Beer Consumption Increases Human Attractiveness to Malaria Mosquitoes

Background

Malaria and alcohol consumption both represent major public health problems. Alcohol consumption is rising in developing countries and, as efforts to manage malaria are expanded, understanding the links between malaria and alcohol consumption becomes crucial. Our aim was to ascertain the effect of beer consumption on human attractiveness to malaria mosquitoes in semi field conditions in Burkina Faso.

Methodology/Principal Findings

We used a Y tube-olfactometer designed to take advantage of the whole body odour (breath and skin emanations) as a stimulus to gauge human attractiveness to Anopheles gambiae (the primary African malaria vector) before and after volunteers consumed either beer (n = 25 volunteers and a total of 2500 mosquitoes tested) or water (n = 18 volunteers and a total of 1800 mosquitoes). Water consumption had no effect on human attractiveness to An. gambiae mosquitoes, but beer consumption increased volunteer attractiveness. Body odours of volunteers who consumed beer increased mosquito activation (proportion of mosquitoes engaging in take-off and up-wind flight) and orientation (proportion of mosquitoes flying towards volunteers'' odours). The level of exhaled carbon dioxide and body temperature had no effect on human attractiveness to mosquitoes. Despite individual volunteer variation, beer consumption consistently increased attractiveness to mosquitoes.

Conclusions/Significance

These results suggest that beer consumption is a risk factor for malaria and needs to be integrated into public health policies for the design of control measures.     

Species Composition of Bacterial Communities Influences Attraction of Mosquitoes to Experimental Plant Infusions

In the container habitats of immature mosquitoes, catabolism of plant matter and other organic detritus by microbial organisms produces metabolites that mediate the oviposition behavior of Aedes aegypti and Aedes albopictus. Public health agencies commonly use oviposition traps containing plant infusions for monitoring populations of these mosquito species, which are global vectors of dengue viruses. In laboratory experiments, gravid females exhibited significantly diminished responses to experimental infusions made with sterilized white oak leaves, showing that attractive odorants were produced through microbial metabolic activity. We evaluated effects of infusion concentration and fermentation time on attraction of gravid females to infusions made from senescent bamboo or white oak leaves. We used plate counts of heterotrophic bacteria, total counts of 4′,6-diamidino-2-phenylindole-stained bacterial cells, and 16S ribosomal DNA (rDNA) polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) to show that changes in the relative abundance of bacteria and the species composition of bacterial communities influenced attraction of gravid A. aegypti and A. albopictus mosquitoes to infusions. DGGE profiles showed that bacterial species composition in infusions changed over time. Principal components analysis indicated that oviposition responses to plant infusions were in general most affected by bacterial diversity and abundance. Analysis of bacterial 16S rDNA sequences derived from DGGE bands revealed that Proteobacteria (Alpha-, Beta-, Delta-, and Gamma-) were the predominant bacteria detected in both types of plant infusions. Gravid A. aegypti were significantly attracted to a mix of 14 bacterial species cultured from bamboo leaf infusion. The oviposition response of gravid mosquitoes to plant infusions is strongly influenced by abundance and diversity of bacterial species, which in turn is affected by plant species, leaf biomass, and fermentation time.     

The Right Dorsal Habenula Limits Attraction to an Odor in Zebrafish

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Cost-Effectiveness of Long-Lasting Insecticide-Treated Hammocks in Preventing Malaria in South-Central Vietnam

Background

Despite much success in reducing the burden of malaria in Vietnam, pockets of malaria persist and eliminating them remains an important development goal. In central Vietnam, insecticide-treated hammocks have recently been introduced to help counter the disease in the highly forested, mountainous areas, where other measures have so far been unsuccessful. This study assesses the cost-effectiveness of using long-lasting insecticide-treated hammocks in this area.

Methods and Findings

This cost-effectiveness study was run alongside a randomized control trial testing the efficacy of the long-lasting insecticide-treated hammocks. Data were collected through an exit survey, a household survey, expenditure records and key informant interviews. The study estimates that under normal (non-trial) conditions the total net societal cost per malaria episode averted in using long-lasting insecticide-treated hammocks in this area was 126 USD. Cost per hammock, including insecticidal netting, sewing, transport, and distribution was found to be approximately 11.76 USD per hammock. Average savings per episode averted were estimated to be $14.60 USD for the health system and 14.37 USD for households (including both direct and indirect cost savings). The study estimates that the annual financial outlay required of government to implement this type of programme to be 3.40 USD per person covered per year.

