Scorpion neurotoxins: structure/function relationships and application in agriculture

Continued use of non‐specific chemical insecticides poses potential risks to the environment and to human health resulting from non‐target toxicity and increased insect resistance to these agents. Scorpions produce anti‐insect selective polypeptide toxins that bind to and modulate voltage‐sensitive ion channels in excitable tissues, thus offering alternative, environmentally safer means for insect pest control. Despite this potential, little is known about their structural elements dictating anti‐insect preference, which may be useful for the design of selective insecticides. We used a bacterial system for expression and genetic dissection of two pharmacologically distinct scorpion toxins: alpha and excitatory. By exploiting a multi‐disciplinary approach consisting of mutagenesis, protein chemistry, electrophysiology, binding and toxicity assays, and structural studies, we elucidated the bioactive surface of two anti‐insect toxins, LqhαIT and Bj‐xtrIT. In both polypeptides the bioactive surface is composed of residues surrounding the C‐terminal region. In addition, a direct, immediate approach in using the toxin genes was demonstrated by engineering baculoviruses with cDNAs encoding LqhIT2 (depressant toxin), and LqhIT1 (excitatory toxin) resulting in viral vectors with significantly improved insecticidal efficacy. © 2000 Society of Chemical Industry     

Ribavirin, a nucleoside with potential insecticidal activity

BACKGROUND: The triazole-based antiviral nucleoside ribavirin exhibited significant in vitro insecticidal effects against Spodoptera litura, a finding that has prompted further investigations into its in vivo insecticidal properties and mode of action against this insect pest. RESULTS: Ribavirin showed delayed insecticidal effects on third-instar larvae of S. litura at a concentration of 1 mg mL⁻¹. When incorporated into an artificial diet, ribavirin caused various effects, depending on the concentration used. Compared with controls, food intake was lower, larval growth was reduced and larval development prolonged. Pupae weight was reduced, and adult morphology was also affected. Relative to the untreated group, treated groups showed higher mortality at larval and pupal stages, which was generally caused by moult disruption. Results of this study with ribavirin resemble those found in treatment with insect growth regulators. CONCLUSION: This work shows that ribavirin merits further study as a lead compound in a novel class of potential insect control agents or for managing field populations of Lepidoptera pests on cruciferous crops. Copyright © 2012 Society of Chemical Industry     

Development and characterisation of transgenic rice expressing two Bacillus thuringiensis genes

BACKGROUND: Transgenic crops that produce insecticidal toxins from the bacterium Bacillus thuringiensis Berliner (Bt) were first commercialised in 1996. The risk that pests have the potential to evolve resistance to Bt toxins is one of the most serious challenges to this technology. Gene stacking, pyramiding two Bt genes into one variety, is considered to be an effective insect resistance management (IRM) strategy. In this study, insect‐resistant rice expressing two Bt genes was developed by sexual crossing, and then characterised. RESULTS: Homozygous rice lines of two pyramided Bt genes were obtained in the F₃ generation. Quantification of Bt toxin showed that protein concentrations of Cry1Ab, Cry1Ac and Cry2A in the two‐gene lines were comparable with their single‐gene parents, while the expression of cry1C gene decreased after gene stacking. Four two‐gene lines showed higher activity to striped stem borer (Chilo suppressalis Walker) than parental lines in the laboratory bioassay. All pyramided lines and their hybrids exhibited excellent efficacy against stemborers and leaffolders in field evaluation, while most pyramided lines had no significant differences from original variety in yield under spraying of insecticide. CONCLUSION: These results demonstrate that the two‐gene lines have commercial potential and could serve as a valuable IRM strategy. Copyright © 2011 Society of Chemical Industry     

Novel use of nanostructured alumina as an insecticide

BACKGROUND: The worldwide need to produce an inexpensive and abundant food supply for a growing population is a great challenge that is further complicated by concerns about risks to environmental stability and human health triggered by the use of pesticides. The result is the ongoing development of alternative pest control strategies, and new, lower‐risk insecticidal molecules. Among the recent technological advances in agricultural science, nanotechnology shows considerable promise, although its development for use in crop protection is in its initial stages. RESULTS: This study reports for the first time the insecticidal effect of nanostructured alumina. Two species were used as model organisms, Sitophilus oryzae L. and Rhyzopertha dominica (F.), which are major insect pests in stored food supplies throughout the world. Both species experienced significant mortality after 3 days of continuous exposure to treated wheat. Nine days after treatment, the median lethal doses (LD₅₀) observed ranged from 127 to 235 mg kg^?1. CONCLUSION: Comparison of these results with recommended rates for commercial insecticidal dusts suggests that inorganic nanostructured alumina may provide a cheap and reliable alternative for control of insect pests. This study expands the frontiers for nanoparticle‐based technologies in pest management. Further research is needed to identify its mode of action and its non‐target toxicity, and to determine the potential of other nanostructured materials as pest control options for insects. Copyright © 2010 Society of Chemical Industry     

