The Phlebotomine sand fly fauna of Switzerland revisited.

Sand flies (Diptera: Psychodidae, Phlebotominae; Newstead, 1911) are widespread in Europe, being particularly common in the Mediterranean region but rare north of the Alps. Thus, Switzerland is an opportune place to investigate the sand fly fauna on both sides of the Alpine crest, in southern sub-Mediterranean climate and northern oceanic temperate climate. We reinvestigated the Swiss sand fly fauna with the aim to assess changes in composition, altitudinal distribution, abundance and seasonality. Thirty-eight sites were investigated with light traps and/or interception sticky traps in 4 years. Ninety and 380 specimens were caught by light traps and sticky traps, respectively, at 15 collecting sites. Four species were identified. Phlebotomus mascittii (Grassi, 1908), Phlebotomus perniciosus (Newstead, 1911) and Sergentomyia minuta (Rondani, 1843) were confirmed in Ticino, and P. mascittii for the first time in neighbouring Grisons. Also, Phlebotomus neglectus (Tonnoir, 1921) is for the first time reported, though at a very low density compared to P. perniciosus at the same site. Its presence in Ticino supports the northward spread observed in Italy. Sand flies were detected north of the Alps at one site only, endorsing a historical report. Overall, the low density of P. perniciosus and very low density of P. neglectus suggest that canine leishmaniosis may not be an important disease risk in Switzerland.

Thermal preference of Culicoides biting midges in laboratory and semi-field settings.

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are hematophagous insects, and some species can transmit a plethora of pathogens, e.g., bluetongue virus and African horse sickness virus, that mainly affect animals. The transmission of vector-borne pathogens is strongly temperature dependent, and recent studies pointed to the importance of including microclimatic data when modelling disease spread. However, little is known about the preferred temperature of biting midges. The present study addressed the thermal selection of field-caught Culicoides with two experiments. In a laboratory setup, sugar-fed or blood-fed midges were video tracked for 15 min while moving inside a 60 × 30 × 4 cm setup with a 15-25 °C temperature gradient. Culicoides spent over double the time in the coldest zone of the setup compared to the warmest one. This cold selection was significantly stronger for sugar-fed individuals. Calculated preferred temperatures were 18.3 °C and 18.9 °C for sugar-fed and blood-fed Culicoides, respectively. The effect of temperature on walking speed was significant but weak, indicating that their skewed distribution results from preference and not cold trapping. A second experiment consisted of a two-way-choice-setup, performed in a 90 × 45 × 45 cm net cage, placed outdoors in a sheltered environment. Two UV LED CDC traps were placed inside the setup, and a mean temperature difference of 2.2 °C was created between the two traps. Hundred-fifty Culicoides were released per experiment. Recapture rates were negatively correlated with ambient temperature and were on average three times higher in the cooled trap. The higher prevalence of biting midges in cooler environments influences fitness and ability to transmit pathogens and should be considered in models that predict Culicoides disease transmission.

Thermal preference of adult mosquitoes (Culicidae) and biting midges (Ceratopogonidae) at different altitudes in Switzerland.

Mosquitoes (Diptera: Culicidae) and biting midges (Diptera: Ceratopogonidae) are among the most important vectors of human and veterinary pathogens. For modelling the distribution of these pathogens, entomological aspects are essential, which in turn are highly dependent on environmental factors, such as temperature. In this study, mosquitoes and biting midges were sampled in multiple microclimates at two low (360, 480 meters above sea level, m.a.s.l.) and two high (1250, 1530 m.a.s.l.) altitude locations in Switzerland. Sets of various traps (CO2 -baited CDC, LED-UV, resting boxes, oviposition cups) equipped with dataloggers were placed in transects at five sites with similar vegetation at each location. Only the CDC and the LED-UV traps collected enough insects for analyses. Taxonomic diversity was greater for mosquitoes but lower for biting midges at lower altitudes. Both mosquitoes and biting midges had a thermal preference. Culicoides preferred the traps with warmer microclimate, especially at lower altitudes, whereas mosquito preferences depended on the species, but not on altitude. Relative humidity had a significant positive impact on catches of biting midges but not mosquitoes. To obtain better data on thermal preferences of resting and ovipositing vectors in addition to host seeking individuals, new and improved collecting methods are needed.

