Mosquito odour-baited mass trapping reduced malaria transmission intensity: a result from a controlled before-and-after intervention study.

BACKGROUND: Conventional vector control strategies have significantly reduced the malaria burden. The sustainability of these methods is currently challenged. Odour-based traps are emerging technologies that can complement the existing tools. Implementation of odour-based traps for mass trapping is limited due to the restricted range of vectors caught with available carbon dioxide-dependent lures, and the lack of comprehensive field studies. The objective of this study was to assess the impact of odour-mediated mass trapping targeting outdoor vectors, using a synthetic cattle urine lure that attracts a wide range of vector species in a variety of physiological states, on malaria prevalence and entomological parameters to determine malaria transmission intensities. METHODS: A controlled before-and-after study was conducted in two rural communities in southern Ethiopia. Baseline monthly entomological and seasonal cross-sectional malaria prevalence surveys were conducted in both communities for a year. Then, mass trapping of mosquitoes was conducted in one of the villages, while the monthly entomological surveillance and seasonal malaria prevalence surveys continued in both villages. Generalised linear mixed models were constructed and tested to determine which factors were significantly affected by the intervention. RESULTS: Mass trapping contributed to the reduction of the population of the principal malaria vector, Anopheles arabiensis, and the associated entomological indicators, the human bite rate (HBR) and the entomological inoculation rate (EIR), in the intervention village compared to the control village. The intervention village had an average HBR by An. arabiensis of 3.0 (95% CI 1.4-4.6) during the peak malaria transmission season, compared to 10.5 (95% CI - 0.5-21.5; P < 0.0001) in the control village. The intervention village (mean 0.02, 95% CI - 0.05-0.4.8) had a daily EIR eight times lower than the control village (mean 0.17, 95% CI), which likely contributed to the reduced malaria prevalence in the intervention community following its introduction by ca. 60% (95% CI 55-63). CONCLUSIONS: The combined use of odour-based mass trapping and conventional control strategies coincided with a reduction of human-vector contact and malaria prevalence, providing support for odour-baited technologies as a viable option for next-generation vector control tools. Further cluster-randomised control studies are recommended in different eco-epidemiological settings with varying malaria transmission intensities.

Knockout of OR39 reveals redundancy in the olfactory pathway regulating the acquisition of host seeking in Anopheles coluzzii.

The attraction of anthropophilic mosquitoes to human host cues, such as body odour and carbon dioxide, gradually increases during adult maturation. This acquisition of host-seeking behaviour correlates with age-dependent changes in odorant receptor (OR) transcript abundance and sensitivity of olfactory sensory neurons (OSNs). One OR gene of the human malaria vector, Anopheles coluzzii, AcolOR39, is significantly downregulated in mature females, and a cognate ligand of AcolOR39, sulcatone, a major component of human emanations, mediates the observed behavioural inhibition of newly emerged (teneral) females to human body odour. Knockout of AcolOR39, using CRISPR-Cas9 mutagenesis, selectively abolished sulcatone detection in OSNs, housed in trichoid sensilla. However, knockout of AcolOR39 altered neither the response rate nor the flight behaviour of teneral females in a wind tunnel, indicating the involvement of other genes, and thus a redundancy, in regulating the acquisition of host seeking in mosquitoes.

Adenosine triphosphate overrides the aversive effect of antifeedants and toxicants: a model alternative phagostimulant for sugar-based vector control tools.

