Characterization of the mode of action of eCry1Gb.1Ig, a fall armyworm (Spodoptera frugiperda) active protein, with a novel site of action.

Insect pests cause immense agronomic losses worldwide. One of the most destructive of major crops is the Fall Armyworm (Spodoptera frugiperda, FAW). The ability to migrate long distances, a prodigious appetite, and a demonstrated ability to develop resistance to insecticides, make it a difficult target to control. Insecticidal proteins, for example those produced by the bacterium Bacillus thuringiensis, are among the safest and most effective insect control agents. Genetically modified (GM) crops expressing such proteins are a key part of a successful integrated pest management (IPM) program for FAW. However, due to the development of populations resistant to commercialized GM products, new GM traits are desperately needed. Herein, we describe a further characterization of the newly engineered trait protein eCry1Gb.1Ig. Similar to other well characterized Cry proteins, eCry1Gb.1Ig is shown to bind FAW midgut cells and induce cell-death. Binding competition assays using trait proteins from other FAW-active events show a lack of competition when binding FAW brush border membrane vesicles (BBMVs) and when utilizing non-pore-forming versions as competitors in in vivo bioassays. Similarly, insect cell lines expressing SfABCC2 and SfABCC3 (well characterized receptors of existing commercial Cry proteins) are insensitive to eCry1Gb.1Ig. These findings are consistent with results from our previous work showing that eCry1Gb.1Ig is effective in controlling insects with resistance to existing traits. This underscores the value of eCry1Gb.1Ig as a new GM trait protein with a unique site-of-action and its potential positive impact to global food production.

Genomic-proteomic analysis of a novel Bacillus thuringiensis strain: toxicity against two lepidopteran pests, abundance of Cry1Ac5 toxin, and presence of InhA1 virulence factor.

Bacillus thuringiensis (Bt) is a biological alternative to the indiscriminate use of chemical insecticides in agriculture. Due to resistance development on insect pests to Bt crops, isolating novel Bt strains is a strategy for screening new pesticidal proteins or strains containing toxin profile variety that can delay resistance. Besides, the combined genomic and proteomic approaches allow identifying pesticidal proteins and virulence factors accurately. Here, the genome of a novel Bt strain (Bt TOL651) was sequenced, and the proteins from the spore-crystal mixture were identified by proteomic analysis. Toxicity bioassays with the spore-crystal mixture against larvae of Diatraea saccharalis and Anticarsia gemmatalis, key pests of sugarcane and soybean, respectively, were performed. The toxicity of Bt TOL651 varies with the insect; A. gemmatalis (LC50 = 1.45 ng cm-2) is more susceptible than D. saccharalis (LC50 = 73.77 ng cm-2). Phylogenetic analysis of the gyrB gene indicates that TOL651 is related to Bt kenyae strains. The genomic analysis revealed the presence of cry1Aa18, cry1Ac5, cry1Ia44, and cry2Aa9 pesticidal genes. Virulence factor genes such as phospholipases (plcA, piplc), metalloproteases (inhA), hemolysins (cytK, hlyIII, hblA, hblC, hblD), and enterotoxins (nheA, nheB, nheC) were also identified. The combined use of the genomic and proteomic data indicated the expression of Cry1Aa18, Cry1Ac5, and Cry2Aa9 proteins, with Cry1Ac5 being the most abundant. InhA1 also was expressed and may contribute to Bt TOL651 pathogenicity. These results provide Bt TOL651 as a new tool for the biocontrol of lepidopteran pests.

Abundance, distribution, and expression of nematicidal crystal protein genes in Bacillus thuringiensis strains from diverse habitats.

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that accumulates pesticidal proteins (Cry and Cyt) in parasporal crystals. Proteins from the Cry5, App6 (formerly Cry6), Cry12, Cry13, Cry14, Cry21, and Xpp55 (formerly Cry55) families have been identified as toxic to nematodes. In this study, a total of 846 Bt strains belonging to four collections were analyzed to determine the diversity and distribution of the Bt Cry nematicidal protein genes. We analyzed their presence by PCR, and positives were confirmed by sequencing. As a result, 164 Bt isolates (20%) contained at least one gene coding for nematicidal Cry proteins. The cry5 and cry21 genes were enriched in collection 1 and were often found together in the same strain. Differently, in collection 4, obtained from similar habitats but after 10 years, cry14 was the gene most frequently found. In collection 2, cry5 and app6 were the most abundant genes, and collection 3 had a low incidence of any of these genes. The results point to high variability in the frequencies of the studied genes depending on the timing, geographical origins, and sources. The occurrence of cry1A, cry2, and cry3 genes was also analyzed and showed that the nematicidal Cry protein genes were frequently accompanied by cry1A + cry2. The expression of the genes was assessed by mass spectrometry showing that only 14% of the positive strains produced nematicidal proteins. To our knowledge, this is the first comprehensive screening that examines the presence and expression of genes from the seven known Bt Cry nematicidal families.

