillus thuringiensis (Bt) Cry toxins have been widely used in the development of genetically modified organisms (GMOs) for pest control. This work aimed to establish more cost effective and broader detection methods for commonly used Cry toxins. Using ligand blot and bio-layer interferometry, we confirmed that a recombinant toxin-binding fragments derived from Helicoverpa armigera cadherin-like protein (HaCad-TBR) could broadly bind Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab with the affinity of 0.149, 0.402, 120, and 4.12 nM, respectively. Based on the affinity results, a novel receptor-antibody sandwich assay broadly detecting Cry1A and Cry2 toxins was developed by using HaCad-TBR as capture molecules, and anti-Cry1A/Cry2A polyclonal antibodies (pAbs) as the detection antibodies. The detection limit (LOD) for Cry1Ab, Cry1Ab, Cry2Aa, and Cry2Ab were 5.30, 5.75, 30.83 and 13.70 ng/mL. To distinguish Cry1A and Cry2A toxins in a singular test, anti-Cry1A pAbs and anti-Cry2A pAbs were labelled with different quantum dots (QDs). The LOD for the four toxins by receptor-QDs-pAbs sandwich assay were calculated to be 1.36, 4.71, 17.48, and 7.54 ng/mL, respectively. The two developed methods were validated by spiked rice and corn samples, suggesting they may potentially be used in monitoring and quantifying Cry toxins in food and environment.