Sub-lethal dose of Bacillus sphaericus suppresses vector competence of Anopheles dirus for Plasmodium yoelii by up-regulating the Imd signaling pathway

Objective: To investigate the effect of sublethal dose of Bacillus sphaericus (Bs) 2 362 strain on the malaria transmission ability of Anopheles (An.) dirus (Hainan strain) and its molecular mechanism. Methods: Firstly, the fourth instar larvae of An. dirus was treated with sublethal dose of Bacillus sphaericus. The surviving larvae were then collected and placed into mosquito cages, where they were hatched into adult mosquitoes. These mosquitoes were recorded as Bs group. Meanwhile, the control group was set without any treatment. Then, for infection with Plasmodium yoelii BY265 RFP, 3- to 5-day old female adults were kept at 24 °C and fed on Plasmodium yoelii BY265 RFP-infected Kunming mice with a gametocytemia above 0.5%. On day 9-11 post infection, mosquitoes were dissected, and the oocysts on the midguts were examined under a fluorescence microscope. Thirdly, total RNA was extracted from mosquitoes of Bs group and the control group at different time-points respectively, and the cDNA were synthesized later. Finally, SYBR quantitative PCR was conducted to investigate the expression of Imd pathway anti-malaria molecules at different time-points, including TEP1 and Rel2, in Bs and control group mosquitoes. Results: Bs treatment remarkably reduced the infection rate of Plasmodium from 23.71% (124/523) to 16.23% (87/536) (Chi-square test, P=0.002 0.05). Additionally, the intensities of melanized oocysts were compared between the two groups, and no significant difference was found, either (P=0.566>0.05). Interestingly, compared with the control group, the expression levels of TEP1 and Rel2 in Bs group were obviously up-regulated in larval, adult and infected mosquitoes. Especially in 3 dpi and 7 dpi, the expression level of TEP1 in Bs group was nearly 4 times higher than that of the control group, while Rel2 reached to approximately 7 times. Conclusions: We firstly found that the sublethal dose of Bs significantly suppressed the vector competence of An. dirus to malaria parasites, which revealed a new important role of Bs on the basis of killing mosquito larvae. Furthermore, the Imd signaling pathway might play an effective way in Bs impacting the vector competence of An. dirus through upregulating the expression of NF-kB transcription factor Rel2, enhancing the expression of TEP1, which killed the Plasmodium, but not through melanization.

Evaluation of entomopathogenic Bacillus sphaericus isolated from Lombok beach area against mosquito larvae

Objective: To isolate, characterize and evaluate toxicity of Bacillus sphaericus (B. sphaericus) from beach area of Lombok Island. Methods: Soil was collected from determined locations and suspended in sterile physiological saline water. After heat shock was applied, suspension was spread on NYSM agar medium. Colonies grown were then observed and isolated. Colony, cell morphology, and biochemical/physiological characteristics were tested and compared to B. sphaericus 2362 as standard. Initial toxicity testing was done against three species of mosquito larvae (Culex quinquefasciatus, Anopheles aconitus and Aedes aegypti) and isolates that showed more than 50% larvae killing will be assayed to obtain LC

Isolation of mosquitocidal bacteria (Bacillus thuringiensis, B.sphaericus and B.cereus) from excreta of arid birds.

Mosquitocidal bacteria are environmentally friendly alternatives to chemical insecticides for controlling mosquitoes and therefore, there have been tremendous world-wide efforts to identify novel mosquitocidal bacteria from natural environment. In the present study, excreta from arid-birds were analyzed for identifying mosquitocidal bacteria. The selection of sample for bacterial screening is significant, because, arid-birds are the unique living species and gathering the foods from variety of sources from environment. Out of 1000 samples examined, twelve bacterial strains were identified as mosquitocidal and the 16S rRNA gene sequence alignment depicted that these isolates belonged to Bacillus species (Bacillus thuringiensis, B.sphaericus and B.cereus). Toxicity assay against mosquito vectors have shown that these isolates are potential. The B. sphaericus VCRC-B547 (NCBI: JN377789) has shown a higher toxicity against Cx. quinquefasciatus, An. stephensi, and Aed. aegypti. Result from SDS-PAGE has shown that there was considerable difference in the protein profiles among the new bacterial isolates. Phylogenetic tree with branch length 0.05 revealed three distinct groups with homology among the closely related Bacillus strains. This study therefore throws considerable interest on the diversity of microbial organisms from arid birds and its application in mosquito control.