钙粘蛋白片段与苏云金杆菌晶体毒素协同作用研究进展

从钙粘蛋白介导苏云金杆菌晶体(Bt Cry)毒素对害虫的毒杀过程、钙粘蛋白重复区和近膜区与Bt Cry毒素的分子间作用涉及的结合位点及可能的互作机制等方面,综述了钙粘蛋白片段与Bt Cry毒素协同作用的最新研究进展。昆虫钙粘蛋白某些片段在非折叠状态时,可与Bt Cry毒素形成寡聚体,从而增加Bt Cry毒素对靶标害虫的毒杀活性。相关研究成果有助于提高Bt Cry毒素毒杀害虫的能力,克服害虫抗药性,具有一定的应用前景。     

Prohibitin,an essential protein for Colorado potato beetle larval viability,is relevant to Bacillus thuringiensis Cry3Aa toxicity

Bacillus thuringienesis (Bt) Cry toxins constitute the most extensively used environmentally safe biopesticide and their mode of action relies on the interaction of the toxins with membrane proteins in the midgut of susceptible insects that mediate toxicity and insect specificity. Therefore, identification of Bt Cry toxin interacting proteins in the midgut of target insects and understanding their role in toxicity is of great interest to exploit their insecticidal action. Using ligand blot, we demonstrated that Bt Cry3Aa toxin bound to a 30 kDa protein in Colorado potato beetle (CPB) larval midgut membrane, identified by sequence homology as prohibitin-1 protein. Prohibitins comprise a highly conserved family of proteins implicated in important cellular processes. We obtained the complete CPB prohibitin-1 DNA coding sequence of 828 pb, in silico translated into a 276-amino acid protein. The analysis at the amino acid level showed that the protein contains a prohibitin-homology domain (Band7_prohibitin, cd03401) conserved among prohibitin proteins. A striking feature of the CPB identified prohibitin-1 is the predicted presence of cadherin elements, potential binding sites for Cry toxins described in other Bt susceptible insects. We also showed that CPB prohibitin-1 protein partitioned into both, detergent soluble and insoluble membrane fractions, as well as a prohibitin-2 homologous protein, previously reported to form functional complexes with prohibitin-1 in other organisms. Prohibitin complexes act as membrane scaffolds ensuring the recruitment of membrane proteases to facilitate substrate processing. Accordingly, sequestration of prohibitin-1 by an anti-prohibitin-1 antibody impaired the Cry3Aa toxin inhibition of the proteolytic cleavage of a fluorogenic synthetic substrate of an ADAM-like metalloprotease previously reported to proteolize this toxin. In this work, we also demonstrated that prohibitin-1 RNAi silencing in CPB larvae produced deleterious effects and together with a LD50 Cry3Aa toxin treatment resulted in a highly efficient short term response since 100% larval mortality was achieved just 5 days after toxin challenge. Therefore, the combination of prohibitin RNAi and Cry toxin reveals as an effective strategy to improve crop protection.     

Muscarinic Agonists as Insecticides and Acaricides

A series of known agonists of the mammalian muscarinic receptor were prepared and evaluated for their insecticidal potential. It was discovered that pests such as Nilaparvata lugens (brown planthopper), Nephotettix cincticeps (green leafhopper), Tetranychus urticae (two-spotted spider mite) and Aphis gossypii (cotton aphid) were particularly sensitive to most of these compounds. Several analogs proved to be extremely active, surpassing commercial standards in some of the laboratory bioassays. These compounds exhibited a range of potencies for the insect (Musca) muscarinic receptor. Addition of GTP significantly reduced the affinity of the most potent analog for the Musca mAChR, indicating the compound functions as an agonist in insect tissue. Regression analysis indicated that significant relationships exist between displacement of [³H]QNB at the Musca muscarinic receptor and whole organism toxicity to three insect and one mite species. The results suggested that the insect muscarinic receptor represents a viable target site for insecticidal action. © 1997 SCI.     

Insecticidal toxins from the bacterium Photorhabdus luminescens: gene cloning and toxin histopathology

Four toxin complexes, Tca, Tcb, Tcc and Tcd from the culture broth of Photorhabdus luminescens have been purified and the four toxin complex encoding loci, tca, tcb, tcc and tcd, cloned. Genetic knockout of either tca or tcd reduced oral toxicity to Manduca sexta, and knockout of both loci eliminated activity. Purified Tca specifically affected the insect midgut, despite its putative normal delivery directly into the insect haemocel. These Photorhabdus toxins may form useful alternatives to other orally active bacterial protein toxins such as those from Bacillus thuringiensis.     

