Renate C. Smallegange

Development and field evaluation of a synthetic mosquito lure that is more attractive than humans.

Background Disease transmitting mosquitoes locate humans and other blood hosts by identifying their characteristic odor profiles. Using their olfactory organs, the mosquitoes detect compounds present in human breath, sweat and skins, and use these as cues to locate and obtain blood from the humans. These odor compounds can be synthesized in vitro, then formulated to mimic humans. While some synthetic mosquito lures already exist, evidence supporting their utility is limited to laboratory settings, where long-range stimuli cannot be investigated. Methodology and Principal Findings Here we report the development and field evaluation of an odor blend consisting of known mosquito attractants namely carbon dioxide, ammonia and carboxylic acids, which was optimized at distances comparable with attractive ranges of humans to mosquitoes. Binary choice assays were conducted inside a large-cage semi-field enclosure using attractant-baited traps placed 20 m apart. This enabled high-throughput optimization of concentrations at which the individual candidate attractants needed to be added so as to obtain a blend maximally attractive to laboratory-reared An. gambiae. To determine whether wild mosquitoes would also be attracted to this synthetic odor blend and to compare it with whole humans under epidemiologically relevant conditions, field experiments were conducted inside experimental huts, where the blend was compared with 10 different adult male volunteers (20-34 years old). The blend attracted 3 to 5 times more mosquitoes than humans when the two baits were in different experimental huts (10–100 metres apart), but was equally or less attractive than humans when compared side by side within same huts. Conclusion and Significance This highly attractive substitute for human baits might enable development of technologies for trapping mosquitoes in numbers sufficient to prevent rather than merely monitor transmission of mosquito-borne diseases.

Infection of malaria (Anopheles gambiae s.s.) and filariasis (Culex quinquefasciatus) vectors with the entomopathogenic fungus Metarhizium anisopliae

BackgroundCurrent intra-domiciliary vector control depends on the application of residual insecticides and/or repellents. Although biological control agents have been developed against aquatic mosquito stages, none are available for adults. Following successful use of an entomopathogenic fungus against tsetse flies (Diptera: Glossinidae) we investigated the potency of this fungus as a biological control agent for adult malaria and filariasis vector mosquitoes.MethodsIn the laboratory, both sexes of Anopheles gambiae sensu stricto and Culex quinquefasciatus were passively contaminated with dry conidia of Metarhizium anisopliae. Pathogenicity of this fungus for An. gambiae was further tested for varying exposure times and different doses of oil-formulated conidia.ResultsComparison of Gompertz survival curves and LT50 values for treated and untreated specimens showed that, for both species, infected mosquitoes died significantly earlier (p < 0.0001) than uninfected control groups. No differences in LT50 values were found for different exposure times (24, 48 hrs or continuous exposure) of An. gambiae to dry conidia. Exposure to oil-formulated conidia (doses ranging from 1.6 × 107 to 1.6 × 1010 conidia/m2) gave LT50 values of 9.69 ± 1.24 (lowest dose) to 5.89 ± 0.35 days (highest dose), with infection percentages ranging from 4.4–83.7%.ConclusionOur study marks the first to use an entomopathogenic fungus against adult Afrotropical disease vectors. Given its high pathogenicity for both adult Anopheles and Culex mosquitoes we recommend development of novel targeted indoor application methods for the control of endophagic host-seeking females.

Composition of Human Skin Microbiota Affects Attractiveness to Malaria Mosquitoes

The African malaria mosquito Anopheles gambiae sensu stricto continues to play an important role in malaria transmission, which is aggravated by its high degree of anthropophily, making it among the foremost vectors of this disease. In the current study we set out to unravel the strong association between this mosquito species and human beings, as it is determined by odorant cues derived from the human skin. Microbial communities on the skin play key roles in the production of human body odour. We demonstrate that the composition of the skin microbiota affects the degree of attractiveness of human beings to this mosquito species. Bacterial plate counts and 16S rRNA sequencing revealed that individuals that are highly attractive to An. gambiae s.s. have a significantly higher abundance, but lower diversity of bacteria on their skin than individuals that are poorly attractive. Bacterial genera that are correlated with the relative degree of attractiveness to mosquitoes were identified. The discovery of the connection between skin microbial populations and attractiveness to mosquitoes may lead to the development of new mosquito attractants and personalized methods for protection against vectors of malaria and other infectious diseases.

