The characterization of the Phlebotomus papatasi transcriptome.

As important vectors of human disease, phlebotomine sand flies are of global significance to human health, transmitting several emerging and re-emerging infectious diseases. The most devastating of the sand fly transmitted infections are the leishmaniases, causing significant mortality and morbidity in both the Old and New World. Here we present the first global transcriptome analysis of the Old World vector of cutaneous leishmaniasis, Phlebotomus papatasi (Scopoli) and compare this transcriptome to that of the New World vector of visceral leishmaniasis, Lutzomyia longipalpis. A normalized cDNA library was constructed using pooled mRNA from Phlebotomus papatasi larvae, pupae, adult males and females fed sugar, blood, or blood infected with Leishmania major. A total of 47 615 generated sequences was cleaned and assembled into 17 120 unique transcripts. Of the assembled sequences, 50% (8837 sequences) were classified using Gene Ontology (GO) terms. This collection of transcripts is comprehensive, as demonstrated by the high number of different GO categories. An in-depth analysis revealed 245 sequences with putative homology to proteins involved in blood and sugar digestion, immune response and peritrophic matrix formation. Twelve of the novel genes, including one trypsin, two peptidoglycan recognition proteins (PGRP) and nine chymotrypsins, have a higher expression level during larval stages. Two novel chymotrypsins and one novel PGRP are abundantly expressed upon blood feeding. This study will greatly improve the available genomic resources for P. papatasi and will provide essential information for annotation of the full genome.

Transcription profiling associated with life cycle of Anopheles gambiae.

Complex biological events occur during the developmental process of the mosquito Anopheles gambiae (Giles). Using cDNA expression microarrays, the expression patterns of 13,440 clones representing 8,664 unique transcripts were revealed from six different developmental stages: early larvae (late third instar/early fourth instar), late larvae (late fourth instar), early pupae (< 30 min after pupation), late pupae (after tanning), and adult female and male mosquitoes (24 h postemergence). After microarray analysis, 560 unique transcripts were identified to show at least a fourfold up- or down-regulation in at least one developmental stage. Based on the expression patterns, these gene products were clustered into 13 groups. In total, eight genes were analyzed by quantitative real-time polymerase chain reaction to validate microarray results. Among 560 unique transcripts, 446 contigs were assigned to respective genes from the An. gambiae genome. The expression patterns and annotations of the genes in the 13 groups are discussed in the context of development including metabolism, transport, protein synthesis and degradation, cellular processes, cellular communication, intra- or extra-cellular architecture maintenance, response to stress or immune-related defense, and spermatogenesis.

A detailed genetic map for the X chromosome of the malaria vector, Anopheles gambiae.

Anopheles gambiae, the primary vector of human malaria in Africa, is responsible for approximately a million deaths per year, mostly of children. Despite its significance in disease transmission, this mosquito has not been studied extensively by genetic or molecular techniques. To facilitate studies on this vector, a genetic map has been developed that covers the X chromosome at an average resolution of 2 centimorgans. This map has been integrated with the chromosome banding pattern and used to localize a recessive, sex-linked mutation (white eye) to within 1 centimorgan of flanking markers.

The white gene of Ceratitis capitata: a phenotypic marker for germline transformation.

Reliable germline transformation is required for molecular studies and ultimately for genetic control of economically important insects, such as the Mediterranean fruit fly (medfly) Ceratitis capitata. A prerequisite for the establishment and maintenance of transformant lines is selectable or phenotypically dominant markers. To this end, a complementary DNA clone derived from the medfly white gene was isolated, which showed substantial similarity to white genes in Drosophila melanogaster and other Diptera. It is correlated with a spontaneous mutation causing white eyes in the medfly and can be used to restore partial eye color in transgenic Drosophila carrying a null mutation in the endogenous white gene.

Quantitative trait loci for refractoriness of Anopheles gambiae to Plasmodium cynomolgi B.

The severity of the malaria pandemic in the tropics is aggravated by the ongoing spread of parasite resistance to antimalarial drugs and mosquito resistance to insecticides. A strain of Anopheles gambiae, normally a major vector for human malaria in Africa, can encapsulate and kill the malaria parasites within a melanin-rich capsule in the mosquito midgut. Genetic mapping revealed one major and two minor quantitative trait loci (QTLs) for this encapsulation reaction. Understanding such antiparasite mechanisms in mosquitoes may lead to new strategies for malaria control.

Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae.

The anopheline mosquito is the target in most malaria control programs, primarily through the use of residual insecticides. A mosquito was studied that is refractory to most species of malaria through a genetically controlled mechanism. A strain of Anopheles gambiae, which was selected for complete refractoriness to the simian malaria parasite Plasmodium cynomolgi, also has varying degrees of refractoriness to most other malaria species examined, including the human parasites P. falciparum, P. ovale, and P. vivax for which this mosquito is the principal African vector. Furthermore, the refractoriness extends to other subhuman primate malarias, to rodent malaria, and to avian malaria. Refractoriness is manifested by encapsulation of the malaria ookinete after it completes its passage through the mosquito midgut, approximately 16 to 24 hours after ingestion of an infective blood meal. Fully encapsulated ookinetes show no abnormalities in parasite organelles, suggesting that refractoriness is due to an enhanced ability of the host to recognize the living parasite rather than to a passive encapsulation of a dead or dying parasite. Production of fully refractory and fully susceptible mosquito strains was achieved through a short series of selective breeding steps. This result indicates a relatively simple genetic basis for refractoriness. In addition to the value these strains may serve in general studies of insect immune mechanisms, this finding encourages consideration of genetic manipulation of natural vector populations as a malaria control strategy.

Anatomical ontologies of mosquitoes and ticks, and their web browsers in VectorBase.

VectorBase, an integrated, relational database that manages genomic and other genetic/biological data pertaining to arthropod vectors of disease, has recently embarked on the construction of ontologies and controlled vocabularies (CVs). It aims, thus, at providing all necessary tools for the complete annotation of vector genomes and, in particular, the annotation of functional genomic data. This task was initiated with the development of anatomical ontologies of mosquitoes and ticks, both of which were made compliant to CARO, the common anatomy reference ontology. The ontologies are complemented by the development of novel web-based browsers that can show figures for anatomical terms, something that is especially helpful for fully illustrating the controlled vocabularies of anatomy.

Distinct families of site-specific retrotransposons occupy identical positions in the rRNA genes of Anopheles gambiae.

Two distinct site-specific retrotransposon families, named RT1 and RT2, from the sibling mosquito species Anopheles gambiae and A. arabiensis, respectively, were previously identified. Both were shown to occupy identical nucleotide positions in the 28S rRNA gene and to be flanked by identical 17-bp target site duplications. Full-length representatives of each have been isolated from a single species, A. gambiae, and the nucleotide sequences have been analyzed. Beyond insertion specificity, RT1 and RT2 share several structural and sequence features which show them to be members of the LINE-like, or non-long-terminal-repeat retrotransposon, class of reverse transcriptase-encoding mobile elements. These features include two long overlapping open reading frames (ORFs), poly(A) tails, the absence of long terminal repeats, and heterogeneous 5' truncation of most copies. The first ORF of both elements, particularly ORF1 of RT1, is glutamine rich and contains long tracts of polyglutamine reminiscent of the opa repeat. Near the carboxy ends, three cysteine-histidine motifs occur in ORF1 and one occurs in ORF2. In addition, each ORF2 contains a region of sequence similarity to reverse transcriptases and integrases. Alignments of the protein sequences from RT1 and RT2 reveal 36% identity over the length of ORF1 and 60% identity over the length of ORF2, but the elements cannot be aligned in the 5' and 3' noncoding regions. Unlike that of RT2, the 5' noncoding region of RT1 contains 3.5 copies of a 500-bp subrepeat, followed by a poly(T) tract and two imperfect 55-bp subrepeats, the second spanning the beginning of ORF1. The pattern of distribution of these elements among five siblings species in the A. gambiae complex is nonuniform. RT1 is present in laboratory and wild A. gambiae, A. arabiensis, and A. melas but has not been detected in A. quadriannulatus or A. merus. RT2 has been detected in all available members of the A. gambiae complex except A. merus. Copy number fluctuates, even among the offspring of individual wild female A. gambiae mosquitoes. These findings reflect a complex evolutionary history balancing gain and loss of copies against the coexistence of two elements competing for a conserved target site in the same species for perhaps millions of years.

An evaluation of evolutionary constraints on microsatellite loci using null alleles.

