Genetic population structure and introgression in Anopheles dirus mosquitoes in South-east Asia

Genetic structure and species relationships were studied in three closely related mosquito species, Anopheles dirus A, C and D in Thailand using 11 microsatellite loci and compared with previous mitochondrial DNA (mtDNA) data on the same populations. All three species were well differentiated from each other at the microsatellite loci. Given the almost complete absence of mtDNA differentiation between An. dirus A and D, this endorses the previous suggestion of mtDNA introgression between these species. The high degree of differentiation between the northern and southern population of An. dirus C (RST = 0.401), in agreement with mtDNA data, is suggestive of incipient species. The lack of genetic structure indicated by microsatellites in four populations of An. dirus A across northern Thailand also concurs with mtDNA data. However, in An. dirus D a limited but significant level of structure was detected by microsatellites over ~400 km in northern Thailand, whereas the mtDNA detected no population differentiation over a much larger area (>1200 km). There is prior evidence for population expansion in the mtDNA. If this is due to a selective sweep originating in An. dirus D, the microsatellite data may indicate greater barriers to gene flow within An. dirus D than in species A. Alternatively, there may have been historical introgression of mtDNA and subsequent demographic expansion which occurred first in An. dirus D so enabling it to accumulate some population differentiation. In the latter case the lack of migration-drift equilibrium precludes the inference of absolute or relative values of gene flow in An. dirus A and D.     

DNA analysis of transferred sperm reveals significant levels of gene flow between molecular forms of Anopheles gambiae

Anopheles gambiae populations in west Africa are complex, being composed of multiple, sympatric subpopulations. Recent studies have failed to reveal significant genetic differences among subpopulations, stimulating a debate regarding the levels of gene flow among them. The observed homogeneity may be the consequence of substantial contemporary gene flow or it may be that reproductive isolation is complete, but too recent for the accumulation of significant levels of genic divergence. Here, we report the results of a study estimating contemporary levels of gene flow between An. gambiae subpopulations by analysing females and transferred sperm removed from their reproductive systems. A total of 251 female and associated sperm extracts was analysed from a single site in Mali. Two molecular forms of An. gambiae, the M- and S-forms, occurred in sympatry at this site. Overall, we found very strong positive assortative mating within forms, however, we did observe significant hybridization between forms. In the M subpopulation 2/195 females (1.03%) contained sperm from S-form males and in 55 S-form females we found one female containing M-form sperm (1.82%). We also identified a mated M xS hybrid adult female. From mating frequencies, we estimate the Nem between the M- and S-form at 16.8, and from the adult hybrid frequency at 5.6. These values are consistent with our earlier estimate, based on FST for 21 microsatellite loci in which Nem = 5.8. We conclude that the general lack of genetic divergence between the M and S subpopulations of An. gambiae can be explained entirely by contemporary gene flow.     

PCR assay for identification of Anopheles quadriannulatus species B from Ethiopia and other sibling species of the Anopheles gambiae complex

Sibling species A and B of Anopheles quadriannulatus (Theobald) are recognized as allopatric members of the Anopheles gambiae Giles complex of Afrotropical mosquitoes (Diptera: Culicidae). Species A represents An. quadriannulatus sensu stricto, widespread in southern Africa, whereas An. quadriannulatus species B occurs in Ethiopia. Because of difficulty of identification, distribution of An. quadriannulatus sensu lato remains poorly known. Cytotaxonomy and the standard DNA polymerase chain reaction (PCR) assay do not distinguish between species A and B of An. quadriannulatus. By optimizing the standard PCR assay (Scott et al., 1993) for identification of members of the An. gambiae complex, we identified two discriminant fragments of 153 bp and 900 bp from DNA of An. quadriannulatus species B, whereas only the 153 bp fragment was amplified for species A from South Africa. This modified PCR assay can therefore be used to distinguish between species A and B of An. quadriannulatus s.l. as well as other members of the An. gambiae complex.     

Transmission dynamics of lymphatic filariasis: density-dependence in the uptake of Wuchereria bancrofti microfilariae by vector mosquitoes

