fatigans larvae collected from the drains around
Chirala in Andhra Pradesh, India (Rao & Mahajan, 1990). The standard strains of B. sphaericus, 1593 and 2362, were obtained from the Pasteur Institute, Paris, France. All bacterial cultures were maintained on a standard nutrient broth (NB) medium supplemented with 0.3% sugar cane molasses (NB medium). A single colony suspension was heat shocked at 80 °C for 12 min to synchronize the culture. This culture was first inoculated in a sterile 50 mL NB medium and incubated as a static culture at room temperature for 16 h. The 5% v/v inoculum from the static culture was transferred to 250 mL NB medium and incubated at 28±2 °C on a rotary shaker at 180 r.p.m. (Orbitek, Sciegenics, Chennai, India). The culture was harvested JAK pathway when it showed >90% sporulation as observed by phase-contrast microscopy
(Axioscop-40, Carl Zeiss, Göttingen, Germany). The culture usually reached a spore count of 1–2 × 109 spores mL−1 at the time of harvest. The pellet was washed twice with sterile-distilled water and lyophilized. The lyophilized powder was stored in an airtight container for further use. The cultures of Culex quinquefasciatus, Culex tritaeniorhynchus, Aedes aegypti and Aedes albopictus were maintained at 28±2 °C and 85% relative humidity in our laboratory. The adults were reared separately in closed cages and fed with a chicken bloodmeal. Eggs were collected and allowed to hatch in plastic bowls containing 1 L of tap water supplemented with 0.13 g of sterilized larval food (13 : 6 : 1 of wheat flour, chickpea flour and yeast extract) (Hadapad et al., 2008). Anti-infection Compound high throughput screening For all bioassays, third-instar larvae of the same age and size were used. The total viable counts (TVC) of all the strains were determined from lyophilized powder as described in Hire et al. (2006). Statistically significant differences between different means were calculated using Fisher’s least significant difference multiple comparison test (spss 12.0 for Windows). For preliminary screening
of the larvicidal activity, the stock suspensions of all B. sphaericus strains were prepared in sterile-distilled water and tested against the third-instar larvae of C. quinquefasciatus. Lck Different concentrations of all these strains, along with the control, were tested in 100 mL tap water containing 20 larvae in each beaker (150 mL), with four replications for each concentration. Based on the preliminary bioassay studies, ISPC-8 was found to be highly toxic and was hence selected to determine the spectrum of larvicidal activity against different mosquito species. The different concentrations of ISPC-8 were tested against third-instar larvae of C. tritaeniorhynchus, A. aegypti and A. albopictus. All the experiments were repeated three times on different days. The total larval mortality was scored after 48 h of treatment.