Christos Louis

Molecular characterization of ribosomal DNA polymorphisms discriminating among chromosomal forms of Anopheles gambiae s.s.

The sequence of a 2.3 kb long DNA segment derived from the 5′‐most end of the ribosomal intergenic spacer was determined in three chromosomal forms of Anopheles gambiae s.s. The analysis revealed that the sequence of the Mopti form differed from that of the Bamako and Savanna forms by a total of ten nucleotide substitutions. Using these sequence variations we set up a diagnostic polymerase chain reaction (PCR) assay to distinguish mosquitoes belonging to the three chromosomal forms, facilitating studies on the distribution and the ecology of these incipient taxa. The assay also allows to distinguish whether a given specimen could represent a heterozygote between Mopti and Savanna or Bamako.

VectorBase: A Data Resource for Invertebrate Vector Genomics

VectorBase (http://www.vectorbase.org) is an NIAID-funded Bioinformatic Resource Center focused on invertebrate vectors of human pathogens. VectorBase annotates and curates vector genomes providing a web accessible integrated resource for the research community. Currently, VectorBase contains genome information for three mosquito species: Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus, a body louse Pediculus humanus and a tick species Ixodes scapularis. Since our last report VectorBase has initiated a community annotation system, a microarray and gene expression repository and controlled vocabularies for anatomy and insecticide resistance. We have continued to develop both the software infrastructure and tools for interrogating the stored data.

Gene expression in insecticide resistant and susceptible Anopheles gambiae strains constitutively or after insecticide exposure.

A microarray containing approximately 20 000 expressed sequence tags (ESTs; 11 760 unique EST clusters) from the malaria vector, Anopheles gambiae, was used to monitor differences in global gene expression in two insecticide resistant and one susceptible strains. Statistical analysis identified 77 ESTs that were differentially transcribed among the three strains. These include the cytochrome P450 CYP314A1, over‐transcribed in the DDT resistant ZAN/U strain, and many genes that belong to families not usually associated with insecticide resistance, such as peptidases, sodium/calcium exchangers and genes implicated in lipid and carbohydrate metabolism. Short‐term (6 and 10 h) effects of exposure of the pyrethroid resistant RSP strain to permethrin were also detected. Several genes belonging to enzyme families already implicated in insecticide or xenobiotic detoxification were induced, including the carboxylesterase COEAE2F gene and members of the UDP‐glucuronosyl transferase and nitrilase families.

VectorBase: improvements to a bioinformatics resource for invertebrate vector genomics

VectorBase (http://www.vectorbase.org) is a NIAID-supported bioinformatics resource for invertebrate vectors of human pathogens. It hosts data for nine genomes: mosquitoes (three Anopheles gambiae genomes, Aedes aegypti and Culex quinquefasciatus), tick (Ixodes scapularis), body louse (Pediculus humanus), kissing bug (Rhodnius prolixus) and tsetse fly (Glossina morsitans). Hosted data range from genomic features and expression data to population genetics and ontologies. We describe improvements and integration of new data that expand our taxonomic coverage. Releases are bi-monthly and include the delivery of preliminary data for emerging genomes. Frequent updates of the genome browser provide VectorBase users with increasing options for visualizing their own high-throughput data. One major development is a new population biology resource for storing genomic variations, insecticide resistance data and their associated metadata. It takes advantage of improved ontologies and controlled vocabularies. Combined, these new features ensure timely release of multiple types of data in the public domain while helping overcome the bottlenecks of bioinformatics and annotation by engaging with our user community.

Plasmodium berghei calcium-dependent protein kinase 3 is required for ookinete gliding motility and mosquito midgut invasion

Apicomplexan parasites critically depend on a unique form of gliding motility to colonize their hosts and to invade cells. Gliding requires different stage and species‐specific transmembrane adhesins, which interact with an intracellular motor complex shared across parasite stages and species. How gliding is regulated by extracellular factors and intracellular signalling mechanisms is largely unknown, but current evidence suggests an important role for cytosolic calcium as a second messenger. Studying a Plasmodium berghei gene deletion mutant, we here provide evidence that a calcium‐dependent protein kinase, CDPK3, has an important function in regulating motility of the ookinete in the mosquito midgut. We show that a cdpk3– parasite clone produces morphologically normal ookinetes, which fail to engage the midgut epithelium, due to a marked reduction in their ability to glide productively, resulting in marked reduction in malaria transmission to the mosquito. The mutant was successfully complemented with an episomally maintained cdpk3 gene, restoring mosquito transmission to wild‐type level. cdpk3– ookinetes maintain their full genetic differentiation potential when microinjected into the mosquito haemocoel and cdpk3– sporozoites produced in this way are motile and infectious, suggesting an ookinete‐limited essential function for CDPK3.

