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.     

The Role of Oxidative Stress in the Longevity and Insecticide Resistance Phenotype of the Major Malaria Vectors Anopheles arabiensis and Anopheles funestus

Oxidative stress plays numerous biological roles, both functional and pathological. The role of oxidative stress in various epidemiologically relevant biological traits in Anopheles mosquitoes is not well established. In this study, the effects of oxidative stress on the longevity and insecticide resistance phenotype in the major malaria vector species An. arabiensis and An. funestus were examined. Responses to dietary copper sulphate and hydrogen peroxide were used as proxies for the oxidative stress phenotype by determining the effect of copper on longevity and hydrogen peroxide lethal dose. Glutathione peroxidase and catalase activities were determined colorimetrically. Oxidative burden was quantified as protein carbonyl content. Changes in insecticide resistance phenotype were monitored by WHO bioassay. Insecticide resistant individuals showed an increased capacity for coping with oxidative stress, mediated by increased glutathione peroxidase and catalase activity. This effect was observed in both species, as well as in laboratory strains and F₁ individuals derived from wild-caught An. funestus mothers. Phenotypic capacity for coping with oxidative stress was greatest in strains with elevated Cytochrome P450 activity. Synergism of oxidative stress defence enzymes by dietary supplementation with haematin, 3-Amino-1, 2, 4-triazole and Sodium diethyldithiocarbamate significantly increased pyrethroid-induced mortality in An. arabiensis and An. funestus. It is therefore concluded that defence against oxidative stress underlies the augmentation of the insecticide resistance phenotype associated with multiple blood-feeding. This is because multiple blood-feeding ultimately leads to a reduction of oxidative stress in insecticide resistant females, and also reduces the oxidative burden induced by DDT and pyrethroids, by inducing increased glutathione peroxidase activity. This study highlights the importance of oxidative stress in the longevity and insecticide resistance phenotype in malaria vectors.     

Impacts of Agricultural Practices on Insecticide Resistance in the Malaria Vector Anopheles arabiensis in Khartoum State,Sudan

Background

Agricultural pesticides may play a profound role in selection of resistance in field populations of mosquito vectors. The objective of this study is to investigate possible links between agricultural pesticide use and development of resistance to insecticides by the major malaria vector Anopheles arabiensis in northern Sudan.

Methodology/Principal Findings

Entomological surveys were conducted during two agricultural seasons in six urban and peri-urban sites in Khartoum state. Agro-sociological data were collected from 240 farmers subjected to semi-structured questionnaires based on knowledge attitude and practice (KAP) surveys. Susceptibility status of An. arabiensis (n=6000) was assessed in all sites and during each season using WHO bioassay tests to DDT, deltamethrin, permethrin, Malathion and bendiocarb. KAP analysis revealed that pesticide application was common practice among both urban and peri-urban farmers, with organophosphates and carbamates most commonly used. Selection for resistance is likely to be greater in peri-urban sites where farmers apply pesticide more frequently and are less likely to dispose of surpluses correctly. Though variable among insecticides and seasons, broad-spectrum mortality was slightly, but significantly higher in urban than peri-urban sites and most marked for bendiocarb, to which susceptibility was lowest. Anopheles arabiensis from all sites showed evidence of resistance or suspected resistance, especially pyrethroids. However, low-moderate frequencies of the L1014F kdr allele in all sites, which was very strongly associated with DDT, permethrin and deltamethrin survivorship (OR=6.14-14.67) suggests that resistance could increase rapidly.

Conclusions

Ubiquitous multiple-resistance coupled with presence of a clear mechanism for DDT and pyrethroids (kdr L1014F) in populations of An. arabiensis from Khartoum-Sudan suggests careful insecticide management is essential to prolong efficacy. Our findings are consistent with agricultural insecticide use as a source of selection for resistance and argue for coordination between the integrated vector control program and the Ministry of Agriculture to permit successful implementation of rational resistance management strategies.     

