RESUMEN

The resistance to the insecticidal protein Cry1Ac from the bacterium Bacillus thuringiensis (Bt) in the cabbage looper, Trichoplusia ni, has previously been identified to be associated with a frameshift mutation in the ABC transporter ABCC2 gene and with altered expression of the aminopeptidase N (APN) genes APN1 and APN6, shown as missing of the 110-kDa APN1 (phenotype APN1¯) in larval midgut brush border membrane vesicles (BBMV). In this study, genetic linkage analysis identified that the APN1¯ phenotype and the ABCC2 mutation in Cry1Ac-resistant T. ni segregated independently, although they were always associated under Cry1Ac selection. The ABCC2 mutation and APN1¯ phenotype were separated into two T. ni strains respectively. Bioassays of the T. ni strains with Cry1Ac determined that the T. ni with the APN1¯ phenotype showed a low level resistance to Cry1Ac (3.5-fold), and the associated resistance is incompletely dominant in the background of the ABCC2 mutation. Whereas the ABCC2 mutation-associated resistance to Cry1Ac is at a moderate level, and the resistance is incompletely recessive in the genetic background of downregulated APN1. Analysis of Cry1Ac binding to larval midgut BBMV indicated that the midgut in larvae with the APN1¯ phenotype had reduced binding affinity for Cry1Ac, but the number of binding sites remained unchanged, and the midgut in larvae with the ABCC2 mutation had both reduced binding affinity and reduced number of binding sites for Cry1Ac. The reduced Cry1Ac binding to BBMV from larvae with the ABCC2 mutation or APN1¯ phenotype correlated with the lower levels of resistance.IMPORTANCEThe soil bacterium Bacillus thuringiensis (Bt) is an important insect pathogen used as a bioinsecticide for pest control. Bt genes coding for insecticidal proteins are the primary transgenes engineered into transgenic crops (Bt crops) to confer insect resistance. However, the evolution of resistance to Bt proteins in insect populations in response to exposure to Bt threatens the sustainable application of Bt biotechnology. Cry1Ac is a major insecticidal toxin utilized for insect control. Genetic mechanisms of insect resistance to Cry1Ac are complex and require to be better understood. The resistance to Cry1Ac in Trichoplusia ni is associated with a mutation in the ABCC2 gene and also associated with the APN expression phenotype APN1¯. This study identified the genetic independence of the APN1¯ phenotype from the ABCC2 mutation and isolated and analyzed the ABCC2 mutation-associated and APN1¯ phenotype-associated resistance traits in T. ni to provide new insights into the genetic mechanisms of Cry1Ac resistance in insects.

RESUMEN

Bacillus thuringiensis (Bt) toxins are potential alternatives to synthetic insecticides for the control of lepidopteran pests. However, the evolution of resistance in some insect pest populations is a threat and can reduce the effectiveness of Bt toxins. In this review, we summarize the results of 161 studies from 20 countries reporting field and laboratory-evolved resistance, cross-resistance, and inheritance, mechanisms, and fitness costs of resistance to different Bt toxins. The studies refer mainly to insects from the United States of America (70), followed by China (31), Brazil (19), India (12), Malaysia (9), Spain (3), and Australia (3). The majority of the studies revealed that most of the pest populations showed susceptibility and a lack of cross-resistance to Bt toxins. Factors that delay resistance include recessive inheritance of resistance, the low initial frequency of resistant alleles, increased fitness costs, abundant refuges of non-Bt, and pyramided Bt crops. The results of field and laboratory resistance, cross-resistance, and inheritance, mechanisms, and fitness cost of resistance are advantageous for predicting the threat of future resistance and making effective strategies to sustain the effectiveness of Bt crops.