Fermenting molasses and a synthetic odour blend to attract blood-fed Anopheles coluzzii.

Collecting blood-fed mosquitoes to monitor pathogen presence or to gather information on the host blood meal is often challenging. Fermenting molasses can be used to produce carbon dioxide to attract host-seeking mosquitoes, however, earlier work indicated that it may also attract blood-fed mosquitoes in the field. In the current study, these field results were validated in an experimental setting using a large cage setup with Anopheles coluzzii (Diptera, Culicidae). Blood-fed mosquitoes were indeed attracted to fermenting molasses with the highest attraction at 72 hours post feeding, which was used for subsequent experiments. Next, it was tested if fermentation of molasses is required for attraction, and whether it acts as an oviposition attractant, increases egg laying, or increases mosquito survival. The compounds that could be responsible for attraction were identified by combined electrophysiology and chemical analyses and formulated into a synthetic blend. Fermenting molasses attracted blood-fed mosquitoes in the large cage study, while fermenting sugar and non-fermenting molasses did not. The fecundity of blood-fed mosquitoes increased after feeding on fermenting molasses, however, compounds emanating from molasses did not trigger oviposition. The synthetic blend attracted blood-fed mosquitoes and may be used to determine mosquito host selection and for xenomonitoring, as 'flying syringes' to detect non-vector borne pathogens.

Temperature preference of sugar- or blood-fed Aedes japonicus mosquitoes under semi-natural conditions.

Mosquito-borne diseases pose a major burden on humans and animals. Temperature strongly influences the physiology and life cycle of mosquitoes and also the pathogens they transmit. Thermoregulatory behaviour of mosquitoes has been addressed in a few laboratory studies. Here, we expand such studies by investigating the thermal preference when resting of Aedes japonicus, an invasive and putative vector species of many pathogens, in a semi-field setup during summers in a temperate climate. Blood-fed or sugar-fed Ae. japonicus females were released in the late afternoon in a large outdoor cage containing three resting boxes. The next morning, temperature treatments were applied to the boxes, creating a "cool" (over all experiments around 18 °C), and a "warm" (around 35 °C) microhabitat in addition to an untreated "ambient" (around 26 °C) one. The mosquitoes resting within the three boxes were counted five times, every 2 h between 9h and 17h. The highest proportions of mosquitoes (e.g. up to 21% of blood-fed ones) were found in the cool box while both blood-fed and sugar-fed mosquitoes avoided the warm box. The mean resting temperatures of Ae. japonicus were below the ambient temperatures measured by a nearby meteorological station, and this was more pronounced at higher outdoor temperatures and in blood-fed as compared to sugar-fed mosquitoes. Thus, over all experiments with blood-fed mosquitoes, the calculated average resting temperature was 4 °C below the outdoor temperature. As mosquitoes prefer cooler resting places than temperatures measured by weather stations in summer, models to predict mosquito-borne disease outbreaks need to account for the thermoregulatory behaviour of mosquitoes, especially in the wake of climate change.

Environment rather than breed or body site shapes the skin bacterial community of healthy sheep as revealed by metabarcoding.

