This proposal focuses on the isolation and characterization of regulatory neuropeptides from heads of Aedes aegypti females. With the adult ecdysiotropic factor (ETF), which stimulates secretion of ecdysteroids by the ovaries, purified to homogeniety, sequencing work will continue. Even a partial sequence can be synthesized to produce an antiserum for localization and quantification of ETF in tissues and hemolymph. Concurrently, purification and sequence determination will be initiated for larval prothoracicotropic hormone, so it too can be localized and quantified, and the conditions and timing of its release studied. Two """"""""Aedes RFamide"""""""" peptides (ARFa I & III), found in specific neurons in the adult brain, have been sequenced. ARFa I, a novel decapeptide, has been synthesized and its gene cloned. However, its physiological function is unknown and will be investigated in a series of bioassays (diuresis, midgut contraction, allatotropic action, stimulation or inhibition of trypsin or vitellogenin synthesis, feeding behavior, etc). Antiserum to ARFa I will be produced for immmunolocalization and labelled ARFa I prepared for in situ receptor studies; both should provide important clues to its function. Sequence analysis of a third head peptide (ARFa II) will be continued. Our mass-rearing facility produces one million mosquitoes per week, giving sufficient material for purification of peptides to homogeneity. In vivo and in vitro bioassays and radioimmunoassays are used to detect activity in chromatographic fractions. Recently, fractions with antidiuretic activity were found and will be purified. Testing fractions in the bioassays will likely identify other neuropeptides. While each peptide brings opportunities for detailed studies of the regulation of specific physiological processes, some may have application in novel insect control systems now being developed, i.e., microencapsulation and protein engineering of peptides as stomach poisons and insertion of genes for regulatory peptides in species-specific microorganisms.