Genetic linkage between malathion and dieldrin resistance in Anopheles arabiensis

A strain of Anopheles arabiensis resistant to both malathion and dieldrin was crossed and backcrossed to a susceptible strain. The progeny were tested on each insecticide in turn. Less than 50% mortality in the second insecticide exposure among the backcross progeny indicated linkage between the resistance genes. In a backcross of A. gambiae X A. arabiensis hybrids a recombination rate of 7.5% was observed. A Y-translocation strain of A. arabiensis showed less than 2.8% recombination between the resistance genes. It is impossible to confirm the genotype of apparent recombinants using existing stocks, but the two resistance mechanisms are biochemically distinguishable. If the two genes are very closely linked, linkage disequilibrium could influence the consequences of switching to malathion spraying after dieldrin resistance has evolved.     

Physical Map of the Malaria Vector Anopheles Gambiae

Random cDNA clones, cosmid clones and RAPD polymorphic fragments have been localized by in situ hybridization to the ovarian nurse cell polytene chromosomes of the malaria vector Anopheles gambiae. We thus established 85 molecular markers for 110 sites within the whole A. gambiae polytene chromosome complement. The cDNA clones analyzed were isolated at random, and their exact localizations were determined by in situ hybridization. For 15 of the cDNA clones, a partial nucleotide sequence has been obtained; for nine of them sequence searches in the GenBank database revealed high degrees of similarity with published sequences. The cosmid clones analyzed were obtained as the result of screening with a few of the aforementioned cDNA clones of particular interest, or taken from a small set of randomly isolated cosmid clones. The RAPD clones are polymorphic fragments, potentially diagnostic for the various chromosomal forms of A. gambiae that are currently being analyzed.     

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.     

Host selection by Anopheles arabiensis and An. quadriannulatus feeding on cattle in Zimbabwe

In the Zambezi valley, mosquito females of the Anopheles gambiae Giles complex (Diptera: Culicidae) were collected from a hut containing pairs of cattle distinguishable by known DNA markers. DNA was extracted from the blood-fed mosquito abdomens and primer sets for ungulate and mosquito DNA loci were used to identify the mosquito sibling species and individual host source(s) of their bloodmeals. The 67 mosquitoes comprised a mixture of An. arabiensis Patton (31%) and An. quadriannulatus Theobald (69%). DNA from one or both of the cattle present in the hut was detected in 91% of samples. When the hut contained an adult and a calf, the percentage of bloodmeals from the adult, the calf and adult + calf were 58%, 27% and 15%, respectively; the trend towards meals from the adult host was consistent but not always significant. When the pair of cattle comprised two adults of roughly equal size and age, then mosquitoes generally showed no significant bias towards feeding from one individual. There was no significant difference in the pattern of host selection made by An. arabiensis and An. quadriannulatus but the former had a significantly higher percentage (20%) of mixed meals than An. quadriannulatus (9%). These two members of the An. gambiae complex appear to be less selective in their choice of cattle hosts compared to day-active Diptera such as tsetse and Stomoxys, possibly because the hosts are generally asleep when Anopheles are active and there is therefore less selective pressure to adapt to host defensive behaviour. The slight bias of Anopheles towards older and/or larger cattle may be related to the host's larger surface area.     

The distribution of two major malaria vectors, Anopheles gambiae and Anopheles arabiensis, in Nigeria.

The distribution of Anopheles gambiae and An. arabiensis across the ecological zones of Nigeria (arid savanna in the north gradually turns into humid forest in the south) was investigated. Results of the present study were compared to the distributions determined from samples of indoor-resting females reported by an earlier study over 20 years ago. Larvae were sampled in the rainy seasons of 1997 and 1999 from 24 localities, 10 of which were sampled in both years. Specimens were identified by the polymerase chain reaction method. Results showed that species composition changed significantly among the 10 localities in both years (chi2=13.62, P = 0.0002), but this change was significant in only four of the 10 localities. The identity of the prevalent (more abundant) species changed between 1997 and 1999 in only three of 10 localities. An. arabiensis was prevalent in several localities in the southern Guinea savanna, an area where it was virtually absent over 20 years ago. The data suggest that An. arabiensis has extend its range, although differences in sampling technique (larval sampling versus adult collection) can not be ruled out as a possible explanation.     

