Evidence for X-linked introgression between molecular forms of Anopheles gambiae from Angola.

The M and S molecular forms of the African malaria vector Anopheles gambiae (Diptera: Culicidae) are morphologically identical incipient species in which reproductive isolation is incomplete, enabling low-level gene flow between forms. In an attempt to find differences between the M and S forms, sequence variation was studied at loci along the X chromosome in adult female An. gambiae from Angola. A high proportion of M form specimens from Angola (79% of the 456 X chromosomes sampled) were found to contain a 16-bp insertion in intron 4 of the X-linked GPRCCK1 locus, relative to the AgamP3 release of the An. gambiae PEST genome sequence. The insertion was in Hardy-Weinberg equilibrium in Angolan M form populations. The same insertion was found in all S form specimens examined, regardless of where in Africa they were sampled, but was absent from a sample of M form specimens collected in Ghana, Bioko and Mali. In M form specimens from Angola, there was an association between alleles at the GPRCCK1 locus and those at a microsatellite locus, AGXH678, close to the centromere of the X chromosome, with significant linkage disequilibrium between loci separated by 0.472 Mbp (P < 0.033). We show that the insertion results from introgression from the S form into the M form, rather than from the retention of an ancestral character. Gene flow from the S to M form could allow genes of adaptive value to be transferred, including those conferring insecticide resistance and others influencing ecology and behaviour, and thus malaria transmission and control. We discuss factors that may have led to this introgression event.

Molecular taxonomy of members of the Anopheles hyrcanus group from Thailand and Indonesia.

During studies of malaria vectors in Indonesia and Thailand, several specimens identified by field staff as members of the Anopheles barbirostris group (Diptera: Culicidae) were found to belong to the Anopheles hyrcanus group, as shown by marked differences in the size of the nuclear rDNA second internal transcribed spacer (ITS2) between the barbirostris (~1500 bp) and hyrcanus (~600 bp) groups. Identification of the species concerned required a more detailed study of ITS2 sequences and subunit I of the mitochondrial DNA cytochrome oxidase gene (COI). A phylogenetic analysis, based on Bayesian methods, revealed that the hyrcanus group specimens comprised five distinct clades, two of which corresponded with known species, Anopheles peditaeniatus and Anopheles sinensis. The remaining specimens formed three additional clades, for which there are no similar sequences in GenBank and which cannot be linked to previously described species. The misidentification of hyrcanus group species has important implications for malaria vector control; more comprehensive studies employing gene sequences are required to clarify the number of species in the group, their distribution and vector status.

Bionomics of Anopheles stephensi Liston in the malarious area of Hormozgan province, southern Iran, 2002.

Anopheles stephensi Liston is an important malaria vector in Hormozgan province, where it is the most prevalent anopheline mosquito. It shows two annual activity peaks, one in spring and another in the autumn. In mountainous areas the second peak starts earlier than in coastal regions. Adults are endophilic and endophagic, but in the hot season when people sleep outside buildings they frequently bite outdoors. Larvae are found in a wide-range of habitats, both natural and man-made. All three biological forms of the species, occur in the province, i.e. An. stephensi stephensi (type form), An. stephensi mysorensis, and the intermediate form. An. stephensi mysorensis is found only in rural-mountainous areas, whereas the type and intermediate forms occur in urban-coastal regions and the rural plains, with the type form predominant. The presence of the type form in urban areas and mysorensis in rural areas is consistent with the available epidemiological data for malaria in the region and with the finding in India that the type form is an efficient malaria vector inhabiting urban areas whereas mysorensis is rural and has a lower vectorial capacity. Insecticide susceptibility tests on field collected adult mysorensis and adults from laboratory strains of the type and intermediate forms were carried out according to WHO standard methods. These showed that all three forms are susceptible to bendiocarb, propoxur, malathion, fenitrothion, deltamethrin, permethrin, cyfluthrin, and lambdacyhalothrin, but are resistant to DDT and show low level of tolerance to dieldrin. Examination of the larvicidal activity of malathion, fenitrothion, temephos and chlorpyrifos at diagnostic doses showed that these stephensi forms are susceptible to all larvicides except fenitrothion. Irritability tests to pyrethroid insecticides showed high levels of irritability to permethrin and lambdacyhalothrin, but low irritability to cyfluthrin and deltamethrin. The importance of these findings for the epidemiology and control of malaria in the region are discussed.