Evaluación preliminar de la eficiencia de Bacillus sphaericus en un área endémica a malaria del estado Bolívar, Venezuela / Preliminary evaluation of the effectiveness of Bacillus sphaericus in an malaria-endemic region in Bolívar state, Venezuela

El municipio Sifontes del estado Bolívar es la principal área endémica a malaria de Venezuela. Durante los cinco años anteriores al estudio se reportaron allí un promedio anual de 14 mil casos, alcanzándose en 2009 la cifra de 19.408, constituyendo una situación de epidemia. En la búsqueda de estrategias alternativas al uso de insecticidas, se evaluó la eficiencia para el control de malaria de una formulación comercial de Bacillus sphaericus Neide (VectoLex® CG 7,5%). Entre mayo 2010 y mayo de 2011 se aplicó biolarvicida con motomochila (Solo®) a dosis de 20-30 kg/ha a frecuencia mensual en todos los criaderos de Anopheles darlingi, An. marajoara y An. nuneztovari ubicados alrededor de las viviendas de cuatro comunidades de la parroquia San Isidro de este municipio, acompañadas de colectas de anofelinos adultos con atrayente humano y trampa de luz CDC y ultravioleta. Después de 48 semanas de aplicación, la reducción de 80% de la malaria en el área, más la reducción de la densidad de larvas en los criaderos, muestran que el biolarvicida es eficiente para controlar malaria. Los resultados también sugieren que la prevalencia de larvas es un buen indicador en un programa de control de vectores basado en la aplicación de biolarvicida, no así la de adultos, cuya abundancia no se corresponde con la de larvas. Es la primera vez que se evalúa la eficiencia de B. sphaericus para controlar malaria en Venezuela, considerándose estos resultados prometedores, si el tratamiento de criaderos con biolarvicida es selectivo y se hace a la cobertura, frecuencia y dosis correctas, e integrado a otras estrategias de control de vectores.

The Sifontes municipality in Bolivar state is the principal malaria-endemic region in Venezuela. During the five years prior to the study an annual average of 14,000 cases was reported reaching an epidemic level of 19,408 cases in 2009. As part of the search for vector control measures that could be used as alternatives to insecticides, a commercial formulation of Bacillus sphaericus Neide (VectoLex® CG 7.5%) was evaluated. Between May 2010 and May 2011 monthly dosages of 20-30 kg/ha of Vectolex were sprayed using a motorized backpack (Solo®) in larval Anopheles darlingi, An. marajoara and An. nuneztovari habitats close to houses in four communities in the parish of San Isidro, Sifontes municipality. In parallel, adult anophelines were captured using human baits, CDC light traps and ultraviolet light traps. After 48 weeks of application there was an 80% reduction in malaria as well as a decrease in larval density demonstrating the effectiveness of B. sphaericus for the control of this disease. Moreover, the results suggest that larval prevalence is good indicator for biolarvicide based malaria control programs whereas the abundance of adults, which does not correlate with larval abundance, is not. This is the first time that the effectiveness of B. sphaericus for malaria control has been evaluated in Venezuela. These results show that spraying larval habitats for malaria control can be effective when integrated with other vector control strategies if the treatment is selective and carried out at the correct frequency and dosage in order to ensure adequate coverage of the larval habitat.

Hexavalent chromium reduction by immobilized cells of Bacillus sphaericus AND 303

Bacillus sphaericus AND 303, a Cr(VI)-resistant and reducing bacterium reported from serpentine outcrops of Andaman was evaluated for Cr(VI) reduction using immobilized cells under batch culture. Screening of inert matrices for entrapment of whole cells indicated that polyvinyl alchohol-alginate was the most effective one reducing 87.5% of 20 µM Cr(VI) in 24 h. The rate of chromate reduction was dependent on initial Cr(VI) and biomass concentrations. The PVA cell beads were recycled three times without cell leakage and disintegration. The reduction efficiency was improved in the presence of glucose and glycerol as electron donors leading to complete reduction. However, the presence of additional metal ions was inhibitory to Cr(VI) reduction. It could be emphasized that PVA-alginate immobilized cells of B. sphaericus AND 303 could be used as a continuous bioprocess in treating Cr(VI) contaminated effluents.