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     

Structure of the G119S Mutant Acetylcholinesterase of the Malaria Vector Anopheles gambiae Reveals Basis of Insecticide Resistance

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Widespread Pyrethroid and DDT Resistance in the Major Malaria Vector Anopheles funestus in East Africa Is Driven by Metabolic Resistance Mechanisms

Background

Establishing the extent, geographical distribution and mechanisms of insecticide resistance in malaria vectors is a prerequisite for resistance management. Here, we report a widespread distribution of insecticide resistance in the major malaria vector An. funestus across Uganda and western Kenya under the control of metabolic resistance mechanisms.

Methodology/Principal Findings

Female An. funestus collected throughout Uganda and western Kenya exhibited a Plasmodium infection rate between 4.2 to 10.4%. Widespread resistance against both type I (permethrin) and II (deltamethrin) pyrethroids and DDT was observed across Uganda and western Kenya. All populations remain highly susceptible to carbamate, organophosphate and dieldrin insecticides. Knockdown resistance plays no role in the pyrethroid and DDT resistance as no kdr mutation associated with resistance was detected despite the presence of a F1021C replacement. Additionally, no signature of selection was observed on the sodium channel gene. Synergist assays and qRT-PCR indicated that metabolic resistance plays a major role notably through elevated expression of cytochrome P450s. DDT resistance mechanisms differ from West Africa as the L119F-GSTe2 mutation only explains a small proportion of the genetic variance to DDT resistance.

Conclusion

The extensive distribution of pyrethroid and DDT resistance in East African An. funestus populations represents a challenge to the control of this vector. However, the observed carbamate and organophosphate susceptibility offers alternative solutions for resistance management.     

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.     

Melanotic Pathology and Vertical Transmission of the Gut Commensal Elizabethkingia meningoseptica in the Major Malaria Vector Anopheles gambiae

Background

The resident gut flora is known to have significant impacts on the life history of the host organism. Endosymbiotic bacterial species in the Anopheles mosquito gut are potent modulators of sexual development of the malaria parasite, Plasmodium, and thus proposed as potential control agents of malaria transmission.

Results

Here we report a melanotic pathology in the major African malaria vector Anopheles gambiae, caused by the dominant mosquito endosymbiont Elizabethkingia meningoseptica . Transfer of melanised tissues into the haemolymph of healthy adult mosquitoes or direct haemolymph inoculation with isolated E . meningoseptica bacteria were the only means for transmission and de novo formation of melanotic lesions, specifically in the fat body tissues of recipient individuals. We show that E . meningoseptica can be vertically transmitted from eggs to larvae and that E . meningoseptica -mono-associated mosquitoes display significant mortality, which is further enhanced upon Plasmodium infection, suggesting a synergistic impact of E . meningoseptica and Plasmodium on mosquito survival.

Conclusion

The high pathogenicity and permanent association of E . meningoseptica with An. Gambiae through vertical transmission constitute attractive characteristics towards the potential design of novel mosquito/malaria biocontrol strategies.     

Embryonic Development and Rates of Metabolic Activity in Early and Late Hatching Eggs of the Major Malaria Vector Anopheles gambiae

Anopheles gambiae eggs generally hatch at the completion of embryo development; two-three days post oviposition. However, staggered or delayed hatching has been observed whereby a single batch of eggs shows marked variation in time-to-hatch, with some eggs hatching 18 days post oviposition or later. The mechanism enabling delayed hatch has not been clearly elucidated but is likely mediated by environmental and genetic factors that either induce diapause or slow embryo development. This study aimed to compare metabolic activity and embryonic development between eggs collected from sub-colonies of the baseline Anopheles gambiae GAH colony previously selected for early or late time-to-hatch. Egg batches from early and late hatch sub-colonies as well as from the baseline colony were monitored for hatching. For both time-to-hatch selected sub-colonies and the baseline colony the majority of eggs hatched on day two post oviposition. Nevertheless, eggs produced by the late hatch sub-colony showed a significantly longer mean time to hatch than those produced by the early hatch sub-colony. The overall proportions that hatched were similar for all egg batches. CO₂ output between eggs from early and late hatch sub-colonies showed significant differences only at 3 and 7 days post oviposition where eggs from the early hatch and the late hatch sub-colony were more metabolically active, respectively. No qualitative differences were observed in embryo development between the sub-colonies. It is concluded that all viable embryos develop to maturity at the same rate and that a small proportion then enter a state of diapause enabling them to hatch later. As it has previously been shown that it is possible to at least partially select for late hatch, this characteristic is likely to involve genetic as well as environmental factors. Delayed hatching in An. gambiae is likely an adaptation to maximise reproductive output despite the increased risk of desiccation in an unstable aquatic environment.     