Population differentiation and Wolbachia phylogeny in mosquitoes of the Aedes scutellaris group.

Mosquito species of the Aedes (Stegomyia) scutellaris (Walker) group (Diptera: Culicidae) are distributed across many islands of the South Pacific and include major regional vectors of filariasis, such as Aedes polynesiensis (Marks). Analysis of populations of Ae. polynesiensis at the extremes of its range, from Fiji and from Moorea, French Polynesia, using the rDNA ITS2 (internal transcribed spacer 2) region and six microsatellite markers showed considerable genetic differentiation between them (F(ST) = 0.298-0.357). Phylogenetic analysis of the Wolbachia endosymbionts in three members of the complex revealed that based on the wsp gene they are all very similar and belong to the Mel subgroup of the A clade, closely related to the Wolbachia strain present in the gall wasp Callyrhytis glandium (Giraud) (Hymenoptera: Cynipidae). By contrast they are only distantly related to the A-clade Wolbachia in Aedes albopictus (Skuse), a species closely allied to the Ae. scutellaris group. There was very low differentiation between the Wolbachia in the Moorea and Fiji populations of Ae. polynesiensis.

Exploiting the potential of vector control for disease prevention.

Although vector control has proven highly effective in preventing disease transmission, it is not being used to its full potential, thereby depriving disadvantaged populations of the benefits of well tried and tested methods. Following the discovery of synthetic residual insecticides in the 1940s, large-scale programmes succeeded in bringing many of the important vector-borne diseases under control. By the late 1960s, most vector-borne diseases--with the exception of malaria in Africa--were no longer considered to be of primary public health importance. The result was that control programmes lapsed, resources dwindled, and specialists in vector control disappeared from public health units. Within two decades, many important vector-borne diseases had re-emerged or spread to new areas. The time has come to restore vector control to its key role in the prevention of disease transmission, albeit with an increased emphasis on multiple measures, whether pesticide-based or involving environmental modification, and with a strengthened managerial and operational capacity. Integrated vector management provides a sound conceptual framework for deployment of cost-effective and sustainable methods of vector control. This approach allows for full consideration of the complex determinants of disease transmission, including local disease ecology, the role of human activity in increasing risks of disease transmission, and the socioeconomic conditions of affected communities.

Multiplex PCR assay for malaria vector Anopheles minimus and four related species in the Myzomyia Series from Southeast Asia.

Mosquitoes (Diptera: Culicidae) of the Anopheles (Cellia) Myzomyia Series are important malaria vectors in Africa, India and Southeast Asia. Among 10 named species of Myzomyia known from the Oriental Region, seven form the An. minimus group. Even for expert taxonomists, the adults of these species remain difficult to identify morphologically. For technical staff of malaria control programmes, confusion may extend to misidentification of species that are not formally within the minimus group. For identification of specimens from Indochina (Cambodia, Laos, Vietnam), we describe a multiplex polymerase chain reaction (PCR) assay, based on rDNA internal transcribed spacer 2 (ITS2) sequences, that employs a cocktail of primers to identify An. minimus Theobald sibling species A and C (sensu; Green et al., 1990) and three other species in the An. minimus group (An. aconitus Dönitz, An. pampanai Büttiker & Beales, An. varuna Iyengar), as well as An. jeyporiensis James, also belonging to the Myzomyia Series. As the test is DNA-based, it can be applied to all life stages of these mosquitoes for ecological investigations and vector incrimination studies. This PCR assay is simpler, quicker, cheaper and more readily interpreted than previous assays.

Molecular evidence for a kdr-like pyrethroid resistance mechanism in the malaria vector mosquito Anopheles stephensi.