苏云金芽胞杆菌在马铃薯甲虫防治上的研究进展

马铃薯甲虫是重要的入侵害虫,严重威胁着我国粮食作物马铃薯的生产。苏云金芽胞杆菌是重要的农业害虫生防细菌,对马铃薯甲虫有良好的防治效果。本文围绕苏云金芽胞杆菌在马铃薯甲虫防治上的研究进展与应用进行综述。主要从马铃薯甲虫的入侵与防治手段、苏云金芽胞杆菌的晶体蛋白结构与杀虫机制、对马铃薯甲虫有活性的Bt毒蛋白研究进展、Bt毒蛋白对马铃薯甲虫的作用机制以及马铃薯甲虫对Bt毒蛋白的抗性机制等方面进行了综述。最后,从Bt新基因的挖掘和杀虫机理方面对苏云金芽胞杆菌在马铃薯甲虫防治上的研究进行了展望。     

Bacillus thuringiensis vegetative insecticidal protein family Vip3A and mode of action against pest Lepidoptera

Vip3A proteins are widely used for controlling pest Lepidoptera. Different binding sites with different receptors in the insect midgut membrane and lack of cross‐resistance with crystal (Cry) proteins enhance their applicability, as both single proteins and proteins pyramided with Cry proteins in transgenic Bt crops. Vip3A proteins are effective but there is relatively little information about their structure, function, activation, specificity, and mode of action. In addition, the mechanism of insect resistance to these proteins is unknown. Phylogenetic analysis and multiple sequence alignment showed that Vip3A proteins are genetically distant from Cry proteins. The mode of action and insecticidal activity of Vip3A proteins are discussed in this review. This review also provides detailed information about the Vip3A protein family that may aid in the design of more efficient pest management strategies in response to insect resistance to insecticidal proteins. © 2020 Society of Chemical Industry     

Genetics of Heliothis and Helicoverpa resistance to chemical insecticides and to Bacillus thuringiensis

Genetic linkage maps of Heliothis virescens and Helicoverpa armigera are being used to identify and characterize resistance-conferring genes. The insensitive acetylcholinesterase conferring resistance to organophosphorus insecticides and the insensitive sodium channel conferring resistance to pyrethroids have both been mapped in H. virescens. The linkage mapping approach permits a genetic dissection of resistance, even when the mode of action and lethal target are not precisely known, such as for the insecticidal toxins from the bacterium Bacillus thuringiensis (Bt). We have identified and mapped a major Bt-resistance locus in a strain of H. virescens exhibiting up to 10000-fold resistance to Cry1Ac toxin and are currently developing a linkage map for H. armigera with a set of ‘anchor’ loci to facilitate comparison with H. virescens. Both species are currently experiencing their first significant selective pressure in the field by transgenic cotton expressing Cry1Ac, and timely identification of resistance mechanisms and their underlying genetic basis will be essential in successfully managing the Bt resistance that will eventually appear. ©1997 SCI     

双氧木脂素E对东方粘虫幼虫体壁肌超微结构及Na+-K+-ATPase、Ca2+-ATPase的影响

双氧木脂素E（haedoxane E）是从杀虫植物透骨草Phryma leptostachya L.中分离出的一种对多种农林害虫和卫生害虫具有杀虫活性的化合物。本研究以东方粘虫Mythimna separata为供试昆虫,对双氧木脂素E对其幼虫杀虫作用部位进行了初步研究。电镜观察表明,双氧木脂素E对东方粘虫幼虫体壁肌具有致毒作用,能使肌细胞发生明显病变,细胞核皱缩变形,染色质浓缩,线粒体肿胀、大面积空泡化,肌质网扩张,肌原纤维排列紊乱,胞质内出现大量多泡体。酶活力测定结果也显示,双氧木脂素E能不同程度抑制Na+-K+-ATPase和Ca2+-ATPase活力。因此推测双氧木脂素E是一种作用于东方粘虫肌肉组织的肌肉毒剂。     

植物蛋白酶抑制剂抗虫基因工程研究进展

蛋白酶抑制剂抗虫基因工程为提高植物的抗虫能力提供了一种新的策略。与其它来源的抗虫蛋白相比，植物蛋白酶抑制剂有很多独特的优点。蛋白酶抑制剂的抗虫机理尚不完全清楚，影响其抗虫效果的因素也很多。文中列举分析了PI抗虫作用的机理以及目前研究的新进展，提出植物蛋白酶抑制剂抗虫基因工程中存在的问题和进一步解决的办法。     

Differential toxicity of Bacillus thuringiensis strains and their crystal toxins against high-altitude Himalayan populations of diamondback moth, Plutella xylostella L

BACKGROUND: Diamondback moth, Plutella xylostella (L.), is a major insect pest of crucifers in the biodiversity‐rich north‐western Indian Himalayan hills. The present investigation was aimed at determining the susceptibility pattern of P. xylostella populations collected from different locations of this region to autochthonous and standard Bacillus thuringiensis strains. RESULTS: Among the reference as well as indigenous B. thuringiensis strains tested, sub spp. kurstaki HD‐1, kurstaki HD‐73, galleriae HD‐8, local galleriae/colmeri strain BtOa1 and some of their Cry1 class toxins were found to be highly toxic. Surprisingly, the sub sp. tolworthi HD‐125, local tolworthi strain BtHa1 and Cry9 class toxins were found to be non‐toxic. Midgut homogenate from fourth‐instar larvae was found to activate 130 kDa protoxin from the local tolworthi strain BtHa1 into 68 kDa toxin, but failed to exert any larval mortality, probably owing to lack of receptor binding. CONCLUSION: The present study provides valuable baseline susceptibility data for the deployment of B. thuringiensis‐based control methods, as well as for future monitoring of development of resistance in P. xylostella to B. thuringiensis in this ecologically sensitive region. Copyright © 2008 Society of Chemical Industry     

Towards area‐wide control of Bactrocera invadens: prospects of the sterile insect technique and molecular entomology

It is 10 years since the first detection of the invader fruit fly, Bactrocera invadens, in sub‐Saharan Africa. The pest continues to hamper fruit production and create barriers to trade. Strategies currently employed to control B. invadens are insufficient, and more effective area‐wide strategies are needed. The sterile insect technique and molecular entomology approaches have high potential and could help to bring about effective area‐wide control of the pest if adopted and used as components of area‐wide integrated pest management. © 2013 Society of Chemical Industry     

Purification and characterisation of proteins secreted by the entomopathogenic fungus Metarhizium anisopliae with insecticidal activity against adults of the Mediterranean fruit fly,Ceratitis capitata (Diptera: Tephritidae)

BACKGROUND: The control of the Mediterranean fruit fly (medfly) Ceratitis capitata (Wied) is usually performed with protein bait sprays incorporating chemical insecticides that may have adverse effects on humans, non‐target organisms and the environment. In recent years, scientists have sought more environmentally friendly insecticides for medfly control, such as plant‐ and microorganism‐derived compounds. Among these compounds, entomopathogenic fungi are an unexplored source of natural insecticides. RESULTS: The crude soluble protein extract (CSPE) of the entomopathogenic fungus Metarhizium anisopliae (Mestch.) (strain EAMa 01/58‐Su) shows chronic insecticidal activity when administered per os. Mortality in flies exhibits a dose response. The CSPE produces an antifeedant effect in adult flies, a result probably due to a progressive deterioration of the fly midgut after ingestion of the extract. Protease and temperature treatments show that insecticidal activity against C. capitata is due to proteinaceous compounds that are highly thermostable. Four monomeric proteins from this crude extract have been purified by liquid chromatography and gel electroelution. Although all four monomers seem to be involved in the insecticidal activity of the CSPE, the 15 kDa and the 11 kDa proteins appear to be mainly responsible for the observed insecticidal effect. CONCLUSIONS: Four new fungal proteins with insecticidal activity have been purified and identified. These proteins might be combined with insect baits for C. capitata biocontrol. Copyright © 2009 Society of Chemical Industry     

四种Bt毒素蛋白对二点委夜蛾幼虫毒力测定及中肠组织病理学变化

为更好地了解苏云金芽胞杆菌Bacillus thuringiensis毒素蛋白对二点委夜蛾Athetis lepigone的毒力以及作用机理,通过饲喂含有Cry1Ac、Cry1Ab、Cry2Ab和Vip3Aa四种不同Bt毒素蛋白饲料,测定Bt毒素蛋白对二点委夜蛾幼虫的毒力,并观察取食4种毒素蛋白后幼虫中肠组织的病理学变化。结果显示,二点委夜蛾幼虫取食毒素蛋白后72 h,Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫的杀虫活性较高,校正死亡率为84.7%和76.4%;Vip3Aa和Cry2Ab毒素蛋白的毒力较弱。二点委夜蛾幼虫取食4种Bt毒素蛋白后,中肠柱状细胞微绒毛脱落,杂乱地分散在肠腔内,杯状细胞变形和腔内微绒毛脱落,线粒体和内质网等变形破裂,细胞核的核膜消失、核质凝聚和形状发生变化,经Cry1Ab和Cry1Ac毒素蛋白处理后中肠细胞的病变症状和速度明显高于Cry2Ab和Vip3Aa毒素蛋白处理。表明Cry1Ab和Cry1Ac毒素蛋白对二点委夜蛾幼虫杀虫活性较高,显著高于Cry2Ab和Vip3Aa毒素蛋白,且对其中肠细胞的破坏作用也较强。     

