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.

Real-time jellyfish classification and detection algorithm based on improved YOLOv4-tiny and improved underwater image enhancement algorithm.

The outbreak of jellyfish blooms poses a serious threat to human life and marine ecology. Therefore, jellyfish detection techniques have earned great interest. This paper investigates the jellyfish detection and classification algorithm based on optical images and deep learning theory. Firstly, we create a dataset comprising 11,926 images. A MSRCR underwater image enhancement algorithm with fusion is proposed. Finally, an improved YOLOv4-tiny algorithm is proposed by incorporating a CBMA module and optimizing the training method. The results demonstrate that the detection accuracy of the improved algorithm can reach 95.01%, the detection speed is 223FPS, both of which are better than the compared algorithms such as YOLOV4. In summary, our method can accurately and quickly detect jellyfish. The research in this paper lays the foundation for the development of an underwater jellyfish real-time monitoring system.

Identifying the Epitopes of Bacillus thuringiensis Cry2Aa Toxin Involved in Cadherin Interaction by a Monoclonal Antibody.

Antibodies are a useful tool for assistance to map the binding epitopes in Bacillus thuringiensis Cry toxins and their receptors, and even determine how receptors promote toxicity. In this work, a monoclonal antibody (mAb-1D2) was produced by the hybridoma cell line raised against Cry2Aa toxins, with a half inhibition concentration (IC50) of 9.16 µg/mL. The affinity constant of two recombinant toxin-binding fragments derived from Helicoverpa armigera and Plutella xylostella cadherin-like protein (HaCad-TBR or PxCad-TBR) to Cry2Aa toxin was measured to be 1.21 µM and 1.24 µM, respectively. Competitive ELISA showed that mAb-1D2 competed with HaCad-TBR or PxCad-TBR binding to Cry2Aa. Meanwhile, the toxicity of the Cry2Aa toxin to the H. armigera and P. xylostella larvae were greatly reduced when the toxin was mixed with mAb-1D2, which indicated that cadherin may play an important functional role in the toxicity of Cry2Aa. After transforming mAb-1D2 to a single-chain variable fragment (scFv), the hot spot residues of Cry2Aa with 1D2-scFv, PxCad-TBR, and HaCad-TBR were analyzed by molecular docking. It was demonstrated that the hot spot residues of Cry2Aa involving with 1D2-scFv interaction were mainly in Domain II, and some residues in Domain I. Moreover, mAb-1D2 and the two cadherin fragments shared the common hot spot residues on Cry2Aa, which could explain mAb-1D2 inhibited Cry2Aa binding with cadherin fragments. This monoclonal antibody could be a useful tool for identifying the binding epitopes between Cry2Aa and cadherin, and even assist to analyze the roles of cadherin in Cry2Aa toxicity.

Generation of Human Domain Antibody Fragments as Potential Insecticidal Agents against Helicoverpa armigera by Cadherin-Based Screening.

New insecticidal genes and approaches for pest control are a hot research area. In the present study, we explored a novel strategy for the generation of insecticidal proteins. The midgut cadherin of Helicoverpa armigera (H. armigera) was used as a target to screen materials that have insecticidal activity. After three rounds of panning, the phage-displayed human domain antibody B1F6, which not only binds to the H. armigera cadherin CR9-CR11 but also significantly inhibits Cry1Ac toxins from binding to CR9-CR11, was obtained from a phage-displayed human domain antibody (DAb) library. To better analyze the relevant activity of B1F6, soluble B1F6 protein was expressed by Escherichia coli BL21 (DE3). The cytotoxicity assays demonstrated that soluble B1F6 induced Sf9 cell death when expressing H. armigera cadherin on the cell membrane. The insect bioassay results showed that soluble B1F6 protein (90 µg/cm2) caused 49.5 ± 3.3% H. armigera larvae mortality. The midgut histological results showed that soluble B1F6 caused damage to the midgut epithelium of H. armigera larvae. The present study explored a new strategy and provided a basic material for the generation of new insecticidal materials.

Identification of single domain antibodies with insect cytotoxicity using phage-display antibody library screening and Plutella xylostella ATP-binding cassette transporter subfamily C member 2 (ABCC2) -based insect cell expression system.

