Swarming and mating activity of Anopheles gambiae mosquitoes in semi-field enclosures.

Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) is the major Afro-tropical vector of malaria. Novel strategies proposed for the elimination and eradication of this mosquito vector are based on the use of genetic approaches, such as the sterile insect technique (SIT). These approaches rely on the ability of released males to mate with wild females, and depend on the application of effective protocols to assess the swarming and mating behaviours of laboratory-reared insects prior to their release. The present study evaluated whether large semi-field enclosures can be utilized to study the ability of males from a laboratory colony to respond to natural environmental stimuli and initiate normal mating behaviour. Laboratory-reared males exhibited spatiotemporally consistent swarming behaviour within the study enclosures. Swarm initiation, peak and termination time closely tracked sunset. Comparable insemination rates were observed in females captured in copula in the semi-field cages relative to females in small laboratory cages. Oviposition rates after blood feeding were also similar to those observed in laboratory settings. The data suggest that outdoor enclosures are suitable for studying swarming and mating in laboratory-bred males in field-like settings, providing an important reference for future studies aimed at assessing the comparative mating ability of strains for SIT and other vector control strategies.

An Anopheles gambiae salivary gland promoter analysis in Drosophila melanogaster and Anopheles stephensi.

Regulatory regions driving gene expression in specific target organs of the African malaria vector Anopheles gambiae are of critical relevance for studies on Plasmodium-Anopheles interactions as well as to devise strategies for blocking malaria parasite development in the mosquito. In order to identify an appropriate salivary gland promoter we analysed the transactivation properties of genomic fragments located just upstream of the An. gambiae female salivary gland-specific genes AgApy and D7r4. An 800 bp fragment from the AgApy gene directed specific expression of the LacZ reporter gene in the salivary glands of transgenic Anopheles stephensi. However, expression levels were lower than expected and the transgene was expressed in the proximal-rather than in the distal-lateral lobes of female glands. Surprisingly, a promoter fragment from the D7r4 gene conferred strong tissue-specific expression in Drosophila melanogaster but only low transcription levels in transgenic An. stephensi. These results imply a certain conservation of gland-specific control elements between the fruit fly and the mosquito suggesting that an increased degree of complexity, probably connected to the evolution of haematophagy, underlies the regulation of tissue-specific expression in mosquito female salivary glands.

Blood digestion in the malaria mosquito Anopheles gambiae: molecular cloning and biochemical characterization of two inducible chymotrypsins.

The elucidation of digestive processes in the Anopheles gambiae gut leading to the utilization of the blood meal will result in a deeper understanding of the physiology of blood digestion and its impact on parasite-vector interactions. Accordingly, the identification of digestive serine proteases in A. gambiae has implications for the development of alternative strategies for the control of mosquito-borne diseases. We report here on the cDNA and genomic cloning and on the expression analysis of two closely related chymotrypsin genes, Anchym1 and Anchym2. Genomic cloning revealed that Anchym1 and Anchym2, which map on chromosomal division 25D, are clustered in tandem within 6 kb, both genes being interrupted by two short introns. After blood feeding, transcription of Anchym1 and Anchym2 is induced in the midgut epithelium, followed by secretion of the translated products into the midgut lumen where the Anchym1 and Anchym2 zymogens are activated by partial tryptic digestion. The amino-acid residues forming the substrate pocket of Anchym1 and Anchym2 suggested chymotryptic cleavage specificity. This was confirmed by mass spectrometry analysis and Edman degradation sequencing of proteolytic products generated by the recombinant, trypsin-activated Anchym1.

Nuclear factors bind to a conserved DNA element that modulates transcription of Anopheles gambiae trypsin genes.

