Molecular Entomology and Prospects for Malaria Control
Collins, Frank H., Kamau, Luna, Ranson, Hilary A., Vulule, John M., Bulletin of the World Health Organization
Bulletin of the World Health Organization, 2000, 78: 1412-1423.
Voir page 1419 le resume en francais. En la pagina 1420 figura un resumen en espanol.
Plasmodium falciparum malaria remains one of the three most important pathogen-specific causes of human mortality in the world today. The 1998 World Health Report stated that there are now more cases of malaria in the world, perhaps 300-500 million per year (a major underestimate in the minds of many) than there were in 1954 (then estimated at 250 million). More importantly, the annual number of deaths caused by malaria, estimated at between 1.5 and 2.7 million in 1997, seems to have remained stable or even risen over this period (1). The problem of malaria has been exacerbated in recent years by the development and rapid spread of resistance in P. falciparum to the more commonly used and affordable antimalarial drugs. Chloroquine resistance, which first appeared in East Africa in the late 1970s, has now spread throughout most of the continent, and resistance to pyrimethamine--sulfadoxine (Fansidar) has followed rapidly. The emergence of insecticide resistance in African malaria vectors (see below) threatens to exacerbate further the problem.
In 1990, the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), together with the John D. and Catherine T. MacArthur Foundation and the University of Arizona, convened a meeting in Tucson, Arizona. At this meeting, 36 specialists in entomology, genetics and biochemistry were brought together to discuss the prospects for malaria control by genetic modification of the vector competence of natural vector populations (2). In the decade since that meeting, an extraordinary amount of molecular research has been done on malaria vectors, particularly Anopheles gambiae, the principal vector of malaria in sub-Saharan Africa. A fine-scale A. gambiae genetic map based on microsatellite markers and other sequenced tagged sites has been developed (3, 4) and used to map both morphological markers (5) and genes affecting parasite development (6). These markers have also formed the basis of a rapidly increasing number of population genetic and ecological studies of A. gambiae and its sibling species. Studies of innate immunity in Anopheles have revealed an extraordinarily complex defence system, some of whose elements are responsive to malaria parasites (7-19). Several groups are also actively examining the complex interactions between the malaria parasite and its mosquito vector during both the midgut and the salivary gland phases of sporogony (20-31). Other investigators are characterizing genes expressed in the midgut, fat body and salivary, glands, with the long-range goal of developing Plasmodium-inhibiting constructs that can be expressed specifically in these tissues (32, 33).
Indeed, the past year has resulted in two additional important developments in malaria vector research. An efficient technique for Anopheles germ line transformation has been developed (34, 35), and an informal genome project for A. gambiae has been launched. More than 25 000 Anopheles sequences are now in GenBank, more than 6000 of which are cDNAs (36). Around 17 000 of these entries are the end-sequences of a fivefold coverage bacterial artificial chromosome genomic library of A. gambiae (X. Wang, Z. Ke and F. H. Collins, unpublished data), sequenced by the French national genomics centre Genescope. The combined total BAC end-sequence is more than 14 megabases (http://bioweb.pasteur.fr/BBMI), an amount equivalent to about 5.4% of the 260-megabase A. gambiae genome (37). End-sequencing has also begun on a second bacterial artificial chromosome library, containing about 30 000 clones with an almost 20-fold genome coverage (F. H. Collins and H. Zhang, unpublished data). The National Institute of Allergy and Infectious Diseases (National Institutes of Health) has recently reviewed proposals to initiate genomics projects for several new organisms, one of which is A. gambiae (www.niaid.nih.gov/dmid/genomes/ priorities.htm). Thus building on the solid foundation of molecular work already completed, excellent prospects exist that a formal international A. gambiae genome project will soon be launched. The results of such a project, combined with the ongoing human and P. falciparum genome projects, will set the stage for a much deeper understanding of the interactions among the parasite, vector and human host that will almost certainly lead to new approaches for preventing and curing malaria.
Although most scientists in the vector research community have welcomed these developments, some still remain sceptical that molecular research on vectors will have any major impact on malaria control in the near future, and that such projects draw funds that could be better used implementing currently available methods for malaria control (38). There is also a somewhat justifiable concern that much of the current molecular research is too focused on a single molecular-based malaria control strategy, the interruption of malaria parasite transmission by altering the vector competence of natural vector populations (M. Coluzzi, personal communication.).
Rather than attempt to review all recent progress in molecular studies of malaria vectors -- some aspects of which were briefly mentioned above -- we focus on research in three areas that we believe can have important near-term impacts on vector-based malaria control programmes. These areas are molecular assays for mosquito species identification, studies of the molecular basis of insecticide resistance, and improved molecular tools for the study of vector population structure and gene flow. Most of the cited literature was identified from searches of databases and from the literature that the authors track for their own research purposes.
