Renate C. Smallegange

Malaria parasites are thought to influence mosquito attraction to human hosts, a phenomenon that may enhance parasite transmission. This is likely mediated by alterations in host odour because of its importance in mosquito host-searching behaviour. Here, we report that the human skin odour profile is affected by malaria infection. We compared the chemical composition and attractiveness toAnopheles coluzziimosquitoes of skin odours from participants that were infected by Controlled Human Malaria Infection withPlasmodium falciparum. Skin odour composition differed between parasitologically negative and positive samples, with positive samples collected on average two days after parasites emerged from the liver into the blood, being associated with low densities of asexual parasites and the absence of gametocytes. We found a significant reduction in mosquito attraction to skin odour during infection for one experiment, but not in a second experiment, possibly due to differences in parasite strain. However, it does raise the possibility that infection can affect mosquito behaviour. Indeed, several volatile compounds were identified that can influence mosquito behaviour, including 2- and 3-methylbutanal, 3-hydroxy-2-butanone, and 6-methyl-5-hepten-2-one. To better understand the impact of our findings onPlasmodiumtransmission, controlled studies are needed in participants with gametocytes and higher parasite densities.

Behavioral responses of the malaria mosquito Anopheles coluzzii (An. gambiae sensu stricto molecular ‘M form’) to an expanded blend of human-derived volatiles were assessed in a dual-port olfactometer. A previously documented attractive three-component blend consisting of NH₃, (S)-lactic acid, and tetradecanoic acid served as the basis for expansion. Adding 4.5 % CO₂ to the basic blend significantly enhanced its attractiveness. Expansion of the blend with four human-derived C4-volatiles was then assessed, both with and without CO₂. Only when CO₂ was offered simultaneously, did addition of a specific concentration of 3-methyl-1-butanol or 3-methyl-butanoic acid significantly enhance attraction. The functional group at the terminal C of the 3-methyl-substituted C4 compounds influenced behavioral effectiveness. In the absence of CO₂, addition of three concentrations of butan-1-amine caused inhibition when added to the basic blend. In contrast, when CO₂ was added, butan-1-amine added to the basic blend strongly enhanced attraction at all five concentrations tested, the lowest being 100,000 times diluted. The reversal of inhibition to attraction by adding CO₂ is unique in the class Insecta. We subsequently augmented the three-component basic blend by adding both butan-1-amine and 3-methyl-1-butanol and optimizing their concentrations in the presence of CO₂ in order to significantly enhance the attractiveness to An. coluzzii compared to the three- and four-component blends. This novel blend holds potential to enhance malaria vector control based on behavioral disruption.

The insect endosymbiont Wolbachia pipientis (Rickettsiales: Rickettsiaceae) is undergoing field trials around the world to determine if it can reduce transmission of dengue virus from the mosquito Stegomyia aegypti to humans. Two different Wolbachia strains have been released to date. The primary effect of the w Mel strain is pathogen protection whereby infection with the symbiont limits replication of dengue virus inside the mosquito. A second strain, w MelPop, induces pathogen protection, reduces the adult mosquito lifespan and decreases blood feeding success in mosquitoes after 15 days of age. Here we test whether Wolbachia infection affects mosquito attraction to host odours in adults aged 5 and 15 days. We found no evidence of reduced odour attraction of mosquitoes, even for those infected with the more virulent w MelPop. This bodes well for fitness and competitiveness in the field given that the mosquitoes must find hosts to reproduce for the biocontrol method to succeed.

Mosquito fitness is determined largely by body size and nutritional reserves. Plasmodium infections in the mosquito and resultant transmission of malaria parasites might be compromised by the vector’s nutritional status. We studied the effects of nutritional stress and malaria parasite infections on transmission fitness of Anopheles mosquitoes.

