Assessing fitness costs of the resistance of Spodoptera frugiperda (Lepidoptera: Noctuidae) to pyramided Cry1 and Cry2 insecticidal proteins on different host plants

Fall armyworm (FAW), Spodoptera frugiperda (Smith), is one of the major pests targeted by transgenic crops expressing insecticidal proteins from Bacillus thuringiensis (Bt) Berliner. However, FAW presents a high capacity to develop resistance to Bt protein-expressing crop lines, as reported in Brazil, Argentina, Puerto Rico and the southeastern U.S. Here, FAW genotypes resistant to pyramided maize events expressing Cry1F/Cry1A.105/Cry2Ab2 (P-R genotype) and Cry1A.105/Cry2Ab2 (Y-R genotype) from Brazil were used to investigate the interactions between non-Bt hosts (non-Bt maize, non-Bt cotton, millet and sorghum) and fitness costs. We also tested a FAW genotype susceptible to Bt maize and F1 hybrids of the resistant and susceptible genotypes (heterozygotes). Recessive fitness costs (i.e., costs affecting the resistant insects) were observed for pupal and neonate to adult survival of the P-R genotype on non-Bt cotton; larval developmental time of the P-R genotype on millet and sorghum; larval and neonate-to-adult developmental time of the Y-R genotype on non-Bt cotton and sorghum; the fecundity of the Y-R genotype on non-Bt cotton; and mean generation time of both resistant genotypes. However, on non-Bt cotton and non-Bt maize, the P-R genotype had a higher fitness (i.e., fitness benefits), displaying greater fecundity and rates of population increases than the Sus genotype. Non-recessive fitness costs (i.e., costs affecting heterozygotes) were found for fecundity and population increases on millet and sorghum. These findings suggest that, regardless of the disadvantages of the resistant genotypes in some hosts, the resistance of FAW to Cry1 and Cry2 Bt proteins is not linked with substantial fitness costs, and may persist in field conditions once present.

Structural and functional insights into the first Bacillus thuringiensis vegetative insecticidal protein of the Vpb4 fold, active against western corn rootworm

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major maize pest in the United States causing significant economic loss. The emergence of field-evolved resistant WCR to Bacillus thuringiensis (Bt) traits has prompted the need to discover and deploy new insecticidal proteins in transgenic maize. In the current study we determined the crystal structure and mode of action (MOA) of the Vpb4Da2 protein (formerly known as Vip4Da2) from Bt, the first identified insecticidal Vpb4 protein with commercial level control against WCR. The Vpb4Da2 structure exhibits a six-domain architecture mainly comprised of antiparallel β-sheets organized into β-sandwich layers. The amino-terminal domains 1–3 of the protein share structural homology with the protective antigen (PA) PA14 domain and encompass a long β-pore forming loop as in the clostridial binary-toxB module. Domains 5 and 6 at the carboxyl-terminal half of Vpb4Da2 are unique as this extension is not observed in PA or any other structurally-related protein other than Vpb4 homologs. These unique Vpb4 domains adopt the topologies of carbohydrate-binding modules known to participate in receptor-recognition. Functional assessment of Vpb4Da2 suggests that domains 4–6 comprise the WCR receptor binding region and are key in conferring the observed insecticidal activity against WCR. The current structural analysis was complemented by in vitro and in vivo characterizations, including immuno-histochemistry, demonstrating that Vpb4Da2 follows a MOA that is consistent with well-characterized 3-domain Bt insecticidal proteins despite significant structural differences.