BACKGROUND: The skin is inhabited by a variety of micro-organisms, with bacteria representing the predominant taxon of the skin microbiome. In sheep, the skin bacterial community of healthy animals has been addressed in few studies, only with culture-based methods or sequencing of cloned amplicons. OBJECTIVES: The objectives of this study were to determine the sheep skin bacterial community composition by using metabarcoding for a detailed characterisation and to determine the effect of body part, breed and environment. MATERIALS AND METHODS: Overall, 267 samples were taken from 89 adult female sheep, belonging to three different breeds and kept on nine different farms in Switzerland. From every individual, one sample each was taken from belly, left ear and left leg and metabarcoding of the 16S rRNA V3-V4 hypervariable region was performed. RESULTS: The main phyla identified were Actinobacteriota, Firmicutes, Proteobacteria and Bacteriodota. The alpha diversity as determined by Shannon's diversity index was significantly different between sheep from different farms. Beta diversity analysis by principal coordinate analysis (PCoA) showed clustering of the samples by farm and body site, while breed had only a marginal influence. A sparse partial least squares discriminant analysis (sPLS-DA) revealed seven main groups of operational taxonomic units (OTUs) of which groups of OTUs were specific for some farms. CONCLUSIONS AND CLINICAL RELEVANCE: These findings indicate that environment has a larger influence on skin microbial variability than breed, although the sampled breeds, the most abundant ones in Switzerland, are phenotypically similar. Future studies on the sheep skin microbiome may lead to novel insights in skin diseases and prevention.

Video analysis of the locomotory behaviour of Aedes aegypti and Ae. japonicus mosquitoes under different temperature regimes in a laboratory setting.

Mosquito-borne diseases impose a high burden on human and animal health. Temperature strongly influences the physiology and life cycle of mosquitoes, but also the development and/or propagation of the pathogens they transmit. Thus, the vector capacity of mosquitoes depends strongly on temperature and their behavioural thermoregulation through microhabitat selection. Expanding on a previous study of static thermal preferences, the locomotory dynamics of temperate Aedes japonicus (reared from eggs collected in the field) and tropical Ae. aegypti (from a laboratory colony) was investigated at constant temperatures (10 °C, 25 °C, 40 °C) and in temperature gradients (10-20 °C, 20-30 °C, 30-40 °C). Blood-fed or non-blood-fed female mosquitoes were released in groups of 15 individuals into a Plexiglas box positioned on two thermoregulators connected by an aluminium plate to automatically monitor by video analysis mosquito flying, walking and resting duration, covered distances and velocity. Mosquitoes were predominantly resting, followed by walking and flying. At constant 10 °C, flights were rare and brief, and walking was slow. Most activity was observed at 25 °C for Ae. japonicus and 40 °C for Ae. aegypti. In the 30-40 °C gradient, activity of Ae. aegypti increased towards the cold end, suggesting active avoidance of very high temperatures. In the 20-30 °C gradient, edge effects were prominent, nevertheless revealing a greater proportion of mosquitoes gathered at the cooler end. Video analysis showed that this effect was not caused by a cold trap but represents true thermal preference. In the coolest gradient (10-20 °C), mosquitoes were active in all sectors without displaying a preference for either side. Overall, both the tropical and temperate mosquito species preferred cooler temperatures and actively avoided the hottest temperatures. Further studies with infected mosquitoes should provide important insights for developing models of vector-borne disease outbreaks.

Plasmodium-associated changes in human odor attract mosquitoes.

Malaria parasites (Plasmodium) can change the attractiveness of their vertebrate hosts to Anopheles vectors, leading to a greater number of vector-host contacts and increased transmission. Indeed, naturally Plasmodium-infected children have been shown to attract more mosquitoes than parasite-free children. Here, we demonstrate Plasmodium-induced increases in the attractiveness of skin odor in Kenyan children and reveal quantitative differences in the production of specific odor components in infected vs. parasite-free individuals. We found the aldehydes heptanal, octanal, and nonanal to be produced in greater amounts by infected individuals and detected by mosquito antennae. In behavioral experiments, we demonstrated that these, and other, Plasmodium-induced aldehydes enhanced the attractiveness of a synthetic odor blend mimicking "healthy" human odor. Heptanal alone increased the attractiveness of "parasite-free" natural human odor. Should the increased production of these aldehydes by Plasmodium-infected humans lead to increased mosquito biting in a natural setting, this would likely affect the transmission of malaria.

Thermal preferences of subtropical Aedes aegypti and temperate Ae. japonicus mosquitoes.