Asunto(s)

Toxinas de Bacillus thuringiensis , Bacillus thuringiensis , Resistencia a los Insecticidas , Control Biológico de Vectores , Animales , Resistencia a los Insecticidas/genética , Bacillus thuringiensis/genética , Lepidópteros/efectos de los fármacos , Aptitud Genética , Insecticidas/farmacología , Endotoxinas/genética

RESUMEN

The fall armyworm (FAW) poses a significant global threat to food security, and economics. Timely detection is crucial, and this research explores innovative techniques like data analysis, remote sensing, satellite imagery, and AI with machine learning algorithms for predicting and managing outbreaks. Emphasizing the importance of community engagement and international collaboration, social network analysis (SNA) is employed to uncover collaborative networks in FAW management research. The study analyzes a decade of research, revealing trends, influential institutions, authors, and countries, providing insights for efficient FAW management strategies. The research highlights a growing interest in Spodoptera frugiperda (Smith and Abbott 1797) research, focusing on biological control, chemical insecticides, plant extracts, and pest resistance. Co-Citation analysis identifies key research concepts, while collaboration analysis emphasizes the contributions of actors and institutions, such as China, the USA, and Brazil, with international collaboration playing a vital role. Current research trends involve evolving resistance, insecticidal protein gene discovery, and bio-control investigations. Leveraging insights from collaborative networks is essential for formulating effective strategies to manage fall armyworm and ensure global food security. This comprehensive analysis serves as a valuable resource for researchers and stakeholders, guiding efforts to combat this pervasive agricultural pest.

Asunto(s)

Spodoptera , Animales , Insecticidas , Control de Insectos/métodos , Control Biológico de Vectores , Resistencia a los Insecticidas , Investigación , Cooperación Internacional

RESUMEN

Pore-forming toxins (PFTs) are proteins that form lesions in biological membranes. Better understanding of the structure and function of these proteins will be beneficial in a number of biotechnological applications, including the development of new pest control methods in agriculture. When searching for new pore formers, existing sequence homology-based methods fail to discover truly novel proteins with low sequence identity to known proteins. Search methodologies based on protein structures would help us move beyond this limitation. As the number of known structures for PFTs is very limited, it's quite challenging to identify new proteins having similar structures using computational approaches like deep learning. In this article, we therefore propose a sample-efficient graphical model, where a protein structure graph is first constructed according to consensus secondary structures. A semi-Markov conditional random fields model is then developed to perform protein sequence segmentation. We demonstrate that our method is able to distinguish structurally similar proteins even in the absence of sequence similarity (pairwise sequence identity < 0.4)-a feat not achievable by traditional approaches like HMMs. To extract proteins of interest from a genome-wide protein database for further study, we also develop an efficient framework for UniRef50 with 43 million proteins.

Asunto(s)

Bases de Datos de Proteínas , Proteínas Citotóxicas Formadoras de Poros , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Biología Computacional/métodos , Modelos Moleculares , Algoritmos , Cadenas de Markov , Secuencia de Aminoácidos , Estructura Secundaria de Proteína , Aprendizaje Profundo

RESUMEN

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins. These active transporters are involved in the export of different substances such as xenobiotics. ABC transporters from subfamily C (ABCC) have also been described as functional receptors for different insecticidal proteins from Bacillus thuringiensis (Bt) in several lepidopteran species. Numerous studies have characterized the relationship between the ABCC2 transporter and Bt Cry1 proteins. Although other ABCC transporters sharing structural and functional similarities have been described, little is known of their role in the mode of action of Bt proteins. For Heliothis virescens, only the ABCC2 transporter and its interaction with Cry1A proteins have been studied to date. Here, we have searched for paralogs to the ABCC2 gene in H. virescens, and identified two new ABC transporter genes: HvABCC3 and HvABCC4. Furthermore, we have characterized their gene expression in the midgut and their protein topology, and compared them with that of ABCC2. Finally, we discuss their possible interaction with Bt proteins by performing protein docking analysis.

