Genetic diversity of Brazilian Bacillus thuringiensis isolates with toxicity against Aedes aegypti (Diptera: Culicidae).

Bacillus thuringiensis (Bt) isolates native to Maranhão (BtMA) that are highly toxic to Aedes aegypti larvae and seven standard subspecies of Bt were analyzed for genetic diversity using the rep-PRC technique with BOX, ERIC, REP, MB1, and GTG5 markers. The rep-PCR technique is considered an extremely reliable, reproducible, fast and highly discriminatory technique that may be used even among populations of the same species. These five markers revealed a total of 38 polymorphic DNA fragments for 30 BtMA isolates. Eight groups were obtained with the dendrogram generated through Pearson's correlation analysis, with four groups formed only with BtMA isolates and four comprised of isolates of BtMA and the standard subspecies toxic to dipterans and lepidopterans. Despite the high genetic diversity of BtMA, a low correlation between the collection site, gene content and mortality against A. aegypti larvae was evidenced. The clustering of the standard subspecies of Bt that were toxic against dipterans with BtMA isolates confirm the mosquitocidal action of the native isolates from Maranhão, and they can be used as an alternative for A. aegypti control and other insects of medical importance and for the control of agricultural pests.

Inheritance and Fitness of Plutella xylostella (Lepidoptera: Plutellidae) Resistance to Chlorfenapyr.

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a key pest of Brassicaceae worldwide. Populations have globally evolved resistance to various insecticides including chlorfenapyr, which was observed at high frequency in Brazil. We report the genetic characterization and fitness costs associated with chlorfenapyr resistance in a field-derived strain. The resistant strain (BZR-RR) and a susceptible strain (REC-SS) were used in both concentration-response bioassays and demography-based approach. Inheritance pattern of chlorfenapyr resistance was determined by conducting reciprocal crosses between susceptible and resistant strains, and by backcrossing. Next, life table analysis for the susceptible, heterozygotes, and resistant strains was performed to assess eventual fitness costs associated with chlorfenapyr resistance. Resistance of P. xylostella (BZR-RR) strain to chlorfenapyr was very high (RR50 = 421.58-fold) and also autosomal (no differences between reciprocal crosses), monofactorial and incompletely dominant (F1 pool DD = 0.26 ± 0.14). Dominance (h) was concentration dependent with 16 mg/l allowing at least 95% survival of the resistant heterozygotes. Recessive fitness cost was observed to be associated with resistance to chlorfenapyr. The relative fitness of heterozygotes (RS) and resistant homozygotes (BZR-RR) in comparison to the susceptible strain (REC-SS) was 0.91 and 0.23, respectively. Significant differences were found for many fitness components in the resistant homozygotes. Altogether, results suggest a rational use of chlorfenapyr in areas where susceptible populations still prevail, in parallel with the use of diagnostic concentrations (e. g., 20 mg chlorfenapyr/l), and rotation with different mode of actions, for which fitness costs of resistance are nonrecessive in P. xylostella.

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.

Lethal and sublethal effects of lufenuron on sugarcane borer Diatraea flavipennella and its parasitoid Cotesia flavipes.

The combination of chemical and biological controls is a historic goal of integrated pest management, but has rarely been achieved due to lethal and sublethal impact of insecticides on natural enemies altering their performance. In this context, the susceptibility of the yellow sugarcane borer, Diatraea flavipennella (Lepidoptera: Crambidae), to the insect growth regulator lufenuron and the consequent effects upon its endoparasitoid Cotesia flavipes (Hymenoptera: Braconidae) encountering exposed but surviving larvae were studied. Neonate and 10-day-old larvae were subjected to one of seven concentrations of lufenuron (1.56, 3.12, 6.25, 12.5, 25.0, 50.0 and 100 mg a.i./L). Further, effects of lufenuron to the host larvae and to the parasitoid were assessed using low lethal LC20 and LC50. Lufenuron at concentrations up to 12.5 mg a.i./L allowed partial survival of borer larvae; and concentrations over 12.5 mg a.i./L caused 100 % larval mortality before pupation in both ages. Neonate larvae exhibited lower pupal weights only at concentrations 12.5 mg a.i./L; while 10-day-old larvae treated with the LC50 exhibited delayed development. Egg viability was reduced for adult borers from surviving larvae of both ages treated with low lethal concentrations. The parasitoid C. flavipes successfully parasitized surviving low lethal treated larvae. Among the studied life history characteristics of C. flavipes, only a delayed development was observed. The results showed that lufenuron can be effective against D. flavipennella at concentrations over 25 mg a.i./L, and that surviving larvae can be successfully parasitized by C. flavipes. The insecticide lufenuron and the parasitoid C. flavipes seem to be compatible for sugarcane borer control.

