FIG 3.

The nonlive Csp_P biopesticide has a low effective dose and a durable active ingredient that is not produced by other common mosquito-associated bacteria. ROCK larvae were fed pellets containing different doses of nonlive Csp_P (A). Doses of 100 and 50 mg killed 100% of larvae, while doses of 25, 12.5, and 6.25 mg yielded partial mortality and delayed pupation. An LD₅₀ was calculated at 11.35 mg per liter of water in the larval habitat. Nonlive Csp_P powder was heat treated at room temperature (22°C), 30, 37, or 54°C (B) and, in independent experiments, at room temperature (22°C) or 70°C (C) in accelerated shelf life tests in order to assess the durability of the active ingredient. The 30, 37, and 54°C treatments did not differ in efficacy from the room temperature treatment (Cox regression; P > 0.05), while the 70°C treatment still killed 100% of the larvae exposed but took significantly longer to do so (Cox regression: P < 0.0001), with these results suggesting that the active ingredient was highly heat stable and likely to have a long shelf life. (D) Three other common mosquito-associated bacterial samples were cultured and dried according to the same protocol used to produce nonlive Csp_P powder. Then, 100 mg of each of these powders was added to attractive pellets and provided to ROCK larvae. None of these three preparations caused mortality that was significantly different from that seen for larvae treated with no-bacterium control pellets (Cox regression: P > 0.05), suggesting that the larvicidal effect we observed in our results was not due to the culturing methods and not universal among all bacteria. In all experiments, larvae were reared in groups of 30 in 300 ml of deionized water. Lines depict the percentages of larvae surviving at each day posttreatment (± SEM) for three experimental replicates, with each containing three cages per treatment.