Identification of vector species
Species identification is clearly critical to any vector control programme that seeks to be efficient as well as effective, yet it has been apparent since as early as the 1920s that some malaria vectors are members of morphologically indistinguishable groups of species (39). For example, in a study of benzene hexachloride insecticide resistance among the malaria vectors in Zimbabwe in the late 1970s, bioassays suggested that most if not all wild specimens of the A. gambiae species complex were susceptible to benzene hexachloride. However, when the specimens tested in these bioassays were subsequently identified to species by a diagnostic isoenzyme assay, it was found that only A. quadriannulatus (constituting most of the test samples) was killed by benzene hexachloride. The few A. arabiensis in the samples survived exposure to the insecticide (40). This was an important finding, as only A. arabiensis is a vector in Zimbabwe; A. quadriannulatus is a highly zoophilic species that is not involved in transmission.
Within the past decade or so it has become apparent that most major vectors are members of such cryptic species complexes. Until the late 1980s, methods for identifying individual members of such complexes were too labour intensive to be used routinely in field studies of vector biology or malaria transmission. Thus, research even on vectors known to be a member of such complexes continued to lead to publications in which the species was identified only to the nominal taxon of the complex. The unfortunate consequence of such work was that because the species under study could be any one species or a mixture of species in a cryptic species complex, the resulting literature was at best imprecise and at worst misleading. The number of malaria vectors that are members of such complexes continues to increase as more vector populations are subject to careful population analysis, suggesting that much of the scientific literature describing the ecology of malaria vectors and their involvement in transmission is to some extent compromised.
Hybridization assays. Most of the first DNA-based assays for identifying cryptic species (ignoring for this discussion analysis of chromosome structure) were based on hybridization assays that detected species-specific differences in highly repetitive sequences. Although somewhat time consuming and technical, these assays were more efficient than previous methods, such as polytene chromosome analysis and isoenzyme gel electrophoresis. The approach, however, generally had two major limitations. The species composition of the cryptic species complex under study had to be known before the assay could be developed, and each species in the complex had to differ from the others in the abundance of one or more repeat sequences. Despite these limitations, repeat sequence hybridization assays were developed and used for field studies of the A. gambiae, A. dirus and A. punctulatus complexes (41-47).
Polymerase chain reaction methods. With the development and refinement of polymerase chain reaction (PCR) technology in the mid and late 1980s, PCR-based assays became more popular. Although randomly amplified polymorphic DNA-PCR has been used in Anopheles species identification (48-50), species-diagnostic PCR assays are more commonly targeted at specific regions of repeat gene families, such as ribosomal DNA (rDNA) that were found to differ among cryptic species. Initially, assays were based on species-specific differences in the nucleotide sequences of rapidly evolving regions such as rDNA internal transcribed spacers (ITS1 and ITS2) or intergenic spacer regions. Thus prior knowledge of the composition of the species complex was required. For example, a diagnostic assay that distinguishes cryptic species in the A. gambiae complex is based on differences in the intergenic spacer (51-54). Similar diagnostic assays based on differences in the ITS1 or ITS2 sequences have been developed for the A. hermsi/A.freeborni cryptic pair of species (55), the A. quadrimaculatus complex (56), the European A. maculipennis complex (57) and the `4. dims complex (58). Generally, because such spacer regions evolve and diverge rapidly, it is usually possible to find diagnostic differences between even the most closely related of species.
PCR amplification of regions of the rDNA, followed by restriction enzyme digestion or by single DNA strand conformational polymorphisms have also been used to develop assays for different chromosomal forms of A. gambiae (59), the A. punctulatus complex (60), the A. funestus group (61) and the A. minimus group (62). Although both these approaches are technically a little more complicated than simple rDNA-PCR (particularly single DNA strand conformational polymorphisms), they do offer several advantages, the most important of which is that sequencing is not a prerequisite to assay development. Because sequencing is not required, the assays can be used as population screening tools to survey for variation that might be indicative of the presence of cryptic species.
Although identification of cryptic species within a complex has traditionally been done by observing mating incompatibilities, it can also be inferred indirectly by showing combinations of molecular and/or biological markers that group nonrandomly in a single population. Drawing …
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Publication information: Article title: Molecular Entomology and Prospects for Malaria Control. Contributors: Collins, Frank H. - Author, Kamau, Luna - Author, Ranson, Hilary A. - Author, Vulule, John M. - Author. Journal title: Bulletin of the World Health Organization. Volume: 78. Issue: 12 Publication date: December 2000. Page number: 1412. © 1990 World Health Organization. COPYRIGHT 2000 Gale Group.
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