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 mosquitoes 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 marker, 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.     

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, F(2) 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.     

Suboptimal Larval Habitats Modulate Oviposition of the Malaria Vector Mosquito Anopheles coluzzii

Selection of oviposition sites by gravid females is a critical behavioral step in the reproductive cycle of Anopheles coluzzii, which is one of the principal Afrotropical malaria vector mosquitoes. Several studies suggest this decision is mediated by semiochemicals associated with potential oviposition sites. To better understand the chemosensory basis of this behavior and identify compounds that can modulate oviposition, we examined the generally held hypothesis that suboptimal larval habitats give rise to semiochemicals that negatively influence the oviposition preference of gravid females. Dual-choice bioassays indicated that oviposition sites conditioned in this manner do indeed foster significant and concentration dependent aversive effects on the oviposition site selection of gravid females. Headspace analyses derived from aversive habitats consistently noted the presence of dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS) and 6-methyl-5-hepten-2-one (sulcatone) each of which unitarily affected An. coluzzii oviposition preference. Electrophysiological assays across the antennae, maxillary palp, and labellum of gravid An. coluzzii revealed differential responses to these semiochemicals. Taken together, these findings validate the hypothesis in question and suggest that suboptimal environments for An. coluzzii larval development results in the release of DMDS, DMTS and sulcatone that impact the response valence of gravid females.     

Integrated Genetic Map of Anopheles Gambiae: Use of Rapd Polymorphisms for Genetic, Cytogenetic and Sts Landmarks

Randomly amplified polymorphic DNA (RAPD) markers have been integrated in the genetic and cytogenetic maps of the malaria vector mosquito, Anopheles gambiae. Fifteen of these markers were mapped by recombination, relative to microsatellite markers that had been mapped previously. Thirty-four gel-purified RAPD bands were cloned and sequenced, generating sequence tagged sites (STSs) that can be used as entry points to the A. gambiae genome. Thirty one of these STSs were localized on nurse cell polytene chromosomes through their unique hybridization signal in in situ hybridization experiments. Five STSs map close to the breakpoints of polymorphic inversions, which are notable features of the Anopheles genome. The usefulness and limitations of this integrated mosquito map are discussed.     

Landscape Movements of Anopheles gambiae Malaria Vector Mosquitoes in Rural Gambia

Background

For malaria control in Africa it is crucial to characterise the dispersal of its most efficient vector, Anopheles gambiae, in order to target interventions and assess their impact spatially. Our study is, we believe, the first to present a statistical model of dispersal probability against distance from breeding habitat to human settlements for this important disease vector.

Methods/Principal Findings

We undertook post-hoc analyses of mosquito catches made in The Gambia to derive statistical dispersal functions for An. gambiae sensu lato collected in 48 villages at varying distances to alluvial larval habitat along the River Gambia. The proportion dispersing declined exponentially with distance, and we estimated that 90% of movements were within 1.7 km. Although a ‘heavy-tailed’ distribution is considered biologically more plausible due to active dispersal by mosquitoes seeking blood meals, there was no statistical basis for choosing it over a negative exponential distribution. Using a simple random walk model with daily survival and movements previously recorded in Burkina Faso, we were able to reproduce the dispersal probabilities observed in The Gambia.

Conclusions/Significance

Our results provide an important quantification of the probability of An. gambiae s.l. dispersal in a rural African setting typical of many parts of the continent. However, dispersal will be landscape specific and in order to generalise to other spatial configurations of habitat and hosts it will be necessary to produce tractable models of mosquito movements for operational use. We show that simple random walk models have potential. Consequently, there is a pressing need for new empirical studies of An. gambiae survival and movements in different settings to drive this development.     

