Microencapsulation of Bacillus thuringiensis strains for the control of Aedes aegypti.

In this study, we investigated the microencapsulation of two strains of the entomopathogenic bacteria Bacillus thuringiensis (B. thuringiensis) (BtMA-750 and BtMA-1114), which are biopesticides of high toxicity for the mosquito vector Aedes aegypti. The encapsulation of different concentrations of microorganisms in starch microparticles was evaluated, and the inverse suspension polymerization technique was explored. It was possible to observe that the higher amounts of the biopesticide caused a slight decrease in the diameter of the particles; however, even when encapsulated, the biopesticide still presents an average diameter that is able to be consumed by the larvae of Aedes aegypti. Furthermore, it was noticed that the presence of both of the B. thuringiensis strains did not affect the thermal stability of the particles. The microencapsulated bacterial strains presented a high number of viable spores and preserved the expression of proteins with molecular masses corresponding to the insecticidal toxins Cry and Cyt, indicating that the encapsulation process was conducted satisfactorily. Finally, the encapsulated strains were tested against Ae. aegypti larvae and maintained 100% larval mortality even after 35 days. Therefore, microencapsulation of B. thuringiensis not only guarantees the bacterial activity, but also prolongs the action of the biopesticide. Collectively, such findings highlight the great potential of the new biopesticides, which may help to reduce the population indices of the mosquito vector Ae. aegypti via a sustainable and environment-friendly route.

Cyt1Aa toxin gene frequency in Bacillus thuringiensis isolates and its relation with pathogenicity for vector mosquitoes.

Bacillus thuringiensis produces several virulence factors, the main ones being the Cry and Cyt toxins, present in the parasporal body produced during sporulation. The Cyt toxins have mechanisms specific for mosquitoes and Cyt1Aa, the most studied cytolytic toxin, is effective for mosquito control by acting in synergism with Cry toxins. The goal of the present work was to study the frequency of the codifying gene for Cyt1Aa in B. thuringiensis native isolates acquired from samples of soil, insect and water, as well as to verify any possible genetic polymorphism. 1,448 B thuringiensis strains were used for DNA extraction and PCR technique, all with the use of a primer that amplifies a fragment of 300 pairs of the cyt1Aa gene. The strains that showed amplification in the PCR reaction were sequenced and compared to each other and to the sequences available at Genbank. 32 (2.3%) strains of B. thuringiensis showed positive amplification for the cyt1Aa gene. The highest frequency of isolates with cyt1Aa gene was acquired from samples coming from the Cerrado biome, both isolates from soil and from insects, equally with 3.4%. The cyt1Aa gene sequencing highlighted that, for that 300 bp region, the gene is conserved and there is no single-base polymorphism.

Molecular characterization of the gene profile of Bacillus thuringiensis Berliner isolated from Brazilian ecosystems and showing pathogenic activity against mosquito larvae of medical importance.

The occurrence of Aedes aegypti, Culex quinquefasciatus, and mosquitoes of the genus Anopheles potentiate the spread of several diseases, such as dengue, Zika, chikungunya, urban yellow fever, filariasis, and malaria, a situation currently existing in Brazil and in Latin America. Control of the disease vectors is the most effective tool for containing the transmission of the pathogens causing these diseases, and the bacterium Bacillus thuringiensis var. israelensis has been widely used and has shown efficacy over many years. However, new B. thuringiensis (Bt) strains with different gene combinations should be sought for use as an alternative to Bti and to prevent the resistant insects selected. Aiming to identify diversity in the Bt in different Brazilian ecosystems and to assess the pathogenicity of this bacterium to larvae of Ae. aegypti, C. quinquefasciatus, and Anopheles darlingi, Bt strains were obtained from the Amazon, Caatinga (semi-arid region), and Cerrado (Brazilian savanna) biomes and tested in pathogenicity bioassays in third-instar larvae of Ae. aegypti under controlled conditions in the laboratory. The isolates with larvicidal activity to larvae of Ae. aegypti were used in bioassays with the larvae of C. quinquefasciatus and An. darlingi and characterized according to the presence of 14 cry genes (cry1, cry2, cry4, cry10, cry11, cry24, cry32, cry44Aa, cry1Ab, cry4Aa, cry4Ba, cry10Aa, cry11Aa, and cry11Ba), six cyt genes (cyt1, cyt2, cyt1Aa, cyt1Ab, cyt2Aa and cyt2Ba), and the chi gene. Four hundred strains of Bt were isolated: 244 from insects, 85 from Amazon soil, and 71 from the Caatinga biome. These strains, in addition to the 153 strains isolated from Cerrado soil and obtained from the Entomopathogenic Bacillus Bank of Maranhão, were tested in bioassays with Ae. aegypti larvae. A total of 37 (6.7%) strains showed larvicidal activity, with positive amplification of the cry, cyt, and chi genes. The most frequently amplified genes were cry4Aa and cry4Ba, both occurring in 59.4% in these strains, followed by cyt1Aa and cyt2Aa, with 56.7% and 48% occurrence, respectively. Twelve (2.2%) strains that presented 100% mortality within 24h were used in bioassays to estimate the median lethal concentration (LC50) for Ae. aegypti larvae. Two strains (BtMA-690 and BtMA-1114) showed toxicity equal to that of the Bti standard strain, and the same LC50 value (0.003mg/L) was recorded for the three bacteria after 48h of exposure. Detection of the presence of the Bt strains that showed pathogenicity for mosquito larvae in the three biomes studied was possible. Therefore, these strains are promising for the control of insect vectors, particularly the BtMA-1114 strain, which presents a gene profile different from that of Bti but with the same toxic effect.