Temperature is an important determinant affecting the capacity of disease vectors like mosquitoes (Culicidae) to transmit disease agents. Although the impact of temperature on vector-borne disease dynamics has been studied intensively, the actual temperature encountered by the vector in a heterogeneous landscape is rarely taken into account. If disease vectors have temperature preferences and therefore select specific microhabitats, this would substantially influence key life history traits that determine transmission intensity. The thermal preferences of subtropical Aedes aegypti and temperate Ae. japonicus mosquitoes were investigated in a temperature gradient set-up consisting of a Plexiglas box on top of an aluminium plate on two thermal regulators. Blood-fed (one day after feeding) and unfed (non-blood-fed) mosquitoes were released in small (15-20 °C, 20-25 °C, 25-30 °C) and large (15-30 °C, 30-45 °C) temperature gradients to assess their thermal preferences after 15 min. Additionally, the effect of humidity was investigated in a two-choice chamber setup. Both mosquito species avoided higher temperatures, pronouncedly dangerously high temperatures in the 30-45 °C gradient. At lower temperatures, blood-fed mosquitoes preferred the cooler sides of the 20-25 °C and 25-30 °C gradient, which were all below their rearing temperature. In the lowest gradient of 15-20 °C, no preferences were found. The thermal preference of unfed mosquitoes was similar to that of the blood-fed mosquitoes. No humidity preference or effect of humidity on temperature preferences was observed within the tested range (40-90%). The set-up allows for assessing the thermal preference of mosquitoes under controlled conditions. The observed preference of mosquitoes for cooler temperatures would increase their longevity and slow down pathogen development. If similar microhabitat selection is observed in the field, vector borne disease models should be adjusted accordingly.

Do apes smell like humans? The role of skin bacteria and volatiles of primates in mosquito host selection.

Anthropophilic mosquitoes are effective vectors of human disease because of their biting preferences. To find their host, these mosquitoes are guided by human odours, primarily produced by human skin bacteria. By analysing the skin bacterial and skin volatile profiles of humans, bonobos, chimpanzees, gorillas, lemurs and cows, we investigated whether primates that are more closely related to humans have a skin bacterial community and odour profile that is similar to that of humans. We then investigated whether this affected discrimination between humans and closely related primates by anthropophilic and zoophilic mosquitoes that search for hosts. Humans had a lower skin bacterial diversity than the other animals and their skin bacterial composition was more similar to that in other primates than it was to the skin bacteria of cows. Like the skin bacterial profiles, the volatile profiles of the animal groups were clearly different from each other. The volatile profiles of cows and lemurs were more closely related to the human profiles than expected. Human volatiles were indeed preferred above cow volatiles by anthropophilic mosquitoes and no preference was observed when tested against non-human primate odour, except for bonobo volatiles, which were preferred over human volatiles. Unravelling the differences between mosquito hosts and their effect on host selection is important for a better understanding of cross-species transmission of vector-borne diseases.

Gametocytemia and Attractiveness of Plasmodium falciparum-Infected Kenyan Children to Anopheles gambiae Mosquitoes.

It has been suggested that Plasmodia manipulate their vertebrate hosts to enhance parasite transmission. Using a dual-choice olfactometer, we investigated the attraction of Anopheles gambiae to 50 Kenyan children (aged 5-12 years) who were naturally infected with Plasmodium falciparum or noninfected controls. Microscopic gametocyte carriers attracted almost 2 times more mosquitoes than children who were parasite free, harbored asexual stages, or had gametocytes at submicroscopic densities. By using highly sensitive stage-specific molecular methods to detect P. falciparum, we show that gametocytes-and not their noninfectious asexual progenitors-induce increased attractiveness of humans to mosquitoes. Our findings therefore support the parasite host manipulation hypothesis.

Mosquito host preferences affect their response to synthetic and natural odour blends.