BACKGROUND: Sugar, when used as the phagostimulant in attractive toxic bait control tools, limits the efficacy and selectivity of this technology. Thus, more potent and selective phagostimulants than sugar are required to improve this technology. The potency of adenosine triphosphate (ATP) as an alternative model phagostimulant was assessed to determine its capacity to override the aversive effects of select antifeedants and toxicants. How ATP and sucrose modulate the rate of toxicity in the yellow fever mosquito Aedes aegypti was also examined. METHODS: A no-choice feeding assay was used to investigate the phagostimulatory ability of ATP to override the aversive effects of structurally divergent antifeedant and toxicant compounds, and to modulate the rate of toxicity over 24 h. Binary combinations of antifeedant and toxicant compounds, at various concentrations, were similarly assessed for enhanced lethal potency. In comparison, no-choice open access and cotton wick feeding assays were used to determine the phagostimulatory role of sucrose in the ingestion of boric acid-laced diets. Dissections of the guts were performed to determine the diet destination as dependant on the phagostimulant. RESULTS: ATP is a potent phagostimulant that dose dependently overrides aversion to antifeedant and toxicant tastants. Feeding on antifeedant- or toxicant-laced diets that was induced by ATP selectively resulted in rapid knockdown (nicotine, lobeline and caffeine) or death (boric acid and propylene glycol), with a combination of the two lethal compounds inducing a synergistic effect at lower concentrations. ATP- and sucrose-induced feeding predominantly directed the antifeedant- or toxicant-laced meals to the midgut and the crop, respectively. CONCLUSIONS: ATP is an efficacious alternative model phagostimulant to sucrose that overrides the aversive effects of antifeedants and toxicants, resulting in rapid toxic effects. Furthermore, this study demonstrates that variation in the rate of toxicity between ATP- and sugar-induced feeding is at least partly regulated by the differential feeding response, volume imbibed and the destination of the meals. Additional research is needed to identify structurally related, stable analogues of ATP due to the ephemeral nature of this molecule. For future applications, the workflow presented in this study may be used to evaluate such analogues for their suitability for use in attractive bait stations designed to target a broad range of haematophagous arthropods and prevent off-target species' feeding.

Odour-mediated oviposition site selection in Aedes aegypti depends on aquatic stage and density.

BACKGROUND: Olfaction plays an important role in the selection and assessment of oviposition sites by mosquitoes. Volatile organic compounds (VOCs) associated with potential breeding sites affect the behaviour of gravid mosquitoes, with VOCs from aquatic stages of conspecific mosquitoes influencing and regulating oviposition. The purpose of this study was to conduct a systematic analysis of the behavioural response of gravid Aedes aegypti to conspecific aquatic stage-conditioned water, to identify the associated bioactive VOCs and to determine how blends of these VOCs regulate oviposition site selection and stimulate egg-laying. METHODS: Using a multi-choice olfactory oviposition assay, controlling for other sensory modalities, the responses of individual females to water conditioned with different densities of conspecific aquatic stages were assessed. The conditioned water samples from the most preferred density of each aquatic stage were subsequently compared to each other using the same oviposition assay and analysed using an analysis of variance (ANOVA) followed by a Tukey post-hoc test. Using combined gas chromatography and electroantennographic detection or mass spectrometry, bioactive VOCs from the preferred density of each aquatic stage were identified. Synthetic blends were prepared based on the identified ratios of bioactive VOCs in the aquatic stages, and then tested to determine the oviposition choice of Ae. aegypti in a dose-dependent manner, against a solvent control, using a dual-choice assay. This dataset was analysed using nominal logistic regression followed by an odds ratio comparison. RESULTS: Gravid Ae. aegypti responded stage- and density-dependently to water conditioned with eggs, second- and fourth-instar larvae, and pupal exuviae, but not to water conditioned with pupae alone. Multi-choice assays demonstrated that gravid mosquitoes preferred to oviposit in water conditioned with fourth-instar larvae, over the other aquatic stage-conditioned water. Gravid Ae. aegypti were attracted, and generally stimulated, to oviposit in a dose-dependent manner to the individual identified synthetic odour blends for the different aquatic stages. CONCLUSIONS: Intraspecific VOCs regulate oviposition site selection in Ae. aegypti in a stage- and density-dependent manner. We discuss the need for further studies to evaluate the identified synthetic blends to modulate the odour-mediated oviposition of Ae. aegypti under field conditions.

Identification of Bioactive Volatile Organic Compounds Using Combined Gas Chromatography and Single Sensillum Recording.

Most arthropods rely heavily on their sense of smell (i.e., olfaction) to locate and discriminate among mates, food, and egg laying sites. The odors emanating from these resources are composed of blends of volatile compounds that are detected by olfactory sensory neurons (OSNs) that are housed in hair-like structures, called sensilla, on the olfactory organs of arthropods. By inserting an electrode into a single sensillum and recording the activity of the OSNs while stimulating with volatile compounds eluting from a gas chromatograph, combined gas chromatography and single sensillum recording (GC-SSR) provides a high-resolution tool to identify bioactive compounds and to functionally characterize the peripheral olfactory system of arthropods.

Combined Gas Chromatography and Single Sensillum Recording for Mosquitoes.

