Interaction of insecticidal proteins from Pseudomonas spp. and Bacillus thuringiensis for boll weevil management.

Cotton crop yields are largely affected by infestations of Anthonomus grandis, which is its main pest. Although Bacillus thuringiensis (Bt) derived proteins can limit insect pest infestations, the diverse use of control methods becomes a viable alternative in order to prolong the use of technology in the field. One of the alternative methods to Bt technology has been the utilization of certain Pseudomonas species highly efficient in controlling coleopteran insects have been used to produce highly toxic insecticidal proteins. This study aimed to evaluate the toxicity of IPD072Aa and PIP-47Aa proteins, isolated from Pseudomonas spp., in interaction with Cry1Ia10, Cry3Aa, and Cry8B proteins isolated from B. thuringiensis, to control A. grandis in cotton crops. The genes IPD072Aa and PIP-47Aa were synthesized and cloned into a pET-SUMO expression vector. Moreover, Cry1Ia10, Cry3Aa, and Cry8B proteins were obtained by inducing recombinant E. coli clones, which were previously acquired by our research group from the Laboratory of Bacteria Genetics and Applied Biotechnology (LGBBA). These proteins were visualized in SDS-PAGE, quantified, and incorporated into an artificial diet to estimate their lethal concentrations (LC) through individual or combined bioassays. The results of individual toxicity revealed that IPD072Aa, PIP-47Aa, Cry1Ia10, Cry3Aa, and Cry8B were efficient in controlling A. grandis, with the latter being the most toxic. Regarding interaction assays, a high synergistic interaction was observed between Cry1Ia10 and Cry3Aa. All interactions involving Cry3Aa and PIP-47Aa, when combined with other proteins, showed a clear synergistic effect. Our findings highlighted that the tested proteins in combination, for the most part, increase toxicity against A. grandis neonate larvae, suggesting possible constructions for pyramiding cotton plants to the manage and the control boll weevils.

Sub-lethal effects of a Bt-based bioinsecticide on the biological conditioning of Anticarsia gemmatalis.

Bioinsecticides based on Bacillus thuringiensis (Bt) Berliner, 1915 are widely used to control lepidopteran in several crops. However, surviving insects exposed to the sub-lethal concentration of Bt-based bioinsecticides can suffer a multitude of effects on the biological conditioning known as hormesis. Here, we aimed to provide a clearer understanding of the biological conditioning of Anticarsia gemmatalis (Hübner, 1818), exposed to different concentrations of a Bt-based bioinsecticide, by assessing life table parameters over three generations. We defined five sub-lethal concentrations (LC5, LC10, LC15, LC20, and LC25) from the response curve estimate of A. gemmatalis. Deionized water was used as a control. We assessed the parameters of eggs-viability and the duration of the stages, incubation, larval, pre-pupal, pupal, adult, pre-oviposition and total biological cycle. Data were used to construct the fertility life table using the two-sex program. The survival curves showed greater variation in the proportion of individuals at each development stage using the LC25. The sub-lethal concentrations did not influence the incubation-eggs period, pre-pupal and pupal. However, the larval and adult stages using LC25 and LC10 were the most affected. Changes in sex ratio were observed using LC20 and LC5. The toxic effect of Bt-based bioinsecticide interfered mainly in the parameters of fertility, sex ratio, net reproduction rate (R0), and gross reproduction rate (GRR).

Synergism of the Bacillus thuringiensis Cry1, Cry2, and Vip3 Proteins in Spodoptera frugiperda Control.

The polyphagous caterpillar, Spodoptera frugiperda, has been controlled with either chemical insecticides or transgenic plants such as Bt maize that expresses the cry and/or vip genes of the Bacillus thuringiensis (Bt) bacterium. Despite the efficiency of Bt toxins in lepidopteran control, populations resistant to Bt plants have emerged in different locations around the world. Thus, understanding how combined proteins interact against pests can assist resistance control and management. This work demonstrated the toxicity of Cry1Ab, Cry1Ac, Cry1Ca, Cry1Ea, Cry2Aa, Cry2Ab, Vip3Aa, and Vip3Ca in single and combined assays against S. frugiperda neonatal larvae. All protein mixtures had synergistic action in the control of the larvae. The Vip3Aa + Cry1Ab mixture had the highest toxicity, sequentially followed by Vip3Aa + Cry2Ab, Cry1Ab + Cry2Ab + Vip3Aa, Cry1Ea + Cry1Ca, Cry1Ab + Cry2Ab, Vip3Ca + Cry1Ea, and Vip3Ca + Cry1Ca. Cry1Ab, Cry1Ac, Cry2Ab, and Vip3Aa bound to more than one site on the brush border membrane vesicles (BBMV) of S. frugiperda. The Cry1Ab and Cry1Ac proteins share binding site, while Cry1Ab does not share binding site with the Cry2Aa and Cry2Ab proteins. The Vip3Aa protein does not share receptors with the tested Cry1 and Cry2. The results suggest that combination these tested proteins may increase toxicity against S. frugiperda neonates.

