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.

New traps for the capture of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) (Diptera: Culicidae) eggs and adults.

The control of arboviruses carried by Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) can be performed with tools that monitor and reduce the circulation of these vectors. Therefore, the efficiency of four types of traps in capturing A. aegypti and A. albopictus eggs and adults, with the biological product Vectobac WG, was evaluated in the field. For this, 20 traps were installed in two locations, which were in the South (Londrina, Paraná) and North (Manaus, Amazonas) Regions of Brazil, from March to April 2017 and January to February 2018, respectively. The UELtrap-E (standard trap) and UELtrap-EA traps captured A. aegypti and A. albopictus eggs: 1703/1866 eggs in Londrina, and 10268/2149 eggs in Manaus, respectively, and presented high ovitraps positivity index (OPI) values (averages: 100%/100% in Londrina, and 100%/96% in Manaus, respectively); and high egg density index (EDI) values (averages: 68/75 in Londrina, and 411/89 in Manaus, respectively), so they had statistically superior efficiency to that of the CRtrap-E and CRtrap-EA traps in both regions, that captured less eggs and adults: 96/69 eggs in Londrina, and 1091/510 eggs in Manaus, respectively. Also presented lower OPI values (averages: 28%/4% in Londrina, and 88%/60% in Manaus, respectively); and lower EDI values (averages: 10.5/9 in Londrina, and 47/30 in Manaus, respectively). The capture ratios of Aedes adults in the UELtrap-EA and CRtrap-EA traps in Londrina and Manaus were 53.3%/29.5% and 0%/9.8%, respectively. UELtrap-EA can be adopted as efficient tool for Aedes monitoring due to their high sensitivity, low cost and ease of use.

Oviposition of Aedes aegypti Linnaeus, 1762 and Aedes albopictus Skuse, 1894 (Diptera: Culicidae) under laboratory and field conditions using ovitraps associated to different control agents, Manaus, Amazonas, Brazil

ABSTRACT The aim of this study was to analyze the effectiveness of different control agents of Aedes aegypti and Aedes albopictus associated with ovitraps under laboratory and field conditions. Five treatments were used: grass infusion + Bacillus thuringiensis israelensis (gI + Bti), grass infusion + Saccharopolyspora spinosa (gI + Ss), grass infusion + Pyriproxyfen (gI + P), distilled water + Toxorhynchites haemorrhoidalis (dW + Th), and grass infusion (gI) (control). The highest mean number of eggs of both species were obtained with grass infusion in the laboratory. Among control agents, the lowest mean of A. aegypti eggs occurred with gI + Ss and the lowest mean of A. albopictus eggs occurred with dW + Th. There was no difference between treatments in A. aegypti (P = 0.4320) and A. albopictus (P = 0.7179). In the field, the highest mean number of eggs for both species were obtained with gI + Ss, and the lowest values were obtained with gI + P (P = 0.0124). The treatments can be applied to both the surveillance and the control, but ovitraps with biological larvicide Bti were more effective and safer considering the number of eggs laid and selectivity of pathogens for mosquitoes.

Isolation and molecular characterization of Bacillus thuringiensis found in soils of the Cerrado region of Brazil, and their toxicity to Aedes aegypti larvae

ABSTRACT This study investigated the potential of Bacillus thuringiensis isolates obtained in the Cerrado region of the Brazilian state of Maranhão for the biological control of Aedes aegypti larvae. The isolates were obtained from soil samples and the identification of the B. thuringiensis colonies was based on morphological characteristics. Bioassays were run to assess the pathogenicity and toxicity of the different strains of the B. thuringiensis against third-instar larvae of A. aegypti. Protein profiles were obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polymerase chain reaction assays were used to detect the toxin genes found in the bacterial isolates. Overall, 12 (4.0%) of the 300 isolates obtained from 45 soil samples were found to present larvicidal activity, with the BtMA-104, BtMA-401 and BtMA-560 isolates causing 100% of mortality. The BtMA-401 isolate was the most virulent, with the lowest median lethal concentration (LC50) (0.004 × 107 spores/mL), followed by the Bacillus thuringiensis var. israelensis standard (0.32 × 107 spores/mL). The protein profiles of BtMA-25 and BtMA-401 isolates indicated the presence of molecular mass consistent with the presence of the proteins Cry4Aa, Cry11Aa and Cyt1, similar to the profile of Bacillus thuringiensis var. israelensis IPS-82. Surprisingly, however, none of the cry and cyt genes analyzed were amplified in the isolate BtMA-401. The results of the present study revealed the larvicidal potential of B. thuringiensis isolates found in the soils of the Cerrado region from Maranhão, although further research will be necessary to better elucidate and describe other genes associated with the production of insecticidal toxins in these isolates.

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.