Combined gas chromatography and single sensillum recording (GC-SSR) joins together the established volatile compound separation and relative quantification techniques of GC with the ability to functionally screen olfactory sensory neurons (OSNs) for volatile selectivity and sensitivity using SSR. With minimal equipment modification, including a splitter and a heated transfer line, half of the effluent from the gas chromatograph column is directed to the flame ionization detector and half to the mosquito antennae. This GC-SSR combination provides a bioassay capable of determining the salient natural ligands for individual OSNs and of providing accurate and comparable dose-response curves irrespective of differences in the volatility of the compounds.

Golgi Silver Staining of Mosquito Central Neuropils.

The Golgi silver staining procedure relies on three interdependent stages: fixation, chromation, and metal impregnation. Each of these stages can be modified. This protocol describes a method demonstrated to stain neurons within the mosquito central nervous system. The resulting preparations are stable at room temperature.

The Golgi Method.

Silver staining by the Golgi method is a classical procedure to identify the three-dimensional morphology of individual neurons. Although the method was developed in the 1870s, it is still used to study the morphological characteristics of neuron types in the central nervous system, either alone or in combination with other neuroanatomical techniques. The neuropil of insects is fully accessible to the original refractive staining procedure, and modifications of the Golgi technique have paved the path for modern structural research on the insect central nervous system. Here, we provide an introduction to this easy and low-cost method.

Regulation of the antennal transcriptome of the dengue vector, Aedes aegypti, during the first gonotrophic cycle.

BACKGROUND: In the light of dengue being the fastest growing transmissible disease, there is a dire need to identify the mechanisms regulating the behaviour of the main vector Aedes aegypti. Disease transmission requires the female mosquito to acquire the pathogen from a blood meal during one gonotrophic cycle, and to pass it on in the next, and the capacity of the vector to maintain the disease relies on a sustained mosquito population. RESULTS: Using a comprehensive transcriptomic approach, we provide insight into the regulation of the odour-mediated host- and oviposition-seeking behaviours throughout the first gonotrophic cycle. We provide clear evidence that the age and state of the female affects antennal transcription differentially. Notably, the temporal- and state-dependent patterns of differential transcript abundance of chemosensory and neuromodulatory genes extends across families, and appears to be linked to concerted differential modulation by subsets of transcription factors. CONCLUSIONS: By identifying these regulatory pathways, we provide a substrate for future studies targeting subsets of genes across disparate families involved in generating key vector behaviours, with the goal to develop novel vector control tools.

Malaria hotspots explained from the perspective of ecological theory underlying insect foraging.

Hotspots constitute the major reservoir for residual malaria transmission, with higher malaria incidence than neighbouring areas, and therefore, have the potential to form the cornerstone for successful intervention strategies. Detection of malaria hotspots is hampered by their heterogenous spatial distribution, and the laborious nature and low sensitivity of the current methods used to assess transmission intensity. We adopt ecological theory underlying foraging in herbivorous insects to vector mosquito host seeking and modelling of fine-scale landscape features at the village level. The overall effect of environmental variables on the density of indoor mosquitoes, sporozoite infected mosquitoes, and malaria incidence, was determined using generalized linear models. Spatial analyses were used to identify hotspots for malaria incidence, as well as malaria vector density and associated sporozoite prevalence. We identify household occupancy and location as the main predictors of vector density, entomological inoculation rate and malaria incidence. We propose that the use of conventional vector control and malaria interventions, integrated with their intensified application targeting predicted hotspots, can be used to reduce malaria incidence in endemic and residual malaria settings.

Dengue infection modulates locomotion and host seeking in Aedes aegypti.

Pathogens may manipulate their human and mosquito hosts to enhance disease transmission. Dengue, caused by four viral serotypes, is the fastest-growing transmissible disease globally resulting in 50-100 million infections annually. Transmission of the disease relies on the interaction between humans and the vector Aedes aegypti and is largely dependent on the odor-mediated host seeking of female mosquitoes. In this study, we use activity monitors to demonstrate that dengue virus-1 affects the locomotion and odor-mediated behavior of Ae. aegypti, reflecting the progression of infection within the mosquito. Mosquitoes 4-6 days post-infection increase locomotion, but do not alter their odor-driven host-seeking response. In contrast, females 14-16 days post-infection are less active, yet more sensitive to human odors as assessed by behavioral and electrophysiological assays. Such an increase in physiological and behavioral sensitivity is reflected by the antennal-specific increase in abundance of neural signaling transcripts in 14 days post-infection females, as determined by transcriptome analysis. This suggests that the sensitivity of the mosquito peripheral olfactory system is altered by the dengue virus by enhancing the overall neural responsiveness of the antenna, rather than the selective regulation of chemosensory-related genes. Our study reveals that dengue virus-1 enhances vector-related behaviors in the early stages post-infection that aid in avoiding predation and increasing spatial exploration. On the other hand, at the later stages of infection, the virus enhances the host-seeking capacity of the vector, thereby increasing the risk of virus transmission. A potential mechanism is discussed.

