Mosquito Larvicide Screening of Sea Lily

The toxicity study used Yellow-fever mosquito larvae. This species is commonly used by the World Health Organization for insecticide evaluation (WHO 1981 ). Sea lily samples were weighed out and mixed with appropriate amounts of methanol resulting in a 1% stock solution. These 1% solutions were then serially diluted (1:1) with methanol to 12 concentrations ranging from 10,000 to 4.9 ppm. The toxicity assays were performed in a 96-well microtitre plate with third instar larva of A. aegypti according to the procedure described by Chio (2007) . Exactly, 100 µl of sea lily sample was transferred to a 96-well plate and allowed to evaporate to complete dryness by a heat block. Samples were reconstituted with 100 µl of 0.01% Keflin solution followed by another 100 µl of water containing several third instar mosquito larvae. Methanol and 0.5% permethrin were adopted as negative and positive controls, respectively. Larvae mobility and mortality 24-hr post-treatment were recorded with “+” for all kill, “±” for partial kill, and “−” for no kill. Four replicates were adopted for each treatment group. Any samples that completely killed the mosquitoes at 156 ppm or higher concentrations were retested at lower concentrations. Another serial dilution (1:1) starting at 200 ppm was used for the retesting. Methanol and 0.5% permethrin were again adopted as negative and positive controls for the retesting. The minimum concentration that provided the complete control of the mosquito larvae was defined as the minimum inhibition concentration (MIC). The MIC method has been demonstrated to match well with that of LC₅₀ by comparison side-by-side with the LC₅₀ values of five common mosquito larvacides (Chio 2007 ). This study adopts the MIC method instead of the LC₅₀ method, because MIC is simpler than LC₅₀, and can handle higher sample volumes.