BACKGROUND: The anthropophilic malaria mosquito Anopheles gambiae sensu stricto (hereafter termed Anopheles gambiae) primarily takes blood meals from humans, whereas its close sibling Anopheles arabiensis is more opportunistic. Previous studies have identified several compounds that play a critical role in the odour-mediated behaviour of An. gambiae. This study determined the effect of natural and synthetic odour blends on mosquitoes with different host preferences to better understand the host-seeking behaviour of mosquitoes and the potential of synthetic odour blends for standardized monitoring. METHODS: Odour blends were initially tested for their attractiveness to An. gambiae and An. arabiensis in a semi-field system with MM-X traps baited with natural and synthetic odours. Natural host odours were collected from humans, cows and chickens. The synthetic odour blends consisted of three or five previously identified compounds released with carbon dioxide. These studies were continued under natural conditions where odour blends were tested outdoors to determine their effect on species with different host preferences. RESULTS: In the semi-field experiments, human odour attracted significantly higher numbers of both mosquito species. However, An. arabiensis was also attracted to cow and chicken odours, which confirms its opportunistic behaviour. A five-component synthetic blend was highly attractive to both mosquito species. In the field, the synthetic odour blend caught significantly more An. funestus than traps baited with human odour, while no difference was found for An. arabiensis. Catches of An. arabiensis and Culex spp. contained large numbers of blood-fed mosquitoes, mostly from cows, which indicates that these mosquitoes had fed outdoors. CONCLUSIONS: Different odour baits elicit varying responses among mosquito species. Synthetic odour blends are highly effective for trapping mosquitoes; however, not all mosquitoes respond equally to the same odour blend. Combining fermenting molasses with synthetic blends in a trap represents the most effective tool to catch blood-fed mosquitoes outside houses, which is essential for understanding outdoor malaria transmission.

Mosquito Attraction: Crucial Role of Carbon Dioxide in Formulation of a Five-Component Blend of Human-Derived Volatiles.

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.

Modification of the Suna Trap for Improved Survival and Quality of Mosquitoes in Support of Epidemiological Studies.

Monitoring adult mosquito populations provides information that is critical for assessing risk of vector-borne disease transmission. The recently developed Suna trap was found to be a very effective trap when baited with an attractive odor blend. A modification of this trap was tested to improve its function as a tool for monitoring mosquito populations, including Anopheles coluzzii (An. gambiae sensu stricto molecular form M), Aedes aegypti, and Culex pipiens. The modified Suna trap (Suna-M) was altered by changing the position of the catch bag and the inclusion of a holding chamber in attempts to increase trapping efficacy and enhance the survival of mosquitoes. Each adaptation was tested in a dual-choice setup in a climate-controlled room against the original Suna trap and against 4 standard monitoring methods: the BG-sentinel (BGS), Centers for Disease Control and Prevention (CDC) light trap, Mosquito Magnet X (MM-X) trap, and human landing catch (HLC). No differences in trapping efficacy were observed between the original Suna trap and modified version; however, a version in which the funnel was extended with a box and supplemented with moistened cotton wool increased mosquito survival from 6.5% to 78.0% over 24 h. The HLC and BGS trap outperformed the Suna-M trap, whereas the MM-X and commonly used CDC light trap performed significantly less well than the Suna-M trap in the dual-choice setup. The performance of the Suna-M trap equaled the performance of the original Suna trap and could therefore be used for monitoring purposes. Although the HLC and BGS trap achieved higher catch sizes, the Suna trap has the advantage that it is standardized, does not place humans at risk, and is weather resistant. Field studies should be conducted to confirm that the Suna-M trap, baited with the odor blend, is an efficient and standardized tool to measure both indoor and outdoor disease transmission risk for a range of vector-borne diseases.

Host preferences of blood-feeding mosquitoes.

Mosquitoes use plant sugars and vertebrate blood as nutritional resources. When searching for blood hosts, some mosquitoes express preferential behavior for selected species. Here, we review the available knowledge on host preference, as this is expected to affect the life history and transmission of infectious pathogens. Host preference is affected by myriad extrinsic and intrinsic factors. Inherent factors are determined by genetic selection, which appears to be controlled by adaptive advantages that result from feeding on certain host species. Host preference of mosquitoes, although having a genetic basis, is characterized by high plasticity mediated by the density of host species, which by their abundance form a readily accessible source of blood. Host-selection behavior in mosquitoes is an exception rather than the rule. Those species that express strong and inherent host-selection behavior belong to the most important vectors of infectious diseases, which suggests that this behavioral trait may have evolved in parallel with parasite-host evolution.