Flower vs. Leaf Feeding by Pieris brassicae: Glucosinolate-Rich Flower Tissues are Preferred and Sustain Higher Growth Rate

Interactions between butterflies and caterpillars in the genus Pieris and plants in the family Brassicaceae are among the best explored in the field of insect–plant biology. However, we report here for the first time that Pieris brassicae, commonly assumed to be a typical folivore, actually prefers to feed on flowers of three Brassica nigra genotypes rather than on their leaves. First- and second-instar caterpillars were observed to feed primarily on leaves, whereas late second and early third instars migrated via the small leaves of the flower branches to the flower buds and flowers. Once flower feeding began, no further leaf feeding was observed. We investigated growth rates of caterpillars having access exclusively to either leaves of flowering plants or flowers. In addition, we analyzed glucosinolate concentrations in leaves and flowers. Late-second- and early-third-instar P. brassicae caterpillars moved upward into the inflorescences of B. nigra and fed on buds and flowers until the end of the final (fifth) instar, after which they entered into the wandering stage, leaving the plant in search of a pupation site. Flower feeding sustained a significantly higher growth rate than leaf feeding. Flowers contained levels of glucosinolates up to five times higher than those of leaves. Five glucosinolates were identified: the aliphatic sinigrin, the aromatic phenyethylglucosinolate, and three indole glucosinolates: glucobrassicin, 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Tissue type and genotype were the most important factors affecting levels of identified glucosinolates. Sinigrin was by far the most abundant compound in all three genotypes. Sinigrin, 4-hydroxyglucobrassicin, and phenylethylglucosinolate were present at significantly higher levels in flowers than in leaves. In response to caterpillar feeding, sinigrin levels in both leaves and flowers were significantly higher than in undamaged plants, whereas 4-hydroxyglucobrassicin leaf levels were lower. Our results show that feeding on flower tissues, containing higher concentrations of glucosinolates, provides P. brassicae with a nutritional benefit in terms of higher growth rate. This preference appears to be in contrast to published negative effects of volatile glucosinolate breakdown products on the closely related Pieris rapae.

A novel synthetic odorant blend for trapping of malaria and other African mosquito species.

Estimating the biting fraction of mosquitoes is of critical importance for risk assessment of malaria transmission. Here, we present a novel odor-based tool that has been rigorously assessed in semi-field assays and traditional African villages for estimating the number of mosquitoes that enter houses in search of a blood meal. A standard synthetic blend (SB) consisting of ammonia, (S)-lactic acid, tetradecanoic acid, and carbon dioxide was complemented with isovaleric acid, 4,5 dimethylthiazole, 2-methyl-1-butanol, and 3-methyl-1-butanol in various combinations and concentrations, and tested for attractiveness to the malaria mosquito Anopheles gambiae. Compounds were released through low density polyethylene (LDPE) material or from nylon strips (nylon). Studies were done in a semi-field facility and two traditional villages in western Kenya. The alcohol 3-methyl-1-butanol significantly increased the attraction of SB. The other compounds proved less effective or inhibitory. Tested in a village, 3-methyl-1-butanol, released from LDPE, increased the attraction of SB. Further studies showed a significantly enhanced attraction of adding 3-methyl-1-butanol to SB compared to previously-published attractive blends both under semi-field and village conditions. Other mosquito species with relevance for public health were collected with this blend in significantly higher numbers as well. These results demonstrate the advent of a novel, reliable odor-based sampling tool for the collection of malaria and other mosquitoes. The advantage of this odor-based tool over existing mosquito sampling tools is its reproducibility, objectiveness, and relatively low cost compared to current standards of CDC light traps or the human landing catch.

Cultured skin microbiota attracts malaria mosquitoes

BackgroundHost-seeking of the African malaria mosquito, Anopheles gambiae sensu stricto, is guided by human odours. The precise nature of the odours, and the composition of attractive blends of volatiles, remains largely unknown. Skin microbiota plays an important role in the production of human body odours. It is hypothesized that host attractiveness and selection of An. gambiae is affected by the species composition, density, and metabolic activity of the skin microbiota. A study is presented in which the production and constituency of volatile organic compounds (VOCs) by human skin microbiota is examined and the behavioural responses of An. gambiae to VOCs from skin microbiota are investigated.MethodsBlood agar plates incubated with skin microbiota from human feet or with a reference strain of Staphylococcus epidermidis were tested for their attractiveness to An. gambiae in olfactometer bioassays and indoor trapping experiments. Entrained air collected from blood agar plates incubated with natural skin microbiota or with S. epidermidis were analysed using GC-MS. A synthetic blend of the compounds identified was tested for its attractiveness to An. gambiae. Behavioural data were analysed by a χ2-test and GLM. GC-MS results were analysed by fitting an exponential regression line to test the effect of the concentration of bacteria.ResultsMore An. gambiae were caught with blood agar plates incubated with skin bacteria than with sterile blood agar plates, with a significant effect of incubation time and dilution of the skin microbiota. When bacteria from the feet of four other volunteers were tested, similar effects were found. Fourteen putative attractants were found in the headspace of the skin bacteria. A synthetic blend of 10 of these was attractive to An. gambiae.ConclusionsThe discovery that volatiles produced by human skin microorganisms in vitro mediate An. gambiae host-seeking behaviour creates new opportunities for the development of odour-baited trapping systems. Additionally, identification of bacterial volatiles provides a new method to develop synthetic blends, attractive to An. gambiae and possibly other anthropophilic disease vectors.

