Biomedical subjects

O Marinotti

Publications and source records attributed to O Marinotti.

At least 19 recordsLinked to original sources

PubMed2006

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.

Aging↗

PubMed2005

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.

Animals↗

PubMed2005

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.

Animals↗

PubMed2005

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/.

Animals↗

PubMed2004

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.

Amino Acid Sequence↗

PubMed2004

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.

Actins↗

PubMed2004

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/

Animals↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed2003

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.

Aedes↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed2002

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.

Amino Acid Sequence↗

PubMed2002

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.

Aedes↗

PubMed2001

Analysis of salivary gland proteins of the mosquito Anopheles darlingi (Diptera: Culicidae).

The salivary proteins of Anopheles darlingi Root, the principal vector of malaria in the Amazon Region, Brazil, were analyzed. Comparison of the protein profiles between adult males and females revealed that most of the polypeptides are present in both sexes, but female-specific polypeptides also were observed. SDS-PAGE analysis of sugar-fed female mosquitoes with ages varying from 1 to 10 d after adult emergence indicated that the proteins start to be accumulated in the first day of life and are present throughout the period analyzed. Analysis of blood-fed mosquitoes showed no differences in salivary proteins when compared with sugar fed ones, suggesting that there is no specific protein induced by blood. The protein profiles of the salivary glands dissected from wild-caught female mosquitoes from different geographical regions of Brazil were compared and some differences were observed.

Animals↗

PubMed2000

Salivary gland proteins of the mosquito Culex quinquefasciatus.

Several properties of the salivary glands of Culex quinquefasciatus mosquitoes were analysed. The amount of protein in female salivary glands increased from 0.26 microg on day one after emergence to about 1.4 microg on day seven. The major polypeptides found in the female salivary glands had molecular weights of 35.7, 28.3, and 20.5 kDa. Antibodies produced by mice immunized by bites of Culex quinquefasciatus female mosquitoes reacted with the 35.7 and 28.3 kDa polypeptides, showing that these molecules were secreted by mosquitoes during blood feeding. The salivary glands of C. Quinquefasciatus females displayed the same morphological and biochemical organization as that of Aedes aegypti mosquitoes, accumulating apyrase in the distal portions and alpha-glucosidase in the proximal portions of the gland. Arch.

Animals↗

PubMed2000

Expression patterns of the larval and adult hexamerin genes of Musca domestica.

Hexamerins are proteins found in high abundance in the haemolymph of larval and adult insects. The expression patterns of the genes encoding the house fly, Musca domestica, hexamerins were determined by Northern analyses using cDNAs as probes. A cDNA, A1, hybridized to a fat body-specific messenger RNA (mRNA) which is detectable in larvae until pupation. Antibodies raised to the larval-specific hexamerin, Hex-L, bind recombinant protein encoded by a 5' rapid amplification of cDNA ends (RACE) product of A1, A2, indicating that the A cDNAs likely represent the genes encoding Hex-L. The F1, F2 and F3 cDNAs, corresponding to genes encoding an adult, female-enriched hexamerin, Hex-F, hybridized with an mRNA isolated from protein-fed females which has a temporal expression profile similar to that observed for the accumulation of Hex-F. Furthermore, expression of the mRNAs hybridizing to the F cDNAs is correlated with the abundance of Hex-F protein during the gonotrophic cycles. The mRNA transcription profiles indicate that the Hex-L and Hex-F genes are regulated in a sex-, tissue- and developmental phase-dependent manner. This stage-specific expression of hexamerins contrasts with the expression patterns of hexamerins seen in other insects. The conceptual translation products of larval hexamerin cDNAs showed identity with larval serum protein 1 (LSP1)-type hexamerins while the deduced products of the female hexamerin cDNAs showed the highest identity with LSP2-type hexamerins. Genomic analyses showed that the larval hexamerin and female hexamerin genes from M. domestica belong to two distinct multigenic families.

Amino Acid Sequence↗

PubMed1999

Morphological and biochemical analyses of the salivary glands of the malaria vector, Anopheles darlingi.

Adult Anopheles darlingi salivary glands are paired organs located on either side of the esophagus. The male glands consist of a single small lobe. The female gland is composed of two lateral lobes, with distinct proximal and distal portions, and a medial lobe. The lobes are acinar structures, organized as a unicellular epithelium that surrounds a salivary canal. The general cellular architecture is similar among the lobes, with secretory material appearing as large masses that push the cellular structures to the periphery of the organ. Cells of the proximal-lateral lobes show asynchronous cycles of secretory activity and contain secretory masses with finely filamentous aspect. In the distal-lateral lobes, cells display synchronous cycles of activity, and have a dense secretory product with mottled pattern. Cells of the medial lobe have secretory masses uniformly stained and highly electrondense. Biochemical analysis of the adult female salivary glands revealed apyrase, alpha-glucosidase and lysozyme activities. Alpha-glucosidase and lysozyme activities are detected mostly in the proximal lobes while apyrase is mainly accumulated in the distal lobes. This differential distribution of the analyzed enzymes reflects a specialization of different regions for sugar and blood feeding. Thus, the morphological differences observed in the lobes correlate with functional ones.

Animals↗

PubMed1999

Analysis of ITS2 DNA sequences from Brazilian Anopheles darlingi (Diptera: Culicidae).

Specimens of Anopheles darlingi Root, the major vector of malaria in Brazil, were collected from several states in Brazil: Sao Paulo (Dourado), Bahia (Itabela), Rondônia (Porto Velho), Roraima (Boa Vista), and Acre (Plácido de Castro). Sequence divergence in the 2nd internal transcribed spacer (ITS2) was examined. The ITS2 sequences of mosquitoes captured in the Amazon region (Porto Velho, Boa Vista and Plácido de Castro) and in the northeast of Brazil (Itabela) were almost identical; however, a 4-5% sequence divergence was observed in the ITS2 of mosquitoes captured in the southeast (Dourado). Further analysis is needed to determine if these differences indicate that Dourado population may be a separate species.

Animals↗

PubMed1999

Sequence analysis of the second internal transcribed spacer of ribosomal DNA in Anopheles oswaldoi (Diptera: Culicidae).

Sequence divergence in the second internal transcribed spacer (ITS2) of ribosomal DNA was examined for female specimens of Anopheles oswaldoi Peryassu from 7 localities in South America. The lengths of ITS2 for all mosquitoes ranged from 348 to 356 nucleotides. After alignment of these sequences, similarity ranged from 87 to 100%. Divergence was within the range of inter-specific differences for members of anopheline species complexes. Therefore, specimens were placed into 4 groups that may correspond to at least 4 cryptic species. One is probably related to An. oswaldoi sensu stricto and another to Anopheles konderi Galvão & Damasceno. The other 2 groups may correspond to species for which morphological identification remains to be clarified. These data provide evidence that An. oswaldoi comprise a complex of cryptic species and that DNA identification may help to resolve the taxonomic questions related to this group.

Animals↗