Altered thermal preferences of infected or immune-challenged Aedes aegypti and Aedes japonicus mosquitoes.

Temperature is a critical factor shaping physiology, life cycle, and behaviour of ectothermic vector insects, as well as the development and multiplication of pathogens within them. However, the influence of pathogen infections on thermal preferences (behavioural thermoregulation) is not well-understood. The present study examined the thermal preferences of mosquitoes (Aedes aegypti and Ae. japonicus) infected with either Sindbis virus (SINV) or Dirofilaria immitis over 12 days post exposure (p.e.) or injected with a non-pathogenic Sephadex bead over 24 h in a thermal gradient (15-30 °C). SINV-infected Ae. aegypti preferred 5 °C warmer temperatures than non-infected ones at day 6 p.e., probably the time of highest innate immune response. In contrast, D. immitis-infected Ae. japonicus preferred 4 °C cooler temperatures than non-infected ones at day 9 p.e., presumably a stress response during the migration of third instar larvae from their development site to the proboscis. Sephadex bead injection also induced a cold preference in the mosquitoes but to a level that did not differ from control-injections. The cold preference thus might be a strategy to escape the risk of desiccation caused by the wound created by piercing the thorax. Further research is needed to uncover the genetic and physiological mechanisms underlying these behaviours.

Unexpected behavioural adaptation of yellow fever mosquitoes in response to high temperatures.

Temperature is a major ecological driver of mosquito-borne diseases as it influences the life-history of both the mosquito and the pathogen harboured within it. Understanding the mosquitoes' thermal biology is essential to inform risk prediction models of such diseases. Mosquitoes can respond to temperatures by microhabitat selection through thermal preference. However, it has not yet been considered that mosquitoes are likely to adapt to changing temperatures, for example during climate change, and alter their preference over evolutionary time. We investigated this by rearing six cohorts of the yellow fever mosquito Aedes aegypti at two temperatures (24 °C, 30 °C) for 20 generations and used these cohorts to explicitly separate the effects of long-term evolution and within-generation acclimation on their thermal preferences in a thermal gradient of 20-35 °C. We found that warm-evolved mosquitoes spent 31.5% less time at high temperatures, which affects their efficiency as a vector. This study reveals the complex interplay of experimental evolution, rearing temperatures, and thermal preference in Ae. aegypti mosquitoes. It highlights the significance of incorporating mosquito microhabitat selection in disease transmission models, especially in the context of climate change.

Sense the moist and feel the heat: mosquito host and oviposition site detection.

Laursen et al. identified coreceptor Ir93a that is required for humidity and thermal sensing in the mosquito species Anopheles gambiae and Aedes aegypti. Behavioural studies with mutant mosquitoes with disrupted Ir93a revealed that they were less attracted to a blood meal source and oviposition site at close distance.

Effects of host blood on mosquito reproduction.

Female mosquitoes require blood from their host for egg development. However, the relationship between the composition of host blood and mosquito reproduction, and whether and how this is linked to host selection, remain unclear. A better understanding of these issues is beneficial for mass-rearing of mosquitoes for vector control. This review provides an overview of the currently known effects of blood constituents on mosquito reproduction. Furthermore, it highlights knowledge gaps and proposes new avenues for investigation. We recommend that research efforts be focused on physiological differences between generalist and specialist mosquito species as models to investigate if and how host preference correlates with reproductive output.

Skin bacterial volatiles: propelling the future of vector control.

The skin microbiota plays an essential role in the protection against pathogens. It is our skin microbiota that makes us smell different from each other, rendering us more or less attractive to mosquitoes. Mosquitoes exploit skin bacterial odours to locate their hosts and are vectors of pathogens that can cause severe diseases such as malaria and dengue fever. A novel solution for long-lasting protection against insect vectors of disease could be attained by manipulating the bacterial commensals on human skin. The current options for protection against biting insects usually require topical application of repellents that evaporate within hours. We discuss possible routes for the use of commensal bacteria to create a microbial-based repellent.