The mosquito Anopheles stephensi Liston (Diptera: Culicidae) is the urban vector of malaria in several countries of the Middle East and Indian subcontinent. Extensive use of residual insecticide spraying for malaria vector control has selected An. stephensi resistance to DDT, dieldrin, malathion and other organophosphates throughout much of its range and to pyrethroids in the Middle East. Metabolic resistance mechanisms and insensitivity to pyrethroids, so-called knockdown resistance (kdr), have previously been reported in An. stephensi. Here we provide molecular data supporting the hypothesis that a kdr-like pyrethroid-resistance mechanism is present in An. stephensi. We found that larvae of a pyrethroid-selected strain from Dubai (DUB-R) were 182-fold resistant to permethin, compared with a standard susceptible strain of An. stephensi. Activities of some enzymes likely to confer pyrethroid-resistance (i.e. esterases, monooxygenases and glutathione S-transferases) were significantly higher in the permethrin-resistant than in the susceptible strain, but the use of synergists--piperonyl butoxide (PBO) to inhibit monooxygenases and/or tribufos (DEF) to inhibit esterases--did not fully prevent resistance in larvae (permethrin LC50 reduced by only 51-68%), indicating the involvement of another mechanism. From both strains of An. stephensi, we obtained a 237-bp fragment of genomic DNA encoding segment 6 of domain II of the para type voltage-gated sodium channel, i.e. the putative kdr locus. By sequencing this 237 bp fragment, we identified one point mutation difference involving a single A-T base change encoding a leucine to phenylalanine amino acid substitution in the pyrethroid-resistant strain. This mutation appears to be homologous with those detected in An. gambiae and other insects with kdr-like resistance. A diagnostic polymerase chain reaction assay using nested primers was therefore designed to detect this mechanism in An. stephensi.

Wolbachia as a potential tool for suppressing filarial transmission.

There is currently a great deal of interest in Wolbachia because of their wide distribution in arthropods and filarial nematodes and their striking effects on the biology of their hosts, including a possible role in speciation. They manipulate the reproduction of arthropod hosts through various effects on their hosts' biology, particularly cytoplasmic incompatibility (CI), to increase the proportion of infected individuals in the population, often to the point of fixation. This ability of Wolbachia to sweep through host populations indicates several potential applications of Wolbachia in the control of mosquito-borne disease. One uses Wolbachia-induced CI as a form of sterile-insect technique, to suppress mosquito populations. Another envisages the application of CI for population replacement, with the intention of preventing the transmission of human pathogens, by substituting desirable genotypes, including those carried in transgenes. A third possibility is to use Wolbachia to reduce the survival of mosquito populations and thereby reduce their ability to transmit the infection. This article provides an overview of the biological effects of Wolbachia on arthropod hosts, with discussion of the possible future exploitation of these effects in the control of filariasis.

rDNA-ITS2 polymerase chain reaction assay for the sibling species of Anopheles fluviatilis.

Species-specific differences in the nucleotide sequences of the second internal transcribed spacer (ITS2) region of the ribosomal DNA (rDNA) were used to develop a diagnostic polymerase chain reaction (PCR) assay for two of the sibling species of the Anopheles fluviatilis complex, members of which are major vectors of malaria in central and northern parts of India. This assay consisted of a three primer reaction, which could amplify the DNA of both the species producing fragments of two distinct sizes, 350 bp for species X and 450 bp for species Y, respectively. The assay was found to be highly specific and sensitive.

Evidence for extensive genetic differentiation among populations of the malaria vector Anopheles arabiensis in Eastern Africa.

We describe the geographical population structure of the malaria vector Anopheles arabiensis in Eastern Africa. Allelic variation at eight microsatellite loci was scored in samples from nine localities along a 4500 km transect from Sudan to Mozambique. Highly significant differences in genotype frequencies were found between all populations separated by more than 200 km. Populations within Malawi separated by 191 km were indistinguishable, as were those within Sudan separated by 134 km. FST and rhoST gave significant estimates of isolation by distance. These data, lead us to conclude that there are extensive barriers to gene flow in this region. The high estimates of Nm (9.4 from FST and 5.2 from rhoST) indicate recent range expansion in this species rather than extensive contemporary gene flow.

Population structure in the malaria vector, Anopheles arabiensis patton, in East Africa.