Insights from the Genome Annotation of Elizabethkingia anophelis from the Malaria Vector Anopheles gambiae

Elizabethkingia anophelis is a dominant bacterial species in the gut ecosystem of the malaria vector mosquito Anopheles gambiae. We recently sequenced the genomes of two strains of E. anophelis, R26^T and Ag1, isolated from different strains of A. gambiae. The two bacterial strains are identical with a few exceptions. Phylogenetically, Elizabethkingia is closer to Chryseobacterium and Riemerella than to Flavobacterium. In line with other Bacteroidetes known to utilize various polymers in their ecological niches, the E. anophelis genome contains numerous TonB dependent transporters with various substrate specificities. In addition, several genes belonging to the polysaccharide utilization system and the glycoside hydrolase family were identified that could potentially be of benefit for the mosquito carbohydrate metabolism. In agreement with previous reports of broad antibiotic resistance in E. anophelis, a large number of genes encoding efflux pumps and β-lactamases are present in the genome. The component genes of resistance-nodulation-division type efflux pumps were found to be syntenic and conserved in different taxa of Bacteroidetes. The bacterium also displays hemolytic activity and encodes several hemolysins that may participate in the digestion of erythrocytes in the mosquito gut. At the same time, the OxyR regulon and antioxidant genes could provide defense against the oxidative stress that is associated with blood digestion. The genome annotation and comparative genomic analysis revealed functional characteristics associated with the symbiotic relationship with the mosquito host.     

Distinct Olfactory Signaling Mechanisms in the Malaria Vector Mosquito Anopheles gambiae

Anopheles gambiae is the principal Afrotropical vector for human malaria, in which olfaction mediates a wide range of both adult and larval behaviors. Indeed, mosquitoes depend on the ability to respond to chemical cues for feeding, host preference, and mate location/selection. Building upon previous work that has characterized a large family of An. gambiae odorant receptors (AgORs), we now use behavioral analyses and gene silencing to examine directly the role of AgORs, as well as a newly identified family of candidate chemosensory genes, the An. gambiae variant ionotropic receptors (AgIRs), in the larval olfactory system. Our results validate previous studies that directly implicate specific AgORs in behavioral responses to DEET as well as other odorants and reveal the existence of at least two distinct olfactory signaling pathways that are active in An. gambiae. One system depends directly on AgORs; the other is AgOR-independent and requires the expression and activity of AgIRs. In addition to clarifying the mechanistic basis for olfaction in this system, these advances may ultimately enhance the development of vector control strategies, targeting olfactory pathways in mosquitoes to reduce the catastrophic effects of malaria and other mosquito-borne diseases.     

A Wickerhamomyces anomalus Killer Strain in the Malaria Vector Anopheles stephensi

The yeast Wickerhamomyces anomalus has been investigated for several years for its wide biotechnological potential, especially for applications in the food industry. Specifically, the antimicrobial activity of this yeast, associated with the production of Killer Toxins (KTs), has attracted a great deal of attention. The strains of W. anomalus able to produce KTs, called “killer” yeasts, have been shown to be highly competitive in the environment. Different W. anomalus strains have been isolated from diverse habitats and recently even from insects. In the malaria mosquito vector Anopheles stephensi these yeasts have been detected in the midgut and gonads. Here we show that the strain of W. anomalus isolated from An. stephensi, namely WaF17.12, is a killer yeast able to produce a KT in a cell-free medium (in vitro) as well as in the mosquito body (in vivo). We showed a constant production of WaF17.12-KT over time, after stimulation of toxin secretion in yeast cultures and reintroduction of the activated cells into the mosquito through the diet. Furthermore, the antimicrobial activity of WaF17.12-KT has been demonstrated in vitro against sensitive microbes, showing that strain WaF17.12 releases a functional toxin. The mosquito-associated yeast WaF17.12 thus possesses an antimicrobial activity, which makes this yeast worthy of further investigations, in view of its potential as an agent for the symbiotic control of malaria.     

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.     