It is extremely imminent to study a new strategy to manage agricultural pest like Plutella xylostella (P. xylostella) which is currently resistant to most of pesticides, including three domain-Cry toxins from Bacillus thuringiensis (Bt). In this study, we reported a phage displayed single domain antibody screening from human domain antibody (DAb) library targeted on Spodoptera frugiperda 9 (Sf9) cells expressed Cry1Ac toxin receptor, ATP-dependent binding cassette transporter C2 in P. xylostella (PxABCC2). After three rounds of panning, three cytotoxic antibodies (1D2, 2B7, 3C4) were obtained from thirty-eight antibodies and displayed high binding ability towards PxABCC2-expressed Sf9 cells. Through homology modeling and molecular docking, the interaction mode indicated that the most cytotoxic 1D2 of the three antibodies presented the lowest binding free energy required and had the most hydrogen bond formed with PxABCC2 in molecular docking analysis. Functional assay of key regions in 1D2 via Alanine replacement indicated that complementarity-determining region (CDR) 3 played a crucial role in antibody exerts binding activity and cytotoxicity. This study provides the first trial for discovering of potential cytotoxic antibodies from the human antibody library via specific receptor-expressed insect cell system biopanning.

Real-Time Jellyfish Classification and Detection Based on Improved YOLOv3 Algorithm.

In recent years, jellyfish outbreaks have frequently occurred in offshore areas worldwide, posing a significant threat to the marine fishery, tourism, coastal industry, and personal safety. Effective monitoring of jellyfish is a vital method to solve the above problems. However, the optical detection method for jellyfish is still in the primary stage. Therefore, this paper studies a jellyfish detection method based on convolution neural network theory and digital image processing technology. This paper studies the underwater image preprocessing algorithm because the quality of underwater images directly affects the detection results. The results show that the image quality is better after applying the three algorithms namely prior defogging, adaptive histogram equalization, and multi-scale retinal enhancement, which is more conducive to detection. We establish a data set containing seven species of jellyfishes and fish. A total of 2141 images are included in the data set. The YOLOv3 algorithm is used to detect jellyfish, and its feature extraction network Darknet53 is optimized to ensure it is conducted in real-time. In addition, we introduce label smoothing and cosine annealing learning rate methods during the training process. The experimental results show that the improved algorithms improve the detection accuracy of jellyfish on the premise of ensuring the detection speed. This paper lays a foundation for the construction of an underwater jellyfish optical imaging real-time monitoring system.

Neuropeptide F from endocrine cells in Plutella xylostella midgut modulates feeding and synergizes Cry1Ac action.

With the wide cultivation of transgenic plants throughout the world and the rising risk of resistance to Bacillus thuringiensis crystal (Cry) toxins, it is essential to design an adaptive resistance management strategy for continued use. Neuropeptide F (NPF) of insects has proven to be valuable for the production of novel-type transgenic plants via its important role in the control of feeding behavior. In this study, the gene encoding NPF was cloned from the diamondback moth, Plutella xylostella, an important agricultural pest. Real-time quantitative reverse transcription-polymerase chain reaction and in situ hybridization showed a relatively high expression of P. xylostella-npf (P. x-npf) in endocrine cells of the midgut of fourth instar larvae, and it was found to participate in P. xylostella feeding behavior and Cry1Ac-induced feeding inhibition. Prokaryotic expression and purification provided structure unfolded P. x-npf from inclusion bodies for diet surface overlay bioassays and the results demonstrated a significant synergistic effect of P. x-npf on Cry1Ac toxicity by increasing intake of noxious food which contains Cry toxins, especially quick death at an early stage of feeding. Our findings provided a potential new way to efficiently control pests by increasing intake of lower dose Cry toxins and a novel hint for the complex Cry toxin mechanism.

Synergism of Bacillus thuringiensis Toxin Cry1Ac by a Fragment of Toxin-Binding Polycalin from Plutella xylostella.