Conclusion

The study finds that the use of a hammock intervention could represent good value for money to help prevent malaria in more remote areas, where traditional control measures such as insecticide-treated bednets and indoor residual spraying are insufficient or inappropriate to control malaria. However, the life span of the hammock–the number of years over which it effectively deters mosquitoes–has a significant impact on the cost-effectiveness of the intervention and study results should be interpreted in light of the evidence on effectiveness gathered in the years to come.     

A Periodically-Forced Mathematical Model for the Seasonal Dynamics of Malaria in Mosquitoes

We describe and analyze a periodically-forced difference equation model for malaria in mosquitoes that captures the effects of seasonality and allows the mosquitoes to feed on a heterogeneous population of hosts. We numerically show the existence of a unique globally asymptotically stable periodic orbit and calculate periodic orbits of field-measurable quantities that measure malaria transmission. We integrate this model with an individual-based stochastic simulation model for malaria in humans to compare the effects of insecticide-treated nets (ITNs) and indoor residual spraying (IRS) in reducing malaria transmission, prevalence, and incidence. We show that ITNs are more effective than IRS in reducing transmission and prevalence though IRS would achieve its maximal effects within 2 years while ITNs would need two mass distribution campaigns over several years to do so. Furthermore, the combination of both interventions is more effective than either intervention alone. However, although these interventions reduce transmission and prevalence, they can lead to increased clinical malaria; and all three malaria indicators return to preintervention levels within 3 years after the interventions are withdrawn.     

General Visual and Contingent Thermal Cues Interact to Elicit Attraction in Female Aedes aegypti Mosquitoes

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Protein Kinase C-Dependent Signaling Controls the Midgut Epithelial Barrier to Malaria Parasite Infection in Anopheline Mosquitoes

Anopheline mosquitoes are the primary vectors of parasites in the genus Plasmodium, the causative agents of malaria. Malaria parasites undergo a series of complex transformations upon ingestion by the mosquito host. During this process, the physical barrier of the midgut epithelium, along with innate immune defenses, functionally restrict parasite development. Although these defenses have been studied for some time, the regulatory factors that control them are poorly understood. The protein kinase C (PKC) gene family consists of serine/threonine kinases that serve as central signaling molecules and regulators of a broad spectrum of cellular processes including epithelial barrier function and immunity. Indeed, PKCs are highly conserved, ranging from 7 isoforms in Drosophila to 16 isoforms in mammals, yet none have been identified in mosquitoes. Despite conservation of the PKC gene family and their potential as targets for transmission-blocking strategies for malaria, no direct connections between PKCs, the mosquito immune response or epithelial barrier integrity are known. Here, we identify and characterize six PKC gene family members – PKCδ, PKCε, PKCζ, PKD, PKN, and an indeterminate conventional PKC − in Anopheles gambiae and Anopheles stephensi. Sequence and phylogenetic analyses of the anopheline PKCs support most subfamily assignments. All six PKCs are expressed in the midgut epithelia of A. gambiae and A. stephensi post-blood feeding, indicating availability for signaling in a tissue that is critical for malaria parasite development. Although inhibition of PKC enzymatic activity decreased NF-κB-regulated anti-microbial peptide expression in mosquito cells in vitro, PKC inhibition had no effect on expression of a panel of immune genes in the midgut epithelium in vivo. PKC inhibition did, however, significantly increase midgut barrier integrity and decrease development of P. falciparum oocysts in A. stephensi, suggesting that PKC-dependent signaling is a negative regulator of epithelial barrier function and a potential new target for transmission-blocking strategies.     

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.     

