Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74∆sp chitinase inclusions, Cry crystals and spores for applied use.

BACKGROUND: The endochitinase ChiA74 is a soluble secreted enzyme produced by Bacillus thuringiensis that synergizes the entomotoxigenecity of Cry proteins that accumulate as intracellular crystalline inclusion during sporulation. The purpose of this study was to produce alkaline-soluble ChiA74∆sp inclusions in B. thuringiensis, and to determine its effect on Cry crystal production, sporulation and toxicity to an important agronomical insect, Manduca sexta. To this end we deleted the secretion signal peptide-coding sequence of chiA74 (i.e. chiA74∆sp) and expressed it under its native promoter (pEHchiA74∆sp) or strong chimeric sporulation-dependent cytA-p/STAB-SD promoter (pEBchiA74∆sp) in Escherichia coli, acrystalliferous B. thuringiensis (4Q7) and B. thuringiensis HD1. RESULTS: Based on mRNA analyses, up to ~9-fold increase in expression of chiA74∆sp was observed using the cytA-p/STAB-SD promoter. ChiA74∆sp (~70 kDa) formed intracellular inclusions that frequently accumulated at the poles of cells. ChiA74∆sp inclusions were dissolved in alkali and reducing conditions, similar to Cry crystals, and retained its activity in a wide range of pH (5 to 9), but showed a drastic reduction (~70%) at pH 10. Chitinase activity of E. coli-pEHchiA74∆sp was ~150 mU/mL, and in E. coli-pEBchiA74∆sp, 250 mU/mL. 4Q7-pEBchiA74∆sp and 4Q7-pEHchiA74∆sp had activities of ~127 mU/mL and ~41 mU/mL, respectively. The endochitinase activity in HD1-pEBchiA74∆sp increased 42x when compared to parental HD1 strain. HD1-pEBchiA74∆sp and HD1 harbored typical bipyramidal Cry inclusions, but crystals in the recombinant were ~30% smaller. Additionally, a 3x increase in the number of viable spores was observed in cultures of the recombinant strain when compared to HD1. Bioassays against first instar larvae of M. sexta with spore-crystals of HD1 or spore-crystal-ChiA74∆sp inclusions of HD1-pEBchiA74∆sp showed LC50s of 67.30 ng/cm² and 41.45 ng/cm², respectively. CONCLUSIONS: Alkali-labile ChiA74∆sp inclusion bodies can be synthesized in E. coli and B. thuringiensis strains. We demonstrated for the first time the applied utility of synthesis of ChiA74∆sp inclusions, Cry crystals and spores in the same sporangium of HD1, a strain used successfully worldwide to control economically significant lepidopteran pests of agriculture. Our findings will allow to us develop strategies to modify expression of ChiA74∆sp while maximizing Cry crystal synthesis in commercial strains of B. thuringiensis.

Behavioral report of Chrysomya rufifacies in response to substrate infestation by Lucilia sericata and Lucilia cuprina using a tetrahedron olfactometer device.

Calliphoridae are widespread globally and can inhabit a variety of habitats. In this brief report, we assessed the appeal of Chrysomya rufifacies to resources that were previously inhabited by Lucilia sericata and L. cuprina, both common carrion colonizers. Two hundred adult male and female (1:1) C. rufifacies were released under controlled conditions into clear plastic cages (45 x 45 x 45 cm) with four types of substrates: beef-liver; liver with 100 third-instar larvae of L. cuprina; (III) liver containing 100 third-instar larvae of L. sericata; and liver containing 100 third-instar larvae of C. rufifacies. Each substrate was left in place for 24 hours at the end of a tube connected to the cage, where sticky traps were positioned to capture flies that might have been attracted to a specific substrate. The results indicate variations in the attraction of flies to different types of livers colonized by larvae of various species. It is suggested that flies may have specific preferences depending on the species of larvae present in the substrate. The liver without larvae was the preferred choice, while beef liver with C. rufifacies larvae was the least attractive. Results of statistical tests indicated that there is independence between attractiveness preference and the presence of C. rufifacies flies. Although there is a trend among certain levels of the variables in the correspondence analysis, these relationships are not statistically significant. However, they indicate specific patterns of association between different groups of flies and species of larvae. This study demonstrated that C. rufifacies does not show reduced attraction to any of the resources. A tetrahedron olfactometer device has been used for the first time in a behavioral study of C. rufifacies flies. This may enable future studies to enhance the understanding of fly behavior.