Gaining a better understanding of parasite infection dynamics in the vector mosquito (Diptera: Culicidae) population is central to improving knowledge regarding the transmission, persistence and hence control of lymphatic filariasis. Here, we use data on mosquito feeding experiments collated from the published literature to examine the available evidence regarding the functional form of the first component of this parasite-vector relationship for Wuchereria bancrofti (Filarioidea: Onchocercidae) causing Bancroftian filariasis, i.e. the rate of microfilariae (mf) uptake from the blood of infected humans by the feeding mosquito vector. Using a simple logarithmic regression model for describing the observed relationships between the mean numbers of mf ingested per mosquito and parasite load in humans in each study, and a linear mixed-effects meta-analytical framework for synthesizing the observed regressions across studies, we show here for the first time clear evidence for the existence of density-dependence in this process for all the three major filariasis transmitting mosquito vectors. An important finding of this study is that this regulation of mf uptake also varies significantly between the vector genera, being weakest in Culex, comparatively stronger in Aedes and most severe and occurring at significantly lower human mf loads in Anopheles mosquitoes. The analysis of the corresponding mf uptake prevalence data has further highlighted how density-dependence in mf uptake may influence the observed distributions of mf in vector populations. These results show that whereas strong regulation of mf uptake, especially when it leads to saturation in uptake at low human parasite intensities, can lead to static distributions of mf per mosquito with host parasite intensity, a weaker regulation of mf ingestion can give rise to changes in both mean mf loads and in the frequency distribution of parasites/mosquito with increasing human parasite intensity. These findings highlight the importance of considering local vector infection dynamics when attempting to predict the impacts of community-based filariasis control. They also emphasize the value of developing and applying robust meta-analytic methods for estimating functional relationships regarding parasitic infection from population ecological data.     

Extensive multiple test centre evaluation of the VecTest malaria antigen panel assay

To determine which species and populations of Anopheles transmit malaria in any given situation, immunological assays for malaria sporozoite antigen can replace traditional microscopical examination of freshly dissected Anopheles. We developed a wicking assay for use with mosquitoes that identifies the presence or absence of specific peptide epitopes of circumsporozoite (CS) protein of Plasmodium falciparum and two strains of Plasmodium vivax (variants 210 and 247). The resulting assay (VecTest Malaria) is a rapid, one-step procedure using a 'dipstick' test strip capable of detecting and distinguishing between P. falciparum and P. vivax infections in mosquitoes. The objective of the present study was to test the efficacy, sensitivity, stability and field-user acceptability of this wicking dipstick assay. In collaboration with 16 test centres world-wide, we evaluated more than 40 000 units of this assay, comparing it to the standard CS ELISA. The 'VecTest Malaria' was found to show 92% sensitivity and 98.1% specificity, with 97.8% accuracy overall. In accelerated storage tests, the dipsticks remained stable for > 15 weeks in dry conditions up to 45 degrees C and in humid conditions up to 37 degrees C. Evidently, this quick and easy dipstick test performs at an acceptable level of reliability and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.     

Stream-bank shade and larval distribution of the Philippine malaria vector Anopheles flavirostris

The principal malaria vector in the Philippines, Anopheles flavirostris (Ludlow) (Diptera: Culicidae), is regarded as 'shade-loving' for its breeding sites, i.e. larval habitats. This long-standing belief, based on circumstantial observations rather than ecological analysis, has guided larval control methods such as 'stream-clearing' or the removal of riparian vegetation, to reduce the local abundance of An. flavirostris. We measured the distribution and abundance of An. flavirostris larvae in relation to canopy vegetation cover along a stream in Quezon Province, the Philippines. Estimates of canopy openness and light measurements were obtained by an approximation method that used simplified assumptions about the sun, and by hemispherical photographs analysed using the program HEMIPHOT. The location of larvae, shade and other landscape features was incorporated into a geographical information system (GIS) analysis. Early larval instars of An. flavirostris were found to be clustered and more often present in shadier sites, whereas abundance was higher in sunnier sites. For later instars, distribution was more evenly dispersed and only weakly related to shade. The best predictor of late-instar larvae was the density of early instars. Distribution and abundance of larvae were related over time (24 days). This pattern indicates favoured areas for oviposition and adult emergence, and may be predictable. Canopy measurements by the approximation method correlated better with larval abundance than hemispherical photography, being economical and practical for field use. Whereas shade or shade-related factors apparently have effects on larval distribution of An. flavirostris, they do not explain it completely. Until more is known about the bionomics of this vector and the efficacy and environmental effects of stream-clearing, we recommend caution in the use of this larval control method.     

Comparative insecticidal power of three pyrethroids on netting

Adult mosquitoes, Anopheles gambiae Giles and Culex quinquefasciatus Say (Diptera: Culicidae), were exposed for 3 min to replicate samples of polyester netting cut from replicate bednets treated with pyrethroid insecticide formulations at the recommended concentration (alphacypermethrin SC at 40mg ai/m2; cyfluthrin EW at 50 mg ai/m2; deltamethrin WT at 25 mg ai/m2), or treated with only a quarter of those dosages. After 4 months domestic use of the bednets in Malawi, chemical assays showed that pyrethroid deposits on the netting were somewhat less than the target concentrations. Comparing the pyrethroid bioassay results with Anopheles at both treatment concentrations, deltamethrin gave significantly higher mortality (99.7-100%) than the other compounds (alphacypermethrin 94-96%, cyfluthrin 80-89%). Culex bioassay mortality was lower (alphacypermethrin 56-74%; cyfluthrin 63-65%; deltamethrin 50-81 %) and results with the three pyrethroid insecticides at their recommended doses did not differ significantly.     

