Mitochondrial Genomes of Anopheles (Kerteszia) (Diptera: Culicidae) From the Atlantic Forest, Brazil.

Mitochondrial genome sequences are widely used as molecular markers for phylogenetic studies of mosquito species complexes, such as the Anopheles albitarsis complex. Except for a few studies that employed a limited number of nuclear or mitochondrial loci to address the genetic structure and species status of Anopheles cruzii, Anopheles bellator, and Anopheles homunculus, little is known about genetic markers that can be employed in studies focusing on Kerteszia species. The complete mitochondrial genomes of seven specimens of An. bellator, An. cruzii, An. homunculus, and Anopheles laneanus were sequenced using long-range polymerase chain reaction and Illumina sequencing. The mitochondrial genomes varied from 15,446 to 15,738 bp in length and contained 37 genes (13 protein-encoding genes, 2 rRNA genes [12S rRNA and 16S rRNA] and 22 tRNA genes), and the AT-rich control region, as all do other Anopheles mitochondrial genomes sequenced to date. Specimens from four populations of An. cruzii showed differences in codon composition.

Mapping the Anopheles gambiae odorant binding protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays.

The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics.

Transgene-mediated suppression of dengue viruses in the salivary glands of the yellow fever mosquito, Aedes aegypti.

Controlled sex-, stage- and tissue-specific expression of antipathogen effector molecules is important for genetic engineering strategies to control mosquito-borne diseases. Adult female salivary glands are involved in pathogen transmission to human hosts and are target sites for expression of antipathogen effector molecules. The Aedes aegypti 30K a and 30K b genes are expressed exclusively in adult female salivary glands and are transcribed divergently from start sites separated by 263 nucleotides. The intergenic, 5'- and 3'-end untranslated regions of both genes are sufficient to express simultaneously two different transgene products in the distal-lateral lobes of the female salivary glands. An antidengue effector gene, membranes no protein (Mnp), driven by the 30K b promoter, expresses an inverted-repeat RNA with sequences derived from the premembrane protein-encoding region of the dengue virus serotype 2 genome and reduces significantly the prevalence and mean intensities of viral infection in mosquito salivary glands and saliva.

Comparative fitness assessment of Anopheles stephensi transgenic lines receptive to site-specific integration.

Genetically modified mosquitoes that are unable to transmit pathogens offer opportunities for controlling vector-borne diseases such as malaria and dengue. Site-specific gene recombination technologies are advantageous in the development of these insects because antipathogen effector genes can be inserted at integration sites in the genome that cause the least alteration in mosquito fitness. Here we describe Anopheles stephensi transgenic lines containing phi C31 attP'docking' sites linked to a fluorescent marker gene. Chromosomal insertion sites were determined and life-table parameters were assessed for transgenic mosquitoes of each line. No significant differences in fitness between the transgenic and nontransgenic mosquitoes were detected in this study. These transgenic lines are suitable for future site-specific integrations of antiparasite transgenes into the attP sites.

Gene structure and expression of nanos (nos) and oskar (osk) orthologues of the vector mosquito, Culex quinquefasciatus.

The products of the maternal-effect genes, nanos (nos) and oskar (osk), are important for the development of germ cells in insects. Furthermore, these genes have been proposed as candidates for donating functional DNA regulatory sequences for use in gene drive systems to control transmission of mosquito-borne pathogens. The nos and osk genes of the cosmopolitan vector mosquito, Culex quinquefasciatus, encode proteins with domains common to orthologues found in other mosquitoes. Expression analyses support the conclusion that the role of these genes is conserved generally among members of the nematocera. Hybridization in situ analyses reveal differences in mRNA distribution in early embryos in comparison with the cyclorraphan, Drosophila melanogaster, highlighting a possible feature in the divergence of the clades each insect represents.

Taxonomic and phylogenetic relationships between neotropical species of ticks from genus Amblyomma (Acari: Ixodidae) inferred from second internal transcribed spacer sequences of rDNA.

The accurate specific identification of ticks is essential for the study, control and prevention of tick-borne diseases. Herein, we determined ribosomal nucleotide sequences of the second internal transcribed spacer (ITS2) of 15 Neotropical hard tick species of the genus Amblyomma Koch found in Brazil. Most of the studied ticks accidentally parasite humans and potentially act as vectors of zoonoses. Lengths of the ITS2 sequences ranged from 956 to 1,207 bp, whereas GC content varied from 62.4 to 66.9%. A matrix of ITS2 divergence was calculated with the ITS2 sequence data obtained showing divergence levels varying from 1.5 to 28.8%. The analysis indicated that this molecular marker can be useful for Amblyomma-specific identification. Phylogenetic inferences based on the ITS2 sequences were used to assess some issues in subgenus taxonomy.

Genome-wide analysis of gene expression in adult Anopheles gambiae.

With their genome sequenced, Anopheles gambiae mosquitoes now serve as a powerful tool for basic research in comparative, evolutionary and developmental biology. The knowledge generated by these studies is expected to reveal molecular targets for novel vector control and pathogen transmission blocking strategies. Comparisons of gene-expression profiles between adult male and nonblood-fed female Anopheles gambiae mosquitoes revealed that roughly 22% of the genes showed sex-dependent regulation. Blood-fed females switch the majority of their metabolism to blood digestion and egg formation within 3 h after the meal is ingested, in detriment to other activities such as flight and response to environment stimuli. Changes in gene expression are most evident during the first, second and third days after a blood meal, when as many as 50% of all genes showed significant variation in transcript accumulation. After laying the first cluster of eggs (between 72 and 96 h after the blood meal), mosquitoes return to a nongonotrophic stage, similar but not identical to that of 3-day-old nonblood-fed females. Ageing and/or the nutritional state of mosquitoes at 15 days after a blood meal is reflected by the down-regulation of approximately 5% of all genes. A full description of the large number of genes regulated at each analysed time point and each biochemical pathway or biological processes in which they are involved is not possible within the scope of this contribution. Therefore, we present descriptions of groups of genes displaying major differences in transcript accumulation during the adult mosquito life. However, a publicly available searchable database (http://www.angagepuci.bio.uci.edu/) has been made available so that detailed analyses of specific groups of genes based on their descriptions, functions or levels of gene expression variation can be performed by interested investigators according to their needs.

Microarray analysis of genes showing variable expression following a blood meal in Anopheles gambiae.

A microarray analysis of 14 900 genes of the malaria vector mosquito, Anopheles gambiae, shows that as many as 33% (4924) of their corresponding transcription products vary in abundance within 24 h after a blood meal. Approximately half (2388) of these products increase in their accumulation and the remainder (2536) decrease. Expression dynamics of 80% of the genes analysed by expressed sequence tag (EST) projects reported previously are consistent with the observations from this microarray analysis. Furthermore, the microarray analysis is more sensitive in detecting variation in abundance of gene products expressed at low levels and is more sensitive overall in that a greater number of regulated genes are detected. Major changes in transcript abundance were seen in genes encoding proteins involved in digestion, oogenesis and locomotion. The microarray data and an electronic hyperlinked version of all tables are available to the research community at http://www.angagepuci.bio.uci.edu/1/.

Amazonian malaria vector anopheline relationships interpreted from ITS2 rDNA sequences.

Species identification of anopheline mosquitoes (Diptera: Culicidae) can be problematic because many of them belong to complexes of morphologically similar species, often with contrasted ecology, behaviour and vectorial importance. The application of DNA-based diagnostics has proved to be useful for distinguishing between such species. We determined ribosomal DNA sequences of the second internal transcribed spacer (ITS2) from samples of 16 species of Anopheles captured in the Amazon Basin, Brazil. Length of the ITS2 varied from 323 to 410 base pairs, with GC content ranging from 50.7% to 66.5% and sequence identity from 25% to 99% between species. Maximum-likelihood paup analysis separated two distinct groups of species conforming with the recognized subgenera Anopheles (represented by eiseni, mattogrossensis, mediopunctatus and peryassui) and Nyssorhynchus (represented by 12 spp.). For the latter group, the neighbour-joining tree generated from rDNA sequence ITS2 relationships is compatible with the morphological taxonomic key established for these Amazonian species: albitarsis, aquasalis, benarrochi, braziliensis, darlingi, deaneorum, dunhami, evansae, nuneztovari, oswaldoi, rangeli and triannulatus. These ITS2 sequence data proved to be a useful tool for species identification and, potentially, to solve taxonomic problems.

The accumulation of specific mRNAs following multiple blood meals in Anopheles gambiae.

One approach to genetic control of transmission of the parasites that cause human malaria is based on expressing effector genes in mosquitoes that disable the pathogens. Endogenous mosquito promoter and other cis-acting DNA sequences are needed to direct the optimal tissue-, stage- and sex-specific expression of the effector molecules. The mRNA accumulation profiles of eight different genes expressed specifically in the midgut, salivary glands or fat body tissues of the malaria vector, Anopheles gambiae, were characterized as a measure of their suitability to direct the expression of effector molecules designed to disable specific stages of the parasites. RT-PCR techniques were used to determine the abundance of the gene products and their duration following multiple blood meals. Transcription from the midgut-expressed carboxypeptidase-encoding gene, AgCP, follows a cyclical, blood-inducible expression pattern with maximum accumulation every 3 h post blood meal. Other midgut-expressed genes encoding a trypsin and chymotrypsin, Antryp2 and Anchym1, respectively, and the fat body-expressed genes, Vg1 and Cathepsin, also show a blood-inducible pattern of expression with maximum accumulation 24 h after every blood meal. Expression of the Lipophorin gene in the fat body and apyrase and D7-related genes (AgApy and D7r2) in the salivary glands is constitutive and not significantly affected by blood meals. Promoters of the midgut- and fat body-expressed genes may lead to maximum accumulation of antiparasite effector molecule transcripts after multiple blood meals. The multiple feeding behaviour of An. gambiae thus can be an advantage to express high levels of antiparasite effector molecules to counteract the parasites throughout most of adult development.

The AeAct-4 gene is expressed in the developing flight muscles of female Aedes aegypti.

Population reduction of mosquitoes is an effective method for controlling dengue fever and malaria transmission. Recent developments in control techniques include proposals to construct transgenic strains of mosquitoes carrying dominant, conditional-lethal genes under the control of sex- and stage-specific promoters. In order to identify such promoters, subtractive cDNA libraries derived from male and female pupal mRNA of the yellow fever mosquito, Aedes aegypti, were constructed and screened. A cDNA clone, F49, corresponds to a gene expressed specifically in female pupae. Sequence analyses revealed that this gene belongs to the actin gene family, and therefore was designated Aedes Actin-4 (AeAct-4). Transcription analyses demonstrated that this gene is expressed predominantly in the indirect flight muscles and, to a lesser extent, the legs of developing female mosquitoes. The promoter of this gene may be a useful tool for developing conditional lethal strains of mosquitoes.

The transcriptome of adult female Anopheles darlingi salivary glands.

Anopheles (Nyssorhynchus) darlingi is an important malaria vector in South and Central America; however, little is known about molecular aspects of its biology. Genomic and proteomic analyses were performed on the salivary gland products of Anopheles darlingi. A total of 593 randomly selected, salivary gland-derived cDNAs were sequenced and assembled based on their similarities into 288 clusters. The putative translated proteins were classified into three categories: (S) secretory products, (H) housekeeping products and (U) products with unknown cell location and function. Ninety-three clusters encode putative secreted proteins and several of them, such as an anophelin, a thrombin inhibitor, apyrases and several new members of the D7 protein family, were identified as molecules involved in haematophagy. Sugar-feeding related enzymes (alpha-glucosidases and alpha-amylase) also were found among the secreted salivary products. Ninety-nine clusters encode housekeeping proteins associated with energy metabolism, protein synthesis, signal transduction and other cellular functions. Ninety-seven clusters encode proteins with no similarity with known proteins. Comparison of the sequence divergence of the S and H categories of proteins of An. darlingi and An. gambiae revealed that the salivary proteins are less conserved than the housekeeping proteins, and therefore are changing at a faster evolutionary rate. Tabular and supplementary material containing the cDNA sequences and annotations are available at http://www.ncbi.nlm.nih.gov/projects/Mosquito/A_darlingi_sialome/

Primary characterization and basal promoter activity of two hexamerin genes of Musca domestica.

Hexamerins are high molecular-weight proteins found in the hemolymph of insects and have been proposed to function as storage proteins. In previous studies, two Musca domestica hexamerins, designated Hex-L and Hex-F were characterized. Hex-L is synthesized exclusively by the larval fat bodies, is secreted into the hemolymph and likely provides a source of amino acids and energy during metamorphosis. Hex-F synthesis is induced by a proteinaceous meal and occurs only in the adult insect fat bodies. Hex-F also is secreted into the hemolymph and it has been suggested that in females it may be an amino acid reservoir to be used during the final stages of egg formation. Genomic clones containing full-length copies of the genes MdHexL1 and MdHexF1, encoding subunits of the larval and the adult female hexamerin, respectively, were isolated. Complete nucleotide sequences, including the 5'-end untranscribed regions, were determined and analyzed for each of the genes. Comparisons of the conceptual translation products of the cloned genes indicated that MdHexL1 and MdHexF1 are related to the larval serum proteins (LSP) 1 and 2 of Calliphora vicina and Drosophila melanogaster. DNA fragments containing the putative promoters of the two hexamerin genes were compared and cloned into a plasmid vector so as to drive the expression of the GFP reporter gene. The constructs were assayed in vitro in transfected S2 Drosophila melanogaster cells demonstrating that the cloned M. domestica DNA fragments exhibit promoter activity.