Isolation of Bacillus thuringiensis from the state of Amazonas, in Brazil, and screening against Aedes aegypti (Diptera, Culicidae)

We investigated the use of Bacillus thuringiensis isolated in the state of Amazonas, in Brazil, for the biological control of the dengue vector Aedes aegypti. From 25 soil samples collected in nine municipalities, 484 bacterial colonies were obtained, 57 (11.78%) of which were identified as B. thuringiensis. Six isolates, IBt-03, IBt-06, IBt-07, IBt-28, IBt-30, and BtAM-27 showed insecticidal activity, and only BtAM-27 presents the five genes investigated cry4Aa, cry4Ba, cry10Aa, cry11Aa, and cry11Ba. The IBt-07 and IBt- 28, with lower LC50 values, showed equal toxicity compared to the standards. The isolates of B. thuringiensis from Amazonas constitute potential new means of biological control for A. aegypti, because of their larvicidal activity and the possibility that they may also contain new combinations of toxins.

Influence of abiotic factors on the effectiveness of Bacillus thuringiensis israelensis (Berliner, 1911) against larvae of Aedes aegypti (Linnaeus, 1762) / Influencia de los factores abióticos en la efectividad de la Bacillus thuringiensis israelensis (Berliner, 1911) contra larvas de Aedes aegypti (Linnaeus, 1762)

Introduction: Bacillus thuringiensis , has been used worldwide for the control of mosquitoes, particularly in combating Aedes aegypti. Various formulations have been made however, the effectiveness of these insecticides vary varies considerably according to the environmental conditions. Objective: to evaluate the influence of abiotic factors on the effectiveness of B. thuringiensis var. israelensis to against larvae of Ae. aegypti. Methods: we conducted bioassay experiments in the campus of the CESC / UEMA. With use of 20 Twenty cement tanks, of these, were used, 10 of which were exposed to environmental factors, and only. Of these, 5 were treated and 5 served as controls. The other 10 tanks were placed in the shade, also again 5 were treated and 5 untreated were not (control). After treatment, the 25 stage L 3 larvae were placed in 25 L 3 each stage tank and mortality. Mortality readings were taken after 24 hours and every 7 days. Weekly water samples were collected from all tanks to measure once a week to check the number of viable spores and evaluate bacterial recycling. Results and discussion: the larvicidal efficacy: The larvicide showed initial efficacy, killing 100 percent of the larvae in tanks that were located both in the sun and in the shade. Environmental factors, sunlight, temperature and rainfall negatively affected the effectiveness of the product, because for there was a reduction in the effectiveness of the product throughout the experiment. It was observed variation in the number of spores during the development of the experiment demonstrating, showing that the bacterium has potential recyclability. Conclusion: these data serve as parameters for improving to in Bti-based insecticides, in order to make the with a view to making formulations more suited toin keeping with the environmental conditions of our region(AU)

Introducción: la Bacillus thuringiensis se ha utilizado en muchas regiones del mundo para el control de los mosquitos, sobre todo en el combate contra el Aedes aegypti. Se han elaborado varias formulaciones; sin embargo, la efectividad de esos insecticidas varía considerablemente según las condiciones ambientales. Objetivo: evaluar la influencia de los factores abióticos en la efectividad de la B. thuringiensis var. israelensis contra larvas de Ae. aegypti. Métodos: se realizaron bioensayos experimentales en el campus de la CESC / UEMA. Se utilizaron 20 tanques de cemento, 10 de los cuales quedaron expuestos a factores ambientales. De ellos, 5 fueron tratados y 5 sirvieron como controles. Los otros 10 tanques fueron ubicados en la sombra, y también en este caso 5 fueron tratados y 5 no (controles). Después del tratamiento, se colocaron 25 larvas en estadio larval L3 en cada tanque. Se realizaron lecturas de mortalidad luego de 24 horas y cada 7 días. Se tomaron muestras de agua de todos los tanques una vez a la semana para comprobar el número de esporas viables y evaluar el reciclaje bacteriano. Resultados y discusión: el larvicida mostró eficacia inicial, matando el 100 por ciento de las larvas de tanques ubicados tanto al sol como en la sombra. Los factores ambientales, la luz solar, la temperatura y la lluvia afectaron negativamente la efectividad del producto, constatándose una reducción de la misma a lo largo del experimento. Se observaron variaciones en el número de esporas durante el experimento, lo que demuestra que la bacteria presenta potencial de reciclaje. Conclusión: los datos obtenidos sirven de parámetros para el mejoramiento de los insecticidas a base de Bti, con vistas a elaborar formulaciones más acordes con las condiciones ambientales de nuestra región(AU)

Variation in Aedes aegypti (Linnaeus) (Diptera, Culicidae) infestation in artificial containers in Caxias, state of Maranhão, Brazil.