Expression of thurincin H, ChiA74 and Cry proteins at the sporulation phase in Bacillus thuringiensis HD1.

AIMS: The objective of this study was to produce thurincin H, ChiA74 and Cry proteins together using Bacillus thuringiensis subsp. kurstaki HD1 as a heterologous host. METHODS AND RESULTS: pSTAB-ThurH and pSTAB-ChiA74 constructs were designed to produce thurincin H and chitinase, respectively, at the sporulation phase. They were transformed into Bt HD1 generating the recombinant strains HD1/pSTAB-ThurH and HD1/pSTAB-ThurH/pSTAB-ChiA74. Antimicrobial and chitinolytic activity tests were performed with recombinant strains. Both strains were able to produce thurincin H up to 72 h with antibacterial activity of ~4000 U mg-1 . The HD1/pSTAB-ThurH/pSTAB-ChiA74 strain also showed chitinolytic activity of ~23 mU mg-1 at 72 h. All B. thuringiensis strains exhibited crystal formation at 72, and 96 h. In addition, the application of thurincin H in corn seeds increased the germination percentage and root length by 7% and 10%, respectively. CONCLUSIONS: We showed that is possible to produce three proteins of biotechnological interest at the sporulation stage in B. thuringiensis, which two of them (thurincin H, and ChiA74) are naturally expressed in the vegetative stage. SIGNIFICANCE AND IMPACT OF THE STUDY: These results form the basis for developing of a biocontrol and biostimulator product that can be used as an alternative for chemical application.

Characterization and mosquitocidal potency of a Bacillus thuringiensis strain of rice field soil of Burdwan, West Bengal, India.

Bacillus thuringiensis is the most popular mosquitocidal bacteria, strains of which are effective against almost all mosquito larvae. It has host specificity and thus, has no adverse effect on non-target species of the ecosystem. Culex tritaeniorhynchus, a vector of Japanese encephalitis (JE), breeds in vast area of rice fields in Burdwan district of West Bengal, India, which has already confronted JE epidemic. Entomological investigation and ecological studies on this vector mosquito showed that JE epidemic may reoccur anytime in the area. A strain of Bt (BU55) was isolated from rice field soil, efficacy was tested against Cx. tritaeniorhynchus and mosquitocidal role was confirmed against Cx. quinquefascistus also. The LC50 of Bacillus thuringiensis BU55 against Cx. tritaeniorhynchus and Cx. quinquefascistus after 72 h was 8.59 ml (final dose 2.49 x107 CFU/ml) and 7.52 ml (final dose 2.20 x 107 CFU/ml), respectively. Insecticidal crystal protein profile of BU55 produced 136.89, 64.80, 43.45, 33.65 and 26.98 kDa bands. Among them 136.89, 64.29, 26.98 kDa proteins are comparable to actual toxins viz. Cry1Ac (138.3 kDa, Lepidoptera specific), Cry4D (68.0 kDa, Diptera specific) and Cyt (27.4 kDa, Diptera specific). The results clearly showed that the Bt strain is a potent dipteran larvicide and can be used against the JE vectors to control the disease.

Degradation of transgenic Bt-rice straw incorporated with two different paddy soils.