Midgut Microbiota of the Malaria Mosquito Vector Anopheles gambiae and Interactions with Plasmodium falciparum Infection

The susceptibility of Anopheles mosquitoes to Plasmodium infections relies on complex interactions between the insect vector and the malaria parasite. A number of studies have shown that the mosquito innate immune responses play an important role in controlling the malaria infection and that the strength of parasite clearance is under genetic control, but little is known about the influence of environmental factors on the transmission success. We present here evidence that the composition of the vector gut microbiota is one of the major components that determine the outcome of mosquito infections. A. gambiae mosquitoes collected in natural breeding sites from Cameroon were experimentally challenged with a wild P. falciparum isolate, and their gut bacterial content was submitted for pyrosequencing analysis. The meta-taxogenomic approach revealed a broader richness of the midgut bacterial flora than previously described. Unexpectedly, the majority of bacterial species were found in only a small proportion of mosquitoes, and only 20 genera were shared by 80% of individuals. We show that observed differences in gut bacterial flora of adult mosquitoes is a result of breeding in distinct sites, suggesting that the native aquatic source where larvae were grown determines the composition of the midgut microbiota. Importantly, the abundance of Enterobacteriaceae in the mosquito midgut correlates significantly with the Plasmodium infection status. This striking relationship highlights the role of natural gut environment in parasite transmission. Deciphering microbe-pathogen interactions offers new perspectives to control disease transmission.     

How the Malaria Vector Anopheles gambiae Adapts to the Use of Insecticide-Treated Nets by African Populations

BackgroundInsecticide treated bed nets have been recommended and proven efficient as a measure to protect African populations from malaria mosquito vector Anopheles spp. This study evaluates the consequences of bed nets use on vectors resistance to insecticides, their feeding behavior and malaria transmission in Dielmo village, Senegal, were LLINs were offered to all villagers in July 2008.MethodsAdult mosquitoes were collected monthly from January 2006 to December 2011 by human landing catches (HLC) and by pyrethroid spray catches (PCS). A randomly selected sub-sample of 15–20% of An. gambiae s.l. collected each month was used to investigate the molecular forms of the An. gambiae complex, kdr mutations, and Plasmodium falciparum circumsporozoite (CSP) rate. Malaria prevalence and gametocytaemia in Dielmo villagers were measured quarterly.ResultsInsecticide susceptible mosquitoes (wild kdr genotype) presented a reduced lifespan after LLINs implementation but they rapidly adapted their feeding behavior, becoming more exophageous and zoophilic, and biting earlier during the night. In the meantime, insecticide-resistant specimens (kdr L1014F genotype) increased in frequency in the population, with an unchanged lifespan and feeding behaviour. P. falciparum prevalence and gametocyte rate in villagers decreased dramatically after LLINs deployment. Malaria infection rate tended to zero in susceptible mosquitoes whereas the infection rate increased markedly in the kdr homozygote mosquitoes.ConclusionDramatic changes in vector populations and their behavior occurred after the deployment of LLINs due to the extraordinary adaptative skills of An. gambiae s. l. mosquitoes. However, despite the increasing proportion of insecticide resistant mosquitoes and their almost exclusive responsibility in malaria transmission, the P. falciparum gametocyte reservoir continued to decrease three years after the deployment of LLINs.     

The Virulent Wolbachia Strain wMelPop Efficiently Establishes Somatic Infections in the Malaria Vector Anopheles gambiae

