Immunity proteins from mosquito cell lines include three defensin A isoforms from Aedesaegypti and a defensin D from Aedesalbopictus

An Aedes aegypti mosquito cell line, Aag-2, exhibits a response to immune stimulation that is qualitatively similar to that of C7–10 cultured cells from the related mosquito, Aedes albopictus. Using SDS polyacrylamide gels, we found that a small peptide was preferentially induced by the treatment of growing cells with heat-killed, Gram-positive bacteria. By an analogy with other studies, this small peptide was postulated to be a member of the defensin family of insect immunity peptides. A differential display was used to obtain partial polymerase chain reaction products corresponding to mRNAs that were preferentially expressed in induced cells. One of these products, which contained the partial sequence of a defensin gene, was used to screen cDNA libraries from Ae. aegypti and Ae. albopictus cells. From Ae. aegypti cells, we found two previously described isoforms (A1 and A4) of mosquito defensin A, as well as a new isoform which we defined as A5. From Ae. albopictus cells, we found a new mature mosquito defensin, named D, which contains proline and isoleucine as the final amino acids. In both Ae. aegypti and Ae. albopictus cell lines, the expression of defensin mRNA was visible on Northern blots as early as 3 h after exposure to heat-killed bacteria, and defensin mRNA abundance was maximal at 12–36 h after induction.     

GP35 ANOAL, an abundant acidic glycoprotein of female Anopheles albimanus saliva

Salivary glands of female mosquitoes produce proteins, not completely described yet, that participate in carbohydrate and blood feeding. Here, we report an acidic glycoprotein of 35 kDa (GP35 ANOAL) secreted in the saliva of the malaria vector mosquito Anopheles albimanus. GP35 ANOAL is produced exclusively in the distal lateral lobes of adult female salivary glands, it has a pI of 4.45 and is negatively stained by regular silver stain. An 888 bp cDNA clone encoding a predicted product of 240 amino acids has a signal peptide, potential post-translational modification sites, and a disintegrin signature RGD. The GP35 ANOAL sequence depicts high similarities with the 30 kDa saliva allergen of Aedes aegypti, 30 kDa allergen-like hypothetical proteins, and GE-rich proteins present in several Anopheles species, as well as in Ae. albopictus and Culex pipiens quinquefasciatus. The function of this protein family is still unknown.     

Developmental- and tissue-specific expression of an inhibitor of apoptosis protein 1 homologue from Aedes triseriatus mosquitoes

We have identified a homologue of the Drosophila inhibitor of apoptosis protein 1 in Aedes triseriatus mosquitoes (designated AtIAP1). The AtIAP1 gene maps to a single locus on chromosome 2. The translation product is a 403 amino acid protein that contains two baculovirus IAP repeat (BIR) domains and a RING finger motif. AtIAP1 mRNA was detectable by RT-PCR amplification in all the mosquito developmental stages (embryos, first-fourth instar larvae, early and late pupae, adults) and adult tissues (midguts, ovaries) examined. In contrast, immunoblots with AtIAP1-specific antibodies revealed that the protein was detectable only in certain developmental stages (first instar larvae, early pupae, adults) and tissues (ovaries). AtIAP1-specific serum also recognized proteins in Ae. aegypti, Ae. albopictus and Culex tritaeniorhynchus. Immunoblot analysis revealed that similar amounts of IAP1 were expressed in LaCrosse virus infected and uninfected Ae. albopictus cell cultures.     

Sequence of a mosquito ribonucleotide reductase cDNA and evidence for gene amplification in hydroxyurea-resistant cells

Ribonucleotide reductase is essential for production of the deoxyribonucleotide substrates required for DNA synthesis in all eukaryotic cells. The full-length cDNA encoding a mosquito ribonucleotide reductase R2 subunit was obtained from Aedes albopictus cells using a polymerase chain reaction (PCR)-based approach. The cDNA contained 1197 nucleotides, and encoded a 398 amino acid R2 protein. Overall, mosquito R2 shared approximately 70% amino acid sequence identity with R2 proteins from vertebrates, but conservation at the N-terminus of the protein was relatively low. The sequence of R2 cDNA was identical in hydroxyurea-resistant mosquito cells and in wild-type cells, while the R2 gene copy number was increased ten to twentyfold in resistant cells.     