There is much evidence that some pathogens manipulate the behaviour of their mosquito hosts to enhance pathogen transmission. However, it is unknown whether this phenomenon exists in the interaction of Anopheles gambiae sensu stricto with the malaria parasite, Plasmodium falciparum - one of the most important interactions in the context of humanity, with malaria causing over 200 million human cases and over 770 thousand deaths each year. Here we demonstrate, for the first time, that infection with P. falciparum causes alterations in behavioural responses to host-derived olfactory stimuli in host-seeking female An. gambiae s.s. mosquitoes. In behavioural experiments we showed that P. falciparum-infected An. gambiae mosquitoes were significantly more attracted to human odors than uninfected mosquitoes. Both P. falciparum-infected and uninfected mosquitoes landed significantly more on a substrate emanating human skin odor compared to a clean substrate. However, significantly more infected mosquitoes landed and probed on a substrate emanating human skin odor than uninfected mosquitoes. This is the first demonstration of a change of An. gambiae behaviour in response to olfactory stimuli caused by infection with P. falciparum. The results of our study provide vital information that could be used to provide better predictions of how malaria is transmitted from human being to human being by An. gambiae s.s. females. Additionally, it highlights the urgent need to investigate this interaction further to determine the olfactory mechanisms that underlie the differential behavioural responses. In doing so, new attractive compounds could be identified which could be used to develop improved mosquito traps for surveillance or trapping programmes that may even specifically target P. falciparum-infected An. gambiae s.s. females.

Synthetic odour baits present an unexploited potential for sampling, surveillance and control of malaria and other mosquito vectors. However, application of such baits is impeded by the unavailability of robust odour delivery devices that perform reliably under field conditions. In the present study the suitability of low density polyethylene (LDPE) and nylon strips for dispensing synthetic attractants of host-seeking Anopheles gambiae mosquitoes was evaluated.

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.

Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) is the most important vector of human malaria in sub‐Saharan Africa, affecting the lives of millions of people. Existing tools such as insecticide‐treated nets and indoor‐residual sprays are not only effective, but also have limitations as a result of the development of resistance to insecticides and behavioural adaptations in biting time. Therefore, novel mosquito‐control tools are needed. Odour‐releasing traps have a potential for both trapping and surveillance purposes. Based on the outcome of ex vivo gene expression assays and in vivo electrophysiological assays on hundreds of volatile organic compounds, 29 ‘candidate behaviourally‐disruptive organic compounds' (cBDOCs) are selected, belonging to 10 chemical categories, to be assayed in the laboratory using dual‐choice olfactometers for the ability to modify the ‘attractiveness’ (i.e. significantly more insects being caught in the associated trap) of a basic volatile blend consisting of ammonia, lactic acid and tetradecanoic acid but without additional carbon dioxide. Compounds that either reduce or increase trap catches by the basic blend in the olfactometer experiments are tested under African conditions in choice experiments in a semi‐field facility in Kenya. The release rates of cBDOCs are determined gravimetrically to allow the calculation of aerial concentrations at the trap outlet. Aerial concentrations in the sub‐p.p.m. range are reported for the first time to influence mosquito behaviour. The results of the olfactometer and semi‐field assays generally correspond. Under semi‐field conditions, three compounds, 3‐heptanol (0.025 p.p.m.), 2‐methylpropanal (0.05 p.p.m.) and 4,5‐dimethylthiazole (0.73 p.p.m.), significantly increase trap catches relative to the basic blend consisting of ammonia, lactic acid, tetradecanoic acid and carbon dioxide. 2‐Acetylthiophene, 2‐nonanone and 2‐phenylethanol decrease the number of mosquitoes caught at all concentrations tested under semi‐field conditions. These compounds hold promise as attractants and spatial repellents to be applied in mosquito control programmes.

Malaria is caused by Plasmodium parasites which are transmitted by mosquitoes. Until recently, human malaria was considered to be caused by human-specific Plasmodium species. Studies on Plasmodium parasites in non-human primates (NHPs), however, have identified parasite species in gorillas and chimpanzees that are closely related to human Plasmodium species. Moreover, P. knowlesi, long known as a parasite of monkeys, frequently infects humans. The requirements for such a cross-species exchange and especially the role of mosquitoes in this process are discussed, as the latter may act as bridge vectors of Plasmodium species between different primates. Little is known about the mosquito species that would bite both humans and NHPs and if so, whether humans and NHPs share the same Plasmodium vectors. To understand the vector-host interactions that can lead to an increased Plasmodium transmission between species, studies are required that reveal the nature of these interactions. Studying the potential role of NHPs as a Plasmodium reservoir for humans will contribute to the ongoing efforts of human malaria elimination, and will help to focus on critical areas that should be considered in achieving this goal.

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.