Wolbachia pipientis bacteria are maternally inherited endosymbionts that are of interest to control the Anopheles mosquito vectors of malaria. Wolbachia does not infect Anopheles mosquitoes in nature, although cultured Anopheles cells can be infected. Here, we show that the virulent Wolbachia strain wMelPop can survive and replicate when injected into female Anopheles gambiae adults, but the somatic infections established are avirulent. These in vivo data suggest that stable Wolbachia infections of Anopheles may be possible.Infecting up to 500 million people per year (with almost 3 million annual deaths), malaria is the most important vector-borne disease in the world (8, 30, 31, 32). The Plasmodium parasites that cause the disease are transmitted to humans by the bite of Anopheles mosquitoes, with Anopheles gambiae being the principle vector in sub-Saharan Africa (6). Malaria control is limited by the lack of a vaccine and by parasite and mosquito evolution of drug and insecticide resistance (9, 28, 31). In light of these problems, there has been a recent concerted effort to develop innovative methods for malaria control based on the genetic modification of Anopheles mosquitoes (transgenesis) or their associated symbiotic microorganisms (paratransgenesis) (5, 10, 11, 13, 15, 23, 25, 27, 36, 37).In many mosquitoes, Wolbachia pipientis symbionts are the causative agents of cytoplasmic incompatibility, a phenomenon where matings between uninfected females and infected males have reduced egg hatch, while matings in the reciprocal cross are fertile. In a mixed population, infected females have a reproductive advantage which can allow Wolbachia to increase rapidly in frequency due to maternal inheritance. The propensity of Wolbachia to “drive” through populations has been investigated using mathematical models and has been validated by both laboratory and field investigations (20, 21, 33, 34, 35, 36).There are three scenarios currently envisioned to use Wolbachia as part of a malaria control strategy: (i) use Wolbachia spread to “drive” refractory transgenes into Anopheles populations, converting the mosquito population into one that cannot maintain transmission of the malaria parasites (18, 21, 24, 29, 33, 36); (ii) release Wolbachia-infected males into uninfected Anopheles populations to reduce population sizes through cytoplasmic incompatibility, similar to the sterile insect technique but without exposing males to radiation or chemical sterilants that could lower their fitness (2, 4, 37); and (iii) release mosquitoes infected with pathogenic or virulent Wolbachia strains that shorten mosquito life span. Pathogens must pass through an extrinsic incubation period in the vector before they are able to be transmitted. By shortening mosquito life span, it is theoretically possible to reduce the number of mosquitoes that live through the extrinsic incubation period and become infectious (14, 15, 17, 20, 24).All of the above strategies require the stable transfer of Wolbachia into Anopheles. Wolbachia symbionts are common in mosquitoes, but no infections have ever been identified in any species of Anopheles (12, 22, 26). The negative infection status of natural Anopheles populations offers good potential for Wolbachia-based malaria control strategies, since preexisting infections can complicate the behavior of introduced infections (33). However, the absence of natural infections in anophelines has led some to suggest that Anopheles mosquitoes may be physiologically/genetically incapable of harboring Wolbachia infections (1, 29). If this hypothesis is true, then Wolbachia-based malaria control strategies are likely doomed to fail. In vitro studies demonstrated that cultured immunocompetent Anopheles gambiae cell lines (Sua5B and Moss55) are fully competent to harbor infections of distinct Wolbachia strains (the “A” supergroup strains wRi and wMelPop from Drosophila simulans and Drosophila melanogaster and the “B” supergroup strain wAlbB from Aedes albopictus) (16, 18). Some cultured infections reached very high levels where 100% of cells were infected at extremely high levels (wAlbB in Sua5B and wMelPop in Moss55) (16, 18), while other strains were eventually eliminated from the cells (wRi in Sua5B cells) (24). The combined results of these experiments, using multiple Wolbachia strains and multiple Anopheles cell lines, indicate that there is no intrinsic genetic block to Wolbachia infection in Anopheles cells, although certain strains of Wolbachia may be more likely to colonize Anopheles than others.In this study, we investigated the establishment of in vivo Wolbachia infections in Anopheles gambiae (Keele strain) mosquitoes by injection of the virulent Wolbachia strain wMelPop into the hemolymph of adult female