Immune activation upregulates lysozyme gene expression in Aedes aegypti mosquito cell culture

After stimulation with heat-killed bacteria, cultured cells from the mosquito Aedes aegypti (Aag-2 cells) secreted an induced protein with a mass of approximately 16 kDa that cross-reacted with antibody to chicken egg lysozyme. To investigate whether lysozyme messenger RNA is induced in bacteria-treated cells, we used polymerase chain reaction-based approaches to obtain the complete lysozyme cDNA from Aag-2 cells. The deduced protein contained 148 amino acids, including a 23 amino acid signal sequence. The calculated mass of the precursor protein is 16 965 Da, which is processed to yield a mature lysozyme of 14 471 Da with a calculated pI of 10.1. The lysozyme from Ae. aegypti shared 50% amino acid identity with lysozymes from Anopheles gambiae and Anopheles darlingi, which in turn shared 70% identity between each other. Northern analysis with the lysozyme cDNA probe showed induction of a 1.3 kb messenger RNA during the first 3 h after treatment of Aag-2 cells with heat-killed bacteria, followed by maximal expression 12-36 h after treatment. Southern analysis suggested that the gene likely occurs as a single copy in the genome of Aag-2 cells.     

Comparative linkage maps for the mosquitoes, Aedes albopictus and Ae. aegypti, based on common RFLP loci

Aedes albopictus and Aedes aegypti are members of the mosquito family Culicidae and share a haploid chromosome complement of three. Although a genetic linkage map based on restriction fragment length polymorphism (RFLP), markers exists for Ae. aegypti , the extent of synteny and linkage order conservation between the two species was unknown. A comparative linkage map for Ae. albopictus was constructed based mainly on cDNA clones from Ae. aegypti. Nearly all Ae. aegypti probes hybridized to Ae. albopictus DNA at high stringency. For eighteen RFLP markers tested, the linkage group and linear order appears to be identical for the two species. 78% of the loci tested exhibited significant deviations from the expected segregation ratio in at least one of the test crosses. An excess of heterozygote genotypes was recovered with most loci. This probably reflects the effects of lethal loci on survival of F₂ progeny homozygous for the parental genotypes. These results demonstrate that comparative linkage maps based on common DNA markers provide a basis for rapidly developing linkage maps for various mosquito species, and the opportunity to examine the significance and function of orthologous quantitative trait loci associated with mosquito vector competence for disease transmission.     

Leveraging genomic databases: from an Aedes albopictus mosquito cell line to the malaria vector Anopheles gambiae via the Drosophila genome project

An important justification for genome sequencing efforts is the anticipation that data from model organisms will provide a framework for the more rapid analysis of other, less studied genomes. In this investigation, we sequenced an internal region of 25 amino acids from a 52 kDa protein that was differentially expressed in 20-hydroxyecdysone-treated Aedes albopictus cells in culture. Within the GenBank non-mouse and non-human expressed sequence tag (EST) database, this "Aedes peptide" uncovered a putative homology to hypothetical translation products from Anopheles gambiae, Caenorhabditis elegans and Drosophila melanogaster. The hypothetical translation product from D. melanogaster, which included 462 amino acids, uncovered five expressed sequence tags (ESTs) from the malaria vector, Anopheles gambiae. When the Anopheles ESTs were aligned against the hypothetical Drosophila protein, we found that in aggregate they covered 324 amino acids, with gaps measuring 19, 30, and 87 amino acids. To approximate the complete amino acid sequence, gaps between translation products from Anopheles ESTs were replaced with corresponding amino acids from Drosophila to arrive at a calculated mass of 51 104 and a pI of 5.84 for the mosquito protein, consistent with the position of the Ae. albopictus protein on two-dimensional polyacrylamide gels. Finally, tandem mass spectrometry of a tryptic digest of the 52 kDa Ae. albopictus protein revealed 33 peptides with masses within 1 Dalton of those predicted from an in silico digestion of the reconstructed Anophleles protein. In addition to providing the first direct evidence that a hypothetical protein in Drosophila is in fact translated, this analysis provides a general approach for maximizing recovery, from existing databases, of information that can facilitate prioritization of efforts among several candidate proteins.     

Genomic and evolutionary analyses of Tango transposons in Aedes aegypti, Anopheles gambiae and other mosquito species

Tango is a transposon of the Tc1 family and was originally discovered in the African malaria mosquito, Anopheles gambiae. Here we report a systematic analysis of the genome sequence of the yellow fever mosquito, Aedes aegypti, which uncovered three distinct Tango transposons. We name the only An. gambiae Tango transposon AgTango1 and the three Ae. aegypti Tango elements AeTango1-3. Like AgTango1, AeTango1 and AeTango2 elements both have members that retain characteristics of autonomous elements such as intact open reading frames and terminal inverted repeats (TIRs). AeTango3 is a degenerate transposon with no full-length members. All full-length Tango transposons contain subterminal direct repeats within their TIRs. AgTango1 and AeTango1-3 form a single clade among other Tc1 transposons. Within this clade, AgTango1 and AeTango1 are closely related and share approximately 80% identity at the amino acid level, which exceeds the level of similarity of the majority of host genes in the two species. A survey of Tango in other mosquito species was carried out using degenerate PCR. Tango was isolated and sequenced in all members of the An. gambiae species complex, Aedes albopictus and Ochlerotatus atropalpus. Oc. atropalpus contains a rich diversity of Tango elements, while Tango elements in Ae. albopictus and the An. gambiae species complex all belong to Tango1. No Tango was detected in Culex pipiens quinquefasciatus, Anopheles stephensi, Anopheles dirus, Anopheles farauti or Anopheles albimanus using degenerate PCR. Bioinformatic searches of the Cx. p. quinquefasciatus (~10 x coverage) and An. stephensi (0.33 x coverage) databases also failed to uncover any Tango elements. Although other evolutionary scenarios cannot be ruled out, there are indications that Tango1 underwent horizontal transfer among divergent mosquito species.     

Mosquito hexamerins: characterization during larval development

We report here the first examination of hexamerins expressed during mosquito larval development. Haemolymph proteins from fourth-instar larvae of six species representing the two major subfamilies of mosquitoes were characterized by immunoblotting using antisera to calliphorin, the major hexamerin of the blowfly, Calliphora vicina , or to LSP1 or LSP2, the two distinct hexamerins of Drosophila melanogaster . In each mosquito species the antisera demonstrated the presence of multiple abundant hexamerin polypeptides of 66–85 kDa in molecular weight. According to the subunit composition of native proteins, the larval hexamerins from both Aedes aegypti and Anopheles gambiae form heterohexamers. Furthermore, the two major Aedes hexamerin subunits (AaHex1 and AaHex2) are neither rich in aromatic amino acids nor methionine. cDNA clones encoding AaHex1 and AaHex2 were isolated and used to show that hexamerin mRNA is uniquely expressed in fourth-instar larvae of both A. aegypti and A. gambiae and disappears rapidly at the onset of pupal development.     

The mosquito ribonucleotide reductase R2 gene: ultraviolet light induces expression of a novel R2 variant with an internal amino acid deletion

Abstract Using RT-PCR, we examined expression of the ribonucleotide reductase R2 subunit (RNR-R2) in Aedes albopictus mosquito cells after treatment with ultraviolet light (UV). In control cells, a predominant band at 1.2 kb corresponded to the full-length cDNA. A smaller 650 bp band was unique to UV-treated cells. Sequence analysis showed that the 650 bp band encoded a protein with an internal deletion of 179 amino acids, relative to Ae. albopictus RNR-R2. The N-terminal twenty amino acids were identical between AalRNR-R2 and AalDeltaR2; downstream of the deletion, the proteins differed at only four residues. In AalDeltaR2, the internal deletion spanned five residues critical to RNR-R2 enzymatic activity, including a key tyrosine residue that generates an essential free radical. The full-length 46 kDa and truncated 25 kDa RNR-R2 proteins were shown to be expressed on Western blots, and to differ in their subcellular localization. Similarly, expression of the two proteins was differentially regulated during the cell cycle, and expression of AalDeltaR2 predominated after UV treatment. AalDeltaR2 resembled a human RNR-R2 variant called p53R2, which was induced by agents that damage DNA. As was the case with p53R2 and its antisense RNA, levels of AalDeltaR2 were diminished after treatment of mosquito cells with RNAi corresponding to p53 from Drosophila melanogaster. Examination of the AalRNR-R2 homologue in the Anopheles gambiae genome suggested that AalDeltaR2 resulted from precise splicing between Exons 1, 4 and 5, eliminating Exons 2 and 3. The likelihood that AalDeltaR2 is a non-enzymatic, functional participant in DNA metabolism is suggested by enhancement of DNA repair in an in vitro system and by the presence of a similar gene (rnr4) in yeast.     

Linkage map organization of expressed sequence tags and sequence tagged sites in the mosquito,Aedes aegypti

A composite genetic linkage map for the yellow fever mosquito Aedes aegypti was constructed based on restriction fragment length polymorphism (RFLP), single nucleotide polymorphism (SNP) and single strand conformation polymorphism (SSCP) markers. The map consists of 146 marker loci distributed across 205 cM, and includes several morphological mutant marker loci. Most of the genetic markers are derived from random cDNAs or Ae. aegypti genes of known function. A number of markers are derived from random genomic DNAs, including several cloned RAPD-PCR fragments, and also several cDNAs from Drosophila melanogaster. Most of the random cDNAs (80.2%) have high BlastX sequence identities to known genes, with the majority of matches to genes from D. melanogaster. Access to sequence data for all markers will facilitate their continued development for use in high-throughput SNP marker analyses and also provides additional physical anchor points for an anticipated genome sequencing effort.     

Identification of odorant-binding proteins of the yellow fever mosquito Aedes aegypti: genome annotation and comparative analyses

The yellow fever mosquito Aedes aegypti is an important human health pest which vectors yellow fever and dengue viruses. Olfaction plays a crucial role in its attraction to hosts and although the molecular basis of this is not well understood it is likely that odorant-binding proteins (OBPs) are involved in the first step of molecular recognition. Based on the OBPs of Drosophila melanogaster and Anopheles gambiae we have defined sequence motifs based on OBP conserved cysteine and developed an algorithm which has allowed us to identify 66 genes encoding putative OBPs from the genome sequence and expressed sequence tags (ESTs) of Ae. aegypti. We have also identified 11 new OBP genes for An. gambiae. We have examined all of the corresponding peptide sequences for the properties of OBPs. The predicted molecular weights fall within the expected range but the predicted isoeletric points are spread over a wider range than found previously. Comparative analyses of the 66 OBP sequences of Ae. aegypti with other dipteran species reveal some mosquito-specific genes as well as conserved homologues. The genomic organisation of Ae. aegypti OBPs suggests that a rapid expansion of OBPs has occurred, probably by gene duplication. The analyses of OBP-containing regions for microsynteny indicate a very high synteny between Ae. aegypti and An. gambiae.     

Mosquito dopa decarboxylase cDNA characterization and blood-meal-induced ovarian expression

Dopa decarboxylase (DDC) functions in insect cate-cholamine biochemistry to produce materials essential for cross-linking reactions that result in tanning and/or melanitation, including tanning of the mosquito egg chorion and encapsulation of parasites. We have cloned Ddc from the mosquito, Aedes aegypti , and studied its expression in response to blood-feeding, which initiates events necessary for egg maturation in mosquitoes. The Ae. aegypti Ddc cDNA was isolated via heterologous screening using a clone from Drosophila melanogaster . A resulting 1.87 kilobase (kb) clone was sequenced to reveal an open reading frame of 1464 bp, as well as 5'- and 3'-untranslated segments. The inferred amino acid sequence of this clone shares 81% identity with the published Drosophila Ddc cDNA, including complete identity with twenty-four contiguous amino acids encompassing the pyridoxal-5-phosphate cofactor binding domain. Analysis of an F2 intercross population derived from a parental cross between two Ae. aegypti strains (Hamburg and Moyo-In-Dry) allowed us to map Ddc to a locus on linkage group 2. Expression studies demonstrated the presence of a 2.1 kb message, the majority of which occurs in the ovaries where Ddc-specific mRNA is up-regulated in response to Ingestion of a blood meal. The potential for egg-tanning in anautogenous mosquitoes as a model for understanding specific genetic events in the regulation of catecholamine metabolism is addressed.     

Molecular analysis of the serine/threonine kinase Akt and its expression in the mosquito Aedes aegypti

A key component of the insulin-signalling pathway, the protein kinase Akt, was identified and cloned as a cDNA from ovaries of the mosquito Aedes aegypti. An ortholog gene was found in the Anopheles gambiae genome database, and like other Akts, both mosquito Akts possess pleckstrin homology domains for membrane binding and a serine/threonine kinase domain. When Ae. aegypti ovaries were treated with bovine insulin in vitro, a putative Akt was threonine-phosphorylated, as expected for Akts. AaegAKT was only expressed in embryos for the first 6 h after oviposition and in ovaries before and during a gonotrophic cycle.     

Characterization of a carboxypeptidase A gene from the mosquito, Aedes aegypti

A gut-specific carboxypeptidase A gene (AeCPA) from the mosquito, Aedes aegypti, was cloned and characterized. The gene has an open reading frame that predicts a protein of 427 amino acids, 61% of which are identical to an Anopheles gambiae carboxypeptidase A sequence. AeCPA messenger RNA (mRNA) was not detected during larval and pupal development. In situ hybridization experiments indicated that AeCPA mRNA is expressed by posterior midgut epithelial cells. In sharp contrast to An. gambiae carboxypeptidase A gene expression, AeCPA mRNA accumulates to high levels only late ( approximately 16-24 h) after ingestion of a blood meal. The temporal profile of AeCPA gene induction is similar to that of Ae. aegypti late trypsin, suggesting the existence of common regulatory elements.     

A cluster of four D7-related genes is expressed in the salivary glands of the African malaria vector Anopheles gambiae

Four genes expressed in the Anopheles gambiae adult female salivary glands and similar in sequence to the Aedes aegypti D7 gene were identified. The genes, called D7-related (D7r), are included in a single cluster encompassing approximately six kilobases on chromosome arm 3R. The deduced proteins contain secretory signals and they are probably injected by the mosquito into the host with the saliva during blood feeding. The region of similarity to D7 encompasses the carboxy-terminal part of the Ae. aegypti protein and the different An. gambiae D7r show a degree of similarity to each other, varying from 53% to 73%. The weak but significant similarity to members of a wide family of insect proteins, including odourant- and pheromone-binding proteins, raises the possibility that the D7r-encoded proteins may bind and/or carry small hydrophobic ligands.     

Molecular cloning and expression of the dihydrofolate reductase (DHFR) gene from adult buffalo fly (Haematobia irritans exigua): effects of antifolates

The folate analogues methotrexate, aminopterin and pyrimethamine were toxic when fed in a blood meal to adult buffalo flies (Haematobia irritans exigua), but aminopterin caused greater mortality than methotrexate, while trimethoprim was not toxic to adult flies. This is the first recorded instance of mortality in adult insects caused by ingestion of folate analogues. In order to investigate the mechanism of this toxicity, the dihydrofolate reductase (DHFR) gene was cloned from adult buffalo fly cDNA using a PCR-based approach. The full-length DHFR coding sequence (BF-DHFR) was 887 bp and contained an open reading frame encoding a protein of 188 amino acids. The deduced protein sequence identities between BF-DHFR and the other known insect DHFR sequences were: Drosophila melanogaster, 75%; Aedes albopictus, 54%; Heliothis virescens, 43%. The BF-DHFR gene has a single 52 bp intron, an organization more similar to Dipteran species (Drosophila and Aedes). The cDNA encoding BF-DHFR was inserted into an Escherichia coli expression vector and the recombinant protein was expressed to levels representing about 25% of total cell protein. The active enzyme was purified by affinity chromatography on methotrexate-agarose and displayed a relatively low affinity (IC50 = 30 nm) for methotrexate.