The continued success of pest control using insecticidal crystal (Cry) proteins from Bacillus thuringiensis (Bt) in transgenic plants was threatened by the evolution of resistance. Previous studies suggested that polycalin from Plutella xylostella could bind to Cry1Ac toxin as a potential receptor. In this study, a fragment of P. xylostella polycalin (Pxpolycalinf, G2209-A2942) containing a carboxyl-terminal GPI-anchored signal peptide was cloned and expressed. Purified Pxpolycalinf retained the binding ability to Cry1Ac and synergized Cry1Ac toxicity to the third larvae of P. xylostella in bioassays. Moreover, the polyclonal antibody of Pxpolycalinf decreased the Cry1Ac activity after being fed together with normal food. Further, the ELISA results showed the concentration-dependent binding of Pxpolycalinf to P. xylostella brush border membrane vesicles (BBMV). Spodoptera frugiperda 9 (Sf9) cells expressing Pxpolycalinf were not susceptive to Cry1Ac, whereas Pxpolycalinf increased Cry1Ac cytotoxicity to Sf9 cells expressing P. xylostella ATP-dependent binding cassette transporter C2 (PxABCC2). Immunolocalization presented the binding of Pxpolycalinf to the Sf9 cell membrane, and ELISA showed the concentration-dependent binding of Pxpolycalinf to Sf9 cell extraction. These results here provide the first evidence that a fragment of P. xylostella polycalin, a potential receptor of Cry1Ac, synergizes Cry1Ac toxicity to P. xylostella larvae and Sf9 cells expressing PxABCC2.

Docking-based generation of antibodies mimicking Cry1A/1B protein binding sites as potential insecticidal agents against diamondback moth (Plutella xylostella).

BACKGROUND: Broad use of insecticidal Cry proteins from Bacillus thuringiensis in biopesticides and transgenic crops has resulted in cases of practical field resistance, highlighting the need for novel approaches to insect control. Previously we described an anti-Cry1Ab idiotypic-antibody (B12-scFv) displaying toxicity against rice leafroller (Cnaphalocrocis medinalis) larvae, supporting the potential of antibodies for pest control. The goal of the present study was to generate insecticidal antibodies against diamondback moth (Plutella xylostella) larvae. RESULTS: Four genetically engineered antibodies (GEAbs) were designed in silico from B12-scFv using three-dimensional (3D) structure and docking predictions to alkaline phosphatase (ALP) as a Cry1Ac receptor in P. xylostella. Among these GEAbs, the GEAb-dVL antibody consisting of two light chains had overlapping binding sites with Cry1A and Cry1B proteins and displayed high binding affinity to P. xylostella midgut brush border membrane (BBM) proteins. Proteins in BBM identified by pull-down assays as binding to GEAb-dVL included an ABC transporter and V-ATPase subunit A protein. Despite lacking the α-helical structures in Cry1A that are responsible for pore formation, ingestion of GEAb-dVL disrupted the P. xylostella larval midgut epithelium and resulted in toxicity. Apoptotic genes were activated in gut cells upon treatment with GEAb-dVL . CONCLUSION: This study describes the first insecticidal GEAb targeting P. xylostella by mimicking Cry proteins. Data support that GEAb-dVL toxicity is associated to activation of intracellular cell death pathways, in contrast to pore-formation associated toxicity of Cry proteins. This work provides a foundation for the design of novel insecticidal antibodies for insect control. © 2021 Society of Chemical Industry.

Synergistic selection of a Helicoverpa armigera cadherin fragment with Cry1Ac in different cells and insects.

The midgut cadherin fragments were extensively studied as Bt synergists in insects, while their synergistic selection modes with Bt toxins in different mechanisms of resistance or insects have never been determined. Here, a soluble Helicoverpa armigera cadherin fragment which corresponds to the Cry1Ac binding region (HaCad-TBR) was expressed in Escherichia coli and its synergism with Cry1Ac toxin in H. armigera and Plutella xylostella larvae as well as Sf9 cells expressing different cadherins was tested. HaCad-TBR exhibited higher synergism factor in P. xylostella larvae (4.84-fold) than in H. armigera larvae (2.45-fold). Among the cells expressing HaCad alleles, HaCad-TBR enhanced the Cry1Ac toxicity only in the cells expressing the mutant lacking the extracellular domain. Moreover, HaCad-TBR had a weak enhancement of Cry1Ac toxicity in Sf9 cells expressing the P. xylostella cadherin. Further researches revealed that the enhancement of toxicity in Sf9 cells was correlated with increased toxin binding. These results suggested that cadherin fragments which have high binding level with Cry1Ac are more likely to enhance toxin toxicity well against the cells or larvae where the cadherin has lower binding level with Cry1Ac, especially in the cases lacking the toxin binding domain.

