A gut-specific serine protease from the malaria vector Aanopheles gambiae is downregulated after blood ingestion

A chymotrypsin-like serine protease gene (AgChyL) was cloned from the mosquito Anopheles gambiae by a polymerase chain reaction (PCR)-based subtractive cDNA cloning strategy. AgChyL messenger RNA (mRNA) is abundant in the adult female gut prior to, and for 8 h following, a blood meal. During the peak of digestion, from 12 to 24 h following a blood meal, AgChyL mRNA abundance decreased to barely detectable levels. AgChyL mRNA was abundant again by 48 h following a blood meal. Recombinant pro-AgChyL was expressed in Escherichia coli. The pro-enzyme can be activated by trypsin. Activated AgChyL cleaves the synthetic chymotrypsin substrate succinyl-L-Ala-Ala-Pro-Phe-nitroanilide, but not two other synthetic chymotrypsin substrates or synthetic trypsin and elastase substrates. The potential role of AgChyL in the coordination of An. gambiae digestion is discussed.     

The mosquito Aedes aegypti (L.) leucokinin receptor is a multiligand receptor for the three Aedes kinins

A cDNA cloned from Aedes aegypti (L.) (Aedae) female Malpighian tubule (AY596453) encodes a 584 amino acid residue protein (65.2 kDa) predicted as a G protein-coupled receptor and orthologue of the drosokinin receptor from Drosophila melanogaster and highly similar to the tick Boophilus microplus myokinin receptor (AF228521). Based on the similarity to this Aedes sequence, we also propose a correction for the Anopheles gambiae protein sequence EAA05450. When expressed in CHO-K1 cells, the Aedes receptor behaved as a multiligand receptor and functionally responded to concentrations > or = 1 nM of Aedae kinins 1-3, respectively, as determined by a calcium bioluminescence plate assay and single cell intracellular calcium measurements by confocal fluorescence cytometry. Estimates of EC50 values by the plate assay were 16.04 nM for Aedae-K-3, 26.6 nM for Aedae-K-2 and 48.8 nM for Aedae-K-1 and were statistically significantly different. These results suggest that the observed differences in physiological responses to the three Aedes kinins in the Aedes isolated Malpighian tubule reported elsewhere could now be explained by differences in intracellular signalling events triggered by the different peptides on the same receptor and not necessarily due to the existence of various receptors for the three Aedes kinins.     

Identification of a point mutation in the voltage-gated sodium channel gene of Kenyan Anopheles gambiae associated with resistance to DDT and pyrethroids

A field trial of permethrin-impregnated bednets and curtains was initiated in Western Kenya in 1990, and a strain of Anopheles gambiae showing reduced susceptibility to permethrin was colonized from this site in 1992. A leucine-phenylalanine substitution at position 1014 of the voltage-gated sodium channel is associated with resistance to permethrin and DDT in many insect species, including Anopheles gambiae from West Africa. We cloned and sequenced a partial sodium channel cDNA from the Kenyan permethrin-resistant strain and we identified an alternative substitution (leucine to serine) at the same position, which is linked to the inheritance of permethrin resistance in the F(2) progeny of genetic crosses between susceptible and resistant individuals. The diagnostic polymerase chain reaction (PCR) developed by Martinez-Torres et al. [(1998) Insect Mol Biol 7: 179-184] to detect kdr alleles in field populations of An. gambiae will not detect the Kenyan allele and hence reliance on this assay may lead to an underestimate of the prevalence of pyrethroid resistance in this species. We adapted the diagnostic PCR to detect the leucine-serine mutation and with this diagnostic we were able to demonstrate that this kdr allele was present in individuals collected from the Kenyan trial site in 1986, prior to the introduction of pyrethroid-impregnated bednets. The An. gambiae sodium channel was physically mapped to chromosome 2L, division 20C. This position corresponds to the location of a major quantitative trait locus determining resistance to permethrin in the Kenyan strain of An. gambiae.     

Cloning and characterization of the white gene from Anopheles gambiae

A 14 kb region of genomic DN A containing the X-linked Anopheles gambiae eye colour gene, white , was cloned and sequenced. Genomic clones containing distinct white + alleles were polymorphic for the insertion of a small transposable element in intron 3, and differed at 1% of nucleotide positions compared. Sequence was also determined from a rare 2914 bp cDNA. Comparison of cDNA and genomic sequences established an intron-exon structure distinct from Drosophila white. Despite a common trend in Anopheles and Drosophila of weak codon bias given low levels of gene expression, codon usage by Anopheles gambiae white was strongly biased. Overall amino acid identity between the predicted mosquito and fruitfly proteins was 64%, but dropped to 14% at the amino terminus. To correlate phenotypically white-eyed strains of A. gambiae with structural lesions in white , five available strains were analysed by PCR and Southern blotting. Although these strains carried allelic mutations, independently generated by gamma radiation (three strains) or spontaneous events (two strains), no white lesions were detected. Significantly, another non-allelic X-linked mutation, causing an identical white-eyed phenotype, has been correlated with a structural defect in the cloned white gene (Benedict et al. , 1995). Taken together, these observations suggest that the white-eyed mutants analysed in the present study carry mutations in a second eye colour gene and are most likely white +.     

