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.     

Biochemical Characterization of Anopheles gambiae SRPN6, a Malaria Parasite Invasion Marker in Mosquitoes

Serine proteinase inhibitors of the serpin family are well known as negative regulators of hemostasis, thrombolysis and innate immune responses. Additionally, non-inhibitory serpins serve functions as chaperones, hormone transporters, or anti-angiogenic factors. In the African malaria mosquito, Anopheles gambiae s.s., at least three serpins (SRPNs) are implicated in the innate immune response against malaria parasites. Based on reverse genetic and cell biological analyses, AgSRPN6 limits parasite numbers and transmission and has been postulated to control melanization and complement function in mosquitoes. This study aimed to characterize AgSRPN6 biophysically and determine its biochemical mode of action. The structure model of AgSRPN6, as predicted by I-Tasser showed the protein in the native serpin fold, with three central β-sheets, nine surrounding α-helices, and a protruding reactive center loop. This structure is in agreement with biophysical and functional data obtained from recombinant (r) AgSRPN6, produced in Escherichia coli. The physical properties of purified rAgSRPN6 were investigated by means of analytical ultracentrifugation, circular dichroism, and differential scanning calorimetry tools. The recombinant protein exists predominantly as a monomer in solution, is composed of a mixture of α-helices and β-sheets, and has a mid-point unfolding temperature of 56°C. Recombinant AgSRPN6 strongly inhibited porcine pancreatic kallikrein and to a lesser extent bovine pancreatic trypsin in vitro. Furthermore, rAgSRPN6 formed inhibitory, SDS-stable, higher molecular weight complexes with prophenoloxidase-activating proteinase (PAP)1, PAP3, and Hemolymph protein (HP)6, which are required for melanization in the lepidopteran model organism, Manduca sexta. Taken together, our results strongly suggest that AgSRPN6 takes on a native serpin fold and is an inhibitor of trypsin-like serine proteinases.     

Olfactory Coding in Antennal Neurons of the Malaria Mosquito, Anopheles gambiae

Olfactory receptor neurons (ORNs) in the antenna of insects serve to encode odors in action potential activity conducted to the olfactory lobe of the deuterocerebrum. We performed an analysis of the electrophysiological responses of olfactory neurons in the antennae of the female malaria mosquito Anopheles gambiae s.s. and investigated the effect of blood feeding on responsiveness. Forty-four chemicals that are known to be present in human volatile emanations were used as odor stimuli. We identified 6 functional types of trichoid sensilla and 5 functional types of grooved-peg sensilla (GP) based on a hierarchical cluster analysis. Generalist ORNs, tuned to a broad range of odors, moderate specialist ORNs and 2 ORNs tuned to only one odor were identified in different sensilla types. Neurons in GP were tuned to more polar compounds including the important behavioral attractant ammonia and its synergist L-lactic acid, responses to which were found only in GP. Combinatorial coding is the most plausible principle operating in the olfactory system of this mosquito species. We document for the first time both up- and downregulation of ORN responsiveness after blood feeding. Modulation of host-seeking and oviposition behavior is associated with both qualitative and quantitative changes in the peripheral sensory system.     

The Mode of Action of Spatial Repellents and Their Impact on Vectorial Capacity of Anopheles gambiae sensu stricto

Malaria vector control relies on toxicity of insecticides used in long lasting insecticide treated nets and indoor residual spraying. This is despite evidence that sub–lethal insecticides reduce human–vector contact and malaria transmission. The impact of sub–lethal insecticides on host seeking and blood feeding of mosquitoes was measured. Taxis boxes distinguished between repellency and attraction inhibition of mosquitoes by measuring response of mosquitoes towards or away from Transfluthrin coils and humans. Protective effective distance of coils and long-term effects on blood feeding were measured in the semi–field tunnel and in a Peet Grady chamber. Laboratory reared pyrethroid susceptible Anopheles gambiae sensu stricto mosquitoes were used. In the taxis boxes, a higher proportion of mosquitoes (67%–82%) were activated and flew towards the human in the presence of Transfluthrin coils. Coils did not hinder attraction of mosquitoes to the human. In the semi–field Tunnel, coils placed 0.3 m from the human reduced feeding by 86% (95% CI [0.66; 0.95]) when used as a “bubble” compared to 65% (95% CI [0.51; 0.76]) when used as a “point source”. Mosquitoes exposed to coils inside a Peet Grady chamber were delayed from feeding normally for 12 hours but there was no effect on free flying and caged mosquitoes exposed in the semi–field tunnel. These findings indicate that airborne pyrethroids minimize human–vector contact through reduced and delayed blood feeding. This information is useful for the development of target product profiles of spatial repellent products that can be used to complement mainstream malaria vector control tools.     