Transgenic Bt-rice is rice that has been genetically modified to produce insecticidal proteins (Cry1Ab/Ac) within the plant. Rice straw is incorporated into paddy soils after harvest for fertilization or to improve the soil structure. The incorporation of straw from transgenic Bt-rice may pose risks to the paddy soil system. The decomposition of Bt-rice straw and degradation of Cry1Ab/Ac proteins from the straw were investigated under laboratory conditions. In addition, effects of the incorporation with chopped rice straw on microbial communities in differently textured paddy soils were studied. The results indicated that the incorporation of straw from transgenic Bt-rice might have a slight influence on soil respiration and CH4 emissions in two paddy soils, i.e. the Silt Loam soil and the Silty Clay soil. Differences were also observed in the cumulative emissions of CO2 between the two amended paddy soils in addition to the well-known increase in emissions of both CO2 and CH4 due to straw incorporation. The Cry1Ab/Ac proteins from straw of transgenic Bt-rice were degraded in paddy soils. The rate of decline in the concentration of Cry1Ab/Ac proteins was different in the two soils. After 29 d of incubation, 61% and 42% of initial Cry1Ab/Ac proteins were detected in the silt loam and silty clay, respectively. As a result of the presence of the rice straw, the abundance of bacteria, archaea, and total cells were increased in two soils. The numbers of bacteria and total cells were 6.4% and 11.5% higher in the silt loam amended with straw of Bt-rice than non-Bt-rice, respectively. The silty clay displayed a similar trend as the silt loam.

Comprehensive in silico allergenicity assessment of novel protein engineered chimeric Cry proteins for safe deployment in crops.

BACKGROUND: Development of chimeric Cry toxins by protein engineering of known and validated proteins is imperative for enhancing the efficacy and broadening the insecticidal spectrum of these genes. Expression of novel Cry proteins in food crops has however created apprehensions with respect to the safety aspects. To clarify this, premarket evaluation consisting of an array of analyses to evaluate the unintended effects is a prerequisite to provide safety assurance to the consumers. Additionally, series of bioinformatic tools as in silico aids are being used to evaluate the likely allergenic reaction of the proteins based on sequence and epitope similarity with known allergens. RESULTS: In the present study, chimeric Cry toxins developed through protein engineering were evaluated for allergenic potential using various in silico algorithms. Major emphasis was on the validation of allergenic potential on three aspects of paramount significance viz., sequence-based homology between allergenic proteins, validation of conformational epitopes towards identification of food allergens and physico-chemical properties of amino acids. Additionally, in vitro analysis pertaining to heat stability of two of the eight chimeric proteins and pepsin digestibility further demonstrated the non-allergenic potential of these chimeric toxins. CONCLUSIONS: The study revealed for the first time an all-encompassing evaluation that the recombinant Cry proteins did not show any potential similarity with any known allergens with respect to the parameters generally considered for a protein to be designated as an allergen. These novel chimeric proteins hence can be considered safe to be introgressed into plants.

Stacked Bt maize and arthropod predators: exposure to insecticidal Cry proteins and potential hazards.

Genetically engineered (GE) crops with stacked insecticidal traits expose arthropods to multiple Cry proteins from Bacillus thuringiensis (Bt). One concern is that the different Cry proteins may interact and lead to unexpected adverse effects on non-target species. Bi- and tri-trophic experiments with SmartStax maize, herbivorous spider mites (Tetranychus urticae), aphids (Rhopalosiphum padi), predatory spiders (Phylloneta impressa), ladybeetles (Harmonia axyridis) and lacewings (Chrysoperla carnea) were conducted. Cry1A.105, Cry1F, Cry3Bb1 and Cry34Ab1 moved in a similar pattern through the arthropod food chain. By contrast, Cry2Ab2 had highest concentrations in maize leaves, but lowest in pollen, and lowest acquisition rates by herbivores and predators. While spider mites contained Cry protein concentrations exceeding the values in leaves (except Cry2Ab2), aphids contained only traces of some Cry protein. Predators contained lower concentrations than their food. Among the different predators, ladybeetle larvae showed higher concentrations than lacewing larvae and juvenile spiders. Acute effects of SmartStax maize on predator survival, development and weight were not observed. The study thus provides evidence that the different Cry proteins do not interact in a way that poses a risk to the investigated non-target species under controlled laboratory conditions.

Dissimilar Crystal Proteins Cry5Ca1 and Cry5Da1 Synergistically Act against Meloidogyne incognita and Delay Cry5Ba-Based Nematode Resistance.

