Molecular Characterization of Mosquitocidal Toxin (Surface Layer Protein, SLP) from Bacillus cereus VCRC B540.

A marine Bacillus cereus (VCRC B540) with mosquitocidal effect was recently reported from red snapper fish (Lutjanus sanguineous) gut and surface layer protein (S-layer protein, SLP) was reported to be mosquito larvicidal factor. In this present study, the gene encoding the surface layer protein was amplified from the genomic DNA and functionally characterized. Amplification of SLP-encoding gene revealed 1,518 bp PCR product, and analysis of the sequence revealed the presence of 1482 bp open reading frame with coding capacity for a polypeptide of 493 amino acids. Phylogenetic analysis revealed with homology among closely related Bacillus cereus groups of organisms as well as Bacillus strains. Removal of nucleotides encoding signaling peptide revealed the functional cloning fragment of length 1398 bp. Theoretical molecular weight (51.7 kDa) and isoelectric point (5.99) of the deduced functional SLP protein were predicted using ProtParam. The amplified PCR product was cloned into a plasmid vector (pGEM-T), and the open reading frame free off signaling peptide was subsequently cloned inpET-28a(+) and expressed in Escherichia coli BL21 (DE3). The isopropyl-ß-D-thiogalactopyranoside (IPTG)-induced recombinant SLP was confirmed using western blotting, and functional SLP revealed mosquito larvicidal property. Therefore, the major findings revealed that SLP is a factor responsible for mosquitocidal activity, and the molecular characterization of this toxin was extensively studied.

Synthesis of silver nanoparticles from Azadirachta indica--a most effective method for mosquito control.

Mosquitoes transmit major communicable diseases such as dengue, malaria, filariasis, Japanese encephalitis, chikungunya, and so on. Vector control is important in epidemic disease situations as there is an urgent need to develop new and improved mosquito control methods that are economical and effective yet safe for non-targeted organisms. In the present study, silver nanoparticles (AgNPs) were synthesized from the aqueous leaf extract of neem plant (Azadirachta indica), and their effects on mosquito vectors (Aedes aegypti and Culex quinquefasciatus) were assessed. The synthesised AgNPs were characterized by UV-vis spectroscopy, scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FT-IR), and X-ray diffraction analysis (XRD). The nanoparticles have maximum absorption at 442 ± 1.5 nm with an average size of 41-60 nm. The XRD data showed six well-defined diffraction peaks, corresponding to a relative intensity of the crystal structure of metallic silver 36.42, 100.00, 53.70, 14.20, 16.05, and 6.79, respectively. The FT-IR data showed strong prominent peaks in different ranges, reflecting its complex nature. The mosquito larvae were exposed to varying concentrations of AgNPs synthesized from the neem leaves under investigation (0.07-25 mg/l) for 24 h; this revealed larvicidal activity of AgNPs with LC50 and LC90 values of 0.006 and 0.04 mg/l for A. aegypti, respectively. Further, the LC50 and LC90 values were also identified as 0.047 and 0.23 mg/l for Cx. quinquefasciatus, respectively. The result obtained from this study presents biosynthesized silver nanoparticle from A. indica as the biolarvicidal agent with the most potential for mosquito control.

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.

Hexamerin a novel protein associated with Bacillus sphaericus resistance in Culex quinquefasciatus.

Bacterial insecticides like, Bacillus sphaericus and Bacillus thuringiensis serovar israelensis, have been used for the control of nuisance and vector mosquitoes for more than two decades. For many years, it was assumed that the use of microbial larvicides based on B. sphaericus would not lead to resistance in mosquitoes. However, recent reports have shown that B. sphaericus toxins are not free from this problem. Therefore, the resistance of mosquito populations to be will seriously threaten the sustainability of current mosquito control programme using these microbial insecticides. In the present study, we have characterised a novel protein responsible for resistance development in the filariasis vector of Culex quinquefasciatus. Laboratory selection experiments with B. sphaericus against the larvae were carried out up to 17 generations, and the occurrence of resistance was reported (resistance ratio (RR) at lethal concentration (LC)50 and LC90 = 1,987 and 2,051 folds, respectively). The protein profiles of B. sphaericus-resistant and susceptible population have confirmed with the expression of a new polypeptide (80 kDa) in the resistant strain only. Sequence result revealed that the newly expressed protein was 'hexamerin', and this factor might conceivably be responsible for the inheritance of resistance. This study is therefore valuable for comprehending the underlining factor and management of B. sphaericus resistance problem in mosquito population.