Bioinformatics-based identification of chemosensory proteins in African Malaria Mosquito, Anopheles gambiae

Chemosensory proteins (CSPs) are identifiable by four spatially conserved Cys-teine residues in their primary structure or by two disulfide bridges in their tertiary structure according to the previously identified olfactory specific-D related proteins. A genomics- and bioinformatics-based approach is taken in the present study to identify the putative CSPs in the malaria-carrying mosquito, Anopheles     

Synergy in Efficacy of Fungal Entomopathogens and Permethrin against West African Insecticide-Resistant Anopheles gambiae Mosquitoes

Background

Increasing incidences of insecticide resistance in malaria vectors are threatening the sustainable use of contemporary chemical vector control measures. Fungal entomopathogens provide a possible additional tool for the control of insecticide-resistant malaria mosquitoes. This study investigated the compatibility of the pyrethroid insecticide permethrin and two mosquito-pathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, against a laboratory colony and field population of West African insecticide-resistant Anopheles gambiae s.s. mosquitoes.

Methodology/Findings

A range of fungus-insecticide combinations was used to test effects of timing and sequence of exposure. Both the laboratory-reared and field-collected mosquitoes were highly resistant to permethrin but susceptible to B. bassiana and M. anisopliae infection, inducing 100% mortality within nine days. Combinations of insecticide and fungus showed synergistic effects on mosquito survival. Fungal infection increased permethrin-induced mortality rates in wild An. gambiae s.s. mosquitoes and reciprocally, exposure to permethrin increased subsequent fungal-induced mortality rates in both colonies. Simultaneous co-exposure induced the highest mortality; up to 70.3±2% for a combined Beauveria and permethrin exposure within a time range of one gonotrophic cycle (4 days).

Conclusions/Significance

Combining fungi and permethrin induced a higher impact on mosquito survival than the use of these control agents alone. The observed synergism in efficacy shows the potential for integrated fungus-insecticide control measures to dramatically reduce malaria transmission and enable control at more moderate levels of coverage even in areas where insecticide resistance has rendered pyrethroids essentially ineffective.     

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.     

Chromosomal study of Anopheles gambiae and Anopheles arabiensis in Ouagadougou (Burkina Faso) and various neighboring villages

Adult females of Anopheles gambiae s.1. were collected by pyrethrum spray catch in Ouagadougou (Burkina Faso, formerly Upper Volta) and in four neighbouring villages. The collections have been carried out mostly during the 1984 rainy season. Monthly collections in some sampling sites allowed a preliminary longitudinal study. By analysis of nurse cell polytene chromosomes in adult females, An. gambiae s.str. and An. arabiensis were identified in the study area. Both species showed polymorphisms for various paracentric inversions. In all samples of An. arabiensis the frequencies of the alternative karyotypes were in Hardy-Weinberg equilibrium, suggesting panmictic conditions. Conversely, An. gambiae s.str. showed a different situation, since most of its samples had strong deficiency of certain expected heterokaryotypes. This same phenomenon was already observed in Mali, leading to the splitting of gambiae s.str. into different chromosomal forms, partially or totally reproductively isolated from each other. Each chromosomal form is characterized by different chromosomal polymorphisms. Two of these forms, Mopti and Savanna, were detected in the study area. Mopti chromosomal form is apparently associated with the presence of permanent waters (i.e. the "barrages" north of the town), while Savanna is usually found in situations where breeding places are mainly dependent from rain (e.g. in villages far from "barrages" or at the town's center).     

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.     

New selenoproteins identified in silico from the genome of Anopheles gambiae

Selenoprotein is biosynthesized by the incorporation of selenocysteine into proteins,where the TGA codon in the open reading frame does not act as a stop signal but is translated into selenocysteine.The dual functions of TGA result in mis-annotation or lack of selenoproteins in the sequenced genomes of many species.Available computational tools fail to correctly predict selenoproteins.Thus,we devel-oped a new method to identify selenoproteins from the genome of Anopheles gambiae computationally.Based on released genomic information,several programs were edited with PERL language to identify selenocysteine insertion sequence(SECIS)element,the coding potential of TGA codons,and cys-teine-containing homologs of selenoprotein genes.Our results showed that 11365 genes were termi-nated with TGA codons,918 of which contained SECIS elements.Similarity search revealed that 58 genes contained Sec/Cys pairs and similar flanking regions around in-frame TGA codons.Finally,7 genes were found to fully meet requirements for selenoproteins,although they have not been anno-tated as selenoproteins in NCBI databases.Deduced from their basic properties,the newly found se-lenoproteins in the genome of Anopheles gambiae are possibly related to in vivo oxidation tolerance and protein regulation in order to interfere with anopheles' vectorial capacity of Plasmodium.This study may also provide theoretical bases for the prevention of malaria from anopheles transmission.     