Toxicity of Cry2 proteins from Bacillus thuringiensis subsp. thuringiensis strain T01-328 against Aedes aegypti (Diptera: Culicidae) / Toxicidade das proteínas Cry2 do Bacillus thuringiensis subsp. thuringiensis T01-328 contra Aedes aegypti (Diptera: Culicidae)

Bacillus thuringiensis subsp. israelensis has been used to control the Aedes aegypti (Diptera: Culicidae) mosquito larvae, the vector of virus diseases such as dengue, Chikungunya and Zika fever, which have become a major public health problem in Brazil and other tropical countries since the climate favors the proliferation and development of the transmitting vector. Because B. thuringiensis has shown potential for controlling insects of the Diptera order, this work aimed at testing the Bacillus thuringiensis subsp. thuringiensis strain T01-328 and its proteins Cry2Aa and Cry2Ab for control A. aegypti and at comparing the results to the B. thuringiensis subsp. israelensis specific dipteran strain. To this end, bioassays using spore-crystal of both strains, and Cry2Aa and Cry2Ab proteins from the heterologous expression in Escherichia coli, were performed against A. aegypti larvae. The results showed that the B. thuringiensis thuringiensis ­T01-328 has insecticidal activity against the larvae, but it is less toxic than B. thuringiensis subsp. israelensis. Cry2Aa and Cry2Ab proteins expressed heterologously were effective for controlling A. aegypti larvae. Therefore, the results indicate that the Cry2Aa and Cry2Ab proteins of the B. thuringiensis thuringiensis T01-328 can be used as an alternative to assist in the control of A. aegypti.(AU)

Bacillus thuringiensis subsp. israelensis vem sendo empregada no controle do díptero Aedes aegypti, vetor do vírus causador de doenças como dengue, febre Chikungunya e Zika, que se tornou um dos grandes problemas de saúde pública no Brasil e em outros países de clima tropical, que favorece a proliferação e o desenvolvimento do transmissor. Em virtude do potencial de B. thuringiensis no controle de dípteros, a proposta deste trabalho foi testar as proteínas Cry2Aa e Cry2Ab da linhagem de Bacillus thuringiensis subsp. thuringiensis T01-328 no controle de A. aegypti, em comparação à linhagem díptero específica B. thuringiensis subsp. israelensis. Para tanto, foram realizados bioensaios com larvas de A. aegypti com o esporo-cristal de ambas as linhagens, bem como com as proteínas Cry2Aa e Cry2Ab com expressão heteróloga em Escherichia coli. A linhagem B. thuringiensis thuringiensis T01-328 apresentou atividade inseticida contra as larvas, porém foi menos tóxica que a B. thuringiensis subsp. israelensis. As proteínas Cry2Aa e Cry2Ab expressas de forma heteróloga foram eficazes no controle de A. aegypti. Os resultados obtidos sugerem as proteínas Cry2Aa e Cry2Ab da linhagem B. thuringiensis thuringiensis T01-328 como alternativas para contribuir no controle do A. aegypti.(AU)

Cry1Ac and Vip3Aa proteins from Bacillus thuringiensis targeting Cry toxin resistance in Diatraea flavipennella and Elasmopalpus lignosellus from sugarcane.

The biological potential of Vip and Cry proteins from Bacillus is well known and widely established. Thus, it is important to look for new genes showing different modes of action, selecting those with differentiated entomotoxic activity against Diatraea flavipennella and Elasmopalpus lignosellus, which are secondary pests of sugarcane. Therefore, Cry1 and Vip3 proteins were expressed in Escherichia coli, and their toxicities were evaluated based on bioassays using neonate larvae. Of those, the most toxic were Cry1Ac and Vip3Aa considering the LC50 values. Toxins from E. coli were purified, solubilized, trypsinized, and biotinylated. Brush Border Membrane Vesicles (BBMVs) were prepared from intestines of the two species to perform homologous and heterologous competition assays. The binding assays demonstrated interactions between Cry1Aa, Cry1Ac, and Vip3Aa toxins and proteins from the BBMV of D. flavipennella and E. lignosellus. Homologous competition assays demonstrated that binding to one of the BBMV proteins was specific for each toxin. Heterologous competition assays indicated that Vip3Aa was unable to compete for Cry1Ac toxin binding. Our results suggest that Cry1Ac and Vip3Aa may have potential in future production of transgenic sugarcane for control of D. flavipennella and E. lignosellus, but more research is needed on the potential antagonism or synergism of the toxins in these pests.

Synergism and antagonism between Bacillus thuringiensis Vip3A and Cry1 proteins in Heliothis virescens, Diatraea saccharalis and Spodoptera frugiperda.

Second generation Bt crops (insect resistant crops carrying Bacillus thuringiensis genes) combine more than one gene that codes for insecticidal proteins in the same plant to provide better control of agricultural pests. Some of the new combinations involve co-expression of cry and vip genes. Because Cry and Vip proteins have different midgut targets and possibly different mechanisms of toxicity, it is important to evaluate possible synergistic or antagonistic interactions between these two classes of toxins. Three members of the Cry1 class of proteins and three from the Vip3A class were tested against Heliothis virescens for possible interactions. At the level of LC50, Cry1Ac was the most active protein, whereas the rest of proteins tested were similarly active. However, at the level of LC90, Cry1Aa and Cry1Ca were the least active proteins, and Cry1Ac and Vip3A proteins were not significantly different. Under the experimental conditions used in this study, we found an antagonistic effect of Cry1Ca with the three Vip3A proteins. The interaction between Cry1Ca and Vip3Aa was also tested on two other species of Lepidoptera. Whereas antagonism was observed in Spodoptera frugiperda, synergism was found in Diatraea saccharalis. In all cases, the interaction between Vip3A and Cry1 proteins was more evident at the LC90 level than at the LC50 level. The fact that the same combination of proteins may result in a synergistic or an antagonistic interaction may be an indication that there are different types of interactions within the host, depending on the insect species tested.