Field evaluation of biosurfactants, surfactin and di-rhamnolipid produced by Bacillus subtilis subsp. subtilis (VCRC B471) and Pseudomonas fluorescens (VCRC B426) against immature stages of the urban malaria vector Anopheles stephensi.

BACKGROUND & OBJECTIVES: Bacillus subtilis subsp. subtilis (VCRC B471) and Pseudomonas fluorescens (B426) produce mosquitocidal biosurfactant, surfactin and di-rhamnolipid. The objective of the study was to carry out a small-scale field evaluation of the two biosurfactants to determine the efficacy, application dosage, residual activity and frequency of application against Anopheles stephensi immatures in selected sites in Goa, India. METHODS: Surfactin (VCRC B471) and di-rhamnolipid (VCRC B426) were formulated as aqueous suspensions (5% AS), and were applied at the dosages of 34, 51 and 68 mL/m2 and 27, 41 and 54 mL/m2 respectively. Two experiments were carried out with the two formulations. RESULTS: Surfactin (VCRC B471) formulation was effective at all the dosages and there was sustained reduction (>80%) in immature density in the treated sites up to 18 days in experiment 1 and up to 15 days in experiment 2. No pupae were found in the treated sites throughout the study. Di-rhamnolipid (VCRC B426) formulation was also found to reduce the immature density in the treated sites up to 14 days in experiment 1 and up to 15 days in experiment 2. INTERPRETATION & CONCLUSION: For VCRC B471, the optimum application dosage determined was 51 mL/m2 and for VCRC B426, 27mL/m2. The formulations are to be applied fortnightly for effective control of Anopheles. The application dosage determined in the present study can be used for large scale field evaluation to assess their suitability for use in public health programmes for the control of Anopheles mosquitoes vectoring malaria.

Genetic structure and connectivity among Aedes aegypti populations within Madurai city in Southern India.

We investigated the genetic variability and differentiation among 12 Ae. aegypti populations collected within the Madurai city in Tamil Nadu state of Southern India. Genotyping of 12 microsatellite markers in 353 individual samples showed moderate levels of genetic diversity among 12 populations. UPGMA tree, hierarchical clustering, Bayesian clustering and Discriminant Analysis on Principal Components roughly divided these populations into two genetic clusters: main city populations and the populations located at the border of the corporation limit. Significant positive correlation between genetic and geographic distance was observed among 12 populations, however, the correlation was non-significant within each genetic cluster. Population assignment and divMigrate graph depicted less migration between two groups. Overall, the findings of this study provided an overview of Ae. aegypti population structure within an urban setting in India that have implications in effective implementation of vector control in the city area.

Bionomics of Anopheles fluviatilis and Anopheles culicifacies (Diptera: Culicidae) in Relation to Malaria Transmission in East-Central India.

The southern districts of Odisha State in east-central India have been highly endemic for falciparum malaria for many decades. However, there is no adequate information on the abundance of the vector species or their bionomics in relation to space and time in these districts. Therefore, a study was carried out on the entomological aspects of malaria transmission to generate such information. Collections of mosquitoes were made once during each of the three seasons in 128 villages selected from eight districts. Villages within the foot-hill ecotype had a significantly greater abundance of Anopheles fluviatilis James s. l., whereas the abundance of Anopheles culicifacies Giles s. l. was significantly greater in the plain ecotype. The abundance of An. fluviatilis was maximum during the cold season, whereas An. culicifacies abundance was highest during summer and rainy seasons. The maximum likelihood estimation of the malaria infection rate in An. fluviatilis was 1.78%, 6.05%, and 2.6% in Ganjam, Kalahandi, and Rayagada districts, respectively. The infection rate of An. culicifacies was 1.39% only in Kandhamal district; infected females were not detected elsewhere. Concurrently, the annual malaria parasite incidence (MPI) was significantly higher in hill-top (17.6) and foot-hill (14.4) villages compared to plain villages (4.1). The districts with more villages in hill-top and foot-hill ecotypes also had a greater abundance of An. fluviatilis, the major malaria vector, and exhibited a higher incidence of malaria than villages within the plain ecotype, where An. culicifacies was the most abundant vector.

Species-diagnostic polymerase chain reaction assays for Phlebotomus argentipes and Phlebotomus papatasi, vectors of Leishmania.

Species-specific differences encountered in the nucleotide sequences of a highly variable region of the 18S rRNA gene were used to design a multiplex polymerase chain reaction (PCR) assay for the identification of Phlebotomus papatasi Scopoli and Phlebotomus argentipes An-nandale & Brunetti, vectors of Leishmania. This multiplex PCR assay uses a common forward primer and two reverse primers, which are specific for the two species. Amplification of a PCR product of size 788 bp indicates the presence of P. papatasi, whereas a product of size 677 bp indicates the presence of P. argentipes. The assay was found to be highly specific and sensitive.

rDNA-ITS2-PCR assay for grouping the cryptic species of Anopheles culicifacies complex (Diptera: Culicidae).

Anopheles culicifacies, a predominant vector of malaria in India exists as a complex of five sibling species A, B, C, D and E, of which, except species B, all the rest are vectors with varying vectorial capacities. With a combination of PCR assays, it is possible to identify all the five members of this species complex. These assays include amplification of the rDNA-ITS2 region followed by digestion of the ITS2 amplicon using restriction enzyme, Rsa I which groups the five members of the An. culicifacies complex into two categories: species A and D forming one category and species B, C and E forming another. The samples grouped thus are then subjected to two allele-specific PCR assays (AD-PCR and BCE-PCR), which has been designed using sequence differences in the mitochondrial cytochrome oxidase II (CO II) subunit. The AD-PCR assay distinguishes species A and D, whereas the BCE-PCR assay distinguishes species B, C and E. In the present study, the differences in the ITS2 region of the five species was used to design a PCR assay which groups the five members into the same two categories as obtained after digestion of the ITS2-PCR product. This assay uses a common forward primer based on the 5.8S region and two reverse primers, which is specific for the two categories. Amplification of a PCR product of size 253bp indicates the presence of species A/D, while a product of size 409bp indicates the presence of species B/C/E. By using this ITS2 PCR assay, the three-step procedure is reduced to two cutting down the time and cost involved. The ITS2 PCR assay has been validated on specimens collected from different regions of India and the results confirm to the earlier reports on the distribution of the members of the An. culicifacies complex.