Mosquito larval consumption of toxic arborescent leaf-litter,and its biocontrol potential

Previously we described the mosquito larvicidal properties of decomposed leaf-litter from deciduous trees, especially the alder Alnus glutinosa (L) Gaertn., due to toxic polyphenols and other secondary compounds. To further examine the biocontrol potential of toxic leaf-litter for mosquito control, feeding rates of third-instar mosquito larvae were assessed for examples of three genera: Anopheles stephensi Liston, Aedes aegypti (L) and Culex pipiens L. (Diptera: Culicidae). When immersed in a suspension of non-toxic leaf-litter particles (approximately 0.4 mm), pre-starved larvae of all three species ingested sufficient material in 30 min to fill the anterior gut lumen (thorax plus two to three abdominal segments). Gut filling peaked after 1-2 h ingestion time, filling the intestine up to six to seven abdominal segments for Ae. aegypti, but maxima of five abdominal segments for Cx. pipiens and An. stephensi. Using three methods to quantify consumption of three materials by third-instar larvae of Ae. aegypti, the average amount of leaf-litter (non-toxic 0.4 mm particles) ingested during 3 h was determined as approximately 20 microg/larva (by dry weight and by lignin spectrophotometric assay). Consumption of humine (approximately 100 microm particles extracted from leaf-litter) during 3 h was approximately 80 microg/larva for Ae. aegypti, but only approximately 30 microg/larva for Cx. pipiens and 15 microg/larva for An. stephensi, with good concordance of determinations by dry weight and by radiometric assay. Cellulose consumption by Ae. aegypti was intermediate: approximately 40 microg/larva determined by radiometric assay. Apparent differences between the amounts of these materials ingested by Ae. aegypti larvae (humine four-fold, cellulose two-fold more than leaf-litter) may be attributed to contrasts in palatability (perhaps related to particle size or form), rather than technical discrepancies, because there was good concordance between results of both methods used to determine the amounts of humine and leaf-litter ingested. Bioassays of toxic leaf-litter (decomposed 10 months) with 4-h exposure period (ingestion time) ranked the order of sensitivity: Ae. aegypti (LC50 < 0.03 g/L) > An. stephensi (LC50 = 0.35 g/L) > Cx. pipiens (LC20 > 0.4 g/L). When immersed in the high concentration of 0.5 g/L toxic leaf-litter (0.4 mm particles), as little as 15-30 min ingestion time (exposure period) was sufficient to kill the majority of larvae of all three species, as soon as the gut lumen was filled for only the first few abdominal segments. Possibilities for mosquito larval control with toxic leaf-litter products and the need for standardized ingestion bioassays of larvicidal particles are discussed.     

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.     

Breeding of Anopheles mosquitoes in irrigated areas of South Punjab, Pakistan

As part of investigations on potential linkages between irrigation and malaria transmission, all surface water bodies in and around three villages along an irrigation distributary in South Punjab, Pakistan, were surveyed for anopheline mosquito larvae (Diptera: Culicidae) from April 1999 to March 2000. Samples were characterized according to exposure to sunlight, substratum, presence of vegetation, fauna, inorganic matter and physical water condition (clear/turbid/foul). Also water temperature, dissolved oxygen (DO), electroconductivity (EC) and pH of sites were recorded. A total of 37982 Anopheles larvae of six morphological types were collected from 2992 samples taken from irrigation/agricultural and village/domestic aquatic habitats. Anopheles subpictus Grassi sensu lato was by far the most abundant (74.3%), followed by An. culicifacies Giles s.l. (4.1%), An. stephensi Liston s.l. (2.6%), An. pulcherrimus Theobald (1.8%), An. peditaeniatus Leicester (0.3%) and An. nigerrimus Giles (0.1%). The four most abundant species were significantly associated with waterlogged fields and communal village drinking-water tanks. Habitat characteristics most correlated with occurrence of anophelines were the physical water condition and the absence/presence of fauna, particularly predators. Occurrence and abundance of Anopheles immatures were not significantly correlated with water temperature, DO, EC or pH. Malaria vectors of the Anopheles culicifacies complex occurred at relatively low densities, mainly in irrigated and waterlogged fields. In South Punjab, where rainfall is very low, it should be possible to reduce anopheline breeding through water management, as larvae develop mainly in water bodies that are directly or indirectly related to the extensive canal-irrigation system.