A test to evaluate constraints on the evolution of single microsatellite loci is described. The test assumes that microsatellite alleles that share the same flanking sequence constitute a series of alleles with a common descent that is distinct from alleles with a mutation in the flanking sequence. Thus two or more different series of alleles at a given locus represent the outcomes of different evolutionary processes. The higher rate of mutations within the repeat region (10(-3) or 10(-4)) compared with that of insertion/deletion or point mutations in adjacent flanking regions (10(-9)) or with that of recombination between the repeat and the point mutation (10(-6) for sequences 100 bp long) provides the rationale for this assumption. Using a two-phase, stepwise mutation model we simulated the evolution of a number of independent series of alleles and constructed the distributions of two similarity indices between pairs of these allele series. Applying this approach to empirical data from locus AG2H46 of Anopheles gambiae resulted in a significant excess of similarity between the main and the null series, indicating that constraints affect allele distribution in this locus. Practical considerations of the test are discussed.

Mapping a quantitative trait locus involved in melanotic encapsulation of foreign bodies in the malaria vector, Anopheles gambiae.

A Plasmodium-refractory strain of Anopheles gambiae melanotically encapsulates many species of Plasmodium, whereas wild-type mosquitoes are usually susceptible. This encapsulation trait can also be observed by studying the response of refractory and susceptible strains to intrathoracically injected CM-Sephadex beads. We report the results of broad-scale quantitative trait locus (QTL) mapping of the encapsulation trait using the bead model system. Interval mapping using the method of maximum likelihood identified one major QTL, Pen1. The 13.7-cM interval containing Pen1 was defined by marker AGH157 at 8E and AGH46 at 7A on 2R. Pen1 was associated with a maximum LOD score of 9.0 and accounted for 44% of the phenotypic variance in the distribution of phenotypes in the backcross. To test if this QTL is important for encapsulation of Plasmodium berghei, F2 progeny were infected with P. berghei and evaluated for degree of parasite encapsulation. For each of the two markers that define the interval containing Pen1, a significant difference of encapsulation was seen in progeny with at least one refractory allele in contrast with homozygous susceptible progeny. These results suggest that Pen1 is important for melanotic encapsulation of Plasmodium as well as beads.

An integrated genetic map of the African human malaria vector mosquito, Anopheles gambiae.

We present a genetic map based on microsatellite polymorphisms for the African human malaria vector, Anopheles gambiae. Polymorphisms in laboratory strains were detected for 89% of the tested microsatellite markers. Genotyping was performed for individual mosquitos from 13 backcross families that included 679 progeny. Three linkage groups were identified, corresponding to the three chromosomes. We added 22 new markers to the existing X chromosome map, for a total of 46 microsatellite markers spanning a distance of 48.9 cM. The second chromosome has 57 and the third 28 microsatellite markers spanning a distance of 72.4 and 93.7 cM, respectively. The overall average distance between markers is 1.6 cM (or 1.1, 1.2, and 3.2 cM for the X, second, and third chromosomes, respectively). In addition to the 131 microsatellite markers, the current map also includes a biochemical selectable markers, Dieldrin resistance (Dl), on the second chromosome and five visible markers, pink-eye (p) and white (w) on the X, collarless (c) and lunate (lu) on the second, and red-eye (r) on the third. The cytogenetic locations on the nurse cell polytene chromosomes have been determined for 47 markers, making this map an integrated tool for cytogenetic, genetic, and molecular analysis.

Patterns of mitochondrial variation within and between African malaria vectors, Anopheles gambiae and An. arabiensis, suggest extensive gene flow.

Anopheles gambiae and An. arabiensis are mosquito species responsible for most malaria transmission in sub-Saharan Africa. They are also closely related sibling species that share chromosomal and molecular polymorphisms as a consequence of incomplete lineage sorting or introgressive hybridization. To help resolve these processes, this study examined the partitioning of mtDNA sequence variation within and between species across Africa, from both population genetic and phylogeographic perspectives. Based on partial gene sequences from the cytochrome b, ND1 and ND5 genes, haplotype diversity was high but sequences were very closely related. Within species, little or no population subdivision was detected, and there was no evidence for isolation by distance. Between species, there were no fixed nucleotide differences, a high proportion of shared polymorphisms, and eight haplotypes in common over distances as great as 6000 km. Only one of 16 shared polymorphisms led to an amino acid difference, and there was no compelling evidence for nonneutral variation. Parsimony networks constructed of haplotypes from both species revealed no correspondence of haplotype with either geography or taxonomy. This trend of low intraspecific genetic divergence is consistent with evidence from allozyme and microsatellite data and is interpreted in terms of both extensive gene flow and recent range expansion from relatively large, stable populations. We argue that retention of ancestral polymorphisms is a plausible but insufficient explanation for low interspecific genetic divergence, and that extensive hybridization is a contributing factor.