The mitochondrial genome of the Mediterranean fruit fly, Ceratitis capitata

The complete sequence of the mitochondrial genome of Ceratitis capitata has been determined. The circular genome is 15 980 bp long and contains a standard gene complement, i.e. the large and small ribosomal RNA subunits, twenty‐two transfer RNA (tRNA) genes and thirteen genes encoding mitochondrial proteins. When comparing the sequence to fragments previously sequenced from other isolates it becomes apparent that interstrain polymorphisms are not rare. These differences are potentially useful for the development of diagnostic tools for population analysis applications, such as determining the source of recent introductions. Moreover, they could help obtain a solution to the long‐lasting controversy on the possible eradication of the Medfly from certain locations.

hobo is responsible for the induction of hybrid dysgenesis by strains of Drosophila melanogaster bearing the male recombination factor 23.5MRF

The male recombination factor 23.5MRF, isolated ten years ago from a natural Greek population of Drosophila melanogaster, has been shown to induce hybrid dysgenesis when crossed to some M strains, in a fashion slightly different from that of most P strains. Furthermore, it was recently shown that 23.5MRF can also induce GD sterility when crossed to specific P strain females (e.g., Harwich, pi 2 and T-007). In these experiments, the P strains mentioned behaved like M strains in that they did not induce sterility in the reciprocal crosses involving 23.5MRF. We extended the analysis to show that 23.5MRF does not destabilize snW(M) and that a derivative with fewer full-length P elements behaves like an M strain toward the same P strains and still retains its dysgenic properties in the reciprocal crosses. We show that there is a strong correlation between the site of dysgenic chromosomal breakpoints induced by 23.5MRF and the localization of hobo elements on the second chromosome, and also that hobo elements are found associated with several 23.5MRF induced mutations. These results suggest that hobo elements are responsible for the aberrant dysgenic properties of this strain, and that they may express their dysgenic properties independent of the presence of P elements.

VectorBase: a home for invertebrate vectors of human pathogens

VectorBase () is a web-accessible data repository for information about invertebrate vectors of human pathogens. VectorBase annotates and maintains vector genomes providing an integrated resource for the research community. Currently, VectorBase contains genome information for two organisms: Anopheles gambiae, a vector for the Plasmodium protozoan agent causing malaria, and Aedes aegypti, a vector for the flaviviral agents causing Yellow fever and Dengue fever.

Mapping and identification of essential gene functions on the X chromosome of Drosophila

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large‐scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large‐scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X‐chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P‐element‐tagged enhancers is described and discussed.

SOAP, a novel malaria ookinete protein involved in mosquito midgut invasion and oocyst development

An essential, but poorly understood part of malaria transmission by mosquitoes is the development of the ookinetes into the sporozoite‐producing oocysts on the mosquito midgut wall. For successful oocyst formation newly formed ookinetes in the midgut lumen must enter, traverse, and exit the midgut epithelium to reach the midgut basal lamina, processes collectively known as midgut invasion. After invasion ookinete‐to‐oocyst transition must occur, a process believed to require ookinete interactions with basal lamina components. Here, we report on a novel extracellular malaria protein expressed in ookinetes and young oocysts, named secreted ookinete adhesive protein (SOAP). The SOAP gene is highly conserved amongst Plasmodium species and appears to be unique to this genus. It encodes a predicted secreted and soluble protein with a modular structure composed of two unique cysteine‐rich domains. Using the rodent malaria parasite Plasmodium berghei we show that SOAP is targeted to the micronemes and forms high molecular mass complexes via disulphide bonds. Moreover, SOAP interacts strongly with mosquito laminin in yeast‐two‐hybrid assays. Targeted disruption of the SOAP gene gives rise to ookinetes that are markedly impaired in their ability to invade the mosquito midgut and form oocysts. These results identify SOAP as a key molecule for ookinete‐to‐oocyst differentiation in mosquitoes.