New selenoproteins identified in silico from the genome of Anopheles gambiae

Selenoprotein is biosynthesized by the incorporation of selenocysteine into proteins, where the TGA codon in the open reading frame does not act as a stop signal but is translated into selenocysteine. The dual functions of TGA result in mis-annotation or lack of selenoproteins in the sequenced genomes of many species. Available computational tools fail to correctly predict selenoproteins. Thus, we developed a new method to identify selenoproteins from the genome of Anopheles gambiae computationally.Based on released genomic information, several programs were edited with PERL language to identify selenocysteine insertion sequence (SECIS) element, the coding potential of TGA codons, and cysteine-containing homologs of selenoprotein genes. Our results showed that 11365 genes were terminated with TGA codons, 918 of which contained SECIS elements. Similarity search revealed that 58genes contained Sec/Cys pairs and similar flanking regions around in-frame TGA codons. Finally, 7genes were found to fully meet requirements for selenoproteins, although they have not been annotated as selenoproteins in NCBI databases. Deduced from their basic properties, the newly found selenoproteins in the genome of Anopheles gambiae are possibly related to in vivo oxidation tolerance and protein regulation in order to interfere with anopheles' vectorial capacity of Plasmodium. This study may also provide theoretical bases for the prevention of malaria from anopheles transmission.     

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.     

Salivary Gland Proteome Analysis Reveals Modulation of Anopheline Unique Proteins in Insensitive Acetylcholinesterase Resistant Anopheles gambiae Mosquitoes

Insensitive acetylcholinesterase resistance due to a mutation in the acetylcholinesterase (ace) encoding ace-1 gene confers cross-resistance to organophosphate and carbamate insecticides in Anopheles gambiae populations from Central and West Africa. This mutation is associated with a strong genetic cost revealed through alterations of some life history traits but little is known about the physiological and behavioural changes in insects bearing the ace-1^R allele. Comparative analysis of the salivary gland contents between An. gambiae susceptible and ace-1^R resistant strains was carried out to charaterize factors that could be involved in modifications of blood meal process, trophic behaviour or pathogen interaction in the insecticide-resistant mosquitoes. Differential analysis of the salivary gland protein profiles revealed differences in abundance for several proteins, two of them showing major differences between the two strains. These two proteins identified as saglin and TRIO are salivary gland-1 related proteins, a family unique to anopheline mosquitoes, one of them playing a crucial role in salivary gland invasion by Plasmodium falciparum sporozoites. Differential expression of two other proteins previously identified in the Anopheles sialome was also observed. The differentially regulated proteins are involved in pathogen invasion, blood feeding process, and protection against oxidation, relevant steps in the outcome of malaria infection. Further functional studies and insect behaviour experiments would confirm the impact of the modification of the sialome composition on blood feeding and pathogen transmission abilities of the resistant mosquitoes. The data supports the hypothesis of alterations linked to insecticide resistance in the biology of the primary vector of human malaria in Africa.     

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.     

Genetic Structure of a Local Population of the Anopheles gambiae Complex in Burkina Faso

Members of the Anopheles gambiae species complex are primary vectors of human malaria in Africa. Population heterogeneities for ecological and behavioral attributes expand and stabilize malaria transmission over space and time, and populations may change in response to vector control, urbanization and other factors. There is a need for approaches to comprehensively describe the structure and characteristics of a sympatric local mosquito population, because incomplete knowledge of vector population composition may hinder control efforts. To this end, we used a genome-wide custom SNP typing array to analyze a population collection from a single geographic region in West Africa. The combination of sample depth (n = 456) and marker density (n = 1536) unambiguously resolved population subgroups, which were also compared for their relative susceptibility to natural genotypes of Plasmodium falciparum malaria. The population subgroups display fluctuating patterns of differentiation or sharing across the genome. Analysis of linkage disequilibrium identified 19 new candidate genes for association with underlying population divergence between sister taxa, A. coluzzii (M-form) and A. gambiae (S-form).     