Cry proteins of Bacillus thuringiensis (Bt) have been successfully used as biopesticides and in transgenic crops throughout the world. However, resources against the most serious agricultural pathogens, plant root-knot nematodes, are limited. The genomes of several highly nematicidal virulent Bt strains from our laboratory have been sequenced, facilitating the identification of novel Cry proteins and other virulence factors. We identified two novel Cry proteins, Cry5Ca1 and Cry5Da1, that exhibit high toxicity against Meloidogyne incognita Using the Caenorhabditis elegans model, the two Cry5 toxins were shown to negatively affect nematode life span, fertility, and survival. The 50% lethal concentrations (LC50s) of Cry5Ca1 and Cry5Da1 were 57.22 µg/ml and 36.69 µg/ml, respectively. Moreover, a synergistic effect (synergism factor, 1.61 to 2.04) was observed for nematicidal toxicity of Cry5Ca1 and Cry5Da1, which is accordant with the phylogenetic results suggesting that domain II of the two novel Cry5 toxins evolved into two independent clades. Through comparison of the depressed degree of toxicity in the ß-methylgalactoside detoxification test, we found that the novel toxin Cry5D possesses a different galactose-binding epitope; meanwhile, the finding that Cry5D does not share a motif (GXXXE) in the corresponding loop of domain II with Cry5B could explain the different galactose binding performance. Additionally, low-level cross-resistance of C. elegans bre mutant strains was evident between Cry5B and Cry5D. These results suggest that Cry5D can be used as an alternative to delay the potential resistance of nematodes to Cry5B.IMPORTANCE Although proper gene resources for Bt crops against the most serious agricultural pathogens, plant root-knot nematodes, are limited, we have identified two novel nematicidal toxins, Cry5Ca1 and Cry5Da1, against M. incognita, which have supplied more gene candidates for Bt crops designed against nematodes. Moreover, the association of the dissimilarity between Cry5Da1 and Cry5Ba1 and their low cross-resistance can be attributed not only to a low sequence similarity of domain II but also to the structural difference of the key motif and receptor-binding epitope in the loops. This association facilitates the selection of a proper candidate for the prospective design of pyramided Bt crops that can delay potential resistance.

Toxicity and Binding Studies of Bacillus thuringiensis Cry1Ac, Cry1F, Cry1C, and Cry2A Proteins in the Soybean Pests Anticarsia gemmatalis and Chrysodeixis (Pseudoplusia) includens.

Anticarsia gemmatalis (velvetbean caterpillar) and Chrysodeixis includens (soybean looper, formerly named Pseudoplusia includens) are two important defoliating insects of soybeans. Both lepidopteran pests are controlled mainly with synthetic insecticides. Alternative control strategies, such as biopesticides based on the Bacillus thuringiensis (Bt) toxins or transgenic plants expressing Bt toxins, can be used and are increasingly being adopted. Studies on the insect susceptibilities and modes of action of the different Bt toxins are crucial to determine management strategies to control the pests and to delay outbreaks of insect resistance. In the present study, the susceptibilities of both soybean pests to the Bt toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa have been investigated. Bioassays performed in first-instar larvae showed that both insects are susceptible to all these toxins. Competition-binding studies carried out with Cry1Ac and Cry1Fa 125-iodine labeled proteins demonstrated the presence of specific binding sites for both of them on the midgut brush border membrane vesicles (BBMVs) of both A. gemmatalis and C. includens Competition-binding experiments and specific-binding inhibition studies performed with selected sugars and lectins indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites in the midguts of both insects. Also, the Cry1Ac- or Cry1Fa-binding sites were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity and midgut toxin binding sites in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops.IMPORTANCE In the present study, the toxicity and the mode of action of the Bacillus thuringiensis (Bt) toxins Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa in Anticarsia gemmatalis and Chrysodeixis includens (important defoliating pests of soybeans) have been investigated. These studies are crucial for determining management strategies for pest control. Bioassays showed that both insects were susceptible to the toxins. Competition-binding studies demonstrated the presence of Cry1Fa- and Cry1Ac-specific binding sites in the midguts of both pests. These results, together with the results from binding inhibition studies performed with sugars and lectins, indicated that Cry1Ac and Cry1Fa share some, but not all, binding sites, and that they were not shared with Cry1Ca or Cry2Aa in either soybean pest. This study contributes to the knowledge of Bt toxicity in A. gemmatalis and C. includens and sheds light on the cross-resistance potential of Cry1Ac, Cry1Fa, Cry1Ca, and Cry2Aa Bt proteins as candidate proteins for Bt-pyramided crops.

Assessment of potential adjuvanticity of Cry proteins.