Larval control of Anopheles (Nyssorhinchus) darlingi using granular formulation of Bacillus sphaericus in abandoned gold-miners excavation pools in the Brazilian Amazon Rainforest

INTRODUCTION: Use of a Bacillus sphaericus based mosquito larvicide was evaluated as an intervention for malaria vector control at a mining site in Amapá, Brazil. Impacts on larval and adult densities of the primary vector Anopheles darlingi were measured over the course of a 52 week study period. METHODS: In Calçoene, State of Amapá, gold mining activity occurs in 19 mining sites in gold-miners of Lourenço. Large pools are formed in mining sites and naturally colonized by Anopheles darlingi. During one year, the impact of applications of VectoLex(r) CG to these larval sources was evaluated. Applications of 20kg/ha were made as needed, based on 10 immature (3rd, 4th instars and pupae) surveillance of health and established thresholds. RESULTS: One hundred percent initial control was observed 48h after each treatment. The pools received from 2-10 (5.3±1.6) treatments during the year. The average re-treatment interval in productive pools was 9.4±4.3 weeks. During weeks 3-52 of the study, mean density of late stage larvae was 78% and pupae were 93% lower in the treated pools than in untreated pools (p< 0.0001, n=51) while reduction of adult mosquitoes was 53% in comparison to the untreated area during the last five months of the study, which were the rainy season (p<0.001). CONCLUSIONS: VectoLex(r) CG reduced immature Anopheles darlingi infestation levels during the entire study period, and reduced adult mosquito populations during the rainy season. .

Pruebas de campo sobre la eficacia y persistencia de tres formulaciones de Bacillus sphaericus contra larvas de Anopheles aquasalis Curry en manglares del municipio Mariño, estado Sucre, Venezuela / Field trials on the efficacy and persistence of three formulations of Bacillus sphaericus against larvae of Anopheles aquasalis Curry in mangroves of Mariño municipality, Sucre state, Venezuela

Anopheles aquasalis Curry es el principal vector de malaria de la región nororiental de Venezuela. Un estudio sobre el control biológico de la especie fue realizado en Rio Chiquito Abajo, municipio Mariño, estado Sucre. El objetivo del mismo fue evaluar en condiciones de campo, la eficacia y persistencia de tres formulaciones comerciales de Bacillus sphaericus contra larvas de An. aquasalis. Los criaderos típicos de esta especie son humedales de agua dulce con vegetación herbácea emergente y manglares salobres estacionales con vegetación arbórea (Avicennia germinans). El hábitat seleccionado para realizar los bioensayos fue un típico manglar de Avicennia germinans. En este criadero de An. aquasalis, se evaluaron estas formulaciones de B. sphaericus: dos formulaciones liquidas (GRISELESF-Cepa 2362 y VECTOLEX® WGD-51,2%) y una formulación granulada (VECTOLEX® CG-7,5%). En cada prueba, se aplicó un diseño completamente al azar con 4 repeticiones por tratamiento y por control. Los resultados fueron expresados como porcentajes de reducción larval, después de aplicar la formula de Mulla. De acuerdo con los resultados obtenidos, la formulación granulada VECTOLEX® CG-7,5% con dosis de 30 Kg/ha (3 g/m²), resultó ser altamente eficaz hasta 7 días post-aplicación con 92,3% de reducción, la misma fue menos efectiva después de 14 días post-aplicación con 74,8%. Al aumentar la dosis a 40 Kg/ha (4 g/m²), la eficacia después de 7 días de la aplicación fue de 93,9 %, es decir muy similar que a la dosis anterior. Ambas dosis presentaron la misma eficacia contra larvas de An. aquasalis. Por otra parte, la otra formulación VECTOLEX® WGD-51,2%, con la dosis de 2,0 Kg/ ha, resultó ser eficaz hasta 8 días post-aplicación con 85,2% de reducción larval; la misma produjo 13,3% de reducción a los 16 días post-tratamiento; sin embargo, esta formulación con la dosis de 2,5 Kg/ha, fue altamente eficaz hasta 16 días postaplicación con más de 90% de reducción larval. En cambio, en el mismo manglar el producto GRISELESF fue menos eficaz tanto a 2,0 Kg/ha como a 2,5 Kg/ha, obteniéndose porcentajes de reducción muy bajos a los 8 y 16 días post-aplicación.

