2013 Annual Report
1a.Objectives (from AD-416):
To devise innovative methods for the suppression of insects that transmit diseases to U.S. military deployed abroad.
1b.Approach (from AD-416):
Identify and test compounds with insecticidal activity that makes them especially beneficial in the rapid control of mosquitoes, flies, and other arthopods responsible for carrying disease agent. Devise innovative means of applying control agents especially suited for use in combat or deployment conditions. Identify and test new classes of topical and area repellents for use by troops.
Several natural products (essential oils) have been identified as having potent insecticidal properties against sand flies using a glass vial bioassay technique. A static air repellency bioassay technique has also been used successfully in evaluating the repellency of a number of essential oils and several new synthetic compounds against adult female sand flies. Their relative potencies in repelling sand flies in bioassays are compared DEET and permethrin. A single amino acid substitution in a recombinant sand fly acetylcholinesterase (rPpAChE1) was generated, and the altered recombinant protein was expressed in the baculovirus system. The recombinant rPpAChE1 containing the single amino acid substitution replacing glycine with serine at amino acid position 256 (rPpAChE1-G256S) exhibited 1000-fold to 500,000-fold reduced sensitivity to tested organophosphate (OP) and carbamate (PRC-408) inhibitors, respectively, in comparison to the recombinant enzyme without the amino acid substitution (rPpAChE1). PCR-based genotyping assays are under development to test for the specific nucleotide sequence giving rise to the mutation. Initial results suggest that the amino acid substitution may be likely to develop from a single nucleotide mutation in sequence polymorphisms that may exist in nature. This is of particular value because the mutation in the rPpAChE1-G256S corresponds to the most prevalent mutation causing organophosphate resistance in mosquitoes transmitting malaria and other important diseases of humans and animals. Availability of the recombinant mutant enzyme will allow in vitro screening of novel carbamates and organophosphates to identify potentially efficacious control agents in collaboration with researchers at the University of Florida, Virginia Tech, Southwest Research Institute, and the Mayo Clinic. For example, a synthetic carbamate PRC-408, identified and synthesized by collaborators at Virginia Tech and University of Florida was evaluated in vitro for inhibition of recombinant acetylcholinesterases (rAChEs) of horn fly, stable fly, and sand fly. PRC-408 is believed to offer improved safety compared to other AChE inhibitors, because it exhibited approximately 300-fold higher specificity for arthropod AChEs compared to mammalian AChEs (bovine and human). In addition, PRC-408 was evaluated by in vivo bioassay against horn flies, stable flies, and sand flies, exhibiting insecticidal activity comparable to that of carbaryl (an efficacious insecticide).