An Anopheles gambiae cDNA predicts a protein similar to a yeast Suil translation factor.

The nucleotide (nt) sequence of a cDNA cloned from the mosquito Anopheles gambiae was determined. The amino acid (aa) sequence of the deduced protein was 56% identical (60/108 aa) to the recently discovered translation initiation factor Suil of yeast, suggesting that the two proteins are homologs and have similar functions. Database searches also revealed strong similarity to other sequences, including the deduced gene products of cDNAs from organisms as diverse as nematodes, humans and plants. The functions of these putative proteins are unknown, but their homology to Suil suggests that they represent an important component of the eukaryotic translation initiation complex.

A new dominant selectable marker for genetic transformation; Hsp70-opd.

New P element plasmids containing the organophosphate-degrading gene opd as a dominant selectable marker were tested as transformation vectors in Drosophila melanogaster. One of these vectors was modified by the addition of the D. melanogaster mini-white gene as a comarker. When transformed individuals were identified using paraoxon selection for opd alone, results were similar to those obtained with mini-white. No false positives were recovered, however one strain contained the mini-white gene but inadequate resistance to survive our screening regimen due to a defective Hsp70-opd gene. Results suggest that Hsp70-opd is similar to mini-white for distinguishing transformed individuals but does not require time-consuming individual examination. Due to the mode of action of organophosphorus nerve agents, Hsp70-opd has potential as a selectable marker in numerous animals beside fruit flies.

P elements are found in the genomes of nematoceran insects of the genus Anopheles.

We report the identification of genomic sequences in various anopheline mosquitoes (family Culicidae: suborder Nematocera: order Diptera) showing homology to the class II, short inverted-terminal-repeat (ITR) transposable element P from Drosophila melanogaster (family Drosophilidae; suborder Brachycera: order Diptera). Anopheles gambiae appears to have at least six distinct P elements. Other anopheline species, including four additional members of the An. gambiae species complex (An. arabiensis, An. merus, An. melas and An. quadriannulatus), Anopheles stephensi (all subgenus Cellia), An. quadrimaculatus (subgenus Anopheles) and Anopheles albimanus (subgenus Nyssorhynchus) also possess P elements similar to those found in An. gambiae. Ten distinct P element types were identified in the genus Anopheles. At least two of the An. gambiae elements appears to be intact and potentially functional. Phylogenetic analysis of the anopheline P elements reveals them to belong to a distinctly different clade from the brachyceran P elements.

The Tryptophan oxygenase gene of Anopheles gambiae.

The Anopheles gambiae gene encoding tryptophan oxygenase, a homolog of the Drosophila melanogaster vermilion gene, has been molecularly cloned and characterized. Unlike Drosophila, where it is X-linked, the A. gambiae gene maps to chromosome 2R, subdivision 12E, by in situ hybridization to the polytene chromosomes. Of the six introns present, four are positioned identically to those of the Drosophila homolog, one is similarly positioned, and one is novel. A 1 955 nt cDNA potentially encodes a 392 amino acid protein of an estimated 45 kDa. Amino acid comparisons between the deduced protein and previously known tryptophan oxygenases revealed 74% identity between Anopheles and Drosophila, and 53% identity between Anopheles and nematode or mammalian proteins. Northern analysis detected a developmentally regulated transcript about 2 kb in length. Since this gene is known to control adult eye color in other flies, its cloning from A. gambiae provides the basis for a dominant phenotypic marker for germline transformation, one whose expression, unlike that of white, is not cell autonomous.

A family of pupal-specific cuticular protein genes in the mosquito Anopheles gambiae.