Genetically modified (GM) crops have achieved success in the marketplace and their benefits extend beyond the overall increase in harvest yields to include lowered use of insecticides and decreased carbon dioxide emissions. The most widely grown GM crops contain gene/s for targeted insect protection, herbicide tolerance, or both. Plant expression of Bacillus thuringiensis (Bt) crystal (Cry) insecticidal proteins have been the primary way to impart insect resistance in GM crops. Although deemed safe by regulatory agencies globally, previous studies have been the basis for discussions around the potential immuno-adjuvant effects of Cry proteins. These studies had limitations in study design. The studies used animal models with extremely high doses of Cry proteins, which when given using the ig route were co-administered with an adjuvant. Although the presumption exists that Cry proteins may have immunostimulatory activity and therefore an adjuvanticity risk, the evidence shows that Cry proteins are expressed at very low levels in GM crops and are unlikely to function as adjuvants. This conclusion is based on critical review of the published literature on the effects of immunomodulation by Cry proteins, the history of safe use of Cry proteins in foods, safety of the Bt donor organisms, and pre-market weight-of-evidence-based safety assessments for GM crops.

Susceptibility of Grapholita molesta (Busck, 1916) to formulations of Bacillus thuringiensis, individual toxins and their mixtures.

The Oriental fruit moth, Grapholita molesta (Lepidoptera: Tortricidae), is a major pest of fruit trees worldwide, such as peach and apple. Bacillus thuringiensis has been shown to be an efficient alternative to synthetic insecticides in the control of many agricultural pests. The objective of this study was to evaluate the effectiveness of B. thuringiensis individual toxins and their mixtures for the control of G. molesta. Bioassays were performed with Cry1Aa, Cry1Ac, Cry1Ca, Vip3Aa, Vip3Af and Vip3Ca, as well as with the commercial products DiPel® and XenTari®. The most active proteins were Vip3Aa and Cry1Aa, with LC50 values of 1.8 and 7.5ng/cm2, respectively. Vip3Ca was nontoxic to this insect species. Among the commercial products, DiPel® was slightly, but significantly, more toxic than XenTari®, with LC50 values of 13 and 33ng commercial product/cm2, respectively. Since Vip3A and Cry1 proteins are expressed together in some insect-resistant crops, we evaluated possible synergistic or antagonistic interactions among them. The results showed moderate to high antagonism in the combinations of Vip3Aa with Cry1Aa and Cry1Ca.

Toxicity of Bacillus thuringiensis (L.) Cry proteins against summer fruit tortrix (Adoxophyes orana - Fischer von Rösslerstamm).

The activity of seven Cry1, one Cry9 and one hybrid Cry1 protoxins against neonate larvae of summer fruit tortrix (Adoxophyes orana - Fischer von Rösslerstamm) has been investigated. Cry1Ia is identified as the most toxic protein, followed by Cry1Aa and Cry1Ac. Cry1Ca, Cry1Cb, Cry1Da and Cry1Fa were less active, while SN19 (Cry1 hybrid protein with domain composition 1Ba/1Ia/1Ba) and Cry9Aa exhibited negligible toxicity against A. orana. In vitro trypsin-activated Cry1Ac is still less active than Cry1Ia protoxin, suggesting that toxicity of Cry1Ia is most probably due to more complex differences in further downstream processing, toxin-receptor interactions and pore formation in A. orana's midgut epithelium.

Development of Bt Rice and Bt Maize in China and Their Efficacy in Target Pest Control.

Rice and maize are important cereal crops that serve as staple foods, feed, and industrial material in China. Multiple factors constrain the production of both crops, among which insect pests are an important one. Lepidopteran pests cause enormous yield losses for the crops annually. In order to control these pests, China plays an active role in development and application of genetic engineering (GE) to crops, and dozens of GE rice and GE maize lines expressing insecticidal proteins from the soil bacterium Bacillus thuringiensis (Bt) have been developed. Many lines have entered environmental release, field testing, and preproduction testing, and laboratory and field experiments have shown that most of the Bt rice and Bt maize lines developed in China exhibited effective control of major target lepidopteran pests on rice (Chilo suppressalis, Scirpophaga incertulas, and Cnaphalocrocismedinalis) and maize (Ostrinia furnacalis), demonstrating bright prospects for application. However, none of these Bt lines has yet been commercially planted through this writing in 2016. Challenges and perspectives for development and application of Bt rice and maize in China are discussed. This article provides a general context for colleagues to learn about research and development of Bt crops in China, and may shed light on future work in this field.

Bacillus thuringiensis subsp. israelensis producing endochitinase ChiA74Δsp inclusions and its improved activity against Aedes aegypti.