Plasmodium falciparum gametocyte-induced volatiles enhance attraction of Anopheles mosquitoes in the field.

BACKGROUND: Plasmodium parasites manipulate the interaction between their mosquito and human hosts. Patients infected with gametocytes attract anopheline mosquitoes differentially compared to healthy individuals, an effect associated with an increased release of attractive volatile cues. This odour-driven manipulation is partly mediated by the gametocyte-specific metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which induces increased release of select aldehydes and terpenes from red blood cells and results in the enhanced attraction of host-seeking mosquitoes, which are vectors of malaria. This study investigates the effect of the HMBPP-induced volatiles on the attraction of wild Anopheles mosquitoes to humans under field conditions. METHODS: The efficacy of the HMBPP-induced odour blend to attract Anopheles was evaluated in a 4 × 6 Latin rectangular study design indoors using baited Suna traps. Furthermore, to assess the efficacy of the HMBPP-induced odour blend in (1) augmenting the attractiveness of human odour, and (2) attracting Anopheles mosquitoes in the absence of human odour, a two-choice assay using host decoy traps (HDTs) was used and evaluated using binomial generalized regression. RESULTS: Traps baited with the HMBPP-induced odour blend attracted and caught both Anopheles arabiensis and Anopheles pharoensis females in a dose-dependent manner in the presence of background human odour, up to 2.5 times that of an unbaited trap. Given a choice between human odour and human odour laden with the HMBPP-induced odour blend, mosquitoes differentially preferred traps augmented with the HMBPP-induced odour blend, which caught twice as many female An. arabiensis. Traps baited with the HMBPP-induced odour blend but lacking the background of human odour were not effective in attracting and catching mosquitoes. CONCLUSION: The findings of the present study revealed that the HMBPP-induced odour blend, when augmented with human body odour, is attractive to anopheline mosquitoes and could be used as a complementary vector control tool along with existing strategies.

Malaria mosquito chemical ecology.

The field of mosquito chemical ecology has shifted focus over the past five years, driven by the recognition that odour-mediated behaviours are regulated by distinct chemical codes, that is, odour blends emanating from the natural environment. As a research community, we have shifted from our anthropocentric focus to include other behaviours, including plant seeking and oviposition site seeking, in order to develop new tools to combat residual malaria in the wake of the increased insecticide and behavioural resistance in mosquitoes across sub-Saharan Africa. In this short review, we will outline the progress made, and the future directions, in understanding blend recognition and chemical parsimony, and their implications for preadaptation of the odour coding system in malaria mosquitoes.

Sex and age modulate antennal chemosensory-related genes linked to the onset of host seeking in the yellow-fever mosquito, Aedes aegypti.

The mosquito Aedes aegypti is the primary vector for the fastest growing infectious disease in the world, dengue fever. Disease transmission heavily relies on the ability of female mosquitoes to locate their human hosts. Additionally, males may be found in close proximity to humans, where they can find mates. Host seeking behaviour of both sexes is dependent on adult sexual maturation. Identifying the molecular basis for the onset of host seeking may help to determine targets for future vector control. In this study, we investigate modulation of the host seeking behaviour and the transcript abundance of the main chemoreceptor families between sexes and across ages in newly-emerged mosquitoes. Attraction to human odour was assessed using a Y-tube olfactometer, demonstrating that both males and females display age-dependent regulation of host seeking. The largest increase in transcript abundance was identified for select chemosensory genes in the antennae of young adult Ae. aegypti mosquitoes and reflects the increase in attraction to human odour observed between 1 and 3 day(s) post-emergence in both males and females. Future functional characterisation of the identified differentially abundant genes may provide targets for the development of novel control strategies against vector borne diseases.