Asunto(s)

Toxinas de Bacillus thuringiensis , Proteínas Bacterianas , Endotoxinas , Proteínas Hemolisinas , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Animales , Toxinas de Bacillus thuringiensis/metabolismo , Endotoxinas/metabolismo , Endotoxinas/genética , Endotoxinas/química , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Proteínas de Insectos/química , Mariposas Nocturnas/metabolismo , Mariposas Nocturnas/genética , Bacillus thuringiensis/metabolismo , Bacillus thuringiensis/genética , Simulación del Acoplamiento Molecular , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/química

RESUMEN

Paralipsa gularis (Zeller) is a storage pest; however, in recent years it has evolved into a considerable maize pest during the late growth stage in the border region between China and other Southeast Asian countries. Bt transgenic insect-resistant maize is an effective measure in controlling a wide range of lepidopteran pests, but there is a lack of research on the toxic effects of storage pests. We tested the toxicity of Bt-Cry1Ab, Vip3Aa, and their complex proteins against P. gularis via bioassay and investigated the efficiency of Bt-(Cry1Ab+Vip3Aa) maize in controlling P. gularis during the late growth stage of maize in the period 2022-2023. The bioassay results show that the susceptibilities of P. gularis to the two Bt proteins and their complex proteins were significantly different. The LC50 values of DBNCry1Ab ("DBN9936" event), DBNVip3Aa ("DBN9501" event), DBN Cry1Ab+Vip3Aa ("DBN3601T" event), and Syngenta Cry1Ab+Vip3Aa ("Bt11" event × "MIR162" event) were 0.038 µg/g, 0.114 µg/g, 0.110 µg/g, and 0.147 µg/g, and the GIC50 values were 0.014 µg/g, 0.073 µg/g, 0.027 µg/g, and 0.026 µg/g, respectively. Determination of the expression content of the insecticidal protein in different tissues of Bt-(Cry1Ab+Vip3Aa) maize shows that the total Bt protein content in different tissues was in the following order: stalk > bract > cob > kernel. However, the bioassay results show that the mortalities of P. gularis feeding on Bt-(Cry1Ab+Vip3Aa) maize in different tissues at different growth stages were all above 93.00%. The field trial indicates that the occurrence density of larvae and plant damage rate for conventional maize were 422.10 individuals/100 plants and 94.40%, respectively, whereas no larvae were found on Bt-(Cry1Ab+Vip3Aa) maize. In summary, this study implies that Bt-(Cry1Ab+Vip3Aa) maize has a high potential for control of P. gularis, providing a new technical measure for the management of the pest.

Asunto(s)

Bacillus thuringiensis , Lepidópteros , Humanos , Animales , Zea mays/genética , Zea mays/metabolismo , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Endotoxinas/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/toxicidad , Proteínas Bacterianas/genética , Proteínas Hemolisinas/toxicidad , Proteínas Hemolisinas/genética , Control Biológico de Vectores/métodos , Lepidópteros/metabolismo , Larva

RESUMEN

Entomopathogenic nematodes from the genus Steinernema (Nematoda: Steinernematidae) are capable of causing the rapid killing of insect hosts, facilitated by their association with symbiotic Gram-negative bacteria in the genus Xenorhabdus (Enterobacterales: Morganellaceae), positioning them as interesting candidate tools for the control of insect pests. In spite of this, only a limited number of species from this bacterial genus have been identified from their nematode hosts and their insecticidal properties documented. This study aimed to perform the genome sequence analysis of fourteen Xenorhabdus strains that were isolated from Steinernema nematodes in Argentina. All of the strains were found to be able of killing 7th instar larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae). Their sequenced genomes harbour 110 putative insecticidal proteins including Tc, Txp, Mcf, Pra/Prb and App homologs, plus other virulence factors such as putative nematocidal proteins, chitinases and secondary metabolite gene clusters for the synthesis of different bioactive compounds. Maximum-likelihood phylogenetic analysis plus average nucleotide identity calculations strongly suggested that three strains should be considered novel species. The species name for strains PSL and Reich (same species according to % ANI) is proposed as Xenorhabdus littoralis sp. nov., whereas strain 12 is proposed as Xenorhabdus santafensis sp. nov. In this work, we present a dual insight into the biocidal potential and diversity of the Xenorhabdus genus, demonstrated by different numbers of putative insecticidal genes and biosynthetic gene clusters, along with a fresh exploration of the species within this genus.