Anopheles aquasalis Curry is the main vector of malaria in northeastern Venezuela. A study of biological control of An. aquasalis larvae was carried out in Río Chiquito Abajo, Sucre State, Venezuela. The objective of this study was to evaluate effectivity and persistence of three formulations of Bacillus sphaericus against larval populations of An. aquasalis. The main breeding-sites of An. aquasalis are seasonal brackish mangrove with Avicennia germinans and semi-permanent freshwater wetlands with herbaceous emergent vegetation. A typical seasonal brackish mangrove with Avicennia germinans was selected. In these breeding-sites of An. aquasalis, three commercial formulations of B. sphaericus: VECTOLEX® CG-7.5%, VECTOLEX® WGD-51.2% and GRISELESF were evaluated in field trials. According to the results obtained VECTOLEX® CG-7.5% was effective against An. aquasalis larvae during 7 days post-treatment at rates not lower than 30 Kg/ha (3 g/m²), producing a larval reduction greater than 92.3%. This granular formulation provided very good larval control during 7 days. The two dosages (30 and 40 Kg/ha) had the same efficacy against An. aquasalis larvae. The second commercial formulation VECTOLEX® WGD-51.2% was effective against An. aquasalis larvae at dose of 2.0 Kg/ ha, producing a larval reduction greater than 85 % during 8 days post-treatment, and only 13.3 % larval reduction at 16 days after treatment; but the same formulation with at dose of 2.5 Kg/ha was more effective, producing a larval reduction greater than 90 % during 16 days posttreatment. In the same brackish mangrove, GRISELESF at rates not lower than 2.0 and 2.5 Kg/ha, appears to be less effective against An. aquasalis larvae, producing very low larval reduction at 8 and 16 days post-treatment.

Production and characterization of poly-3-hydroxybutyrate from crude glycerol by Bacillus sphaericus NII 0838 and improving its thermal properties by blending with other polymers

The aim of this work was to study the production of poly-3-hydroxybutyrate (PHB) under nitrogen limited conditions by Bacillus sphaericus NII 0838 using crude glycerol from biodiesel industry as sole carbon source. Effect of various process parameters on PHB production such as glycerol concentration, inoculum size and pH of the medium were optimized. Characterization of extracted PHB was carried out by FT-IR, ¹H and 13C NMR. Results showed that the bacterial culture accumulated about 31 percent PHB in crude glycerol medium. The extracted PHB was blended with other polymers to improve its physical characteristics. The thermal properties of the polymer like melting temperature (Tm) and heat of fusion (ΔHf) were determined using DSC.

Transfer of toxin - coding megaplasmid of bacillus thuringiensis subsp. thompsoni into bacillus sphaericus.

Mosquito borne diseases form a major component of vector borne diseases from all over the world. Several control strategies have been adopted to control diseases transmitted by mosquitoes. Bacterial insecticides have been used for the control of nuisance and vector mosquitoes for more than two decades. Nevertheless, due primarily to their high cost and often only moderate efficacy, these insecticides remain of limited use in tropical countries where mosquito-borne diseases are prevalent. Recently, however, recombinant DNA techniques have been used to improve bacterial insecticide efficacy by markedly increasing the synthesis of mosquitocidal proteins and by enabling new endotoxin combinations from different bacteria to be produced within single strains. Both Bacillus sphaericus and Bacillus thuringiensis subsp.thompsoni produce insecticidal toxins during sporulation and are extensively used in the field for control of mosquito populations. All the known toxins of the latter organism are known to be encoded on its megaplasmids. In an attempt to combine the best properties of the two bacteria, a cry proteins (namely 34- and 40-kDa) encoding megaplasmid (~ 100 MDa) of Bt subsp.thompsoni was transferred to Bacillus sphaericus by conjugation. Many of the transconjugants reacted with antibody to the 34- and 40-kDa Bt subsp.thompsoni crystal toxins in western blotting and were more toxic to Aedes mosquitoes than the wild type B. sphaericus. The toxicity of the transconjugants was maintained through many transfers in the absence of selective pressure.