Interindividual variation in the attractiveness of human odours to the malaria mosquito Anopheles gambiae s.s.

Abstract Differences between human individuals in their attractiveness to female mosquitoes have been reported repeatedly, but the underlying mechanisms are not well understood. Skin emanations from 27 human individuals, collected on glass marbles, were tested against ammonia in a dual‐choice olfactometer to establish their degrees of attractiveness to anthropophilic Anopheles gambiae s.s. Giles (Diptera: Culicidae) mosquitoes. Ammonia was used as a standard odour source because of its proven attractiveness to An. gambiae s.s. Skin emanations from most volunteers attracted significantly more mosquitoes than ammonia. There were clear differences in the attractiveness of skin emanations from different volunteers relative to that of ammonia, as well as in the strength of the trap entry response. Consistent differences were observed when emanations from the three most and the three least attractive volunteers were tested pairwise. No gender or age effect was found for relative attractiveness or trap entry response. Emanations from volunteers with higher behavioural attractiveness elicited higher electroantennogram response amplitudes in two pairs, but in a third pair a higher electroantennogram response was found for the less attractive volunteer. These results confirm that odour contributes to the differences in attractiveness of humans to mosquitoes.

Sweaty skin: an invitation to bite?

Anopheles gambiae sensu stricto and Aedes aegypti have a preference for human blood, which determines their importance as vectors of pathogens responsible for human diseases. Volatile organic chemicals are the principal cues by which humans are being located. Human sweat contains components that are attractive to anthropophilic mosquito species, and variation in sweat composition causes differential attractiveness to mosquitoes within and between individuals and also between humans and other mammals. Characteristics of skin glands and skin microbiota define the odorous organic compounds emitted by sweat, thereby the degree of attractiveness of the host to mosquitoes. Carboxylic acids in particular appear to characterize humans. Thus sweat-associated human volatiles are probably the primary determinant factor in the host preference of anthropophilic mosquitoes.

Differential attraction of malaria mosquitoes to volatile blends produced by human skin bacteria.

The malaria mosquito Anopheles gambiae sensu stricto is mainly guided by human odour components to find its blood host. Skin bacteria play an important role in the production of human body odour and when grown in vitro, skin bacteria produce volatiles that are attractive to A. gambiae. The role of single skin bacterial species in the production of volatiles that mediate the host-seeking behaviour of mosquitoes has remained largely unknown and is the subject of the present study. Headspace samples were taken to identify volatiles that mediate this behaviour. These volatiles could be used as mosquito attractants or repellents. Five commonly occurring species of skin bacteria were tested in an olfactometer for the production of volatiles that attract A. gambiae. Odour blends produced by some bacterial species were more attractive than blends produced by other species. In contrast to odours from the other bacterial species tested, odours produced by Pseudomonas aeruginosa were not attractive to A. gambiae. Headspace analysis of bacterial volatiles in combination with behavioural assays led to the identification of six compounds that elicited a behavioural effect in A. gambiae. Our results provide, to our knowledge, the first evidence for a role of selected bacterial species, common on the human skin, in determining the attractiveness of humans to malaria mosquitoes. This information will be used in the further development of a blend of semiochemicals for the manipulation of mosquito behaviour.

Attractiveness of MM-X Traps Baited with Human or Synthetic Odor to Mosquitoes (Diptera: Culicidae) in The Gambia

Abstract Chemical cues play an important role in the host-seeking behavior of blood-feeding mosquitoes (Diptera: Culicidae). A field study was carried out in The Gambia to investigate the effects of human odor or synthetic odor blends on the attraction of mosquitoes. MM-X traps baited with 16 odor blends to which carbon dioxide (CO2) was added were tested in four sets of experiments. In a second series of experiments, MM-X traps with 14 odor blends without CO2 were tested. A blend of ammonia and L-lactic acid with or without CO2 was used as control odor in series 1 and 2, respectively. Centers for Disease Control and Prevention (CDC) traps were placed in a traditional house and an experimental house to monitor mosquito densities during the experiments. The MM-X traps caught a total number of 196,756 mosquitoes, with the most abundant species belonging to the genera Mansonia (70.6%), Anopheles (17.5%), and Culex (11.5%). The most abundant mosquito species caught by the CDC traps (56,290 in total) belonged to the genera Mansonia (59.4%), Anopheles (16.0% An. gambiae s.l. Giles, and 11.3% An. ziemanni Grünberg), and Culex (11.6%). MM-X traps baited with synthetic blends were in many cases more attractive than MM-X traps baited with human odors. Addition of CO2 to synthetic odors substantially increased the catch of all mosquito species in the MM-X traps. A blend of ammonia + L-lactic acid + CO2 + 3-methylbutanoic acid was the most attractive odor for most mosquito species. The candidate odor blend shows the potential to enhance trap collections so that traps will provide better surveillance and possible control.