Asunto(s)

Mariposas Nocturnas , Nematodos , Xenorhabdus , Animales , Xenorhabdus/genética , Filogenia , Argentina , Nematodos/genética , Mariposas Nocturnas/genética , Análisis de Secuencia , Simbiosis

RESUMEN

Bacillus thuringiensis (Bt) is an entomopathogenic bacterium that produces crystalline (Cry and Cyt) and soluble (vegetative insecticidal proteins or Vips) proteins during the sporulation and vegetative growth phases, respectively. Combining Cry and Vip proteins could delay insect resistance development and exhibit synergistic activity against various insect pests. This study aims to screen Bt isolates collected from Thailand for high Vip3A and Cry protein production levels and high thermostability to control Spodoptera spp. Among the selected Bt isolates with high target protein synthesis, Bt isolate 506 was found to be safe for further biopesticide formulation due to the absence of non-specific metabolite, as determined by the detection of thermo-stable ß-exotoxin I based on biological assays and PCR analysis. Bt isolate 506 showed the presence of Cry1A, Cry2A, and Vip3A-type proteins identified as Cry1Aa45, Cry2Aa22, and Vip3A87, respectively. The insecticidal activity of whole culture extracts containing Vip3A and Cry mixtures and culture supernatants containing secreted Vip3A protein was evaluated against the second-instar larvae of S. exigua and S. frugiperda. The Bt isolate 506 showed high toxicity against both insects, and the insecticidal proteins produced by this isolate retained their activity after heating at 50 °C. This Bt isolate is a promising candidate for further development as a biopesticide against lepidopteran pests.

Asunto(s)

Bacillus thuringiensis , Insecticidas , Animales , Bacillus thuringiensis/metabolismo , Spodoptera/metabolismo , Agentes de Control Biológico/metabolismo , Proteínas Bacterianas/metabolismo , Insecticidas/farmacología , Insectos , Larva/metabolismo , Proteínas Hemolisinas/metabolismo , Control Biológico de Vectores , Endotoxinas/metabolismo

RESUMEN

Lepidopterans affect crop production worldwide. The use of transgenes encoding insecticidal proteins from Bacillus thuringiensis (Bt) in crop plants is a well-established technology that enhances protection against lepidopteran larvae. Concern about widespread field-evolved resistance to Bt proteins has highlighted an urgent need for new insecticidal proteins with different modes or sites of action. We discovered a new family of insecticidal proteins from ferns. The prototype protein from Pteris species (Order Polypodiales) and variants from two other orders of ferns, Schizaeales and Ophioglossales, were effective against important lepidopteran pests of maize and soybean in diet-based assays. Transgenic maize and soybean plants producing these proteins were more resistant to insect damage than controls. We report here the crystal structure of a variant of the prototype protein to 1.98 Å resolution. Remarkably, despite being derived from plants, the structure resembles the 3-domain Cry proteins from Bt but has only two out of three of their characteristic domains, lacking the C-terminal domain which is typically required for their activities. Two of the fern proteins were effective against strains of fall armyworm that were resistant to Bt 3-domain Cry proteins Cry1Fa or Cry2A.127. This therefore represents a novel family of insecticidal proteins that have the potential to provide future tools for pest control.

Asunto(s)

Bacillus thuringiensis , Helechos , Insecticidas , Tracheophyta , Animales , Insecticidas/metabolismo , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Control Biológico de Vectores , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Tracheophyta/metabolismo , Zea mays/metabolismo

