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Abstract 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.

Abstract 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.

The human skin bacteria play an important role in the production of volatiles that attract mosquitoes. Using some of the most abundant human skin bacterial species, we created in vitro community models to assess whether increased microbial biodiversity could reduce human attractiveness to females of the dengue fever mosquito Aedes aegypti and whether co-culturing bacterial commensals affects overall attraction. More complex bacterial models were less attractive to female mosquitoes than the simplest models. For instance, the triple bacterial community model was approximately three times less attractive than Staphylococcus epidermidis alone. Our data show, for instance, that an in vitro community model mimicking the skin composition of a highly attractive individual to the anthropophilic Anopheles gambiae was also more attractive to anthropophilic Ae. aegypti than a community model mimicking the skin composition of a poorly attractive individual to An. gambiae. In line with these results, volatile analyses of the blends emitted by the different in vitro community models showed that the more complex models had lower emission overall. Effects on mosquito responses differed sharply when the different bacteria species were sharing the same resources used for growth, showing that either competition or commensalism may influence their relative growth, and that this consequently can influence mosquito responses. We conclude that studies on mosquito responses to skin volatiles need to take the microbial community into account.

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are of huge veterinary importance, mainly as vectors of pathogens, such as Bluetongue virus. Currently, there are no effective methods to protect animals against biting midges, as insecticides have limited or short‐lived efficacy. Biting midges are attracted to hosts by carbon dioxide and by their body odours, which are mainly produced by skin bacteria. In humans, it has been shown that differences between individuals in attractiveness to mosquitoes is mediated by these skin bacterial volatiles. This opens the possibility to protect individuals from biting insects by supplementing their skin microbiome with probiotics. In this study, we investigated this approach by culturing sheep skin bacteria on various media and assessing their effects against field‐caught Culicoides (overwhelmingly Obsoletus group species) as well as laboratory‐reared Culicoides nubeculosus (Meigen). Aerococcus urinaeequi, Bacillus safensis, Bacillus subtilis, Jeotgalicoccus psychrophilus, Micrococcus sp. and Staphylococcus equorum were selected to be tested in a dual‐choice Y‐tube olfactometer, assessing their behavioural effects on biting midges. We revealed an avoidance effect on laboratory‐reared C. nubeculosus when testing B. safensis and B. subtilis. Bacillus safensis and Micrococcus sp. yielded significant repellent potential towards field‐caught Culicoides. These two candidates were subsequently tested in a membrane blood‐feeding assay. When the bacterial species B. safensis was applied to the membrane, a feeding reduction of 83% was observed with field‐caught Culicoides. [ABSTRACT FROM AUTHOR]

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Abstract Background Emerging arboviral diseases like Zika, dengue and chikungunya that are transmitted by Aedes aegypti mosquitoes, are increasingly threatening human health. Blends of human-like synthetic chemical attractants can be used to attract host-seeking mosquitoes. The aim of this study was to test new combinations of traps and odour baits in the laboratory, followed by testing the best candidates in the field to improve Ae. aegypti monitoring and surveillance. Methods First, the BG-Suna trap was evaluated for capturing laboratory-reared Ae. aegypti by testing normal and inverted positions in screen cage tests. Secondly, the attractiveness of the MB5 blend, CO2, and their combination was tested. Thirdly, we tested the attractiveness of different trap types (BG-Suna, BG-Sentinel, MM-X and CDC light trap). Finally, we confirmed laboratory results in the field in Paramaribo, Suriname, using the MB5 and BG-Lure odour blends, CO2 and the BG-Sentinel and BG-Bowl trap using a Latin Square design. Results The MB5 blend in combination with CO2 outperformed traps baited only with CO2 or MB5 in screen cage tests (P

Blood-fed insects can be used to analyse the host blood for circulating vertebrate pathogens or antibodies directed against them. We tested whether naturally acquired antibodies in different host species can be detected by host-specific and pan-specific ELISAs in mosquito blood meals. Cat- and alpaca-specific ELISAs could detect antibodies against Toxoplasma gondii or SARS-CoV-2 in blood meals of Aedes japonicus for 48 and at least 24 h, respectively. In the pan-specific ELISA, a conjugated protein A/G and anti-IgY were used to detect antibodies of mammalian and bird hosts. Thus, Toxoplasma antibodies could be detected in mosquitoes fed on blood from humans, chicken, pig, and sheep up to 72 h after the blood meal. The results, however, demonstrated differences in sensitivities between different host species, and the assay requires further evaluation. Xenosurveillance with antibody detection in mosquito blood meals can be an additional surveillance tool that would especially be helpful when it is difficult to sample the potential animal reservoirs.

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. [ABSTRACT FROM AUTHOR]