Production and characterization of a single-chain variable fragment antibody from a site-saturation mutagenesis library derived from the anti-Cry1A monoclonal antibody.

There are plenty of applications of Cry1A toxins (Cry1Aa, Cry1Ab, Cry1Ac) in genetically modified crops, and it is necessary to establish corresponding detection methods. In this study, a single-chain variable fragment (scFv) with high affinities to Cry1A toxins was produced. First, the variable regions of heavy (VH) and light chain (VL) were amplified from hybridoma cell 5B5 which secrete anti-Cry1A monoclonal antibody (mAb) and then spliced into scFv-5B5 by overlap extension polymerase chain reaction (SOE-PCR). Subsequently, site-saturation mutagenesis was performed after homology modeling and molecular docking, which showed that asparagine35, phenylalanine36, isoleucine104, tyrosine105, and serine196, respectively, located in VH complementarity-determining region (CDR1 and CDR3) and VL framework region (FR3) were key amino acid sites. Then, the mutagenesis scFv library (1.35 × 105 CFU/mL) was constructed and a mutant scFv-2G12 with equilibrium dissociation constant (KD) value of 9.819 × 10-9 M against Cry1Ab toxin, which was lower than scFv-5B5 (2.025 × 10-8 M) was obtained by biopanning. Then, enzyme-linked immunosorbent assay (ELISA) was established with limit of detection (LOD) and limit of quantitation (LOQ) of 4.6-9.2 and 11.1-17.1 ng mL-1 respectively for scFv-2G12, which were lower than scFv-5B5 (12.4-22.0 and 23.6-39.7 ng mL-1). Results indicated the promising prospect of scFv-2G12 used for the detection of Cry1A toxins.

Identification of a Cry1Fa binding site of cadherin in Plutella xylostella through fragment exchanging and molecular docking methods.

Binding to the cadherin in target pests is the primary step in the action mechanism of Cry toxins, but little is known regarding the interaction of Cry1Fa with cadherin. Our previous study suggested that a Plutella xylostella cadherin fragment (PxCad-TBR) can bind to Cry1Fa, while its homologous fragment (HaCad-TBR) in Helicoverpa armigera cannot. In this study, we expressed two cadherin fragments that combine parts of PxCad-TBR and HaCad-TBR in Escherichia coli and tested their binding to the Cry1Fa. The results showed that the fragment containing amino acids T1202-A1341 of P. xylostella cadherin showed binding ability to Cry1Fa. Furthermore, two regions (V1219-E1233 and D1326-F1337) were predicted as hot spot regions that are involved in the interaction of Cry1Fa and PxCad-TBR with computer-aided molecular docking. We then constructed two PxCad-TBR mutations by fragment exchanging based on the molecular docking results and verified the mutations' binding abilities to the Cry1Fa. The results showed that the region that contains amino acids D1326-F1337 was one important binding site to Cry1Fa in P. xylostella cadherin. These results suggested that a combination of computer-aided molecular docking and fragment exchanging is an effective way to locate the key binding sites of Bt toxins in receptors.

Roles of Midgut Cadherin from Two Moths in Different Bacillus thuringiensis Action Mechanisms: Correlation among Toxin Binding, Cellular Toxicity, and Synergism.

The midgut cadherin has been described as one of the main functional receptors for Bacillus thuringiensis (Bt) toxins. Plutella xylostella (P. xylostella) and Helicoverpa armigera (H. armigera) are two major target pests of Bt toxins in China, and the roles of their cadherins in the action of Bt toxins have been only partially studied. Here, we expressed the two cadherins in Sf9 cells and their partial extracellular domains in Escherichia coli and tested them for Bt toxin binding, cellular toxicity, and synergism with toxins. Our results suggested that PxCad might function as a Cry1Ac receptor, although it showed lower binding levels to Cry1Ac and reduced cytotoxicity compared with HaCad. PxCad and HaCad are not receptors for Cry2A, Cry1B, Cry1C, and Cry1F toxins, although some of them can bind to the cadherins. The PxCad-TBR exhibits higher enhancement of Cry1Ac and weak enhancement of Cry1F toxicity in P. xylostella larvae, although it is not the receptor of Cry1F.