RESUMEN

BACKGROUND: Bacillus thuringiensis (Bt) is a gram-positive ubiquitous saprophytic bacterium that produces proteins (Crystal protein, Vegetative insecticidal protein, and Secreted insecticidal protein) toxic to insects during its growth cycle. In the present study, the whole genome of a locally isolated B. thuringiensis strain BA04 was sequenced to explore the genetic makeup and to identify the genes responsible to produce insecticidal proteins including the virulence factors. The strain was isolated from the soil sample of the Kaziranga National Park, Assam, North-Eastern part of India (Latitude: 26°34'39.11''N and Longitude: 93°10'16.04''E). RESULTS: The whole genome sequencing (WGS) of the BA04 strain revealed that it has a circular genome of size 6,113,005 bp with four numbers of plasmids. A total of 6,111 genes including two novel crystal protein-encoding genes (MH753362.1 and MH753363.1) were identified. The BLASTn analysis of MH753362.1 showed 84% similarities (maximum identity) with Cry1Ia (KJ710646.1) gene, whereas MH753363.1 exhibited 66% identity with Insecticidal Crystal Protein (ICP)-6 gene (KM053257.1). At the protein level, MH753362.1 and MH753363.1 shared 79% identity with Cry1Ia (AIW52613.1) and 40% identity with Insecticidal Crystal Protein (ICP)-6 (AJW76687.1) respectively. Three-dimensional structures of these two novel protein sequences revealed that MH753362.1 have 48% structural similarity with Cry8ea1 protein, whereas MH753363.1 showed only 20% structural similarity with Cry4Aa protein. Apart from these insecticidal genes, the strain was also found to contain virulence and virulence-associated factors including the antibiotic resistance genes and Clustered regularly interspaced short palindromic repeat (CRISPR) sequences. CONCLUSION: This is the first report on the whole genome sequence of Bt strain BA04 isolated from Assam, a North-Eastern state of India. The WGS of strain BA04 unveils the presence of two novel types of insecticidal crystal protein-encoding genes which can be used for the development of insect-resistant transgenic crops. Additionally, the strain could be used for the formulations of effective biopesticides. The WGS provides the fastest and cheapest platform for a better understanding of the genetic makeup of a strain and helps to explore the role of virulence genes in pathogenicity against the insect host.

Asunto(s)

Bacillus thuringiensis , Insecticidas , Animales , Bacillus thuringiensis/genética , Insecticidas/farmacología , Insecticidas/metabolismo , Secuenciación Completa del Genoma , Insectos/metabolismo , Plásmidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo

RESUMEN

Bacillus thuringiensis is a Gram-positive bacterium known for its insecticidal proteins effective against various insect pests. However, limited strains and proteins target coleopteran pests like Anthonomous grandis Boheman, causing substantial economic losses in the cotton industry. This study focuses on characterizing a Bacillus sp. strain, isolated from Oncativo (Argentina), which exhibits ovoid to amorphous parasporal crystals and was designated Bt_UNVM-84. Its genome encodes genes for the production of two pairs of binary Vpb1/Vpa2 proteins and three Cry-like proteins showing similarity with different Cry8 proteins. Interestingly, this gene content was found to be conserved in a previously characterized Argentine isolate of B. thuringiensis designated INTA Fr7-4. SDS-PAGE analysis revealed a major band of 130 kDa that is proteolytically processed to an approximately 66-kDa protein fragment by trypsin. Bioassays performed with spore-crystal mixtures demonstrated an interesting insecticidal activity against the cotton boll weevil A. grandis neonate larvae, resulting in 91% mortality. Strain Bt_UNVM-84 is, therefore, an interesting candidate for the efficient biological control of this species, causing significant economic losses in the cotton industry in the Americas.

Asunto(s)

Bacillus thuringiensis , Escarabajos , Insecticidas , Gorgojos , Animales , Humanos , Recién Nacido , Escarabajos/metabolismo , Gorgojos/genética , Gorgojos/metabolismo , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Insecticidas/metabolismo , Proteínas Bacterianas/metabolismo , Larva/metabolismo , Proteínas Hemolisinas/genética , Endotoxinas/genética , Control Biológico de Vectores

RESUMEN

Bacillus thuringiensis (Bt) has been used as sprayable pesticides for many decades. Bt strains utilized in these products produce multiple insecticidal proteins to complement a narrow insect specificity of each protein. In the late 1990s, genes encoding Bt insecticidal proteins were expressed in crop plants such as cotton and corn to protect these crops from insect damage. The first Bt protein used in transgenic cotton was Cry1Ac to control Heliothis virescens (tobacco budworm). Cry1Ab was applied to corn to control Ostrinia nubilalis (European corn borer). Since these insects have developed resistance to Cry1Ac and Cry1Ab, new Bt proteins are required to overcome the resistance. In order to protect corn furthermore, it is desired to control Diabrotica virgifera (Western corn rootworm), Helicoverpa zea (corn earworm) and Spodoptera frugiperda (fall armyworm). Recently, many new Bt insecticidal proteins have been discovered, but most of them require protein engineering to meet the high activity standard for commercialization. The engineering process for higher activity necessary for Bt crops is called optimization. The seed industry has been optimizing Bt insecticidal proteins to improve their insecticidal activity. In this review, several optimization projects, which have led to substantial activity increases of Bt insecticidal proteins, are described.

