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     

Saponins show high entomotoxicity by cell membrane permeation in Lepidoptera

BACKGROUND: In this study, the effects of three saponins and one sapogenin with a triterpenoid or steroid structure in two lepidopteran insect cell lines, ovarian Bm5 and midgut CF‐203 cells, were analysed with regard to cell viability, cell membrane permeation, EcR responsiveness and DNA fragmentation. In addition, the entomotoxic action of Q. saponaria saponin with primary midgut cell cultures and larval stages of the cotton leafworm Spodoptera littoralis was tested. RESULTS: Both lepidopteran cell lines show a high sensitivity to all four sapo(ge)nins, with a concentration‐dependent viability loss and EC₅₀ values of 25–100 µM in MTT bioassays. A trypan blue assay with Q. saponaria saponin confirmed rapid cell membrane permeation to be a cause of cytotoxicity. Saponins caused no EcR activation in Bm5 cells, but a loss of ecdysteroid signalling was observed with IC₅₀ values of 5–10 µM . Lower saponin concentrations induced DNA fragmentation, confirming their potential to induce apoptosis. Finally, Q. saponaria saponin caused cytotoxicity in primary midgut cell cultures of S. littoralis (EC₅₀ = 4.7 µM ) and killed 70–84% of S. littoralis larvae at pupation at 30‐70 mg g^?1, while lower concentrations retarded larval weight gain and development. CONCLUSIONS: The data obtained provide evidence that saponins exert a strong activity on lepidopteran cells, presumably based on a cytotoxic action due to permeation of the cell membrane. Primary midgut cell cultures and larvae of S. littoralis showed high sensitivity to Q. saponaria saponin, indicating the insect midgut as a primary target for entomotoxicity and the potential use of saponins in the control of pest Lepidoptera. Copyright © 2012 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     

Can the mammalian organoid technology be applied to the insect gut?

Mammalian intestinal organoids are multicellular structures that closely resemble the structure of the intestinal epithelium and can be generated in vitro from intestinal stem cells under appropriate culture conditions. This technology has transformed pharmaceutical research and drug development in human medicine. For the insect gut, no biotechnological platform equivalent to organoid cultures has been described yet. Comparison of the regulation of intestinal homeostasis and growth between insects and mammals has revealed significant similarities but also important differences. In contrast to mammals, the differentiation potential of available insect cell lines is limited and can not be exploited for in vitro permeability assays to measure the uptake of insecticides. The successful development of in vitro models could be a result of the emergence of molecular mechanisms of self‐organization and signaling in the intestine that are unique to mammals. It is nevertheless considered that the technology gap is a consequence of vast differences in knowledge, particularly with respect to culture conditions that maintain the differentation potential of insect midgut cells. From the viewpoint of pest control, advanced in vitro models of the insect midgut would be very desirable because of its key barrier function for orally ingested insecticides with hemolymphatic target and its role in insecticide resistance. © 2020 Society of Chemical Industry     

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     

植物病毒与媒介昆虫互作促进其传播的研究进展

近年来植物病毒病频发,严重制约着农作物的产量与品质。绝大多数植物病毒依赖媒介昆虫进行传播,而传播的关键是病毒如何突破昆虫的肠屏障、唾液腺屏障和卵屏障等多个生物屏障。植物病毒一方面利用其外壳蛋白或非结构蛋白突破媒介昆虫的中肠屏障和唾液腺屏障;另一方面则与昆虫体内卵黄原蛋白、共生菌以及精子表面蛋白发生特异性互作,促进病毒跨越卵障碍,最终实现病毒在昆虫体内复制。此外,植物病毒还能通过侵染寄主植物影响其防御性状,间接改变媒介昆虫生理及其行为反应,促进病毒在植物间的传播。该研究对植物病毒突破昆虫生物屏障的分子机制,以及植物病毒-植物-媒介昆虫互作对于病毒传播的影响进行了综述,并对阻断病毒传播的方法进行展望。     