Circadian Regulation of Light-Evoked Attraction and Avoidance Behaviors in Daytime- versus Nighttime-Biting Mosquitoes

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Inhibition of Malaria Infection in Transgenic Anopheline Mosquitoes Lacking Salivary Gland Cells

Malaria is an important global public health challenge, and is transmitted by anopheline mosquitoes during blood feeding. Mosquito vector control is one of the most effective methods to control malaria, and population replacement with genetically engineered mosquitoes to block its transmission is expected to become a new vector control strategy. The salivary glands are an effective target tissue for the expression of molecules that kill or inactivate malaria parasites. Moreover, salivary gland cells express a large number of molecules that facilitate blood feeding and parasite transmission to hosts. In the present study, we adapted a functional deficiency system in specific tissues by inducing cell death using the mouse Bcl-2-associated X protein (Bax) to the Asian malaria vector mosquito, Anopheles stephensi. We applied this technique to salivary gland cells, and produced a transgenic strain containing extremely low amounts of saliva. Although probing times for feeding on mice were longer in transgenic mosquitoes than in wild-type mosquitoes, transgenic mosquitoes still successfully ingested blood. Transgenic mosquitoes also exhibited a significant reduction in oocyst formation in the midgut in a rodent malaria model. These results indicate that mosquito saliva plays an important role in malaria infection in the midgut of anopheline mosquitoes. The dysfunction in the salivary glands enabled the inhibition of malaria transmission from hosts to mosquito midguts. Therefore, salivary components have potential in the development of new drugs or genetically engineered mosquitoes for malaria control.     

The Effective Population Size of Malaria Mosquitoes: Large Impact of Vector Control

Malaria vectors in sub-Saharan Africa have proven themselves very difficult adversaries in the global struggle against malaria. Decades of anti-vector interventions have yielded mixed results—with successful reductions in transmission in some areas and limited impacts in others. These varying successes can be ascribed to a lack of universally effective vector control tools, as well as the development of insecticide resistance in mosquito populations. Understanding the impact of vector control on mosquito populations is crucial for planning new interventions and evaluating existing ones. However, estimates of population size changes in response to control efforts are often inaccurate because of limitations and biases in collection methods. Attempts to evaluate the impact of vector control on mosquito effective population size (N_e) have produced inconclusive results thus far. Therefore, we obtained data for 13–15 microsatellite markers for more than 1,500 mosquitoes representing multiple time points for seven populations of three important vector species—Anopheles gambiae, An. melas, and An. moucheti—in Equatorial Guinea. These populations were exposed to indoor residual spraying or long-lasting insecticidal nets in recent years. For comparison, we also analyzed data from two populations that have no history of organized vector control. We used Approximate Bayesian Computation to reconstruct their demographic history, allowing us to evaluate the impact of these interventions on the effective population size. In six of the seven study populations, vector control had a dramatic impact on the effective population size, reducing N_e between 55%–87%, the exception being a single An. melas population. In contrast, the two negative control populations did not experience a reduction in effective population size. This study is the first to conclusively link anti-vector intervention programs in Africa to sharply reduced effective population sizes of malaria vectors.     

Optimizing Odor-Baited Trap Methods for Collecting Mosquitoes during the Malaria Season in The Gambia

Background

Baited traps are potential tools for removal or surveillance of disease vectors. To optimize the use of counter-flow traps baited with human odor (nylon socks that had been worn for a single day) to capture wild mosquitoes in the Gambia, investigations were conducted at a field experimental site.

Methodology/Principal Findings

Experiments employing Latin square design were conducted with a set of six huts to investigate the effects of the following on overnight mosquito trap catches: (1) placement of traps indoors or immediately outdoors, CO₂ supply, and presence of a human subject in the hut; (2) trap height for collecting mosquitoes immediately outdoors; (3) height and distance from hut; (4) interaction between multiple traps around a single hut and entry of mosquitoes into huts. A total of 106,600 adult mosquitoes (9.1% Anopheles gambiae s.l., 4.0% other Anopheles species) were collected over 42 nights. The high numbers of An. gambiae s.l. and other mosquitoes collected by odor-baited traps required CO₂ but were largely independent of the presence of a person sleeping in the hut or of trap placement indoors or outdoors. For outdoor collection that is considered less intrusive, traps opening 15 cm above the floor of the hut veranda were more highly effective than traps at other heights or further from the hut. There was no significant evidence of saturation or competition by the traps, with multiple traps around a hut each collecting almost as many mosquitoes as single traps and no effect on the numbers of mosquitoes entering the huts.