AIMS: The objective of this study was to produce stable inclusions of chitinase ChiA74Δsp in Bacillus thuringiensis subsp. israelensis (Bti) and to assay its insecticidal activity against Aedes aegypti larvae. METHODS AND RESULTS: Bti was transformed with chiA74Δsp regulated by its own promoter or by the strong chimeric cytAp/STAB-SD promoter system to generate two recombinant Bti strains. These recombinants produced their native parasporal bodies composed of Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa and ChiA74Δsp inclusions, and showed a approx. threefold increase in both endochitinase activity and viable spore count when compared with the parental strain. Both recombinants were approximately twofold more toxic (LC50s 8·02, 9·6 ng ml(-1) ) than parental Bti (19·8 ng ml(-1) ) against 4(th) instars of A. aegypti larvae. CONCLUSIONS: ChiA74Δsp inclusions, together with the insecticidal crystals and spores of Bti increased the toxicity against A. aegypti larvae by at least twofold. SIGNIFICANCE AND IMPACT OF THE STUDY: We report for the first time the engineering of Bti to produce spore-parasporal body-ChiA74∆sp inclusions in the same sporangium, which are released together following autolysis. Our work lays a foundation for engineering Bti to produce more efficacious combinations of Cry4Aa, Cry4Ba, Cry11Aa, Cyt1Aa and chitinase inclusions.

Evaluation of the synergistic activities of Bacillus thuringiensis Cry proteins against Helicoverpa armigera (Lepidoptera: Noctuidae).

With the aim of identifying Cry proteins that would be useful in the management of the economically important lepidopteran pest Helicoverpa armigera, the larvicidal activities of binary combinations (1:1 ratios) of six Cry proteins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ca, Cry2Aa and Cry9Aa) were evaluated against H. armigera neonate larvae using droplet feeding bioassays. Determination of the LD50 values of individual Cry proteins and mixtures of Cry proteins enabled assessment of the nature of the interactions between Cry proteins in H. armigera. There was a more than 6000-fold difference between the LD50 values of the Cry protein mixture with the lowest larvicidal activity and the mixture with the highest larvicidal activity. Cry1Ac and Cry2Aa mixtures and Cry1Ac and Cry1Ca mixtures had the highest larvicidal activity against H. armigera, with Cry1Ac and Cry1Ca interacting synergistically. Differences in the magnitudes of the antagonistic interactions observed for different binary mixtures of Cry1A-class proteins are consistent with a model of more than one binding site for some Cry1A-class proteins in H. armigera. Binary combinations of Cry1A-class and Cry9Aa proteins showed additive interactions in neonate larvae of H. armigera, whereas combinations of Cry1Ca and Cry9Aa were statistically synergistic. The results suggest that products containing mixtures of Cry1Ac and Cry2Aa or Cry1Ac and Cry1Ca may be useful components of H. armigera pest management programs.

Draft genome of neotropical Bacillus thuringiensis UFT038 and its potential against lepidopteran soybean pests.

Bacillus thuringiensis (Bt) is known for its Cry and Vip3A pesticidal proteins with high selectivity to target pests. Here, we assessed the potential of a novel neotropical Bt strain (UFT038) against six lepidopteran pests, including two Cry-resistant populations of fall armyworm, Spodoptera frugiperda. We also sequenced and analyzed the genome of Bt UFT038 to identify genes involved in insecticidal activities or encoding other virulence factors. In toxicological bioassays, Bt UFT038 killed and inhibited the neonate growth in a concentration-dependent manner. Bt UFT038 and HD-1 were equally toxic against S. cosmioides, S. frugiperda (S_Bt and R_Cry1 + 2Ab populations), Helicoverpa zea, and H. armigera. However, larval growth inhibition results indicated that Bt UFT038 was more toxic than HD-1 to S. cosmioides, while HD-1 was more active against Chrysodeixis includens. The draft genome of Bt UFT038 showed the cry1Aa8, cry1Ac11, cry1Ia44, cry2Aa9, cry2Ab35, and vip3Af5 genes. Besides this, genes encoding the virulence factors (inhA, plcA, piplC, sph, and chi1-2) and toxins (alo, cytK, hlyIII, hblA-D, and nheA-C) were also identified. Collectively, our findings reveal the potential of the Bt UFT038 strain as a source of insecticidal genes against lepidopteran pests, including S. cosmioides and S. frugiperda.

Bio-fabricated zinc oxide and cry protein nanocomposites: Synthesis, characterization, potentiality against Zika, malaria and West Nile virus vector's larvae and their impact on non-target organisms.