Feeding-induced changes in allatostatin-A and short neuropeptide F in the antennal lobes affect odor-mediated host seeking in the yellow fever mosquito, Aedes aegypti.

Aedes aegypti is a model species in which the endogenous regulation of odor-mediated host seeking behavior has received some attention. Sugar feeding and host seeking in female A. aegypti are transiently inhibited following a blood meal. This inhibition is partially mediated by short neuropeptide F (sNPF). The paired antennal lobes (ALs), as the first processing centers for olfactory information, has been shown to play a significant role in the neuropeptidergic regulation of odor-mediated behaviors in insects. The expression of sNPF, along with other peptides in the ALs of A. aegypti, indicate parallel neuromodulatory systems that may affect olfactory processing. To identify neuropeptides involved in regulating the odor-mediated host seeking behavior in A. aegypti, we use a semi-quantitative neuropeptidomic analysis of single ALs to analyze changes in the levels of five individual neuropeptides in response to different feeding regimes. Our results show that the level of sNPF-2, allatostatin-A-5 (AstA-5) and neuropeptide-like precursor-1-5 (NPLP-1-5), but not of tachykinin-related-peptides and SIFamide (SIFa), in the AL of female mosquitoes, changes 24 h and 48 h post-blood meal, and are dependent on prior access to sugar. To assess the role of these neuropeptides in modulating host seeking behavior, when systemically injected individually, sNPF-2 and AstA-5 significantly reduced host seeking behavior. However, only the injection of the binary mixture of the two neuropeptides lead to a host seeking inhibition similar to that observed in blood fed females. We conclude that modulation of the odor mediated host seeking behavior of A. aegypti is likely regulated by a dual neuropeptidergic pathway acting in concert in the ALs.

Detection and perception of generic host volatiles by mosquitoes: responses to CO2 constrains host-seeking behaviour.

Natural selection has favoured specialization in anthropophilic mosquito host choice, yet in the absence of human hosts, females feed on a selected range of vertebrates. For host recognition, we hypothesize that mosquitoes primarily rely on generic host volatiles. Detection and perception of such compounds would provide the mosquito with a flexible, yet constrained, odour coding system that could delineate host preference. In this study, we show that the quintessential generic volatile for host-seeking, carbon dioxide, activates and attracts the malaria mosquito, Anopheles coluzzii, and the arbovirus vectors, Aedes aegypti and Culex quinquefasciatus, within boundaries set by the dynamic range and coding capacity of the CO2-sensitive olfactory receptor neurons. These boundaries are sufficiently broad to elicit behavioural responses to various hosts within their preferred host range. This study highlights the significance of the sensitivity of the carbon dioxide detection system and its regulation of host seeking and recognition.

Blood meal induced regulation of the chemosensory gene repertoire in the southern house mosquito.

BACKGROUND: The southern house mosquito, Culex quinquefasciatus, is one of the most prevalent vectors of lymphatic filariasis and flavivirus-induced encephalitis. Its vectorial capacity is directly affected by its reproductive feeding behaviors, such as host seeking, blood feeding, resting, and egg laying. In mosquitoes, these gonotrophic behaviors are odor-mediated and regulated following blood feeding. Immediately after a blood meal, female mosquitoes show reduced olfactory responsiveness and flight activity, as they enter a resting state. Insights into antennal chemosensory gene regulation at this time period can provide a foundation to identify targets involved in the state switch between host seeking and resting. RESULTS: This study used quantitative gene expression analyses to explore blood meal induced regulation of chemosensory gene families in the antennae of 6 days post-emergence C. quinquefasciatus females. Improved annotations for multiple chemosensory gene families, and a quantitative differential gene expression analysis between host seeking and 24 h post- blood fed females of the same age, allowed for the detection of transcripts that potentially play a role in the switch from host seeking to resting, in C. quinquefasciatus. The expression profiles of chemosensory genes varied significantly between the two treatments. CONCLUSIONS: Annotations for chemosensory gene repertoires in C. quinquefasciatus have been manually curated and corrected for 3' exon choice and transcript length, through sequence and transcriptome analyses. The gene expression analyses identified various molecular components of the peripheral olfactory system in C. quinquefasciatus, including odorant receptors, ionotropic receptors, odorant binding proteins and chemosensory proteins, that are regulated in response to blood feeding, and could be critical for the behavioral switch from host seeking to resting. Functional characterization of these proteins in the future can identify targets essential for the females' gonotrophic behaviors, and can be used to design novel vector control strategies.