Chromosome Inversions,Genomic Differentiation and Speciation in the African Malaria Mosquito Anopheles gambiae

The African malaria vector, Anopheles gambiae, is characterized by multiple polymorphic chromosomal inversions and has become widely studied as a system for exploring models of speciation. Near complete reproductive isolation between different inversion types, known as chromosomal forms, has led to the suggestion that A. gambiae is in early stages of speciation, with divergence evolving in the face of considerable gene flow. We compared the standard chromosomal arrangement (Savanna form) with genomes homozygous for j, b, c, and u inversions (Bamako form) in order to identify regions of genomic divergence with respect to inversion polymorphism. We found levels of divergence between the two sub-taxa within some of these inversions (2Rj and 2Rb), but at a level lower than expected and confined near the inversion breakpoints, consistent with a gene flux model. Unexpectedly, we found that the majority of diverged regions were located on the X chromosome, which contained half of all significantly diverged regions, with much of this divergence located within exons. This is surprising given that the Bamako and Savanna chromosomal forms are both within the S molecular form that is defined by a locus near centromere of X chromosome. Two X-linked genes (a heat shock protein and P450 encoding genes) involved in reproductive isolation between the M and S molecular forms of A. gambiae were also significantly diverged between the two chromosomal forms. These results suggest that genes mediating reproductive isolation are likely located on the X chromosome, as is thought to be the case for the M and S molecular forms. We conclude that genes located on the sex chromosome may be the major force driving speciation between these chromosomal forms of A. gambiae.     

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.     

Pyrethroid Resistance in Anopheles gambiae,in Bomi County,Liberia, Compromises Malaria Vector Control

Background

Long Lasting Insecticidal Nets (LLIN) and Indoor Residual Spraying (IRS) have both proven to be effective malaria vector control strategies in Africa and the new technology of insecticide treated durable wall lining (DL) is being evaluated. Sustaining these interventions at high coverage levels is logistically challenging and, furthermore, the increase in insecticide resistance in African malaria vectors may reduce the efficacy of these chemical based interventions. Monitoring of vector populations and evaluation of the efficacy of insecticide based control approaches should be integral components of malaria control programmes. This study reports on entomological survey conducted in 2011 in Bomi County, Liberia.

Methods

Anopheles gambiae larvae were collected from four sites in Bomi, Liberia, and reared in a field insectary. Two to five days old female adult An gambiae s.l. were tested using WHO tube (n = 2027) and cone (n = 580) bioassays in houses treated with DL or IRS. A sample of mosquitoes (n = 169) were identified to species/molecular form and screened for the presence of knock down resistance (kdr) alleles associated with pyrethroid resistance.

Results

Anopheles gambiae s.l tested were resistant to deltamethrin but fully susceptible to bendiocarb and fenithrothion. The corrected mortality of local mosquitoes exposed to houses treated with deltamethrin either via IRS or DL was 12% and 59% respectively, suggesting that resistance may affect the efficacy of these interventions. The presence of pyrethroid resistance was associated with a high frequency of the 1014F kdr allele (90.5%) although this mutation alone cannot explain the resistance levels observed.

Conclusion

High prevalence of resistance to deltamethrin in Bomi County may reduce the efficacy of malaria strategies relying on this class of insecticide. The findings highlight the urgent need to expand and sustain monitoring of insecticide resistance in Liberian malaria vectors, evaluate the effectiveness of existing interventions and develop appropriate resistance management strategies.     

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.