Newly identified APN splice isoforms suggest novel splicing mechanisms may underlie circRNA circularization in moth.

Circular RNA (circRNA) have long been considered by-products of splicing errors, but the coordination of RNA transcription and exon circularization events remains poorly understood. Here, we investigated this question using genes encoding aminopeptidases N (APNs), which are receptors of Bacillus thuringiensis toxins, in the cotton bollworm, Helicoverpa armigera. We cloned and sequenced the cDNA of ten APN genes (HaAPN1-10) located in the same APN gene cluster, and detected 20 and 14 novel splicing isoforms with exon skipping in HaAPN1 and HaAPN3, respectively, whereas no or very few variants were found in the remaining genes. Further study identified 14 and 6 circular RNA (circRNA) in HaAPN1 and HaAPN3, respectively. Neither novel splicing isoforms nor circRNA were detected in HaAPN2 and HaAPN5. Distinct from the conventional GT/AG splicing signal, short co-directional repeats were involved in the splicing of the linear and circular isoforms of HaAPN1 and HaAPN3. Identification of the splice sites revealed that the linear isoforms may be related in some way to the circularization. Moreover, phylogenetic analysis and detection of circRNA of the APN gene of the diamondback moth, Plutella xylostella (PxAPN3), showed that circRNA formation is relatively conserved during the lepidopteran evolutionary process. These results contribute to an improved understanding of lepidopteran APNs and this novel class of insect circRNA.

Epistasis confers resistance to Bt toxin Cry1Ac in the cotton bollworm.

Evolution of resistance by insect pests reduces the benefits of extensively cultivated transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt). Previous work showed that resistance to Bt toxin Cry1Ac, which is produced by transgenic cotton, can be conferred by mutations disrupting a cadherin protein that binds this Bt toxin in the larval midgut. However, the potential for epistatic interactions between the cadherin gene and other genes has received little attention. Here, we report evidence of epistasis conferring resistance to Cry1Ac in the cotton bollworm, Helicoverpa armigera, one of the world's most devastating crop pests. Resistance to Cry1Ac in strain LF256 originated from a field-captured male and was autosomal, recessive, and 220-fold relative to susceptible strain SCD. We conducted complementation tests for allelism by crossing LF256 with a strain in which resistance to Cry1Ac is conferred by a recessive allele at the cadherin locus HaCad. The resulting F1 offspring were resistant, suggesting that resistance to Cry1Ac in LF256 is also conferred by resistance alleles at this locus. However, the HaCad amino acid sequence in LF256 lacked insertions and deletions, and did not differ consistently between LF256 and a susceptible strain. In addition, most of the cadherin alleles in LF256 were not derived from the field-captured male. Moreover, Cry1Ac resistance was not genetically linked with the HaCad locus in LF256. Furthermore, LF256 and the susceptible strain were similar in levels of HaCad transcript, cadherin protein, and binding of Cry1Ac to cadherin. Overall, the results imply that epistasis between HaCad and an unknown second locus in LF256 yielded the observed resistance in the F1 progeny from the complementation test. The observed epistasis has important implications for interpreting results of the F1 screen used widely to monitor and analyze resistance, as well as the potential to accelerate evolution of resistance.

Next-generation transgenic cotton: pyramiding RNAi and Bt counters insect resistance.