mosquitoes. Approximately 200 adult mosquitoes were reared in 30-cm cube cages in a walk-in insectary held at 28°C and 80% relative humidity on a 12:12 h light/dark cycle. Mosquitoes were allowed access to a cotton wick soaked in 10% sucrose as a carbohydrate source. Adults were allowed to blood feed on an anesthetized mouse 5 days postemergence according to JHU animal use protocol MO-03H210. Two days after blood feeding, an oviposition substrate (consisting of a filter paper cone inside a 50-ml beaker half filled with water) was introduced into cages and removed the next day for egg collection. Approximately 250 eggs were placed into a 41- by 34- by 6-cm rearing tray half filled with distilled water and one pellet of dry cat food, with one additional pellet added to each tray daily after day 3. Larvae were removed, and tray water changed if polluted. Pupae were picked with an eyedropper, placed in a cup, and introduced into cages (∼200 pupae/cage) to begin the next generation.wMelPop was cultured in Anopheles gambiae Moss55 cells (16), purified, and assessed for viability as described previously (18, 19). Purified Wolbachia cells were suspended in culture medium and adjusted to a final concentration of 10⁸ bacteria per ml. Using a calibrated glass capillary needle, amounts of 100 to 200 nl suspended Wolbachia cells were injected into the thorax of 2-day-old, cold-immobilized adult Anopheles gambiae females. Injected mosquitoes were held at 18°C for 5 days and then transferred to the 28°C insectary. Mosquitoes were allowed to blood feed on a mouse twice per week.Mosquito genomic DNA was extracted by salt extraction/ethanol precipitation as described previously (21), quantified using a NanoDrop spectrophotometer, and adjusted to 20 ng/μl. Wolbachia infections in individual mosquitoes were detected by PCR amplification of a fragment of the Wolbachia 16S rRNA gene (440 bp) using primers WspecF and WspecR (18). As a control, we amplified a 400-bp fragment from the Anopheles mitochondrial NADH dehydrogenase subunit 4 gene (ND4) (18). Mosquitoes were assayed for Wolbachia infection by PCR at 6, 10, 20, or 30 days postinjection (p.i.). In a second experiment, mosquitoes were assayed at 0, 3, 8, 13, 15, and 21 days p.i. Amplified fragments were separated by 1% agarose gel electrophoresis, stained with ethidium bromide, and viewed under UV light. Template DNA from infected and uninfected Moss55 cells was included as positive and negative controls.Quantitative PCR (qPCR) was used to determine if Wolbachia could survive and replicate in Anopheles by comparing normalized Wolbachia levels in individual mosquitoes at day 6 and day 30 p.i. The relative abundance of wMelPop bacteria in each mosquito was assessed by comparing the abundance of the single-copy Wolbachia ankyrin repeat gene WD_0550 (16) to that of the single-copy Anopheles gambiae ribosomal S7 gene (forward, 5′-TCC-TGG-AGC-TGG-AGA-TGA-AC-3′, and reverse, 5′-GAC-GGG-TCT-GTA-CCT-TCT-GG-3′). For each time point, 14 mosquitoes (biological replicates) were examined. Duplicate reactions were performed for every mosquito, and the results differed by less than 3%, demonstrating consistency of the assay. qPCR was performed using an ABI Prism 7300 detection system (Applied Biosystems) with a QuantiTect SYBR green PCR kit (Qiagen). Determinations of relative abundance of wMelPop in each mosquito and relative changes in wMelPop levels between time points, confidence interval estimation, and statistical analyses were carried out as described by Yuan et al. (38).To test for virulence of wMelPop, 2-day-old adult female mosquitoes were injected with either wMelPop purified from cell culture or filtered lysate of uninfected Moss55 cells (control) and held at 18°C for 2 days as described above. Injected mosquitoes were held an additional 3 days at 28°C to control for mortality due to the injection procedure. At day 5 p.i., mosquitoes were moved into pint-sized cup cages for life table experiments. Approximately 25 mosquitoes were placed in each cage (two replicate control cages and three replicate Wolbachia treatment cages) and the entire experiment replicated two times, for a total of four control cages and six Wolbachia treatment cages. Mosquitoes were provided a cotton pad soaked in 10% sucrose but were not given access to blood. Dead mosquitoes were removed from each cage approximately every other day. For each experiment, mortality data were used to construct treatment-specific cohort life tables (3). Because the data did not conform to parametric assumptions, they were analyzed by the Mann-Whitney U test using STATVIEW (SAS Corporation).In injected mosquitoes, Wolbachia bacteria were detectable by PCR at all time points, as follows [infection frequency (95% exact binomial confidence