植物凝集素在抗刺吸式害虫转基因工程中的应用

植物凝集素是一类具有高度特异性糖结合活性的蛋白,含有1个或多个可与单糖或寡聚糖特异可逆结合的非催化结构域,在农业生产上应用前景广阔。本文简要概述了几种植物凝集素:雪莲凝集素、天南星科植物凝集素、苋菜类植物凝集素在抗刺吸害虫基因工程上的应用,并简单介绍了转双价抗虫基因方面的研究。     

Novel insecticide polymer chemistry to reduce the enzymatic digestion of a protein pesticide, trypsin modulating oostatic factor (TMOF)

A limiting factor in the use of proteins as insecticides, especially when the site of action is in the insect hemocoel, is protease degradation in the digestive system and hemolymph and movement across the midgut ventriculus. Trypsin modulating oostatic factor (TMOF) is a per os mosquito peptidic larvacide which moves across the digestive system and binds to receptors on the hemolymph side of the gut where the hormone inhibits protease synthesis and food utilization ultimately causing death. In the current study, the in vitro degradation of TMOF by the digestive enzyme, leucine aminopeptidase, was inhibited by conjugation of TMOF-K with aliphatic polyethylene glycol (PEG) polymers. Structure activity studies demonstrated a correlation between the molecular weight of the PEG polymer and resistance to digestion and show proof of concept that aliphatic-PEG protein polymerization can be used to prevent protease degradation of a protein insecticide.     

棉铃虫APN基因家族系统进化与功能分析

氨肽酶N（APN）是锌金属蛋白酶M1家族的成员,鳞翅目昆虫中肠刷状缘膜囊泡（brush border membrane vesicles,BBMV）细胞膜上的APN不仅在蛋白消化吸收过程中起着重要的作用,而且是Bt的重要受体蛋白。本研究通过PCR技术克隆得到7条棉铃虫APN基因全长序列,HaAPN1～7（Genbank登录号为MT002819～MT002825）。经生物信息学分析,HaAPN1～7全长为2592～3099 bp,编码863～1032个氨基酸,分子量为110～115 kDa,等电点为4.7～6.4,N端信号肽为15～20 aa。系统进化分析结果表明,HaAPN1～7分属于Class 1～7类别。RT-qPCR结果表明,在敏感棉铃虫5龄幼虫中肠中APN2的表达量最高,APN7的表达量最低;Cry1Ac抗性品系棉铃虫中各类APN的表达趋势与敏感品系一致。取食Cry1Ac、Cry2Ab和Vip3Aa蛋白后棉铃虫中肠APN活性显著降低,取食Cry1Ac和Vip3Aa蛋白后中肠液和BBMV中APN活性都显著降低,而取食Cry2Ab蛋白后只有BBMV上APN活性显著降低;棉铃虫对Cry1Ac、Cry2Ab和Vip3Aa产生耐受性后,中肠液和BBMV中APN活性也都发生显著变化。因此,不同类别的棉铃虫APN在Bt的杀虫机制中可能起到不同的作用,APN活性变化可能与Bt蛋白的降解及抗性演化相关。     

Tomato yellow leaf curl virus accumulates in vesicle-like structures in descending and ascending midgut epithelial cells of the vector whitefly, Bemisia tabaci, but not in those of nonvector whitefly Trialeurodes vaporariorum

Begomoviruses are transmitted by a single species of vector insect, the whitefly Bemisia tabaci, in a circulative manner. However, the mechanisms of this strict vector specificity have not been clarified. By immunoelectron microscopy, we showed that a begomovirus, Tomato yellow leaf curl virus (TYLCV), can enter midgut epithelial cells of the vector whitefly B. tabaci but not those of a nonvector whitefly, Trialeurodes vaporariorum, belonging to the same family. In midgut epithelial cells of viruliferous B. tabaci, the virus was localized in vesicle-like structures, suggesting endocytosis as an entry mechanism. These structures were also observed in midgut cells of nonviruliferous B. tabaci that had fed on healthy plants and in those of the nonvector T. vaporariorum that had fed on virus-infected plants. Vesicles containing TYLCV particles were observed most frequently in cells in the anterior part of the descending midgut, suggesting that this is the major entry site. These results clearly demonstrated that the virus-containing vector and nonvector whiteflies differ in the cellular localization of the virus and strongly suggest that a critical step in determining the vector insect specificity of begomoviruses is the entry of the viruses into midgut epithelial cells.     