The population structure of the malaria vector Anopheles arabiensis was investigated using data from six microsatellite loci in samples from localities in Mozambique and Tanzania. Genotype frequencies were neither significantly different between houses in a village in Tanzania nor between villages within a 20-km radius in Mozambique. Thus a deme has an area greater than 20 km in radius. At five of the six loci the heterozygosity of the population from Mozambique was lower than that from Tanzania, implying a lower effective population size (Ne) at this southern edge of the species range. There were significant differences in genotype frequencies between the Tanzanian and Mozambique populations at five of the six loci (P<0.05). Values for both FST (mean=0.069) and RST (mean=0.025) were significantly different from zero (P<0.05) at four and three out of five loci, respectively, but there was no significant correlation between the two statistics. The wide variation in values of FST and RST across loci suggests that care should be taken in interpreting values derived from averaging across loci. Whether the variation results from sampling effects or selectional constraints on some loci is unclear. Although there is evidence for significant differentiation between these populations, estimates of gene flow (Nm) calculated from mean FST and RST statistics were relatively high, 3.4 and 4.9, respectively. We argue that this is more likely to reflect recent separation of these populations and/or large effective population size rather than large-scale present day migrations.

Malaria: existing methods of vector control and molecular entomology.

In general, the most effective means of malaria vector control is the killing of adult mosquitoes with a residual insecticide applied to bednets or sprayed on house walls and ceilings. Major reductions in all-cause child mortality have been achieved in Africa by these means. In some circumstances, personal protection and larval control may also make a contribution. We discuss the prospects of genetic control by release of sterile male mosquitoes or driving genes for refractoriness to malaria into wild populations. Many major malaria vectors belong to complexes of sibling species which differ in vectorial and biological characteristics. Distinguishing the species by cytogenetic or molecular methods is important for epidemiological studies and could improve the targeting of control.

Use of rDNA-PCR to investigate the ecological distribution of Anopheles bwambae in relation to other members of the An.gambiae complex of mosquitoes in Bwamba County, Uganda.

Environmental relationships were investigated among three species of the Anopheles gambiae complex of mosquitoes associated with the geothermal springs located in Bwamba County, Uganda. The degree of ecological isolation between An.gambiae and An.bwambae, a sibling species known only from the geothermal springs environment, was assessed on the basis of adult distribution and abundance as well as differences in larval habitats. Field data were gathered during June 1995 without knowing which of the species were being collected. Specimens identified subsequently by rDNA-PCR were used to interpret the ecological data. Ten of twenty aquatic sites sampled were found positive for immature stages of the An.gambiae complex. Larvae of An.bwambae were associated with 'springwater' habitats having much higher conductivity, much greater concentrations of dissolved solids and slightly higher temperature and pH than 'normal' fresh water sites inhabited by larvae of An.gambiae. Larval habitats of both species were unshaded: An.bwambae occurred among dense sedge (Cyperus laevigatus) whereas those of An.gambiae were almost devoid of vegetation. One mixed sample showed that larvae of both species occur together in peripheral aquatic sites with intermediate physical and ecological characteristics. In water preference tests, free-flying females were reluctant to lay eggs on bowls of water in cages; gravid females (with one wing amputated) placed on the surface of water in a cup laid eggs on seasoned rainwater (12/51 An.bwambae; 2/3 An.gambiae) as well as spring-water (39/51 An.bwambae; 1/3 An.gambiae). All three An.gambiae oviposited on the first water option, whereas 86% of An.bwambae witheld oviposition until being moved to the other type of water after 5-6 h, and 82% (36/44) of these laid eggs on geothermal water in preference to rainwater. Larval and adult collections showed tha An.gambiae occurs sympatrically with An.bwambae throughout its range in the humid foothill environment of the geothermal springs, whereas the distribution of An.arabiensis overlaps only slightly with An.bwambae towards the savanna environment north of the springs.

Density-dependent processes in the transmission of human onchocerciasis: relationship between microfilarial intake and mortality of the simuliid vector.