interval)]: day 6, 0.875 (0.617 to 0.985; n = 16); day 10, 0.75 (0.401 to 0.968; n = 8); day 20, 0.722 (0.465 to 0.903; n = 18); and day 30, 1.0 (0.794 to 1.0; n = 13). In further experiments, Wolbachia bacteria were easily detectable through day 3 p.i. but were weak or not detectable by conventional PCR by day 8 p.i. After 13 days p.i., Wolbachia bacteria were easily detectable again, and the bands increased in intensity for the remainder of the time series experiment (Fig. ​(Fig.1).1). This initial decrease, followed by an increase, in the apparent infection rate is possibly due to initial clearance of some of the injected bacteria and then establishment of infection and bacterial replication. By qPCR, a highly statistically significant 42-fold increase in the normalized Wolbachia level was observed: on day 6 p.i., there were 23.7 Wolbachia genomes per host genome (95% confidence interval, 10.6 to 52.7; n = 14), and on day 30 p.i., there were 992 Wolbachia genomes per host genome (95% confidence interval, 433.6 to 2,267.4; n = 14) (Mann-Whitney U test, tied Z value = −4.319; P < 0.0001). Since Wolbachia cannot replicate in the extracellular environment (19), these results confirm that injected bacteria are able to infect cells, survive, and replicate in Anopheles gambiae in vivo.Open in a separate windowFIG. 1.Typical results using conventional PCR, showing changes in Wolbachia levels in injected adult Anopheles gambiae females at sequential time points postinjection. Results for mt control (host ND4 mitochondrial gene) indicate that PCR efficiency was approximately equal for all samples. M, 100-bp marker; d, days.wMelPop is a virulent Wolbachia strain that reduces the life span of its host by approximately 50%. While originally found and characterized in a laboratory colony of Drosophila melanogaster, it has similar pathogenic effects when artificially transferred into Drosophila simulans (14), and recently, the yellow fever mosquito Aedes aegypti (15). However, there was no statistically significant difference in survival trajectories between Anopheles gambiae mosquitoes injected with wMelPop and mosquitoes injected with filtered uninfected cell lysate (Mann-Whitney U test, tied Z value = −1.799; P = 0.702) (Fig. ​(Fig.2).2). Although wMelPop replicates to high levels in injected Anopheles (approximately 1,000 bacterial genomes per host genome), these levels do not seem to be associated with virulence. It is possible that the virulence of wMelPop has been attenuated during its culture in Moss55 cells, although during long-term culture in an Aedes aegypti cell line, wMelPop retained its virulent phenotype when reintroduced into either Drosophila or Aedes aegypti in vivo (15, 16). The specific mechanism of wMelPop virulence is not completely understood, but it seems that increased host mortality is not simply due to overreplication and high infection levels but rather to overreplication in and damage to specific host tissues, such as the brain and central nervous system (14, 15, 17). Investigation into the tissue localization of Wolbachia in injected Anopheles mosquitoes is currently ongoing, but in light of these results, it is reasonable to hypothesize that when injected into the hemolymph, Wolbachia bacteria reach high levels in some mosquito tissues but either do not infect or do not replicate in the Anopheles central nervous system. It remains to be seen whether vertically acquired infections will show virulence in Anopheles gambiae.Open in a separate windowFIG. 2.Mean survival trajectories of wMelPop-injected versus cell lysate-injected Anopheles gambiae adult females. Survival trajectories do not differ significantly. Error bars show standard deviations.Previous studies showed that cultured Anopheles gambiae cells can be infected with Wolbachia (16, 18), but no data were available to assess whether the in vitro results could be extrapolated to Anopheles mosquitoes in vivo. The experiments outlined in this paper demonstrate that Wolbachia can infect Anopheles mosquitoes in vivo. However, for a Wolbachia-based malaria control strategy to be effective, simply infecting Anopheles by injection is not sufficient—the infection must be transmitted vertically to offspring. Stable (100%) vertical transmission of Wolbachia after injection into adults has been reported for Drosophila melanogaster (7). A similar phenomenon has been reported for Aedes aegypti, but transmission was unstable (approximately 40%) (27). Experiments to determine whether Wolbachia bacteria injected into the hemolymph of adult Anopheles will be transmitted vertically to offspring are ongoing, and if efficient vertical transmission of the symbionts can be established, Wolbachia-based strategies for malaria control should be possible.     