Evaluación de la persistencia de una formulación comercial de Bacillus sphaericus en criaderos naturales de anofelinos vectores de malaria en estado Bolívar, Venezuela / Evaluation of the persistence of a comercial formulation of Bacillus sphaericus in malaria vectors anopheline larval hábitats in Bolívar state, Venezuela

El municipio Sifontes del estado Bolívar es la principal área endémica a malaria de Venezuela. Durante los últimos cinco años se ha reportado en este municipio un promedio anual cercano a 15 mil casos, alcanzándose durante 2009 la cifra de 20.282, lo que constituye una situación de epidemia. En este municipio se evaluó la persistencia de una formulación comercial de Bacillus sphaericus Neide (Vectolex CG 7,5%) en criaderos naturales de Anopheles marajoara, An. triannulatus y An. braziliensis. Para ello se realizaron dos experimentos en los cuales se evaluaron las dosis de 2 y 3 gr/m2 durante 60 y 90 días. Los resultados indican que hay diferencias en la persistencia por tipo de criadero y especie de anofelino. La tendencia general para todas las especies indica que el porcentaje de reducción de la población larvaria fue de 100% durante la primera semana, luego disminuyó paulatinamente hasta más o menos 40% después de los 28 días, para luego recuperarse a alrededor de 60% a los 45 días, manteniéndose así hasta el día 90 con ambas dosis. Este comportamiento es más evidente para An. marajoara, mientras que con An. triannulatus la eficacia se mantiene alrededor de 80% durante 90 días con 3 gr/m2. Sobre An. braziliensis, la eficacia se mantiene en 100% hasta el día 14 con ambas concentraciones, para luego caer abruptamente sin recuperación. Es la primera vez que se evalúa B. sphaericus sobre estas especies de anofelinos en Venezuela, considerándose estos resultados muy prometedores para el control de vectores

Sifontes municipality in Bolivar state is the main malaria endemic area in Venezuela. During the last five years an annual average of 14,000 cases has been reported, reaching 13,450 during 2008, resulting in an epidemic situation. A commercial formulation of Bacillus sphaericus Neide (Vectolex CG 7.5%), was evaluated in larval habitats of Anopheles marajoara, An. triannulatus and An. braziliensis. Two experiments were carried out in a lagoon and a pasture inundated area, 2 and 3 gr/m2 doses were evaluated during 60 and 90 days. The results point out that there are differences in the persistence depending on the type of larval habitat and anopheline species. The general trend indicated that for all the species the persistence of the Vectolex, therefore the reduction of the larval population was 100% during the first week. However, it felt under 40% after day 28, then recovered by day 45, and remaining around 40% for 90 days with both doses. A similar pattern was observed for An. marajoara, whereas for An. triannulatus the efficacy remained around 80% during 90 days only at 3 gr/m2. With An. braziliensis there was 100% persistence until day 14 with both doses, for then felt abruptly without recovering. This is the first time that B. sphaericus was evaluated for these anopheline species in Venezuela, considering these results promissory for the malaria control program

Base molecular da interação da toxina binária do biolarvicida Bacillus shaericus com a-glicosidases de larvas de culicídios vetores / Molecular basis of interaction of the binary toxin of Bacillus biolarvicide shaericus with a-glucosidase from larvae culicids vectors