Molecular cloning and chromosomal localization of a prophenoloxidase cDNA from the malaria vector Anopheles gambiae

A cDNA clone for prophenoloxidase was isolated from the most important human malaria vector, Anopheles gambiae . The clone encoded a polypeptide of 79341 Da that contains the two copper binding domains common to all invertebrate prophenoloxidases and haemocyanins. Expression of the prophenoloxidase gene was detected throughout all life stages from egg to imago in two strains of A. gambiae ; however, the strongest expression was observed in developing embryos in eggs. The prophenoloxidase gene was mapped to the inversion rich region of the right arm of chromosome-2 in region 13B.     

Inducible immune factors of the vector mosquito Anopheles gambiae: biochemical purification of a defensin antibacterial peptide and molecular cloning of preprodefensin cDNA

Larvae of the mosquito vector of human malaria, Anopheles gambiae , were inoculated wlth bacteria and extracts were biochemically fractionated by reverse-phase HPLC. Multiple induced polypeptides and antibacterial activities were observed following bacterial infection, including a member of the Insect defensin family of antibacterial proteins. A cDNA encoding An. gambiae preprodefensin was isolated using PCR primers based on phyiogeneticaiiy conserved sequences. The mature peptide is highly conserved, but the signal and propeptide segments are not, relative to corresponding defensin sequences of other insects. Defensin expression is Induced in response to bacterial infection, in both adult and larval stages. in contrast, pupae express defensin mRNA constitutively. Defensin expression may prove a valuable molecular marker to monitor the An. gambiae host response to infection by parasitic protozoa of medical importance.     

The white gene from the yellow fever mosquito, Aedes aegypti

We report the cloning and primary characterization of both cDNA and genomic fragments from the white gene of the yellow fever mosquito, Aedes aegypti . Comparisons of the conceptual translation product with white genes from four other species within the order Diptera show that the Ae. aegypti gene is most similar to the white gene of the mosquito vector of human malaria, Anopheles gambiae (86% identity and 92% similarity). The analysis of the primary sequence of genomic DNA at the 5'-end of the coding region revealed the presence of an intron that is also present in An. gambiae , but not in the vinegar fly, Drosophila melanogaster . The isolated clones of the Ae. aegypti white gene will enable the construction of a marker gene for use in the development of a germline transformation system for this species.     

Isolation of cDNA clones encoding putative odourant binding proteins from the antennae of the malaria-transmitting mosquito, Anopheles gambiae

One way of controlling disease transmission by blood-feeding mosquitoes is to reduce the frequency of insect-host interaction, thus reducing the probability of parasite transmission and re-infection. A better understanding of the olfactory processes responsible for allowing mosquitoes to identify human hosts is required in order to develop methods that will interfere with host seeking. We have therefore initiated a molecular approach to isolate and characterize the genes and their products that are involved in the olfactory recognition pathway of the mosquito Anopheles gambiae, which is the main malaria vector in sub-Saharan Africa. We report here the isolation and preliminary characterization of several cDNAs from male and female A. gambiae antennal libraries that encode putative odourant binding proteins. Their conceptual translation products show extensive sequence similarity to known insect odourant binding proteins (OBPs)/pheromone binding proteins (PBPs), especially to those of D. melanogaster. The A. gambiae OBPs described here are expressed in the antennae of both genders, and some of the A. gambiae OBP genes are well conserved in other disease-transmitting mosquito species, such as Aedes aegypti and Culex quinquefasciatus.     

Molecular cloning and characterization of a metal responsive Aedes aegypti intestinal mucin cDNA

We have isolated a cDNA from Aedes aegypti that is transcribed in the larval midgut in response to metal exposure, and in the adult female midgut in response to iron or cadmium exposure, or a blood meal. The cDNA encodes a protein, designated Aedes aegypti intestinal mucin 1 (AEIMUC1), which has similarities with invertebrate intestinal mucins and peritrophins, and vertebrate mucins. Proline, serine and threonine comprise 30% of the amino acid composition of AEIMUC1, a characteristic of mucins. AEIMUC1 contains three cysteine-rich domains, two of which flank a proline/serine/threonine-rich domain, a feature shared by many mucin genes. This is the first report on the isolation of a metal-responsive gene from an aquatic insect.     

Molecular characterization of arrestin family members in the malaria vector mosquito, Anopheles gambiae

Olfaction influences many insect behaviours including mate seeking and host selection. The molecular machinery underlying insect olfactory systems is a G protein-coupled receptor pathway that, in addition to activation, requires adaptation for olfactory sensitivity and discrimination. We have previously identified ARR1 (henceforth AgARR1), a sensory arrestin from the malaria vector mosquito Anopheles gambiae that has been postulated to modulate olfactory adaptation. This report describes three additional arrestin family members including ARR2 (henceforth AgARR2), which is similar to previously characterized insect sensory arrestins and is expressed at significantly higher levels in the antennae of male vs. female A. gambiae mosquitoes. This finding is consistent with the hypothesis that AgARR2 may be important for the regulation of olfactory-driven behaviours particular to male mosquitoes.     