Identification and characterization of a novel marine Bacillus cereus for mosquito control.

Entomopathogenic bacteria to control mosquitoes are a promising environmentally friendly alternative to synthetic pesticides. In the present study, a novel mosquitocidal bacterium was isolated from marine soil collected from east coastal areas at Pondicherry (India). 16S rRNA gene sequence alignment depicted that this isolate belonged to Bacillus cereus VCRC-B520 (NCBI: KC-119192). Biochemical studies on bacterial growth, biomass, and toxin production have revealed that this strain could possibly be helpful in the production of a biopesticide in mosquito control. Toxicity assay with B. cereus against mosquito larvae has shown that the filariasis vector, Culex quinquefasciatus, is more susceptible than the other two species (Anopheles stephensi and Aedes aegypti). The LC50 and LC90 values for C. quinquefasciatus were 0.30 and 2.21 mg/L, respectively. No effect of B. cereus was found on nontargeted organisms. SDS-PAGE analysis and protein purification result from the cell mass of B. cereus have shown that a well-perceptible polypeptide was the dependable factor (85 kDa) for mosquitocidal action. Protein characterization (M/S MALDI-TOF) has shown that it is an endotoxin-specific insecticidal protein, namely "Cry4Aa". Phylogenetic analyses of 16S rDNA gene sequence from this marine isolate have revealed the presence of homology among closely related Bacillus strains. Therefore, considerable interest has been shown on the identification of a potential mosquitocidal bacterium from marine environment (B. cereus), which was not reported earlier in view of the current scenario of the rapid development of resistance to Bacillus sphaericus in mosquito vector control program.

Genotypic diversity of mosquitocidal bacteria (Bacillus sphaericus, B. thuringiensis, and B. cereus) newly isolated from natural sources.

In the present study, to comprehend the genetic diversity of mosquitocidal bacteria, the genotypic analysis of 30 strains of Bacillus species isolated newly from diverse environmental sources has been conducted. Randomly amplified polymorphic DNA polymerase chain reaction was conducted to characterize the genotype diversity between the bacterial strains. The matrix of scores from each bacterial DNA was analyzed, and the correlation between the co-efficients and the similarity matrix data was computed. Clusters from dendrogram showing diversity among isolates could be distinguished genetically based on their origin of isolates. The first major cluster consists of 43 strains grouped under various subclusters (91.489 %). A second cluster contains only four strains (8.511 %). An average similarity value of 0.36 revealed the dendrogram split into 28 distinct "groups" or "clusters," allowing almost a complete separation of strains within the Bacillus group isolated from various sources and thus facilitating assessment of genetic diversity of species and subspecies level. The conclusion from the result was that there was broad diversity among the mosquitocidal strains, and cluster analysis revealed the associations among the isolates based on their origin. A high level of polymorphism with distinct genetic lineages consequent to the source of origin of bacterial strains is the significant impact of this study.

A novel cost-effective medium for the production of Bacillus thuringiensis subsp. israelensis for mosquito control.

Bacillus thuringiensis subsp. israelensis (Bti) has been used for mosquito-control programmes the world-wide. Indeed, the large-scale production of Bti for mosquito control is very expensive due to the high cost of its culture. In the present study, we attempted to widen the scope in developing cost-effective culture medium for Bti production, based on the raw materials available on the biosphere, including coconut cake powder, CCP (Cocos nucifera), neem cake powder, NCP (Azadirachta indica) and groundnut cake powder, GCP (Arachis hypogea). Among these raw materials, the biomass production of Bti, sporulation and toxin synthesizing from 'CCP' in combination with mineral salt (MnCl(2)) was comfortably satisfactory. Bioassays with mosquito species (Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti) and field trials were also satisfactory. The present investigation suggests that coconut cake-based culture medium can be used as an alternative for industrial production of Bti in mosquito-control programme. Therefore, the study is very important from the point of effective production of Bti from cost-effective culture medium for the control of mosquito vectors.

Optimization of medium composition for the production of mosquitocidal toxins from Bacillus thuringiensis subsp. israelensis.