In order to construct an analytical model of onchocerciasis transmission, it is necessary to elucidate the functional relationships of the various population rate processes taking place within the human and vector hosts. Two previous papers have explored the evidence for density-dependent regulation in relation to microfilarial intake by, and larval development within, the Simulium host. This paper investigates the survivorship of wild-caught blackfly samples fed on subjects with different intensities of Onchocerca volvulus microfilarial infection. Analyses were based on data for Guatemalan S. ochraceum s.l. (possessing a well-developed cibarial armature), West African S. damnosum s.l. (forest species), and South Venezuelan S. guianense (the latter two lacking a toothed cibarium). The mean survival times of samples of the 3 species, kept under laboratory conditions, decreased as parasite intake increased, the rate of mortality being dependent on the fly's age (measured as time post-feeding) and on the worm load acquired. An empirical, time-dependent hazard function was fitted to observed death rates/fly/day which rose very shortly after engorgement, declined subsequently, and rose again throughout the extrinsic incubation period of the parasite. The parameters of this hazard model were all positively correlated with the density of microfilariae in the bloodmeal. Expressions of survivorship and life-expectancy as explicit functions of time post-feeding and mean parasite intake were derived. The average expectation of life at engorgement for uninfected flies in the laboratory was estimated to be around 1 week for both, armed and unarmed blackflies. Residual life-expectancy decreased with time post-feeding and microfilarial load in both categories of vectors. This decline (resulting from age- and parasite-dependent mortality rates) was much more pronounced in those species lacking a toothed fore-gut. Whilst a fraction of heavily infected S. ochraceum was able to survive the latent period of the parasite, being therefore potentially capable of transmitting the infection, equivalent worm loads in S. guianense resulted in a drastic reduction of the expectation of infective life. These results provide additional evidence to support the hypothesis that, in the case of intrinsically susceptible vectors, unarmed simuliids are more efficient at low microfilarial loads, when the transmission rate from human to vector host is higher, and parasite-induced fly mortality is negligible. The opposite takes place in armed flies, which perform poorly at low parasite burdens and better at heavier loads, with little parasite-induced vector death.

Full sequence and characterization of two insect defensins: immune peptides from the mosquito Aedes aegypti.

We report the complete amino acid sequence and biological activity of two immune peptides, from the yellow fever mosquito Aedes aegypti, that are induced in response to infection. Both peptides display biological activity against the Gram positive microbe Micrococcus luteus and substantial sequence homology to insect defensins, small heat-stable, antibiotic peptides previously described from several non-vector insects. These mosquito peptides, designated Ae. aegypti defensins A and B, are isoforms. Defensin B is the most abundant antibacterial peptide in this species whereas defensin A is much less abundant and carries two amino acid substitutions compared to defensin B, making it more basic in character. Apparent convergence between isoforms from Ae. aegypti and the fleshfly Phormia terranovae is discussed. The synergistic activity previously described between Ae. aegypti immune haemolymph and lysozyme is not caused by these peptides because synergy occurred only at concentrations far outside the physiological range seen in Ae. aegypti.

Brugia pahangi: the effects of cecropins on microfilariae in vitro and in Aedes aegypti.

Synthetic cecropins, antibacterial peptides from insect haemolymph, have been tested for their ability to attenuate the motility of microfilariae of the filarial nematode Brugia pahangi in an in vitro assay. Fifty micromolar concentrations of these peptides, equivalent to physiological concentrations in immune-stimulated insects, cause significant attenuation of motility compared with untreated microfilariae. Similar results were obtained with cecropins A and B. This is the lowest concentration for which cecropin has been reported to be active against eukaryote organisms. Antiserum to the cecropin homologue sarcotoxin 1A successfully blocked the observed activity. When the same concentration of cecropin B was coinjected with B. pahangi microfilariae into adult females of the mosquito, Aedes aegypti, a significant reduction in the numbers of developing larvae was observed.

Identification by rDNA-PCR of Anopheles bwambae, a geothermal spring species of the An. gambiae complex.

The identification of the malaria vector Anopheles bwambae by rDNA-PCR is described. PCR primers that amplify a region of the intergenic spacer of rDNA of An. gambiae s.s. produce two diagnostic PCR products of 690 bp and 390 bp with An. bwambae.

Purification of an insect defensin from the mosquito, Aedes aegypti.

Using a new, sensitive assay of bacterial growth inhibition, inducible antibacterial activity has been identified in the haemolymph of the mosquito, Aedes aegypti following inoculation with bacteria or with microfilariae of the filarial nematode Brugia pahangi, but not after inoculation with sterile culture medium. A lower level of antibacterial activity has also been observed in untreated individual mosquitoes. Following bacterial inoculation, a basic, inducible antibacterial peptide has been detected using native PAGE at pH 4, which corresponds with a 4.5 kDa peptide detected by tricine SDS-PAGE followed by silver staining. A peptide has been purified from immune haemolymph by ultrafiltration, followed by reversed-phase HPLC, yielding a single major peak with antibacterial activity. Partial amino acid sequence analysis of this fraction has revealed substantial homology with insect defensins. The data are consistent with the peptide being another member of this family, and we propose the name Aedes aegypti defensin.