Combined application of chitinolytic bacterium Paenibacillus sp. D1 with low doses of chemical pesticides for better control of Helicoverpa armigera

Helicoverpa armigera is a serious pest of Cajanus cajan in many parts of world. Rapid development of resistance against number of insecticides and cry toxin-based biocontrol agents has led to search for biocontrol agents with alternative mode of action. The ability of chitinolytic bacteria to degrade vital chitinous structure in insects suggests their potential in insect control. The present investigation was carried out to study insect control potential of a high chitinase producing bacterium, Paenibacillus sp. D1. Biocontrol studies with Helicoverpa larvae showed Paenibacillus sp. D1 and its chitinase to be potent antifeedant that reduced the feeding rate and body weight of the larvae. The decreased body weight was attributed to hydrolysis of the chitinous structures of the larvae. This was evident from decrease in the total chitin content and increased mortality of the larvae fed on the leaves treated with Paenibacillus sp. D1 and chitinase as compared to untreated controls. A combined dose of Paenibacillus sp. D1 or its chitinase with an organophosphate insecticide, acephate, was found to be more lethal than their individual treatments suggesting integrated insect control potential of the bacterium.     

Plant and insect virus-like particles: emerging nanoparticles for agricultural pest management

Virus-like particles (VLPs) represent a biodegradable, biocompatible nanomaterial made from viral coat proteins that can improve the delivery of antigens, drugs, nucleic acids, and other substances, with most applications in human and veterinary medicine. Regarding agricultural viruses, many insect and plant virus coat proteins have been shown to assemble into VLPs accurately. In addition, some plant virus-based VLPs have been used in medical studies. However, to our knowledge, the potential application of plant/insect virus-based VLPs in agriculture remains largely underexplored. This review focuses on why and how to engineer coat proteins of plant/insect viruses as functionalized VLPs, and how to exploit VLPs in agricultural pest control. The first part of the review describes four different engineering strategies for loading cargo at the inner or the outer surface of VLPs depending on the type of cargo and purpose. Second, the literature on plant and insect viruses the coat proteins of which have been confirmed to self-assemble into VLPs is reviewed. These VLPs are good candidates for developing VLP-based agricultural pest control strategies. Lastly, the concepts of plant/insect virus-based VLPs for delivering insecticidal and antiviral components (e.g., double-stranded RNA, peptides, and chemicals) are discussed, which provides future prospects of VLP application in agricultural pest control. In addition, some concerns are raised about VLP production on a large scale and the short-term resistance of hosts to VLP uptake. Overall, this review is expected to stimulate interest and research exploring plant/insect virus-based VLP applications in agricultural pest management. © 2023 Society of Chemical Industry.     

Cyt1Aa protein from Bacillus thuringiensis (Berliner) serovar israelensis is active against the Mediterranean fruit fly,Ceratitis capitata (Wiedemann)

BACKGROUND: Ceratitis capitata (Wiedemann) is one of the world's most destructive fruit pests. The aim of this study was to ascertain insecticidal activity of Bacillus thuringiensis (Berliner) δ‐endotoxins to C. capitata. RESULTS: Among 42 selected Bacillus strains, only B. thuringiensis serovar israelensis (Bti) solubilised protoxins showed biological activity against C. capitata neonate larvae, whereas Bti spore and crystal mixture was inactive. Insecticidal activity of Bti protoxins was significantly enhanced by incubation with Culex pipiens L. gut extracts. Overdigestion of Bti protoxins with Sesamia nonagrioides (Lefebvre) gut extracts suppressed biological activity against C. capitata, and this correlated with degradation of Cyt toxins. Cyt1Aa solubilised protoxin showed the highest toxicity, LC₅₀ after 7 days of 4.93 µg cm^?2, while proteolytical processing of Cyt1Aa protoxins by larval gut extracts did not enhance insecticidal activity. CONCLUSION: The present study provides evidence for the first time of the insecticidal activity of a B. thuringiensis strain against C. capitata and identifies a single δ‐endotoxin with potential for controlling this pest. Copyright © 2010 Society of Chemical Industry