Optimization of chicken feather (CF) based culture medium for the production of Bacillus thuringiensis subsp. israelensis (Bti) biomass in combination with the agro industrial by-product (coconut cake, CC) and manganese chloride (MnCl2) has been evaluated. The biomass yield of Bti spore/crystal toxin was highest (12.06 g/L) from the test medium (CF+CC+MnCl2) compared to the reference medium (Luria Bertani, LB). Toxicity assay with Bti produced from the test medium against mosquito vectors (Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti) was also satisfactory and results were comparable with bacteria produced from LB. The results suggest that Bti can be produced to the maximum extent possible as a potential mosquitocidal activity as suggested by the test medium (CF+CC+MnCl2).

Coffee husk waste for fermentation production of mosquitocidal bacteria.

Coffee husk waste (CHW) discarded as bio-organic waste, from coffee industries, is rich in carbohydrates. The current study emphasizes the management of solid waste from agro-industrial residues for the production of biopesticides (Bacillus sphaericus, and B. thuringiensis subsp. israelensis), to control disease transmitting mosquito vectors. An experimental culture medium was prepared by extracting the filtrates from coffee husk. A conventional culture medium (NYSM) also was prepared. The studies revealed that the quantity of mosquitocidal toxins produced from CHW is at par with NYSM. The bacteria produced in these media, were bioassayed against mosquito vectors (Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti) and it was found that the toxic effect was statistically comparable. Cost-effective analysis have revealed that, production of biopesticides from CHW is highly economical. Therefore, the utilization of CHW provides dual benefits of effective utilization of environmental waste and efficient production of mosquitocidal toxins.

A medium for the production of biopesticides (Bacillus sphaericus and Bacillus thuringiensis subsp. israelensis) in mosquito control.

Chicken feathers, discarded as bioorganic wastes from poultry processing industries, are rich in keratin (structural protein). Deoiled rice bran (DRB), in contrast, is an agrowaste product consisting of protein, fiber, and carbohydrates. This study highlights a new medium for producing biopesticides from these bioorganic wastes, by culturing Bacillus sphaericus (Bs) and B. thuringiensis subsp. israelensis (Bti), to synthesize mosquitocidal toxins. Biochemical studies indicated that the amount of mosquitocidal spore/crystal toxins produced from the experimental culture medium (chicken feather waste [CFW] + DRB) is higher than that from the conventional medium (Nutrient Yeast Extract Salt Medium [NYSM]). The bacteria produced in these media (NYSM, CFW, DRB, and CFW + DRB) were bioassayed against mosquito vectors, and the toxic effect was found to be statistically similar (fiducial limits overlapping). Cost analysis based on the raw materials, indicated that, the use of CFW and DRB, as culture medium, is highly economical, for the industrial production of these mosquito pathogenic bacilli.

Insight into tropical human infectious diseases: an update

Knowledge on infectious diseases encompasses a vast and constantly changing arena; and consistent research work is imperative to understand and combat the new problems resulting from emerging infectious diseases. Public health workers and epidemiologists aim at lowering morbidity and mortality due to diseases by preventing infections. For the rapidly expanding majority of the world's population; who live in the largely tropical areas of Africa; Asia and Latin America; the greatest threats to health remain tropical infectious diseases. Emerging infectious diseases are new; emerging or drug-resistant infections; whose incidence in humans may increase in the near future. With rapidly increasing international travel; the globalization and industrialization of food supply and exploding populations; infectious diseases pose unprecedented threats around the globe. Thus the imperative need is to promptly recognize; isolate and appropriately manage tropical infectious diseases. This review paper has attempted to provide the much needed insight into the different aspects of the major tropical infectious diseases affecting humans throughout the world

Novel fermentation media for production of Bacillus thuringiensis subsp. israelensis.

The production of Bacillus thuringiensis subsp. israelensis (deBarjac) (Bti) as a biopesticide is not cost-effective using existing fermentation technology. In this study, we explored the use of several less expensive alternative culture media (potato, common sugar, and Bengal gram) for the growth and production of Bti. Growth was obtained in all tested media and was comparable to that obtained in conventional medium (Luria-Bertani). Toxicity assays showed that the toxin produced from the novel growth media were effective in killing larvae of Culex quinquefasciatus, Anopheles stephensi, and Aedes aegypti and toxicity was comparable to that produced from Luria-Bertani medium. These observations suggest that potato can be used as a cheap source of culture medium for the production of Bti toxin in mosquito control programs.