The role of mosquito vectors in spreading disastrous diseases to living organisms, especially to humans is inevitable and undeniable. The impacts of the available chemical and synthetic insecticides on non-specific organisms as well as on nature are being the reason behind the search for target-specific, biocompatible and eco-friendly alternatives. The Madhuca longifolia seed extract and cry proteins from Bacillus thuringiensis-based nanocomposites (Cp-Ml-ZnO NCs) were produced to conquer the above-mentioned issues. The Cp-Ml-ZnO NCs (100 µg/mL) expressed better scavenging potentiality on 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radicals than Ml seed extract and Ml-ZnO NPs. The susceptibility of tested vector larvae to the Cp-Ml-ZnO NCs was Ae. aegypti ˃An. stephensi ˃ Cx. quinquefasciatus along with LC50-27.73, 34.81, and 42.54 µg/mL concentration. The target specificity and biocompatibility of Cp-Ml-ZnO NCs were authenticated by the results obtained by evaluating the efficacy on D. similis, A. salina, P. reticulata, G. affinis, and RBCs of goat blood. Thus the Cp-Ml-ZnO NCs could be adopted for the control of vector larvae.

Mpp23Aa/Xpp37Aa Insecticidal Proteins from Bacillus thuringiensis (Bacillales: Bacillaceae) Are Highly Toxic to Anthonomus grandis (Coleoptera: Curculionidae) Larvae.

The beetle Anthonomus grandis Boheman, 1843, is the main cotton pest, causing enormous losses in cotton. The breeding of genetically modified plants with A. grandis resistance is seen as an important control strategy. However, the identification of molecules with high toxicity to this insect remains a challenge. The susceptibility of A. grandis larvae to proteins (Cry1Ba, Cry7Ab, and Mpp23Aa/Xpp37Aa) from Bacillus thuringiensis Berliner, 1915, with toxicity reported against Coleopteran, has been evaluated. The ingestion of different protein concentrations (which were incorporated into an artificial diet) by the larvae was tested in the laboratory, and mortality was evaluated after one week. All Cry proteins tested exhibited higher toxicity than that the untreated artificial diet. These Cry proteins showed similar results to the control Cry1Ac, with low toxicity to A. grandis, since it killed less than 50% of larvae, even at the highest concentration applied (100 µg·g-1). Mpp/Xpp proteins provided the highest toxicity with a 0.18 µg·g-1 value for the 50% lethal concentration. Importantly, this parameter is the lowest ever reported for this insect species tested with B. thuringiensis proteins. This result highlights the potential of Mpp23Aa/Xpp37Aa for the development of a biotechnological tool aiming at the field control of A. grandis.

Bacillus thuringiensis monogenic strains: screening and interactions with insecticides used against rice pests.

The screening of Bacillus thuringiensis (Bt) Cry proteins with high potential to control insect pests has been the goal of numerous research groups. In this study, we evaluated six monogenic Bt strains (Bt dendrolimus HD-37, Bt kurstaki HD-1, Bt kurstaki HD-73, Bt thuringiensis 4412, Bt kurstaki NRD-12 and Bt entomocidus 60.5, which codify the cry1Aa, cry1Ab, cry1Ac, cry1Ba, cry1C, cry2A genes respectively) as potential insecticides for the most important insect pests of irrigated rice: Spodoptera frugiperda, Diatraea saccharalis, Oryzophagus oryzae, Oebalus poecilus and Tibraca limbativentris. We also analyzed their compatibility with chemical insecticides (thiamethoxam, labdacyhalothrin, malathion and fipronil), which are extensively used in rice crops. The bioassay results showed that Bt thuringiensis 4412 and Bt entomocidus 60.5 were the most toxic for the lepidopterans, with a 93% and 82% mortality rate for S. frugiperda and D. saccharalis, respectively. For O. oryzae, the Bt kurstaki NRD-12 (64%) and Bt dendrolimus HD-37 (62%) strains were the most toxic. The Bt dendrolimus HD-37 strain also caused high mortality (82%) to O. poecilus, however the strains assessed to T. limbativentris caused a maximum rate of 5%. The assays for the Bt strains interaction with insecticides revealed the compatibility of the six strains with the four insecticides tested. The results from this study showed the high potential of cry1Aa and cry1Ba genes for genetic engineering of rice plants or the strains to biopesticide formulations.