Detection and perception of generic host volatiles by mosquitoes modulate host preference: context dependence of (R)-1-octen-3-ol.

Natural selection favours a restricted host breadth in disease vector mosquitoes, indicating that there is an adaptive value associated with maintaining plasticity in host preference. One mechanism to maintain such plasticity is via the detection of generic cues by conserved peripheral olfactory pathways, which when perceived in different host odour contexts enable the identification of and discrimination among potential host species. Here, we show that the context of an odour cue shapes host perception in mosquitoes, by altering the release rate of the generic host-related volatile (R)-1-octen-3-ol, within its natural range, and in the background odour of known hosts and non-hosts. This result highlights that host recognition is contextual and dependent on quantitative and qualitative differences in odour blends and the olfactory codes evoked. From the perspective of vector management, understanding the perception of odour blends and their context is essential to the process of developing synthetic blends for the optimal attraction of mosquitoes in efforts to control and monitor populations.

Chicken volatiles repel host-seeking malaria mosquitoes.

BACKGROUND: Anopheles arabiensis is a dominant vector of malaria in sub-Saharan Africa, which feeds indoors and outdoors on human and other vertebrate hosts, making it a difficult species to control with existing control methods. Novel methods that reduce human-vector interactions are, therefore, required to improve the impact of vector control programmes. Investigating the mechanisms underlying the host discrimination process in An. arabiensis could provide valuable knowledge leading to the development of novel control technologies. In this study, a host census and blood meal analysis were conducted to determine the host selection behaviour of An. arabiensis. Since mosquitoes select and discriminate among hosts primarily using olfaction, the volatile headspace of the preferred non-human host and non-host species, were collected. Using combined gas chromatography and electroantennographic detection analysis followed by combined gas chromatography and mass spectrometry, the bioactive compounds in the headspace collections were identified. The efficiency of the identified non-host compounds to repel host-seeking malaria mosquitoes was tested under field conditions. RESULTS: The host census and blood meal analyses demonstrated that An. arabiensis strongly prefers human blood when host seeking indoors, while it randomly feeds on cattle, goats and sheep when found outdoors. However, An. arabiensis avoids chickens despite their relatively high abundance, indicating that chickens are a non-host species for this vector. Eleven bioactive compounds were found in the headspace of the non-host species. Six of these were species-specific, out of which four were identified using combined gas chromatography and mass spectrometry. When tested in the field, the chicken-specific compounds, isobutyl butyrate, naphthalene, hexadecane and trans-limonene oxide, and the generic host compounds, limonene, cis-limonene oxide and ß-myrcene, significantly reduced trap catches within the house compared to a negative control. A significant reduction in trap catch was also observed when suspending a caged chicken next to the trap. CONCLUSIONS: Non-host volatiles repel host-seeking An. arabiensis and thus play a significant role in host discrimination. As such, this study demonstrates that non-host volatiles can provide protection to humans at risk of mosquito-vectored diseases in combination with established control programmes.

Impact of elevated CO2 background levels on the host-seeking behaviour of Aedes aegypti.

Mosquitoes rely on carbon dioxide (CO2) to detect and orient towards their blood hosts. However, the variable and rapid fluctuations of atmospheric CO2 concentrations may have an impact on the host-seeking behaviour of mosquitoes. In this study, we analysed the effect of transient elevated background levels of CO2 on the host-seeking behaviour and the physiological characteristics of the CO2-sensitive olfactory receptor neurones (ORNs) in female yellow fever mosquitoes, Aedes aegypti. We show that the take-off and source contact behaviour of A. aegypti is impeded at elevated background levels of CO2 as a result of masking of the stimulus signal. The mechanism underlying this masking during take-off behaviour is one of sensory constraint. We show that the net response of the CO2-ORNs regulates this CO2-related behaviour. Since these neurones themselves are not habituated or fatigued by the transient elevation of background CO2, we propose that habituation of second-order neurones in response to the elevated CO2-ORN activity could be one mechanism by which the net response is transduced by the olfactory system. The findings from this study may help to predict future shifts in mosquito-host interactions and consequently to predict vectorial capacity in the light of climate change.