Conclusions/Significance

The outdoor trapping protocol is convenient to compare attractiveness of different odors or synthetic chemicals to malaria vectors and other wild mosquitoes. The finding that such traps are reliably attractive in the presence or absence of a human volunteer encourages their potential development as standardised surveillance tools.     

Enhanced UV Resistance and Improved Killing of Malaria Mosquitoes by Photolyase Transgenic Entomopathogenic Fungi

The low survival of microbial pest control agents exposed to UV is the major environmental factor limiting their effectiveness. Using gene disruption we demonstrated that the insect pathogenic fungus Metarhizium robertsii uses photolyases to remove UV-induced cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) photoproducts [(6-4)PPs] from its DNA. However, this photorepair is insufficient to fix CPD lesions and prevent the loss of viability caused by seven hours of solar radiation. Expression of a highly efficient archaeal (Halobacterium salinarum) CPD photolyase increased photorepair >30-fold in both M. robertsii and Beauveria bassiana. Consequently, transgenic strains were much more resistant to sunlight and retained virulence against the malaria vector Anopheles gambiae. In the field this will translate into much more efficient pest control over a longer time period. Conversely, our data shows that deleting native photolyase genes will strictly contain M. robertsii to areas protected from sunlight, alleviating safety concerns that transgenic hypervirulent Metarhizium spp will spread from mosquito traps or houses. The precision and malleability of the native and transgenic photolyases allows design of multiple pathogens with different strategies based on the environments in which they will be used.     

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.     

Heritability of Attractiveness to Mosquitoes

Female mosquitoes display preferences for certain individuals over others, which is determined by differences in volatile chemicals produced by the human body and detected by mosquitoes. Body odour can be controlled genetically but the existence of a genetic basis for differential attraction to insects has never been formally demonstrated. This study investigated heritability of attractiveness to mosquitoes by evaluating the response of Aedes aegypti (=Stegomyia aegypti) mosquitoes to odours from the hands of identical and non-identical twins in a dual-choice assay. Volatiles from individuals in an identical twin pair showed a high correlation in attractiveness to mosquitoes, while non-identical twin pairs showed a significantly lower correlation. Overall, there was a strong narrow-sense heritability of 0.62 (SE 0.124) for relative attraction and 0.67 (0.354) for flight activity based on the average of ten measurements. The results demonstrate an underlying genetic component detectable by mosquitoes through olfaction. Understanding the genetic basis for attractiveness could create a more informed approach to repellent development.     

A New Chromosomal Phylogeny Supports the Repeated Origin of Vectorial Capacity in Malaria Mosquitoes of the Anopheles gambiae Complex

Understanding phylogenetic relationships within species complexes of disease vectors is crucial for identifying genomic changes associated with the evolution of epidemiologically important traits. However, the high degree of genetic similarity among sibling species confounds the ability to determine phylogenetic relationships using molecular markers. The goal of this study was to infer the ancestral–descendant relationships among malaria vectors and nonvectors of the Anopheles gambiae species complex by analyzing breakpoints of fixed chromosomal inversions in ingroup and several outgroup species. We identified genes at breakpoints of fixed overlapping chromosomal inversions 2Ro and 2Rp of An. merus using fluorescence in situ hybridization, a whole-genome mate-paired sequencing, and clone sequencing. We also mapped breakpoints of a chromosomal inversion 2La (common to An. merus, An. gambiae, and An. arabiensis) in outgroup species using a bioinformatics approach. We demonstrated that the “standard” 2R+^p arrangement and “inverted” 2Ro and 2La arrangements are present in outgroup species Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus. The data indicate that the ancestral species of the An. gambiae complex had the 2Ro, 2R+^p, and 2La chromosomal arrangements. The “inverted” 2Ro arrangement uniquely characterizes a malaria vector An. merus as the basal species in the complex. The rooted chromosomal phylogeny implies that An. merus acquired the 2Rp inversion and that its sister species An. gambiae acquired the 2R+^o inversion from the ancestral species. The karyotype of nonvectors An. quadriannulatus A and B was derived from the karyotype of the major malaria vector An. gambiae. We conclude that the ability to effectively transmit human malaria had originated repeatedly in the complex. Our findings also suggest that saltwater tolerance originated first in An. merus and then independently in An. melas. The new chromosomal phylogeny will facilitate identifying the association of evolutionary genomic changes with epidemiologically important phenotypes.