Isolation of Bacillus thuringiensis strains from Saudi Arabia soil and study of their potential efficiency against the lepidopteran pest Ephestia kuehniella

Insect pests represent a major threat to food crops and human health, and therefore have to be combated in several ways, including chemical methods. However, researchers demonstrated that these molecules are dangerous for the farmers, consumers and the environment in general. For this reason, scientists permanently searched environment friendly alternatives such as the use of the bacterium Bacillus thuringiensis classified as one of the best insect pathogens. This microorganism is known by its ability to produce two types of insecticidal proteins, Vegetative insecticidal proteins (Vip) and delta-endotoxins produced during vegetative and sporulation stages of growth, respectively. In the present study, 15 B. thuringiensis strains were isolated from soil collected from different regions in Saudi Arabia (Al Baha, Jeddah, Khulis and Yanbu). B. thuringiensis isolates were then classified according to the shape of their parasporal crystals identified under microscope and proteins content of these crystals. Delta-endotoxins efficiency of the different isolates was investigated and promising strains were identified as very active. After 5 days-treatment, B. thuringiensis isolates 14 and 7 killed Ephestia kuehniella larvae with low LC50 of about 59.18 and 65.67 mg/cm2, respectively. The results described in the present study proved that the new B. thuringiensis isolates could be of a great interest in the control of lepidopteran pests by using their delta-endotoxins in bioinsecticide formulations.

Insekticidni proteini in njihova uporaba za zatiranje koloradskega hrošča (Leptinotarsa decemlineata [Say, 1824])

<p class="042abstractstekst">Plants respond to pest attack, among other mechanisms, by producing specific proteins with insecticidal properties. Proteins with toxic effects on insects have also been discovered in many other organisms, especially fungi and bacteria. Due to their biological function, insecticidal proteins represent an important potential in the development of more environmentally friendly plant protection methods. Increasing knowledge about the mode of action of insecticidal proteins and the identification of genes encoding their synthesis enable the breeding of transgenic plants resistant to insect pests and the development of new bioinsecticidal agents. The Colorado potato beetle (<em>Leptinotarsa decemlineata</em>) is one of the most important pests of potato, so the study of such control methods is crucial for the development of sustainable integrated pest management strategies of potato. This review highlights the properties of some groups of insecticidal proteins and their modes of action, and summarizes examples of studies of their use for the control of Colorado potato beetle.</p>

Structural and functional characterization of Mpp75Aa1.1, a putative beta-pore forming protein from Brevibacillus laterosporus active against the western corn rootworm

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major corn pest of significant economic importance in the United States. The continuous need to control this corn maize pest and the development of field-evolved resistance toward all existing transgenic maize (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins against WCR has prompted the development of new insect-protected crops expressing distinct structural classes of insecticidal proteins. In this current study, we describe the crystal structure and functional characterization of Mpp75Aa1.1, which represents the first corn rootworm (CRW) active insecticidal protein member of the ETX_MTX2 sub-family of beta-pore forming proteins (β-PFPs), and provides new and effective protection against WCR feeding. The Mpp75Aa1.1 crystal structure was solved at 1.94 Å resolution. The Mpp75Aa1.1 is processed at its carboxyl-terminus by WCR midgut proteases, forms an oligomer, and specifically interacts with putative membrane-associated binding partners on the midgut apical microvilli to cause cellular tissue damage resulting in insect death. Alanine substitution of the surface-exposed amino acids W206, Y212, and G217 within the Mpp75Aa1.1 putative receptor binding domain I demonstrates that at least these three amino acids are required for WCR activity. The distinctive spatial arrangement of these amino acids suggests that they are part of a receptor binding epitope, which may be unique to Mpp75Aa1.1 and not present in other ETX_MTX2 proteins that do not have WCR activity. Overall, this work establishes that Mpp75Aa1.1 shares a mode of action consistent with traditional WCR-active Bt proteins despite significant structural differences.

ORFograph: search for novel insecticidal protein genes in genomic and metagenomic assembly graphs

Background Since the prolonged use of insecticidal proteins has led to toxin resistance, it is important to search for novel insecticidal protein genes (IPGs) that are effective in controlling resistant insect populations. IPGs are usually encoded in the genomes of entomopathogenic bacteria, especially in large plasmids in strains of the ubiquitous soil bacteria, Bacillus thuringiensis (Bt). Since there are often multiple similar IPGs encoded by such plasmids, their assemblies are typically fragmented and many IPGs are scattered through multiple contigs. As a result, existing gene prediction tools (that analyze individual contigs) typically predict partial rather than complete IPGs, making it difficult to conduct downstream IPG engineering efforts in agricultural genomics. Methods Although it is difficult to assemble IPGs in a single contig, the structure of the genome assembly graph often provides clues on how to combine multiple contigs into segments encoding a single IPG. Results We describe ORFograph, a pipeline for predicting IPGs in assembly graphs, benchmark it on (meta)genomic datasets, and discover nearly a hundred novel IPGs. This work shows that graph-aware gene prediction tools enable the discovery of greater diversity of IPGs from (meta)genomes. Conclusions We demonstrated that analysis of the assembly graphs reveals novel candidate IPGs. ORFograph identified both already known genes “hidden” in assembly graphs and potential novel IPGs that evaded existing tools for IPG identification. As ORFograph is fast, one could imagine a pipeline that processes many (meta)genomic assembly graphs to identify even more novel IPGs for phenotypic testing than would previously be inaccessible by traditional gene-finding methods. While here we demonstrated the results of ORFograph only for IPGs, the proposed approach can be generalized to any class of genes.

Bacillus thuringiensis as microbial biopesticide: uses and application for sustainable agriculture

Background Bacillus thuringiensis (Bt) has been used in agriculture for a long time because of its insecticidal proteins which make it a valuable environment-friendly biopesticide. However, its use is not only limited to insecticidal properties. Current and previous studies indicate its potential as a biofertilizer for promoting plant growth, the development of transgenic plants, and others. It is the presence of δ-endotoxins, especially cry protein, which attributes the insecticidal property to the bacteria. Besides, there are some vegetative and secreted insecticidal proteins that exert their toxic activity towards specific species. Main body of abstract The present review briefly provides an overview of the Bt uses and application as a biocontrol agent against insect pest for sustainable agriculture. Historical development of Bt as biocontrol, classification of various cry proteins, their mechanisms of actions against different insect-pest, and incorporation of cry genes in the plant for developing transgenic Bt plants such as Bt cotton, potato, and maize. Applications of Bt as biofertilizer and the various bioformulations as biopesticide are also described. Short conclusion Uses of harmful pesticides and chemical cause various health issues and environmental problem; therefore, the Bt served as the best alternative to overcome the above issue. Also, we aim to explore the potential as plant growth-promoting potential and solubilization of minerals and the uses as a biofertilizer, keeping the high specificity and environmental safety of Bt. Its various formulations are commercially available and considered an efficient alternative to chemical pesticides.

Promising approaches to the search for fungal metabolites for management of arthropod pests

Biorational insecticides of natural origin, such as avermectins, spinosins, azadirachtin and afidopyropen, are increasingly used in agriculture. The present paper reviews modern ecological, genomic, and biotechnological approaches to 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 showed that insecticidal metabolites of entomopathogenic fungi had not been sufficiently studied, and the majority of well-characterized compounds had showed 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. Low sensitivity of insect pests of stored products (in particular, of grain) to mycotoxins was recorded. Mushrooms were found to be promising producers of antifeedant compounds as well as insecticidal proteins. It is possible to increase the number of substances with insecticidal properties detected in fungi not only by extension of the screening range but also by exploitation of diverse bioassay sytems and model insect species. Mining genomes for secondary metabolite gene clusters and secreted proteins with their subsequent activation by various methods allows for better understanding of the biosynthetic potential of the prospective strains. Efficacy of these studies can be increased with high-throughput techniques of fungal metabolites extraction and further analysis using chromatography and mass spectrometry. Insecticidal proteins detected in fungi can be used in the technologies for development of transgenic plant varieties resistant to pests, or hypervirulent bioinsecticides.

Correction: Syed, T., et al. Current Insights on Vegetative Insecticidal Proteins (Vip) as Next Generation Pest Killers. Toxins 2020, 12, 522

The authors wish to make the following corrections to their paper [...]