We have cloned and sequenced members of a cuticular protein multi-gene family from the mosquito Anopheles gambiae. Three genes (agcp2a-c), each approximately 1 kb in length, were found in a 17.4 kb genomic phage clone. Analysis of ten cDNAs revealed that at least four related genes are present. The open reading frame of the genes and cDNAs showed 95% sequence identity. Divergence was observed in the sequence of the 3' ends and the number of copies of two repeated coding sequences. In situ hybridizations with a probe prepared from one of these circular protein genes physically mapped to two loci, 26B on chromosome 2L and 37A on 3R. Transcription of these An. gambiae cuticular protein genes appears to be limited to pharate pupae and the expressed protein(s) is found in early pupae. The deduced amino acid sequence of these proteins contains a hydrophilic region with significant similarity to other cuticular proteins including the pupal-specific cuticular protein, EDG84, of Drosophila melanogaster (Apple and Fristrom).

Transient expression of the Drosophila melanogaster cinnabar gene rescues eye color in the white eye (WE) strain of Aedes aegypti.

The lack of eye pigment in the Aedes aegypti WE (white eye) colony was confirmed to be due to a mutation in the kynurenine hydroxylase gene, which catalyzes one of the steps in the metabolic synthesis of ommochrome eye pigments. Partial restoration of eye color (orange to red phenotype) in pupae and adults occurred in both sexes when first or second instar larvae were reared in water containing 3-hydroxykynurenine, the metabolic product of the enzyme kynurenine hydroxylase. No eye color restoration was observed when larvae were reared in water containing kynurenine sulfate, the precursor of 3-hydroxykynurenine in the ommochrome synthesis pathway. In addition, a plasmid clone containing the wild type Drosophila melanogaster gene encoding kynurenine hydroxylase, cinnabar (cn), was also able to complement the kynurenine hydroxylase mutation when it was injected into embryos of the A. aegypti WE strain. The ability to complement this A. aegypti mutant with the transiently expressed D. melanogaster cinnabar gene supports the value of this gene as a transformation reporter for use with A. aegypti WE and possibly other Diptera with null mutations in the kynurenine hydroxylase gene.

Species-diagnostic differences in a ribosomal DNA internal transcribed spacer from the sibling species Anopheles freeborni and Anopheles hermsi (Diptera:Culicidae).

Approximately 460 base pairs (bp) of DNA sequence that included the second internal transcribed spacer (ITS2) and some flanking 5.8S and 28S ribosomal RNA coding regions were compared between the two closely related and morphologically indistinguishable mosquito species Anopheles freeborni and A. hermsi and a third related species, A. occidentalis. Sequences were determined from 14 clones of polymerase chain reaction (PCR)-amplified DNA obtained from four colonies of A. freeborni, two colonies of A. hermsi, and one individual A. occidentalis. Four clones showed independent single bp differences from the consensus for the relevant species. Eleven sites differed between the consensus sequences of A. hermsi and A. freeborni; 28 sites differed between A. hermsi and A. occidentalis. With the exception of a single bp mismatch in the 5.8S and two single bp mismatches near the undetermined junction of the ITS2 and 28S regions, all differences were confined to the ITS2 region. A PCR-based species-diagnostic assay for the cryptic species A. hermsi and A. freeborni was developed; it uses four synthetic oligonucleotides, two derived from areas of interspecies sequence difference in the ITS2, and two derived from highly conserved regions in the flanking coding sequences. Small amounts of mosquito DNA amplified in the presence of these four primers produce fragments of diagnostic size for each species: 900 bp for A. freeborni, 350 bp for A. hermsi, and approximately 1.2-1.4 kb for various other Anopheles species tested. We believe that this general approach to the development of species-diagnostic assays can be extended easily to other complexes of closely related, morphologically indistinguishable species.

Association of a Plasmodium-refractory phenotype with an esterase locus in Anopheles gambiae.

In a genetically selected strain of the malaria vector Anopheles gambiae, most species of Plasmodium parasites are surrounded by melanized capsules and killed in the wall of the mosquito midgut. Genetic studies demonstrate a significant association between the refractory response to Plasmodium cynomolgi B strain and the Est A allele at an autosomal esterase locus. Increased susceptibility to parasites is associated with an alternate Est C allele. Lines selected to be homozygous for the Est A and Est C alleles show enhanced levels of refractoriness and susceptibility when compared to the unselected parental stock. Expression of the refractory phenotype is reduced much more than the susceptible phenotype at high parasite density, suggesting that refractoriness may be due to a positive effector.