Insecticidal activity against Bemisia tabaci biotype B of peel essential oil of Citrus sinensis var. pear and Citrus aurantium cultivated in northeast Brazil.

The fumigant action of peel essential oils of Citrus sinensis var. pear (pear orange = PO) and C. aurantium (bitter orange = BO) from the northeast of Brazil were evaluated against Bemisia tabaci biotype B and compared with eugenol as a positive control. The oil concentration in the PO at 8.5 microL/L of air caused 97% mortality, while the oil concentration of BO at 9.5 microL/L of air caused 99% mortality. However, the LC50 estimates for both oils (LC50 = 3.80 microL/L of air for PO and LC50 = 5.80 microL/L of air for BO) did not differ from each other, but they did when compared with eugenol (LC50 = 0.20 microL/L of air). Regarding their effects on oviposition, the Citrus oils showed concentration-response dependence, reducing the number of eggs as the concentration increased, which was not observed for eugenol. The minimum concentrations of the oils that caused a significant reduction in the egg lay were 3.5 and 7.0 microL/L of air for BO and PO, respectively. These results suggest that oils from PO and BO peels may be promising as models to develop new insecticides that might be applied into the integrated management of whiteflies.

Susceptibility of Anthonomus grandis (cotton boll weevil) and Spodoptera frugiperda (fall armyworm) to a cry1ia-type toxin from a Brazilian Bacillus thuringiensis strain.

Different isolates of the soil bacterium Bacillus thuringiensis produce multiple crystal (Cry) proteins toxic to a variety of insects, nematodes and protozoans. These insecticidal Cry toxins are known to be active against specific insect orders, being harmless to mammals, birds, amphibians, and reptiles. Due to these characteristics, genes encoding several Cry toxins have been engineered in order to be expressed by a variety of crop plants to control insectpests. The cotton boll weevil, Anthonomus grandis, and the fall armyworm, Spodoptera frugiperda, are the major economically devastating pests of cotton crop in Brazil, causing severe losses, mainly due to their endophytic habit, which results in damages to the cotton boll and floral bud structures. A cry1Ia-type gene, designated cry1Ia12, was isolated and cloned from the Bt S811 strain. Nucleotide sequencing of the cry1Ia12 gene revealed an open reading frame of 2160 bp, encoding a protein of 719 amino acid residues in length, with a predicted molecular mass of 81 kDa. The amino acid sequence of Cry1Ia12 is 99% identical to the known Cry1Ia proteins and differs from them only in one or two amino acid residues positioned along the three domains involved in the insecticidal activity of the toxin. The recombinant Cry1Ia12 protein, corresponding to the cry1Ia12 gene expressed in Escherichia coli cells, showed moderate toxicity towards first instar larvae of both cotton boll weevil and fall armyworm. The highest concentration of the recombinant Cry1Ia12 tested to achieve the maximum toxicities against cotton boll weevil larvae and fall armyworm larvae were 230 microg/mL and 5 microg/mL, respectively. The herein demonstrated insecticidal activity of the recombinant Cry1Ia12 toxin against cotton boll weevil and fall armyworm larvae opens promising perspectives for the genetic engineering of cotton crop resistant to both these devastating pests in Brazil.