Protein metabolism in Spodoptera litura (F.) is influenced by the botanical insecticide azadirachtin

Azadirachtin, as a botanical insecticide, affects a wide variety of biological processes, including reduction of feeding, suspension of molting, death of larvae and pupae, and sterility of emerged adults in a dose-dependent manner. However, the mode of action of this toxin remains obscure. By using proteomic techniques, we analyzed changes in protein metabolism of Spodoptera litura (F.) induced by azadirachtin. Following feeding 4th instar larvae of Spodoptera litura (F.) with an artificial diet containing 1 ppm azadirachtin until pupation, 48 h old pupae were collected and protein samples prepared. Total soluble protein content was measured and the results showed that azadirachtin significantly influenced protein level. Moreover, the proteins were separated by 2-DE (two-dimensional polyacrylamide gel electrophoresis) and 10 proteins were significantly affected by azadirachtin treatment when compared to an untreated control. Six of these proteins were identified with peptide mass fingerprinting using MALDI-TOF-MS after in-gel trypsin digestion. These proteins are involved in various cellular functions. One identified protein may function as an ecdysone receptor, which regulates insect development, and reproduction. It is suggested that the botanical insecticide azadirachtin affects protein expression and the azadirachtin-related proteins would be essential for a better understanding of the mechanisms by which neem toxins exert their effects on insects.     

Identifying proteins with insecticidal activity: use of encoding genes to produce insect-resistant transgenic crops

Crops resistant to insect attack offer an alternative strategy of pest control to a total reliance upon chemical pesticides. Transgenic plant technology can be a useful tool in producing resistant crops, by introducing novel resistance genes into a plant species. This technology is seen very much as forming an integral component of a crop management programme. Several different classes of plant proteins have been shown to be insecticidal towards a range of economically important insect pests from different orders; in some cases a role in the defence of specific plant species against phytophagous insects has been demonstrated. Genes encoding insecticidal proteins have been isolated from various plant species and transferred to crops by genetic engineering. Amongst these genes are those that encode inhibitors of proteases (serine and cysteine) and α-amylase, lectins, and enzymes such as chitinases and lipoxygenases. Examples of genetically engineered crops expressing insecticidal plant proteins from different plant species, with enhanced resistance to one or more insect pests from the orders Lepidoptera, Homoptera and Coleoptera are presented. The possibility of ‘pyramiding’ different resistance genes to improve the effectiveness of protection and durability is discussed and exemplified. The number of different crop species expressing such genes is very diverse and ever-increasing. The viability of this approach to crop protection is considered. © 1998 SCI.     

Triterpene saponins of Quillaja saponaria show strong aphicidal and deterrent activity against the pea aphid Acyrthosiphon pisum