INTRODUCTION: Dengue is a serious public health problem worldwide, with cases reported annually in tropical and subtropical regions. Aedes aegypti (Linnaeus, 1762), the main vector of dengue, is a domiciliary species with high dispersal and survival capacities and can use various artificial containers as breeding sites. We assessed potential container breeding sites of A. aegypti in the municipality of Caxias, Maranhão, Brazil. METHODS: In the initial phase, we analyzed 900 properties in 3 neighborhoods during the dry and rainy seasons (August-October 2005 and February-April 2006, respectively). During the second sampling period, September 2006-August 2007, we used 5 assessment cycles for 300 properties in a single neighborhood. RESULTS: During the dry and rainy seasons, water-storage containers comprised 55.7% (n = 1,970) and 48.5% (n = 1,836) of the total containers inspected, and showed the highest productivity of immature A. aegypti; we found 23.7 and 106.1 individuals/container, respectively, in peridomicile sites. In intradomicile sites, water-storage containers were also the most important breeding sites with 86.4% (n = 973) and 85.6% (n = 900) of all containers and a mean of 7.9 and 108.3 individuals/container in the dry and rainy seaso-October 2006 (1,342). The highest number of positives (70) was recorded in May, mostly (94%) in storage containers. CONCLUSIONS: Storage containers are the principal and most productive A. aegypti breeding sites and are a major contributing factor to the maintenance of this vector in Caxias.

Variation in Aedes aegypti (Linnaeus) (Diptera, Culicidae) infestation in artificial containers in Caxias, state of Maranhão, Brazil / Variação da infestação de Aedes aegypti (Linnaeus), (Diptera, Culicidae) em recipientes artificiais em Caxias, estado do Maranhão, Brasil

INTRODUCTION: Dengue is a serious public health problem worldwide, with cases reported annually in tropical and subtropical regions. Aedes aegypti (Linnaeus, 1762), the main vector of dengue, is a domiciliary species with high dispersal and survival capacities and can use various artificial containers as breeding sites. We assessed potential container breeding sites of A. aegypti in the municipality of Caxias, Maranhão, Brazil. METHODS: In the initial phase, we analyzed 900 properties in 3 neighborhoods during the dry and rainy seasons (August-October 2005 and February-April 2006, respectively). During the second sampling period, September 2006-August 2007, we used 5 assessment cycles for 300 properties in a single neighborhood. RESULTS: During the dry and rainy seasons, water-storage containers comprised 55.7% (n = 1,970) and 48.5% (n = 1,836) of the total containers inspected, and showed the highest productivity of immature A. aegypti; we found 23.7 and 106.1 individuals/container, respectively, in peridomicile sites. In intradomicile sites, water-storage containers were also the most important breeding sites with 86.4% (n = 973) and 85.6% (n = 900) of all containers and a mean of 7.9 and 108.3 individuals/container in the dry and rainy seaso-October 2006 (1,342). The highest number of positives (70) was recorded in May, mostly (94%) in storage containers. CONCLUSIONS: Storage containers are the principal and most productive A. aegypti breeding sites and are a major contributing factor to the maintenance of this vector in Caxias.

INTRODUÇÃO: A dengue constitui de um sério problema de saúde pública mundial, com casos sendo registrados anualmente nas regiões tropicais e subtropicais. O Aedes aegypti(Linnaeus, 1762) é o principal vetor da doença, um mosquito domiciliado de fácil dispersão e sobrevivência, com capacidade de utilizar variados recipientes artificiais como criadouro. Em vista disso, neste trabalho investigou-se os tipos de recipientes potenciais criadouros de A. aegypti na Cidade de Caxias, Maranhão, Brasil. MÉTODOS: Inicialmente, a pesquisa foi desenvolvida em três bairros e 900 imóveis analisados no período da seca e das chuvas (agosto a outubro de 2005 e fevereiro a abril de 2006, respectivamente). Um segundo período de amostragem ocorreu de setembro de 2006 a agosto de 2007 em que inspeções de cinco ciclos foram desenvolvidas em um dos bairros com 300 imóveis amostrados. RESULTADOS: Os recipientes de armazenamento de água representaram 55,7% (n=1.970) e 48,5% (n=1.836) do total dos inspecionados, e maior produtividade de imaturos de A. aegypti, com proporção de 23,7 e 106,1 por recipiente no peridomicílio, nas estações seca e chuvosa respectivamente. No intradomicílio, também foi mais representativo, 86,4% (n=973) e 84,6% (n=900), apresentando média de 7,9 e 108,3 indivíduos, nas duas estações. O maior número de recipientes com água foi registrado em setembro e outubro 2006 (1.342), obtendo-se maior número de positivos (70) no mês de maio, sendo (94%) pertencentes ao grupo armazenamento. CONCLUSÕES: Armazenamento são os principais criadouros de A. aegypti, contribuindo na manutenção do vetor na cidade de Caxias, Maranhão.