A 0.59 kilobase DNA fragment cloned from an rDNA cistron of the mosquito Anopheles gambiae can be used as a probe to differentiate between A. gambiae, A. arabiensis, and A. melas, three morphologically identical sibling species in the A. gambiae complex which otherwise can be reliably distinguished only by polytene chromosome banding patterns. Although all are important (and often sympatric) African malaria vectors, their relative roles in malaria transmission have thus far been difficult to assess. The probe, an EcoRI-SalI fragment from the 3' end of the 28S beta coding region of the cistron, is present in all three species, but the species differ uniquely with respect to the location of an EcoRI site in the nontranscribed spacer (NTS) downstream of the fragment. We have routinely used the probe to identify A. gambiae complex mosquitoes to species on the basis of genomic DNA extracted from individual air dried specimens. A single mosquito abdomen provides more than sufficient DNA for the assay, and neither eggs nor a bloodmeal in the abdomen interfere with DNA yield. Moreover, the DNA extraction procedure does not degrade the bloodmeal IgG, so the residual protein pellet can be used to identify the mosquito bloodmeal source. Since the rDNA cistron organization as detected by the probe does not differ between male and female mosquitoes, the probe can be used for either sex. Preliminary experiments show that the probe is equally useful for mosquito larvae and pupae.

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The catalytic subunit gp91phox (Nox2) of the NADPH oxidase of mammalian phagocytes is activated by microbes and immune mediators to produce large amounts of reactive oxygen species (ROS) which participate in microbial killing. Homologs of gp91phox, the Nox and Duox enzymes, were recently described in a range of organisms, including plants, vertebrates, and invertebrates such as Drosophila melanogaster. While their enzymology and cell biology are being extensively studied in many laboratories, little is known about in vivo functions of Noxes. Here, we establish and use an inducible system for RNAi to discover functions of dNox, an ortholog of human Nox5 in Drosophila. We report here that depletion of dNox in musculature causes retention of mature eggs within ovaries, leading to female sterility. In dNox-depleted ovaries and ovaries treated with a Nox inhibitor, muscular contractions induced by the neuropeptide proctolin are markedly inhibited. This functional defect results from a requirement for dNox-for the proctolin-induced calcium flux in Drosophila ovaries. Thus, these studies demonstrate a novel biological role for Nox-generated ROS in mediating agonist-induced calcium flux and smooth muscle contraction.

Chromatographic analysis of pigments extracted from wild-type eyes of the mosquito Anopheles gambiae reveals the presence of the ommatin precursor 3-hydroxykynurenine, its transamination derivative xanthurenic acid, and a dark, red-brown pigment spot that probably is composed of two or more low mobility xanthommatins. No colored or fluorescent pteridines are evident. Mosquitoes homozygous for an autosomal recessive mutation at the red-eye (r) locus have a brick-red eye color in larvae, pupae, and young adults, in contrast to the almost black color of the wild eye. Mosquitoes homozygous for this mutant allele have levels of ommochrome precursors that are indistinguishable from the wild-type, but the low-mobility xanthommatin spot is ochre-brown in color rather than red-brown as in the wild-type. Mosquitoes with two different mutant alleles at the X-linked pink-eye locus (p, which confers a pink eye color, and pw, which confers a white eye phenotype in homozygotes or hemizygous males) have normal levels of ommochrome precursors but no detectable xanthommatins. Mosquitoes homozygous for both the r and p mutant alleles have apricot-colored eyes and show no detectable xanthommatins. Both the pink-eye and red-eye mutations appear to involve defects in the transport into or assembly of pigments in the membrane-bound pigment granules rather then defects in ommochrome synthesis.

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