BACKGROUND: Saponins are a class of secondary plant metabolites consisting of a sugar moiety glycosidically linked to a hydrophobic aglycone (sapogenin) that often possess insecticidal activities. Four saponins were selected: two triterpene saponins, Q. saponaria saponins and aescin, and two steroidal saponins, digitonin and diosgenin. Their effects were investigated on an important pest species and a model piercing‐sucking insect, the pea aphid Acyrthosiphon pisum. The triterpene Q. saponaria saponins bark saponin received special attention because of its high activity. Aphids were challenged by oral and contact exposure to demonstrate aphicidal activities, and in choice experiments to support use as a natural deterrent. RESULTS: When aphids were exposed to supplemented artificial diet for 3 days, a strong aphicidal activity was recorded for three of the four saponins, with an LC₅₀ of 0.55 mg mL^?1 for Q. saponaria saponins, 0.62 mg mL^?1 for aescin and 0.45 mg mL^?1 for digitonin. The LT₅₀ values ranged between 1 and 4 days, depending on the dose. For diosgenin, only low toxicity (14%) was scored for concentrations up to 5 mg mL^?1. In choice experiments with treated diet, a deterrence index of 0.97 was scored for Q. saponaria saponins at 1 mg mL^?1. In contrast, direct contact showed no repellent effect. Spraying of faba bean plants with Q. saponaria saponins resulted in an LC₅₀ of 8.2 mg mL^?1. Finally, histological analysis in aphids fed with Q. saponaria saponins demonstrated strong aberrations of the aphid gut epithelium, and exposure of midgut CF‐203 cell lines to Q. saponaria saponins in vitro confirmed the cytotoxic effect. CONCLUSIONS: The present insect experiments provide strong evidence that saponins, as tested here with triterpene Q. saponaria saponins, can be useful as natural aphicides and deterrents. Furthermore, the insect midgut epithelium is suggested to be a primary target of saponin activity.Copyright © 2011 Society of Chemical Industry     

Effects of camptothecin and hydroxycamptothecin on insect cell lines Sf21 and IOZCAS-Spex-II

BACKGROUND: In recent years, concerns over the potential impact of synthetic pesticides on the environment have made the discovery and development of environmentally friendly pesticides a more pressing issue. Camptothecin (CPT) and its derivatives have attracted much attention owing to their unique mechanisms of action against insects. In this paper, two insect cell lines, Sf21 and IOZCAS‐Spex‐II, were used to conduct a preliminary investigation of the potential of CPT and hydroxycamptothecin (HCPT) as inducers of apoptosis in insect cell lines, and to illustrate the mechanism of action of CPT on insects at the cellular level. RESULTS: The results showed that both CPT and HCPT demonstrate potent cytotoxic effects to the tested insect cell lines in a time‐ and dose‐dependent manner. The DNA fragmentation, activation of caspases and cytochrome c release were observed in both IOZCAS‐Spex‐II and Sf21 treated with CPT and HCPT. There is no significant difference in cytotoxicity and caspase‐3 activation (P < 0.05, except when treated for 2 h) between CPT and HCPT, although the caspase‐3 activation was slightly stronger when treated with HCPT in both Sf21 and IOZCAS‐Spex‐II. CONCLUSION: The results confirm the existence of the mitochondrial‐dependent pathway of apoptosis induced by CPT and HCPT in Sf21 and IOZCAS‐Spex‐II cell lines. Further investigations are required to reveal the mitochondrial mechanisms and regulation of caspase activation during apoptosis. These studies will provide basic knowledge needed to understand the mechanisms of action of CPT and to develop CPT and its derivatives as insecticides. Copyright © 2011 Society of Chemical Industry     

昆虫对生物农药的抗性机制及对策

本文综述了昆虫对B.t等生物杀虫剂的抗性机制及延缓昆虫抗性发展所应采取的措施。昆虫通过下列不同机制产生抗生：1）昆虫的血淋巴对B.t等生物杀虫剂的营养细胞的抑制作用。2）各种来源的蛋白酶对毒素蛋白的过度降解作用。3）昆虫中肠沉淀蛋白对毒素蛋白的沉淀作用。4）中肠上皮修复能力增强。5）中肠的吸附位点对毒素蛋白的亲和力下降。通过加强对B.t菌株的选育，合理科学的用药方式及采用不同的模式进行植物基因操作以提高杀虫蛋白的表达和活性等综合措施，减缓和降低昆虫抗性的发展。     

Managing the risk of insect resistance to transgenic insect control traits: practical approaches in local environments

