Novel application of bioassays and functional importance of minor crystal components of Bacillus thuringiensis serovar israelensis for dipteran pests
- Autores: Daniel Valtierra de Luis
- Directores de la Tesis: Primitivo Caballero (dir. tes.), Maite Villanueva San Martín (codir. tes.)
- Lectura: En la Universidad Pública de Navarra ( España ) en 2020
- Idioma: inglés
- Tribunal Calificador de la Tesis: Colin Berry (presid.), María del Pilar Medina Vélez (secret.), Jesús Murillo Martínez (voc.)
- Programa de doctorado: Programa de Doctorado en Biotecnología por la Universidad Pública de Navarra
- Materias:
- Enlaces
- Tesis en acceso abierto en: Academica-e
- Resumen
Bacillus thuringiensis (Berliner, 1915) (Bt) is a Gram-positive rod-shaped bacterium with the capacity to form resistance spores. Under unfavourable conditions, the bacterium sporulates producing the spore and the parasporal body. The latter is primarily composed by one or more insecticidal proteins, also called δ-endotoxins, which are able to form crystalline inclusions. Bt-based insecticides represent around 80% of all biopesticides in the market. The specific toxicity of crystal proteins against target insects is the basis for the use of Bt as a biopesticide in agriculture, forestry and mosquito control since 1961. Currently, the major alternative for mosquito and blackfly larval control is based on bacterial toxins produced by B. thuringiensis ser. israelensis (Bti).
Simple and repeatable bioassay methods are required to evaluate the toxicity of novel insecticidal compounds for pest management and to determine the presence of resistance traits in pest populations. We used a radioactive tracer based on 32P-ATP to estimate the volume ingested by two dipteran pests: Ceratitis capitata (Tephritidae) and Drosophila suzukii (Drosophilidae). Using blue food dye it was possible to distinguish between individuals that ingested the solution from those that did not. The average volume ingested by C. capitata adults was 1.968 µl. Females ingested a ~20% greater volume of solution than males. Adults of D. suzukii ingested an average of 0.879 and females ingested ~30% greater than males. The droplet feeding method was validated using the naturally-derived insecticide spinosad as the active ingredient (a.i.). For C. capitata, the concentration-mortality response did not differ between the sexes or among three different batches of insects. Lethal dose values were calculated based on mean ingested volumes. For C. capitata LD50 values were 1.462 and 1.502 ng a.i./insect for males and females, respectively, equivalent to 0.274 and 0.271 ng a.i./mg for males and females respectively, when sex-specific variation in body weight was considered. We use the same process for determining the D. suzukii values. This technique could be readily employed for determination of the resistance status and dose-mortality responses of insecticidal compounds in many species of dipteran pests.
B. thuringiensis ser. israelensis forms semi-spherical parasporal inclusion bodies composed of insecticidal proteins, mainly represented by five families (Cry4, Cry10, Cry11, Cyt1 and Cyt2), which are widely used as the basis for microbial larvicides against several dipteran pests. The insecticidal activity of the crystal is high in comparison to the activities of the individual toxins, which is likely due to synergistic interactions among the crystal proteins, particularly those involving Cyt1Aa. In the present study Cry10Aa and Cyt2Ba were cloned from the commercial larvicide VectoBac® 12AS and expressed in the acrystalliferous Bt strain BMB171. These proteins had LC50 values of 299.62 and 279.37 ng/ml, respectively, against A. aegypti second instars but did not show toxic activity against C .capitata adults. When the A. aegypti larvae ingested an equitable mixture (ratio 1:1) of spores and crystals of the Cry10A and Cyt2B recombinant proteins, a strong synergistic activity was observed. It was estimated that the magnitude of this synergistic effect was 34.3, which is among the highest values described so far for the components of the Bti crystal and comparable to those previously reported for Cyt1A with Cry4 and Cry11A. Therefore, Bti minor components have relevance in the overall toxicity of the strain and both proteins can play an important role in synergy and resistance avoidance.