Molecular Characterization of Larval Peripheral Thermosensory Responses of the Malaria Vector Mosquito Anopheles gambiae

Thermosensation provides vital inputs for the malaria vector mosquito, Anopheles gambiae which utilizes heat-sensitivity within a broad spectrum of behaviors, most notably, the localization of human hosts for blood feeding. In this study, we examine thermosensory behaviors in larval-stage An. gambiae, which as a result of their obligate aquatic habitats and importance for vectorial capacity, represents an opportunistic target for vector control as part of the global campaign to eliminate malaria. As is the case for adults, immature mosquitoes respond differentially to a diverse array of external heat stimuli. In addition, larvae exhibit a striking phenotypic plasticity in thermal-driven behaviors that are established by temperature at which embryonic development occurs. Within this spectrum, RNAi-directed gene-silencing studies provide evidence for the essential role of the Transient Receptor Potential sub-family A1 (TRPA1) channel in mediating larval thermal-induced locomotion and thermal preference within a discrete upper range of ambient temperatures.     

Selective Introgression of Paracentric Inversions between Two Sibling Species of the Anopheles Gambiae Complex

The Anopheles gambiae complex includes the major vectors of malaria in sub-Saharan Africa where >80% of all world-wide cases occur. These mosquitoes are characterized by chromosomal inversions associated to the speciation process and to intraspecific ecological and behavioral flexibility. It has been postulated that introgressive hybridization has selectively transferred inversions on the second chromosome between A. gambiae and A. arabiensis, the two most important vectors of malaria. Here we directly test this hypothesis with laboratory experiments in which hybrid populations were established and the fate of chromosomal inversions were followed. Consistent with the hypothesis, ``foreign' X chromosomes were eliminated within two generations, while some ``foreign' second chromosomes persisted for the duration of the experiments and, judging from the excess of heterozygotes, established stable heterotic polymorphisms. Only those second chromosome inversions found naturally in the species could be introgressed.     

Malaria and the Anopheles mosquitoes of Tajikistan

Surveys of Anopheles mosquitoes were conducted in urban, rural, and natural areas of Tajikistan to obtain updated information on their distributions, especially in southern districts of the country where malaria is a prevalent disease. Nine species of Anopheles are found in Tajikistan. Anopheles superpictus, An. claviger, An. hyrcanus, and An. pulcherrimus are the most widespread and abundant species. Investigations in northern Tajikistan confirmed the presence of An. artemievi and the absence of An. martinius, both members of the An. maculipennis complex of malaria vectors. Anopheles barianensis, An. lindesayi, and An. marteri sogdianus, species previously recorded in the country, were not encountered during our surveys. The history of Anopheles and malaria research in Tajikistan is reviewed and bionomical and distributional information is provided for each of the nine species.     

Propeptide-Mediated Specific Inhibition of a Recombinant Serine Protease from Indian Malaria Vector, Anopheles culicifacies

Serine proteases are a class of proteolytic enzymes that are synthesized as enzymically inactive zymogens and when required in the cell, they are activated by the removal of proregion. The role of proregions as potent and specific inhibitors of their associated protease has been established. Here, we investigated the inhibition of a recombinantly expressed and refolded Anopheles c ulicifacies serine protease (ACSP) that was isolated from the body tissue of an Indian malaria vector, A. culicifacies by its own N-terminally located 19 amino acid residue propeptide. The synthetic peptide identical to the propeptide, its three deletion mutants and leupeptin (a general serine protease inhibitor) were tested in vitro for their inhibitory activity towards recombinant ACSP. Amongst the five peptides tested, leupeptin displayed maximum inhibition closely followed by native propeptide. The reduction or loss of inhibitory potential of deletion mutants of propeptide revealed the importance of charged residues present in the propeptide for inhibition of the cognate enzyme.