Successful Human Infection with P. falciparum Using Three Aseptic Anopheles stephensi Mosquitoes: A New Model for Controlled Human Malaria Infection

Controlled human malaria infection (CHMI) is a powerful method for assessing the efficacy of anti-malaria vaccines and drugs targeting pre-erythrocytic and erythrocytic stages of the parasite. CHMI has heretofore required the bites of 5 Plasmodium falciparum (Pf) sporozoite (SPZ)-infected mosquitoes to reliably induce Pf malaria. We reported that CHMI using the bites of 3 PfSPZ-infected mosquitoes reared aseptically in compliance with current good manufacturing practices (cGMP) was successful in 6 participants. Here, we report results from a subsequent CHMI study using 3 PfSPZ-infected mosquitoes reared aseptically to validate the initial clinical trial. We also compare results of safety, tolerability, and transmission dynamics in participants undergoing CHMI using 3 PfSPZ-infected mosquitoes reared aseptically to published studies of CHMI using 5 mosquitoes. Nineteen adults aged 18–40 years were bitten by 3 Anopheles stephensi mosquitoes infected with the chloroquine-sensitive NF54 strain of Pf. All 19 participants developed malaria (100%); 12 of 19 (63%) on Day 11. The mean pre-patent period was 258.3 hours (range 210.5–333.8). The geometric mean parasitemia at first diagnosis by microscopy was 9.5 parasites/µL (range 2–44). Quantitative polymerase chain reaction (qPCR) detected parasites an average of 79.8 hours (range 43.8–116.7) before microscopy. The mosquitoes had a geometric mean of 37,894 PfSPZ/mosquito (range 3,500–152,200). Exposure to the bites of 3 aseptically-raised, PfSPZ-infected mosquitoes is a safe, effective procedure for CHMI in malaria-naïve adults. The aseptic model should be considered as a new standard for CHMI trials in non-endemic areas. Microscopy is the gold standard used for the diagnosis of Pf malaria after CHMI, but qPCR identifies parasites earlier. If qPCR continues to be shown to be highly specific, and can be made to be practical, rapid, and standardized, it should be considered as an alternative for diagnosis.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00744133","term_id":"NCT00744133"}}NCT00744133 {"type":"clinical-trial","attrs":{"text":"NCT00744133","term_id":"NCT00744133"}}NCT00744133     

The Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) Mediates Indole Recognition in the Antennae of Female Mosquitoes

Haematophagous insects are frequently carriers of parasitic diseases, including malaria. The mosquito Anopheles gambiae is the major vector of malaria in sub-Saharan Africa and is thus responsible for thousands of deaths daily. Although the role of olfaction in A. gambiae host detection has been demonstrated, little is known about the combinations of ligands and odorant binding proteins (OBPs) that can produce specific odor-related responses in vivo. We identified a ligand, indole, for an A. gambiae odorant binding protein, AgamOBP1, modeled the interaction in silico and confirmed the interaction using biochemical assays. RNAi-mediated gene silencing coupled with electrophysiological analyses confirmed that AgamOBP1 binds indole in A. gambiae and that the antennal receptor cells do not respond to indole in the absence of AgamOBP1. This case represents the first documented instance of a specific A. gambiae OBP–ligand pairing combination, demonstrates the significance of OBPs in odor recognition, and can be expanded to the identification of other ligands for OBPs of Anopheles and other medically important insects.     

Chromosomal inversions and ecotypic differentiation in Anopheles gambiae: the perspective from whole‐genome sequencing

The molecular mechanisms and genetic architecture that facilitate adaptive radiation of lineages remain elusive. Polymorphic chromosomal inversions, due to their recombination‐reducing effect, are proposed instruments of ecotypic differentiation. Here, we study an ecologically diversifying lineage of Anopheles gambiae, known as the Bamako chromosomal form based on its unique complement of three chromosomal inversions, to explore the impact of these inversions on ecotypic differentiation. We used pooled and individual genome sequencing of Bamako, typical (non‐Bamako) An. gambiae and the sister species Anopheles coluzzii to investigate evolutionary relationships and genomewide patterns of nucleotide diversity and differentiation among lineages. Despite extensive shared polymorphism and limited differentiation from the other taxa, Bamako clusters apart from the other taxa, and forms a maximally supported clade in neighbour‐joining trees based on whole‐genome data (including inversions) or solely on collinear regions. Nevertheless, F_ST outlier analysis reveals that the majority of differentiated regions between Bamako and typical An. gambiae are located inside chromosomal inversions, consistent with their role in the ecological isolation of Bamako. Exceptionally differentiated genomic regions were enriched for genes implicated in nervous system development and signalling. Candidate genes associated with a selective sweep unique to Bamako contain substitutions not observed in sympatric samples of the other taxa, and several insecticide resistance gene alleles shared between Bamako and other taxa segregate at sharply different frequencies in these samples. Bamako represents a useful window into the initial stages of ecological and genomic differentiation from sympatric populations in this important group of malaria vectors.     

Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles stephensi Mosquitoes

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99%. Of those mosquitoes that were infected, we observed a 75–99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity.