In the female mosquito, Aedes aegypti, neurohormones are released from the brain in response to a blood meal and stimulate the ovaries to secrete ecdysteroid hormones, which modulate yolk protein synthesis in the fat body. Neuropeptides with this bioactivity were isolated from head extracts, and partial sequences from these peptides when aligned gave a 31-residue sequence at the amino terminus. Oligonucleotide primers for this sequence were used to amplify with the polymerase chain reaction a genomic DNA product that hybridized to a clone from a head cDNA library. The cDNA encodes a 149-residue preprohormone that is processed into an 86-residue peptide, as indicated by the mass value obtained from the native peptide, with the expected amino-terminal sequence. After modification, the cDNA for the putative neurohormone was expressed in a bacterial system, and the purified peptide had high specific activity in bioassays, thus confirming that it is a steroidogenic gonadotropin, the first to be identified for invertebrates.Among vertebrates, the gonadotropic neurohormones, follicle-stimulating hormone and luteinizing hormone, regulate steroidogenesis during reproductive cycles; for invertebrates, the only characterized steroidogenic neurohormones are the prothoracicotropic hormones, which initiate molting in insect larvae (1). The first insect gonadotropin was discovered by Lea (2, 3), who described a neurohormone controlling reproduction in mosquitoes. For the yellow fever mosquito, Aedes aegypti (Diptera: Culicidae), each reproductive cycle in a female begins with the ingestion of a blood meal and ends with egg deposition. The blood meal, in turn, stimulates release of gonadotropic neurohormones from medial neurosecretory cells in the brain for up to 12 h post ingestion (2, 3). These neurohormones stimulate the ovaries to secrete ecdysteroids and thus are referred to as "ovary ecdysteroidogenic hormones" (OEHs) 1 (4). The ecdysteroids modulate fat body secretion of yolk proteins (5, 6), which are stored selectively in the oocyte for embryonic development.Despite many attempts to purify gonadotropins from mosquitoes (4, 7, 8), only their peptide nature and apparent heterogeneity (3,500 -24,000-Da range) are known. This report describes the purification of OEH I from an extract of six million heads and its structural characterization by a combination of biochemical and molecular techniques. To verify the biological activity of the putative neurohormone, a recombinant OEH I was expressed and purified for testing in vivo and in vitro, and with an OEH I antiserum, the source of the neurohormone was identified. EXPERIMENTAL PROCEDURESBioassays-To test chromatographic fractions and bacterial-expressed protein for bioactivity, samples (0.5 l of saline solution/female) were injected into 3-5-day-old, sugar-fed females (nonoogenic), decapitated within 1 h after a blood meal. After 16 -24 h at 30°C, ovaries were dissected from the females to measure yolk deposition in oocytes. Bioactive peptides induced Ͼ100 m of yolk deposit...

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Using transmission electron microscopy, we investigated the encapsulation of the simian malaria parasite, Plasmodium cynomolgi, in a refractory strain of the mosquito, Anopheles gambiae. After the ookinete penetrates the mosquito midgut epithelium and lodges between the basal membrane and the basal lamina, an electron-dense, melanin-like substance begins to coalesce around the parasite. Completely encapsulated parasites were found as early as 16 hr after the blood meal. Granules of the melanin-like substance often appeared to condense onto the parasite from the fluid in the extracellular spaces of the basal membrane labyrinth. Melanin granules also appeared to condense from the hemolymph onto the basal lamina underlying the parasite. In addition, groups of tubules, vesicles, and membranous whorls often were found in midgut cells that were located next to or were enclosing parasites. These structures were unusually electron-dense, and may have been associated with melanization. Hemocytes rarely were observed near completed capsules and neither hemocytes nor their remnants were components of the capsules. During later stages of encapsulation, parasites appeared abnormal and often were infiltrated with melanin. Although late-stage capsules were usually located basally, completed capsules enclosed by membranes were occasionally observed near the apical border of the midgut. Other capsules associated with cellular debris, were found in the lumen of the midgut from 1 to 6 days after the blood meal.

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