The Anopheles gambiae trypsin family consists of seven genes that are transcribed in the gut of female mosquitoes in a temporal coordinated and mutually exclusive manner, suggesting the involvement of a complex transcription regulatory mechanism. We identified a highly conserved 12-nucleotide motif present in all A. gambiae and Anopheles stephensi trypsin promoters. We investigated the role of this putative trypsin regulatory element (PTRE) in controlling the transcription of the trypsin genes. Gel shift experiments demonstrated that nuclear proteins of A. gambiae cell lines formed two distinct complexes with probes encompassing the PTRE sequence. Mapping of the binding sites revealed that one of the complex has the specificity of a GATA transcription factor. Promoter constructs containing mutations in the PTRE sequence that selectively abolished the binding of either one or both complexes exerted opposite effects on the transcriptional activity of trypsin promoters in A. gambiae and Aedes aegypti cell lines. In addition, the expression of a novel GATA gene was highly enriched in A. gambiae guts. Taken together our data prove that factors binding to the PTRE region are key regulatory elements possibly involved in the blood meal-induced repression and activation of transcription in early and late trypsin genes.

Stable germline transformation of the malaria mosquito Anopheles stephensi.

Anopheline mosquito species are obligatory vectors for human malaria, an infectious disease that affects hundreds of millions of people living in tropical and subtropical countries. The lack of a suitable gene transfer technology for these mosquitoes has hampered the molecular genetic analysis of their physiology, including the molecular interactions between the vector and the malaria parasite. Here we show that a transposon, based on the Minos element and bearing exogenous DNA, can integrate efficiently and stably into the germ line of the human malaria vector Anopheles stephensi, through a transposase-mediated process.

Toward Anopheles transformation: Minos element activity in anopheline cells and embryos.

The ability of the Minos transposable element to function as a transformation vector in anopheline mosquitoes was assessed. Two recently established Anopheles gambiae cell lines were stably transformed by using marked Minos transposons in the presence of a helper plasmid expressing transposase. The markers were either the green fluorescent protein or the hygromycin B phosphotransferase gene driven by the Drosophila Hsp70 promoter. Cloning and sequencing of the integration sites demonstrated that insertions in the cell genome occurred through the action of Minos transposase. Furthermore, an interplasmid transposition assay established that Minos transposase is active in the cytoplasmic environment of Anopheles stephensi embryos: interplasmid transposition events isolated from injected preblastoderm embryos were identified as Minos transposase-mediated integrations, and no events were recorded in the absence of an active transposase. These results demonstrate that Minos vectors are suitable candidates for germ-line transformation of anopheline mosquitoes.

Constitutive and blood meal-induced trypsin genes in Anopheles gambiae.

Trypsin genes in Anopheles gambiae are arranged as a tightly clustered gene family consisting of seven related coding sequences, devoid of introns. The two blood meal-inducible members of this family, Antryp1 and Antryp2, were shown to play a crucial role in the breakdown of the blood meal constituents. The role of Antryp3,4,5,6, and Antryp7 in the process of blood meal digestion remains to be elucidated. We have examined the localization and the expression patterns of these trypsins as well as the functional interactions in blood meal digestion between trypsins and other gut-specific proteases. Northern blot and RT-PCR analysis indicated that the genes Antryp3,4,5,6, and Antryp7 are all constitutively expressed in unfed female mosquitoes. Soon after blood feeding the mRNA of these trypsin genes became undetectable and appeared again at the end of the gonotrophic cycle. The blood meal-inducible trypsin Antryp1 was also constitutively expressed at low level in the gut of adult female mosquitoes. This trypsin was the only member of this gene family to be expressed in the gut of male and female pupae. By using antisera that specifically recognized recombinant Antryp4 we were able to show that the corresponding protein in Anopheles is synthesized and stored in the gut epithelium of unfed females as zymogen. Secretion and activation of this trypsin was shown to occur in the midgut lumen immediately after fluid ingestion and independently of the protein content of the meal. Recombinant trypsins expressed in Escherichia coli, with the exception of Antryp5 and Antryp6, were able to activate in vitro recombinant A. gambiae chymotrypsinogen, thus suggesting that blood meal ingestion is able to trigger a cascade of events leading to the activation of several proteases.