Cloning and analysis of a cecropin gene from the malaria vector mosquito, Anopheles gambiae

Parasites of the genus Plasmodium are transmitted to mammalian hosts by anopheline mosquitoes. Within the insect vector, parasite growth and development are potentially limited by antimicrobial defence molecules. Here, we describe the isolation of cDNA and genomic clones encoding a cecropin antibacterial peptide from the malaria vector mosquito Anopheles gambiae. The locus was mapped to polytene division 1C of the X chromosome. Cecropin RNA was induced by infection with bacteria and Plasmodium. RNA levels varied in different body parts of the adult mosquito. During development, cecropin expression was limited to the early pupal stage. The peptide was purified from both adult mosquitoes and cell culture supernatants. Anopheles gambiae synthetic cecropins displayed activity against Gram-negative and Gram-positive bacteria, filamentous fungi and yeasts.     

The nonvitellogenic female protein of Musca domestica is an adult-specific hexamerin

During Musca domestica vitellogenesis a protein is preferentially synthesized by the female fat body and accumulates in the haemolymph but not in the ovaries. This protein, designated nonvitellogenic female protein (NVFP), was purified and shown to be a hexamer with an M_r = 430 kDa, and subunits of M_r = 70 kDa. The hexamer dissociates into subunits when the pH is elevated from 7.0 to 9.0. Two cDNA clones, F0 and F2, were isolated and analysed. The 2.2 kb F2 clone has an open reading frame that encodes a conceptual translation product that has similarity to the Drosophila melanogaster LSP-2 hexamerin. Recombinant protein from the F2-cDNA is recognized by a specific anti-NVFP serum. The temporal pattern of mRNA expression of the gene represented by the F2 clone follows that determined for the synthesis of NVFP. The data support the conclusion that NVFP is an hexamerin specific to the adult stage of Musca domestica .     

Aquaporin and aquaglyceroporin in silkworms, differently expressed in the hindgut and midgut of Bombyx mori

Two cDNAs similar to aquaporins (AQPs) from other insect species were identified and characterized from the silkworm larva, Bombyx mori. The first cDNA (AQP-Bom1) cloned from the anterior silk gland encodes a 25 900 Da protein similar to insect AQPs isolated from several liquid-feeding insects. The second cDNA (AQP-Bom2) cloned from the posterior midgut encodes a 27 694 Da protein. Northern blot analysis has revealed that the AQP-Bom1 mRNA (2.3 kb) is expressed predominantly in the hindgut (colon and rectum), and moderately or minimally in the silk gland, midgut and Malpighian tubules, while the AQP-Bom2 mRNA (1.3 kb) is mainly expressed in the posterior midgut and Malpighian tubules. Functional analysis in Xenopus oocytes microinjected with the cRNA of these AQPs revealed that the AQP-Bom1 mRNA encodes a water-specific aquaporin, likely involved in the water retrieval function of the hindgut, while the AQP-Bom2 mRNA encodes an aquaglyceroporin, increasing glycerol and urea uptake.     

Modulation of Anopheles gambiae gene expression in response to o'nyong-nyong virus infection

To determine if gene expression of An. gambiae is modulated in response to o'nyong-nyong virus (ONNV) infection, we utilized cDNA microarrays including about 20 000 cDNAs. Gene expression levels of ONNV-infected female mosquitoes were compared to that of the uninfected control females harvested at 14 days postinfection. In response to ONNV infection, expression levels of 18 genes were significantly modulated, being at least two-fold up- or down-regulated. Quantitative real-time PCR analysis (qRT-PCR) further substantiated the differential expression of six of these genes in response to ONNV infection. These genes have similarity to a putative heat shock protein 70, DAN4, agglutinin attachment subunit, elongation factor 1 alpha and ribosomal protein L35. One gene, with sequence similarity to mitochondrial ribosomal protein L7, was down-regulated in infected mosquitoes. The expression levels and annotation of the differentially expressed genes are discussed in the context of host/virus interaction including host translation/replication factors, and intracellular transport pathways.     

A novel lectin with a fibrinogen-like domain and its potential involvement in the innate immune response of Armigeres subalbatus against bacteria

Mosquitoes have an efficient cellular innate immune response that includes phagocytosis of microbial pathogens and encapsulation of metozoan parasites. In this study, we describe a novel lectin in the mosquito, Armigeres subalbatus (aslectin or AL-1). The 1.27 kb cDNA clone for the AL-1 gene (AL-1) encodes a 279 deduced amino acid sequence that contains a C-terminal fibrinogen-like domain. AL-1 is transcribed in all life stages. AL-1 mainly exists in the haemolymph of adult female mosquitoes, and is upregulated following both Escherichia coli and Micrococcus luteus challenge. AL-1 specifically recognizes N-acetyl-d-glucosamine and is able to bind both E. coli and M. luteus. These results suggest that AL-1 might function as a pattern recognition receptor in the immune response in Ar. subalbatus.