Mosquitoes and all other insects so far examined have an abundant haemolymph transferrin (Tsf). The exact function of these proteins has not been determined, but they may be involved in iron transport, in oogenesis and in innate immune defence against parasites and pathogens. The Tsf gene of Aedes aegypti has been cloned and sequenced. It contains a single small intron, which contrasts it to vertebrate Tsf genes that contain up to sixteen introns. The promoter region of the gene is rich in putative NF-kappaB binding sites, which is consistent with the postulated role of Tsf in insect innate immunity. Tsf message levels are very low in embryos and early larvae, but high in late larvae, pupae and adults. Western blotting experiments revealed high levels of Tsf protein in pupae and adults. Late larvae and ovaries of blood-fed mosquitoes have little intact protein, but two prominent proteolytic degradation products. These may represent biologically active peptides, as has been shown for other organisms. Tsf message is down-regulated by inorganic iron in the diet or environment, but up-regulated by a blood meal in the adult female. The up-regulation following a blood meal may, in part, be due to the decrease in juvenile hormone (JH) that is known to follow blood feeding. Treatment of blood-fed females with methoprene, an analogue of JH, resulted in decrease of the Tsf message.

Iron significantly increases the amount of ferritin HCH messages and dramatically shifts the balance toward those messages that lack an IRE and͞or have a short 3 UTR. In the larvae this change takes place in the gut, but not in the fat body. We speculate that this dramatic shift in message distribution may result from an effect of iron on the rate of transcription or message degradation, or from an effect on the splicing process itself. Synthesis of ferritin HCH subunit mRNAs that lack an IRE may be important under conditions of iron overload.

Drosophila melanogaster transferrin cDNA was cloned from an ovarian cDNA library by using a PCR fragment amplified by two primers designed from other dipteran transferrin sequences. The clone (2035 bp) encodes a protein of 641 amino acids containing a signal peptide of 29 amino acids. Like other insect transferrins, Drosophila transferrin appears to have a functional iron-binding site only in the N-terminal lobe. The C-terminal lobe lacks ironbinding residues found in other transferrins, and has large deletions which make it much smaller than functional C-terminal lobes in other transferrins. In-situ hybridization using a digoxigenin labeled transferrin cDNA probe revealed that the gene is located at position 17B1-2 on the X chromosome. Northern blot analysis showed that transferrin mRNA was present in the larval, pupal and adult stages, but was not detectable in the embryo. Iron supplementation of the diet resulted in lower levels of transferrin mRNA. When adult flies were inoculated with bacteria (Escherichia coli), transferrin mRNA synthesis was markedly increased relative to controls.Keywords: transferrin; Drosophila melanogaster; sequence; chromosome; expression; up-regulation; immunity.Iron is an essential nutrient required for nearly all living organisms. Ionic iron is also toxic by virtue of its ability to catalyze the formation of membrane-destroying free radicals. Ionic iron forms oxides that are insoluble at physiological pH. Thus, in order to absorb and transport iron, most organisms have acquired special cofactors and proteins that are capable of maintaining iron in a water soluble state.Iron metabolism has been studied extensively in vertebrates. In vertebrate serum, ferric ion is transported by transferrin, a single chain polypeptide (<80 kDa) consisting of two lobes with similar amino acid sequence, and each with a single ironbinding site [1,2]. Diferric transferrin is taken into cells by receptor-mediated endocytosis, iron is dissociated from transferrin in an acidic endosome, and is transferred to the cytoplasm. In cells, iron is incorporated into metalloproteins or stored in cytoplasm chelated with small molecules or within an iron storage protein, ferritin [3].As in vertebrates, both transferrin and ferritin are found in insects and the cDNAs for each from several insect species have been cloned. Insect transferrins have been reported from four insect species [4±7]. Whereas cockroach (Blaberus discoidalis) transferrin has two iron-binding sites and strongly resembles vertebrate serum transferrins [8], other insect transferrins from a moth (Manduca sexta), flesh fly (Sarcophaga peregrina) and mosquito (Aedes aegypti) have one iron-binding site only in the N-terminal lobe but not in the C-terminal lobe [4,6,7]. The properties of insect transferrins suggest that they may have different, possibly unique, functions in insects. Recently, we found that transferrin is up-regulated when mosquito cells in culture were challenged with heat-killed bacteria. This suggests a possible important role(s) of i...

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