O principal fator larvicida do Bacillus sphaericus (Bs) para culicídeos é a toxina binária (Bin), produzida sob a forma de um cristal, durante a esporulação. Esta toxina, ativada por meio da ação de proteases no lúmen intestinal, reconhece e liga-se a receptores específicos no intestino de larvas por meio de sua subunidade BinB, etapa crítica no seu modo de ação e essencial para a atividade larvicida. Os receptores em Culex pipiens, C. quinquefasciatus e Anopheles gambiae, denominados Cpm1, Cqm1, e Agm3, respectivamente, são alfa glicosidases de 66 kDa ligadas à membrana apical do epitélio intestinal por uma âncora de glicosilfosfatidilinositol (GPI). Larvas de Aedes aegypti expressam a alfaglicosidase Aam1, ortóloga ao receptor Cqm1 que, no entanto, não apresenta capacidade de ligação à toxina Bin. O principal objetivo deste trabalho foi caracterizar a base molecular da interação da toxina Bin do Bs com alfaglicosidases em larvas de culicídeos vetores. Para tal, a sequência protéica de Cqm1 foi alinhada a sequências ortólogas e parálogas de Ae. aegypti e An. gambiae, o que indicou trechos mais conservados na porção N-terminal e divergentes na porção C-terminal. A banálise funcional de fragmentos protéicos de 45 kDa da porção N-terminal de proteínas ortólogas e parálogas à Cqm1 sugeriu que o epitopo de ligação à toxina Bin está potencialmente localizado nesta região e que o estado conformacional nativo das proteínas é determinante para sua funcionalidade. Em seguida, foram avaliadas algumas características das alfa glicosidases Cqm1 e Aam1 de 66 kDa que podem estar envolvidas na sua capacidade de ligação à toxina Bin. Os resultados mostraram que as proteínas possuem diferenças estruturais e conformacionais que podem ter um papel determinante para a interação com a toxina Bin. A análise do padrão de glicosilação das proteínas revelou que a Aam1 posssui glicosilações em sua cadeia polipeptídica ao contrário da Cqm1 e que a ligação do receptor Cqm1...

Polyhydroxybutyrate production using agro-industrial residue as substrate by Bacillus sphaericus NCIM 5149

The aim of this work was to study the production of polyhydroxybutyrate (PHB) using agro- industrial residues as the carbon source. Seven substrates, viz., wheat bran, potato starch, sesame oil cake, groundnut oil cake, cassava powder, jackfruit seed powder and corn flour were hydrolyzed using commercial enzymes and the hydrolyzates assessed for selecting the best substrate for PHB production. Jackfruit seed powder gave the maximum production of PHB under submerged fermentation using Bacillus sphaericus (19 percent) at the initial pH of 7.5.

Laboratory and field evaluation of an oviposition trap for Culex quinquefasciatus(Diptera: Culicidae)

An ovitrap (BR-OVT) based on physical and chemical stimuli for attracting gravid Culex quinquefasciatus (Diptera: Culicidae) females was developed and evaluated under laboratory and field conditions. Attractants were assayed using alternative chamber bioassays prior to being used in the BR-OVT oviposition trap. A significant preference of gravid females for sites containing conspecific egg rafts was observed, as a response to the natural oviposition pheromone, as well as for sites treated with the synthetic pheromone erythro-6-acetoxy-5-hexadecanolide. Five- to 20-day old grass infusion was strongly attractive to gravid females for laying eggs. On the other hand, entomopathogenic Bacillus sphaericus (Bs) did not influence the choice of an oviposition site when used in combination with grass infusion and can therefore be used as a larvicide in ovitraps. Results from field trials showed that the BR-OVT with grass infusion and with or without Bs works as a preferred oviposition site for Cx. quinquefasciatus. The BR-OVT was more effective for egg collection when placed indoors and comparison with the number of egg rafts laid in cesspits over 40 days indicates that this very simple ovitrap may be a useful tool for monitoring populations of the most important of the vectors of bancroftian filariasis.

NATURALLY OCCURING ANTIBIOTIC RESISTANCE IN BACILLUS SPHAERICUS / 微生物学通报

The naturally occuring antibiotic resistance in twentyseven strains from 7 different serotypes of Bacillus sphaericus was investigated. The antibiotics used in this experiment are Streptomycin, Ampicilline, Tetracycline, Chloramphenicol, Kanamycin, Erythromycin, Qingdamycin, Midecamycin, Ribostamycin, Acriflavin, Rifampicin, Sulphanilamide and Azide. All strains are resist to streptomycin and azide at higher concentration and chloramphenicol at lower concentration. Most of strains are resist to tetracycline at lower concentration.