Transgenic crops producing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are extensively cultivated worldwide. To counter rapidly increasing pest resistance to crops that produce single Bt toxins, transgenic plant 'pyramids' producing two or more Bt toxins that kill the same pest have been widely adopted. However, cross-resistance and antagonism between Bt toxins limit the sustainability of this approach. Here we describe development and testing of the first pyramids of cotton combining protection from a Bt toxin and RNA interference (RNAi). We developed two types of transgenic cotton plants producing double-stranded RNA (dsRNA) from the global lepidopteran pest Helicoverpa armigera designed to interfere with its metabolism of juvenile hormone (JH). We focused on suppression of JH acid methyltransferase (JHAMT), which is crucial for JH synthesis, and JH-binding protein (JHBP), which transports JH to organs. In 2015 and 2016, we tested larvae from a Bt-resistant strain and a related susceptible strain of H. armigera on seven types of cotton: two controls, Bt cotton, two types of RNAi cotton (targeting JHAMT or JHBP) and two pyramids (Bt cotton plus each type of RNAi). Both types of RNAi cotton were effective against Bt-resistant insects. Bt cotton and RNAi acted independently against the susceptible strain. In computer simulations of conditions in northern China, where millions of farmers grow Bt cotton as well as abundant non-transgenic host plants of H. armigera, pyramided cotton combining a Bt toxin and RNAi substantially delayed resistance relative to using Bt cotton alone.

Resistance to Bacillus thuringiensis toxin Cry2Ab and survival on single-toxin and pyramided cotton in cotton bollworm from China.

Evolution of Helicoverpa armigera resistance to Bacillus thuringiensis (Bt) cotton producing Cry1Ac is progressing in northern China, and replacement of Cry1Ac cotton by pyramided Bt cotton has been considered to counter such resistance. Here, we investigated four of the eight conditions underlying success of the refuge strategy for delaying resistance to Cry1Ac+Cry2Ab cotton, a pyramid that has been used extensively against H. armigera outside China. Laboratory bioassays of a Cry2Ab-selected strain (An2Ab) and a related unselected strain (An) reveal that resistance to Cry2Ab (130-fold) was nearly dominant, autosomally inherited, and controlled by more than one locus. Strong cross-resistance occurred between Cry2Ab and Cry2Aa (81-fold). Weaker cross-resistance (18- to 22-fold) between Cry2Ab and Cry1A toxins was also present and significantly increased survival of An2Ab relative to An on cotton cultivars producing the fusion protein Cry1Ac/Cry1Ab or Cry1Ac. Survival on Cry1Ac+Cry2Ab cotton was also significantly higher in An2Ab than in An, showing that redundant killing on this pyramid was incomplete. Survival on non-Bt cotton did not differ significantly between An2Ab and An, indicating an absence of fitness costs affecting this trait. These results indicate that a switch to three-toxin pyramided cotton could be valuable for increasing durability of Bt cotton in China.

Identification of a toxic serralysin family protease with unique thermostable property from S. marcescens FS14.

A serralysin family protease (Serralysin-like protease B, SPB) with unique V-shaped thermostable property was isolated and identified from the Serratia marcescens FS14 by biochemical and molecular biological methods. It is the first time to report the isolation of a native serralysin family protease directly from Serratia species except the well-studied serralysin. SPB has an optimum pH at 8.0 and an optimum temperature at 37°C. It shows high proteolytic activities after pretreated at 4-50°C for 10min respectively and almost no detectable activity after pretreated at 60°C. Surprisingly, increasing activities were observed after pretreated at 70-90°C respectively. Further study revealed that the reason behind this phenomenon may be the self-digestion property of SPB with an optimum temperature around 60°C. This self-digestion property may expand the SPB future application in industry. The bioassay using the healthy cotton bollworm Helicoverpa armigera larvae demonstrated that the serralysin and SPB from FS14 are toxic to the H. armigera larvae. This result implied that FS14 strain and/or the SPB and serralysin in FS14 might have a potential application in insect control.

Dual mode of action of Bt proteins: protoxin efficacy against resistant insects.

Transgenic crops that produce Bacillus thuringiensis (Bt) proteins for pest control are grown extensively, but insect adaptation can reduce their effectiveness. Established mode of action models assert that Bt proteins Cry1Ab and Cry1Ac are produced as inactive protoxins that require conversion to a smaller activated form to exert toxicity. However, contrary to this widely accepted paradigm, we report evidence from seven resistant strains of three major crop pests showing that Cry1Ab and Cry1Ac protoxins were generally more potent than the corresponding activated toxins. Moreover, resistance was higher to activated toxins than protoxins in eight of nine cases evaluated in this study. These data and previously reported results support a new model in which protoxins and activated toxins kill insects via different pathways. Recognizing that protoxins can be more potent than activated toxins against resistant insects may help to enhance and sustain the efficacy of transgenic Bt crops.