Abstract Background West Nile virus (WNV) is a mosquito-borne virus that is mainly transmitted among birds by Culex pipiens mosquitoes. The species Cx. pipiens consists of two biotypes named pipiens and molestus, which together can form hybrids. One of the major distinctions between the biotypes is their overwintering behaviour. Adults of biotype pipiens diapause during winter, whereas biotype molestus remains actively blood-feeding. Diapausing may affect survival and vector competence of biotype pipiens. The aims of this study were therefore to identify the biotype composition of diapausing Cx. pipiens mosquitoes, to quantify survival throughout the autumn and winter months, and to determine effects of overwintering on vector competence of emerging Cx. pipiens mosquitoes for WNV. Methods Diapausing mosquitoes were collected at two typical overwintering locations in the Netherlands. A selection of Cx. pipiens mosquitoes was identified to biotype using real-time PCR. Survival of diapausing Cx. pipiens mosquitoes during autumn and winter was monitored by placing cages with either field-collected or laboratory-reared females in houses and sheds. Vector competence of field-collected (diapausing) and laboratory-reared (non-diapausing) Cx. pipiens mosquitoes was determined to gain insight in the effect of overwintering on WNV transmission. Results The majority (92%) of diapausing Cx. pipiens females were identified as biotype pipiens. More than 70% of diapausing Cx. pipiens mosquitoes was able to survive for more than four months in sheds, whereas diapausing in houses resulted in 100% mortality in that same period. In contrast, non-diapausing Cx. pipiens biotype pipiens mosquitoes reared in the laboratory died within a week in both houses and sheds. Vector competence of Cx. pipiens mosquitoes that had diapaused during the autumn and winter months was comparable to non-diapausing laboratory-reared mosquitoes. Conclusions This study confirms that the majority of Cx. pipiens mosquitoes in their typical overwintering site belongs to the pipiens biotype. It shows that more than two-third of diapausing Cx. pipiens mosquitoes is able to survive winter under sheltered winter conditions. Finally, vector competence for WNV of mosquitoes that emerge from overwintering sites is not affected by their relatively old age.

Biting midges (Diptera; Ceratopogonidae; Culicoides spp.) are biological vectors of disease agents, and they cause nuisance and insect bite hypersensitivity. Currently there are no effective means to control biting midges as screening is impractical and the application of insecticides or repellents is of limited efficacy. Spatial repellents have the advantage over contact repellents that they can create a vector-free environment. Studies have shown the efficacy of spatial repellents to protect humans against mosquitoes, also outdoors, but no data are available for biting midges. We tested the spatial repellency and toxicity (knockdown effect) of the volatile pyrethroid transfluthrin against the laboratory-reared biting midges Culicoides nubeculosus (Meigen) and Culicoides sonorensis (Wirth and Jones) and the mosquito Aedes aegypti (Linnaeus) in a high-throughput tube setup. Observations were made 15, 30 and 60 min. after application of the repellent. In addition to transfluthrin, the non-volatile pyrethroid permethrin and DEET, the gold standard of repellents, were included. Spatial repellency by transfluthrin was observed against both biting midge species and Ae. aegypti, already at the first observation after 15 min. and at much lower concentrations than DEET. Permethrin was spatially repellent only to C. sonorensis at the highest concentration tested (10 μg/cm2). Knockdown of biting midges and mosquitoes by transfluthrin, both by vapour or contact toxicity, was observed even at low concentrations. DEET had little to no effect on the knockdown of the insects, neither by direct contact nor vapour toxicity, while permethrin caused a high proportion of knockdown when direct contact was possible. In case these results can be confirmed in field experiments, spatial repellents could become a novel tool in integrated control programmes to reduce biting by Culicoides spp.

Lumpy skin disease (LSD) is a viral disorder of cattle caused by the lumpy skin disease virus (LSDV) which can induce severe infections leading to high economic losses. Being of African origin, the first LSD outbreaks in Europe occurred in Greece and later in the Balkan region. Little is known about the mode of transmission, especially in relation to the potential role of arthropods vectors. The purpose of our study was to investigate the role of Stomoxys calcitrans in the transmission of LSDV and their presence at different farms in Switzerland. Laboratory-reared flies were exposed to LSDV spiked-blood and incubated under a realistic fluctuating temperature regime. Body parts, regurgitated blood, and faecal samples were analysed by qPCR for the presence of viral DNA and infectious virus at different time points post-feeding (p.f.). LSDV DNA was detected in heads, bodies, and regurgitated blood up to three days p.f. and up to two days p.f. in the faeces. Infectious virus was isolated from bodies and faeces up to two days and in the regurgitated blood up to 12 h p.f. There was no increase in viral load, consolidating the role of S. calcitrans as mechanical vectors for LSDV. Stomoxys flies were present at all eight farms investigated, including a farm located at 2128 m asl. The persistence of LSDV in S. calcitrans in combination with the long flight ranges of this abundant and widespread fly might have implications on LSD epidemiology and on implementing control measures during disease outbreaks.

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.

Abstract Background Despite the expanding worldwide distribution of Aedes albopictu s and its increasing relevance as arboviral vector, current methods to collect adult specimens are not optimal. Improved approaches are thus needed to monitor their density and pathogen infections, and to establish baseline data for control interventions. A widely used device is the BG-Sentinel (BG-trap) which mostly targets host-seeking females attracted by release of CO2 and/or a synthetic odour blend (the BG lure). We compared the attractiveness of this blend to that of the Mbita (MB5) lure, a new synthetic blend of proven efficiency in attracting Afrotropical malaria vectors, and evaluated the additional effect of CO2 to the two odour baits. Findings We carried out 6x6 Latin square experiments in two Ae. albopictus-infested areas in Rome, baiting the BG-traps as follows: CO2, BG lure, MB5 lure, BG lure + CO2, MB5 lure + CO2, no bait. CO2 was derived from yeast-fermented sugar. Overall, 949 females and 816 males were collected. Baited traps collected significantly more females than unbaited ones. Traps baited with either lures in combination with CO2 were more effective than those baited with CO2 alone. No significant differences were observed in female captures between traps baited with any of the two lures, nor between the two lures, independently from the addition of CO2. The use of BG lure + CO2 significantly increased males catches compared to unbaited traps. Conclusions The results suggest a broad significance of the MB5 lure for sampling medically important mosquito species and highlight the high efficacy of the combination of lures + CO2 for female Ae. albopictus and of BG lure + CO2 for males, leading to consider CO2 as an essential additional cue for the sampling of this species.