Analysis of the wild-type and mutant genes encoding the enzyme kynurenine monooxygenase of the yellow fever mosquito, Aedes aegypti.

Kynurenine 3-monooxygenase (KMO) catalyses the hydroxylation of kynurenine to 3-hydroxykynurenine. KMO has a key role in tryptophan catabolism and synthesis of ommochrome pigments in mosquitoes. The gene encoding this enzyme in the yellow fever mosquito, Aedes aegypti, is called kynurenine hydroxylase (kh) and a mutant allele that produces white eyes has been designated khw. A number of cDNA clones representative of wild-type and mutant genes were isolated. Sequence analyses of the wild-type and mutant cDNAs revealed a deletion of 162 nucleotides in the mutant gene near the 3'-end of the deduced coding region. RT-PCR analyses confirm the transcription of a truncated mRNA in the mutant strain. The in-frame deletion results in a loss of 54 amino acids, which disrupts a major alpha-helix and which probably accounts for the loss of activity of the enzyme. Recombinant Ae. aegypti KMO showed high substrate specificity for kynurenine with optimum activity at 40 degrees C and pH = 7.5. Kinetic parameters and inhibition of KMO activity by Cl- and pyridoxal-5-phosphate were determined.

The Musca domestica larval hexamerin is composed of multiple, similar polypeptides.

The Musca domestica larval hexamerin (MdHex-L) is a hexameric glycoprotein with an apparent native molecular weight of 500 kDa. Seven different cDNAs that encode MdHex-L subunits were cloned and sequenced. Furthermore, amino acid sequences of isolated subunits were determined by the Edman degradation method and compared to the conceptual translation products derived from the cloned cDNAs. The obtained data indicate the existence of multiple forms of MdHex-L subunits and that these multiple forms may be grouped into three categories according to their percentages of nucleotide sequence identity.

Two digestive trypsins occur in three species of neotropical anophelines.

Trypsin activity increases in the midgut of Anopheles aquasalis, Anopheles albitarsis, and Anopheles darlingi after a bloodmeal. The activity returns to basal levels at the time the blood is completely digested. Affinity chromatography, reversed-phase high performance liquid chromatography (HPLC), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were used to sequentially purify the mosquito trypsins found in the midguts at 24 h after feeding. Aminoterminal sequencing of the purified trypsins showed the occurrence of two distinct trypsins in the midgut of each of the mosquitoes studied. The sequences obtained are similar to those of the trypsins of other hematophagous insects.

The D7 family of salivary proteins in blood sucking diptera.

The D7 subfamily of salivary proteins is widespread in blood sucking Diptera and belongs to the superfamily of pheromone/odourant binding proteins. Although D7 proteins are among the most abundant salivary proteins in adult female mosquitoes and sand flies, their role in blood feeding remains elusive. In the present work we report the sequence of seventeen novel D7 proteins, and propose an evolutionary scenario for the appearance of the several forms of this protein, based on a total of twenty-one sequences from Culex quinquefasciatus, Aedes aegypti, Anopheles gambiae, An. arabiensis, An. stephensi, An. darlingi mosquitoes and Lutzomyia longipalpis and Phlebotomus papatasi sand flies.

The major acid soluble proteins of adult female Anopheles darlingi salivary glands include a member of the D7-related family of proteins.

The salivary gland proteins of adult female Anopheles darlingi were fractionated by reverse-phase HPLC and the five major peaks were submitted for amino-terminal sequencing using automated Edman degradation. The amino acid sequence of one of the purified salivary gland proteins showed similarity with the D7r3 protein of An. gambiae. Cloning and sequencing of two cDNAs allowed the prediction of the complete sequence of the An. darlingi D7 protein. The D7r3 protein is present specifically in adult female salivary glands of An. darlingi and despite being one of the major salivary gland proteins its function is not known. Predictions of secondary and tertiary structures revealed the similarity of the An. darlingi D7 protein to insect odorant binding proteins. This suggests that D7 proteins may act as carriers of hydrophobic molecules in mosquito saliva.