BACKGROUND: Growers have enthusiastically embraced crops genetically modified to express Bacillus thuringiensis (Bt) proteins for insect control because they provide excellent protection from key damaging insect pests around the world. Bt crops also offer superior environmental and health benefits while increasing grower income. However, insect resistance development is an important concern for all stakeholders, including growers, technology providers and seed companies that develop these genetically modified crops. Given the marked benefits associated with Bt crops, insect resistance management (IRM) must be a consideration when cultivating these crops. RESULTS: The technical data and practical experience accumulated with Bt crops in many global regions can inform different aspects of resistance management leading to robust, science‐based IRM plans. A range of elements should be considered in assembling any IRM strategy, including: pest biology/ecology, product deployment patterns, local cropping systems, insect susceptibility monitoring, stakeholder/grower communications, and a remedial action plan should resistance develop. Each of these elements is described in more detail, with specific examples of how these elements can be combined and tailored to the local/regional environments and grower practices. CONCLUSION: IRM plans need to be suitable for the given production situation. What works for large monoculture production systems in North America is unlikely to be appropriate for the small, more diverse agriculture of southeast Asia or Africa. Though it is clear that Bt crops impart considerable value to growers, it is also clear that it is in the best interest of all stakeholders to preserve Bt proteins for the long‐term benefits they provide. Copyright © 2009 Society of Chemical Industry     

V-ATPase H表达量下调增强棉铃虫对Cry1Ac的抗性

棉铃虫Helicoverpa armigera是世界性重要农业害虫。目前防治棉铃虫的主要手段是种植转苏云金芽胞杆菌Bacillus thuringiensis(Bt)杀虫蛋白的转基因作物。本文旨在研究棉铃虫V-ATPase H在Cry1Ac蛋白毒力和抗性中的作用。利用实时荧光定量qRT-PCR技术分析V-ATPase H在Cry1Ac抗、感品系棉铃虫幼虫中肠及敏感品系棉铃虫幼虫受Cry1Ac诱导后的表达情况;在昆虫Sf9细胞中过表达V-ATPase H对其进行细胞定位,通过细胞毒力试验验证其对Cry1Ac毒力的影响。结果发现棉铃虫V-ATPase H基因在抗性品系中低表达,并且V-ATPase H在受到Cry1Ac诱导时也低表达;在Sf9细胞内表达V-ATPase H蛋白发现其在整个细胞中都有分布,过表达该蛋白后增强了细胞对Cry1Ac蛋白的敏感性。结果表明V-ATPase H参与Cry1Ac蛋白的毒力。     

Arabidopsis thaliana resistance to insects, mediated by an earthworm-produced organic soil amendment

BACKGROUND: Vermicompost is an organic soil amendment produced by earthworm digestion of organic waste. Studies show that plants grown in soil amended with vermicompost grow faster, are more productive and are less susceptible to a number of arthropod pests. In light of these studies, the present study was designed to determine the type of insect resistance (antixenosis or antibiosis) present in plants grown in vermicompost‐amended potting soil. Additionally, the potential role of microarthropods, entomopathogenic organisms and non‐pathogenic microbial flora found in vermicompost on insect resistance induction was investigated. RESULTS: Findings show that vermicompost from two different sources (Raleigh, North Carolina, and Portland, Oregon) were both effective in causing Arabidopsis plants to be resistant to the generalist herbivore Helicoverpa zea (Boddie). However, while the Raleigh (Ral) vermicompost plant resistance was expressed as both non‐preference (antixenosis) and milder (lower weight and slower development) toxic effect (antibiosis) resistance, Oregon (OSC) vermicompost plant resistance was expressed as acute antibiosis, resulting in lower weights and higher mortality rates. CONCLUSION: Vermicompost causes plants to have non‐preference (antixenosis) and toxic (antibiosis) effects on insects. This resistance affects insect development and survival on plants grown in vermicompost‐amended soil. Microarthropods and entomopathogens do not appear to have a role in the resistance, but it is likely that resistance is due to interactions between the microbial communities in vermicompost with plant roots, as is evident from vermicompost sterilization assays conducted in this study. Copyright © 2010 Society of Chemical Industry     

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.