RESUMEN

BACKGROUND: Bacillus thuringiensis (Bt) has been widely used as a biological control agent for lepidopteran pests. However, resistance to Bt is a major concern associated with Spodoptera spp. (Noctuidae) and Plutella xylostella (Plutellidae). For efficient control of Noctuidae and Plutellidae, novel Bt strains which have high toxicity and a broad host range are needed. RESULTS: To develop novel Bt strains as used for bio-insecticides, the Bt IMBL-B9 with high toxicity against Spodoptera exigua, Spodoptera frugiperda and P. xylostella was isolated and characterized. The Bt kurstaki IMBL-B9 strain produced bipyramidal and cuboidal crystals consisting of cry toxins with molecular weights of 130 and 65 kDa, respectively. This strain harbors eight crystal protein genes in total, including cry1Ea and one vegetative insecticidal protein gene. The median lethal concentration (LC50 ) values of IMBL-B9 against S. exigua and S. frugiperda were 21.8- and 19.3-fold lower than those of the Bt kusrstaki strain, and 5.6- and 4.9-fold lower than those of Bt aizawai strain, respectively. To evaluate the insecticidal activity of Cry proteins from IMBL-B9, cry gene-sourced recombinant Bt strains were constructed. These strains have insecticidal activity and synergic action against lepidopteran pests. CONCLUSION: In this study, a novel Bt kurstaki IMBL-B9 strain was isolated and this could be useful for the development of new bio-insecticide or cry gene-based recombinant products as an alternative solution against lepidopterans, including Noctuidae and Plutellidae. © 2022 Society of Chemical Industry.

Asunto(s)

Bacillus thuringiensis , Insecticidas , Mariposas Nocturnas , Animales , Bacillus thuringiensis/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Agentes de Control Biológico/metabolismo , Agentes de Control Biológico/farmacología , Endotoxinas/química , Endotoxinas/genética , Endotoxinas/farmacología , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacología , Insecticidas/metabolismo , Insecticidas/farmacología , Mariposas Nocturnas/genética , Mariposas Nocturnas/metabolismo , Control Biológico de Vectores , Spodoptera

RESUMEN

BACKGROUND: The efficacy of Bt crystal proteins has been compromised due to their extensive utilization in the field. The second-generation Bt vegetative insecticidal proteins could be the best-suited alternative to combat resistance build-up due to their broad range affinity with midgut receptors of insects. MATERIAL AND RESULTS: The codon-optimized synthetic vegetative insecticidal proteins (Vip3Aa) gene under the control of CaMV35S promoter was transformed into a locally developed transgenic cotton variety (CKC-01) expressing cry1Ac and cry2A genes. Transformation efficiency of 1.63% was recorded. The highest Vip3Aa expression (51.98-fold) was found in MS3 transgenic cotton plant. Maximum Vip3Aa protein concentration (4.23 µg/mL) was calculated in transgenic cotton plant MS3 through ELISA. The transgenic cotton plant (MS3) showed one copy number on both chromatids in the homozygous form at chromosome 8 at the telophase stage. Almost 99% mortality of H. armigera was recorded in transgenic cotton plants expressing double crystal proteins pyramided with Vip3Aa gene as contrasted to transgenic cotton plant expressing only double crystal protein with 70% mortality. CONCLUSIONS: The results obtained during this study suggest that the combination of Bt cry1Ac, cry2A, and Vip3Aa toxins is the best possible alternative approach to combat chewing insects.

Asunto(s)

Toxinas de Bacillus thuringiensis , Mariposas Nocturnas , Animales , Proteínas Bacterianas/genética , Endotoxinas/genética , Gossypium/genética , Proteínas Hemolisinas/genética , Insectos/genética , Resistencia a los Insecticidas/genética , Larva , Mariposas Nocturnas/genética , Plantas Modificadas Genéticamente/genética

RESUMEN

Biorational insecticides (for instance, avermectins, spinosins, azadirachtin, and afidopyropen) of natural origin are increasingly being used in agriculture. The review considers the chemical ecology approach for the search for new compounds with insecticidal properties (entomotoxic, antifeedant, and hormonal) produced by fungi of various ecological groups (entomopathogens, soil saprotrophs, endophytes, phytopathogens, and mushrooms). The literature survey revealed that insecticidal metabolites of entomopathogenic fungi have not been sufficiently studied, and most of the well-characterized compounds show moderate insecticidal activity. The greatest number of substances with insecticidal properties was found to be produced by soil fungi, mainly from the genera Aspergillus and Penicillium. Metabolites with insecticidal and antifeedant properties were also found in endophytic and phytopathogenic fungi. It was noted that insect pests of stored products are mostly low sensitive to mycotoxins. Mushrooms were found to be promising producers of antifeedant compounds as well as insecticidal proteins. The expansion of the number of substances with insecticidal properties detected in prospective fungal species is possible by mining fungal genomes for secondary metabolite gene clusters and secreted proteins with their subsequent activation by various methods. The efficacy of these studies can be increased with high-throughput techniques of extraction of fungal metabolites and their analysis by various methods of chromatography and mass spectrometry.

RESUMEN

BACKGROUND: A high dose/refuge combination is the main tactic recommended for mitigating resistance selection of target herbivore species in crops expressing insecticidal proteins of the bacterium Bacillus thuringiensis (i.e. Bt proteins). The tactic consists of the simultaneous use of Bt crops expressing high levels of the Bt protein associated with neighboring areas of refuge of the same non-Bt crop species. Nonetheless, the approach faces controversy regarding its effectiveness and scale of adoption, at least in some regions. One concern focuses on its potential impact on the arthropod community, including its short-term and spatially dependent impact considering the likely biota spillover effect between Bt and non-Bt neighboring areas. Thus, the eventual spillover of Bt maize targeted and non-targeted arthropods was surveyed along transects extending from the refuge border to the center of the Bt maize area. RESULTS: Arthropods were collected throughout the maize vegetative and reproductive stages. A total of 85 arthropod species were collected, but their richness and abundance did not vary with distance from the refuge. By contrast, cultivation season played a significant role in distinguishing the arthropod communities. Refuge distance from the sampling point within Bt-fields did not significantly affect the food web metrics, unlike season, which affected the number of nodes integrating each food web. Winter maize cultivation exhibited higher arthropod diversity and combined values of species numeric abundance and biomass at each trophic level. CONCLUSIONS: No arthropod spillover was evident between the refuge edge and Bt maize, adding further controversy to the tactic currently subjected to lower usage in the region with a disputed cost-benefit relationship, because not even the target and its interdependent species were affected. © 2021 Society of Chemical Industry.

Asunto(s)

Artrópodos , Bacillus thuringiensis , Animales , Bacillus thuringiensis/genética , Proteínas Bacterianas/genética , Endotoxinas/genética , Cadena Alimentaria , Proteínas Hemolisinas/genética , Plantas Modificadas Genéticamente/genética , Zea mays/genética

RESUMEN

This paper recommends five criteria to evaluate the reliability of interaction studies with insecticidal proteins. However, these criteria are broadly applicable to an interaction analysis with any type of substance. The recommended criteria reflect the consensus of the literature on interaction analysis from decades of research in fields such as pharmacology and toxicology. The criteria can be used to interrogate the experimental design, assay methodology, data analysis, and interpretation of the results. These criteria will be useful to researchers to help identify the strengths and potential weaknesses of interaction studies and to help define the limits of interpretation of the data. The criteria will also be useful to risk assessors evaluating the reliability of interaction data as part of an environmental risk assessment, and to inform a weight of evidence analysis when there are contradictory results. In addition, these criteria can be used prospectively by researchers to help avoid common pitfalls that are apparent in some interaction studies. Five examples have been provided, with studies from the literature, that demonstrate how these criteria can be objectively and consistently applied to score the reliability of interaction studies with insecticidal proteins that differ in design and methodology.

Asunto(s)

Bacterias/química , Proteínas Bacterianas/química , Endotoxinas/química , Insecticidas/química , Toxinas de Bacillus thuringiensis/química , Proteínas Hemolisinas/química

RESUMEN

Entomopathogenic bacteria have great potential in insect control in the agricultural production because they produce a large variety of protein toxins that can kill their hosts by damaging the insect midgut. However, the mechanisms on how these toxins or specific insecticidal proteins act on insects are very diverse and elusive. Here we select Galleria mellonella larvae as the host to explore the effects of insecticidal proteins on the activities of three protective enzymes (SOD, POD, and CAT) and on the morphology of the midgut tissues. As a result, the activities of the three enzymes consistently increased and then decreased when the host was injected with the insecticidal proteins from the entomopathogenic bacterium Enterobacter cloacae. Moreover, the microscopy analysis showed that tissues, cells, and organelles of the host midgut are all diseased after uptake of the insecticidal proteins. Remarkably, the protein toxins contributed to the deformation of the midgut, blackening of the midgut surface, dissolution of cell membrane, shrinkage of cell nucleus, and chromatin condensation. Our findings will advance the explanation of G. mellonella pathogenesis caused by the insecticidal proteins.

RESUMEN

Cry1C, Cry1F and Cry1Ab are insecticidal proteins from Bacillus thuringiensis (Bt) which are expressed in transgenic crops. Given the entry of these proteins into aquatic environments, it is relevant to evaluate their impacts on aquatic organisms. In this work, we sought to evaluate the effects of Cry1C, Cry1F and Cry1Ab on zebrafish embryos and larvae of a predicted worst-case scenario concentration of these proteins (set to 1.1 mg/L). For that, we coupled a traditional toxicity approach (the zebrafish embryotoxicity test and dosage of enzymatic biomarkers) to gel free proteomics analysis. At the concentration tested, these proteins did not cause adverse effects in the zebrafish early life stages, either by verifying phenotypic endpoints of toxicity or alterations in representative enzymatic biomarkers (catalase, glutathione-S-tranferase and lactate-dehydrogenase). At the molecular level, the Cry proteins tested lead to very small changes in the proteome of zebrafish larvae. In a global way, these proteins upregulated the expression of vitellogenins. Besides that, Cry1C e Cry1F deregulated heterogeneous nuclear ribonucleoproteins (Hnrnpa0l and Hnrnpaba, respectively), implicated in mRNA processing and gene regulation. Overall, these data lead to the conclusion that Cry1C, Cry1F and Cry1Ab proteins, even at a very high concentration, have limited effects in the early stages of zebrafish life.

Asunto(s)

Bacillus thuringiensis , Proteínas Hemolisinas , Animales , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Endotoxinas/toxicidad , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidad , Larva , Plantas Modificadas Genéticamente , Proteómica , Pez Cebra

RESUMEN

The Cry23Aa/Cry37Aa proteins from Bacillus thuringiensis (Bt) have been described toxic to Cylas puncticollis larvae. In general, it is believed that Cry23Aa and Cry37Aa act jointly to exert the insecticidal activity, while there is no evidence of their toxicity individually. Therefore, in the present study, the contribution of each protein in the insecticidal activity toward C. puncticollis larvae has been assessed. The results showed that both proteins were toxic for C. puncticollis larvae when tested individually. Contrary to what was claimed previously, our results suggest that the presence of both proteins is not necessary to exert toxicity against C. puncticollis larvae. Also, the binding behavior of Cry23Aa protein to midgut receptors of C. puncticollis larvae has been determined. According to our results, Cry23Aa binds to C. puncticollis brush border membrane vesicles (BBMV) specifically and independently of Cry37Aa. Due to the lack of common binding sites, Cry23Aa can be pyramided with Cry3Aa protein for better management of C. puncticollis.