Keeping track of the enemy : Flight analyses of the host-seeking malaria mosquito Anopheles gambiae s.s

Female mosquitoes can transmit pathogens to their host during blood feeding and are an important vector of human diseases such as dengue, chikungunya, filariasis and malaria. After 15 years of decline in the number of fatal malaria cases, this decline came to a halt in 2016. Growing resistance against drugs and insecticides pose a serious threat for future human health. This thesis focuses on the behaviour of host-seeking malaria mosquitoes by analysing their flight paths during their approach to different host cues. Fundamental knowledge on the role of selected host cues was acquired. In addition, studies to support successful implementation of vector control interventions were performed in both wind-tunnel settings and the semi-field in Kenya. My research demonstrates that automated tracking systems can strengthen behavioural-ecological studies on disease vectors, in addition to conventional bio-assays such as olfactometers, by providing detailed information on the approach behaviour of mosquitoes to different targets. The attraction towards the host-sensory cue CO2 was investigated in an olfactometer bioassay. Trap catches of female Anopheles gambiae s.s. were enhanced by separation of the CO2 source from the source of human skin emanations. Close-range deterrent effects of CO2 were overcome by the simultaneous presence of skin emanations. Flight path analysis of mosquitoes in a wind tunnel, showed that exposure to human odour resulted in prolonged and highly convoluted flight tracks. The combination of odour with heat was crucial to induce landings of host-seeking mosquitoes. A semi-field study in Kenya revealed that house-entering mosquitoes approached the eave of a house in a wide angle to the house at eave level, where the proportion that entered uninterruptedly (23%) spent just a few seconds around the eave area. The presence of insecticide-treated nets inside a house did not repel mosquitoes as measured by the number of house entries. At close range, in a wind tunnel, free-flight exposure of mosquitoes to deltamethrin-treated nets in combination with human odour did not reveal any (excito-) repellent effect and resulted in lower mortality rates compared to standard bioassays where contact with the treated material is enforced. The knowledge obtained on the behavioural responses of mosquitoes to host cues has indirectly affected vector control tool implementations in the field. For example, in the development of an odour-baited trap, a CO2 release pipe was included that is separated from the attractive odour plume. The role of heat was exploited in the development of a repellent bioassay and a heat source was added to another trap model. Insights in house-entry behaviour and mosquito responses to bed nets support the successful implementation of push-pull systems, installation of eave tubes or implementing house improvement operations to reduce malaria transmission. An integrated vector management approach is required to further develop existing control tools by adding and improving alternative intervention techniques.</p

Chemical Mediation of Oviposition by Anopheles Mosquitoes : a Push-Pull System Driven by Volatiles Associated with Larval Stages

The oviposition behavior of mosquitoes is mediated by chemical cues. In the malaria mosquito Anopheles gambiae, conspecific larvae produce infochemicals that affect this behavior. Emanations from first instar larvae proved strongly attractive to gravid females, while those from fourth instars caused oviposition deterrence, suggesting that larval developmental stage affected the oviposition choice of the female mosquito. We examined the nature of these chemicals by headspace collection of emanations of water in which larvae of different stages were developing. Four chemicals with putative effects on oviposition behavior were identified: dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS) were identified in emanations from water containing fourth instars; nonane and 2,4-pentanedione (2,4-PD) were identified in emanations from water containing both first and fourth instars. Dual-choice oviposition studies with these compounds were done in the laboratory and in semi-field experiments in Tanzania. In the laboratory, DMDS and DMTS were associated with oviposition-deterrent effects, while results with nonane and 2,4-PD were inconclusive. In further studies DMDS and DMTS evoked egg retention, while with nonane and 2,4-PD 88% and 100% of female mosquitoes, respectively, laid eggs. In dual-choice semi-field trials DMDS and DMTS caused oviposition deterrence, while nonane and 2,4-PD evoked attraction, inducing females to lay more eggs in bowls containing these compounds compared to the controls. We conclude that oviposition of An. gambiae is mediated by these four infochemicals associated with conspecific larvae, eliciting either attraction or deterrence. High levels of egg retention occurred when females were exposed to chemicals associated with fourth instar larvae.</p

Field Evaluation of Traditionally Used Plant-Based Insect Repellents and Fumigants Against the Malaria Vector Anopheles darlingi in Riberalta, Bolivian Amazon

Inexpensive insect repellents may be needed to supplement the use of impregnated bed-nets in the Amazon region, where the primary malaria vector, Anopheles darlingi (Root), is exophilic and feeds in the early evening. Three plants that are traditionally used to repel mosquitoes in Riberalta, Bolivian Amazon, were identified by focus group, and then they were tested against An. darlingi as well as Mansonia indubitans (Dyar & Shannon)/Mansonia titillans (Walker). Cymbopogon citratus (Staph), Guatemalan lemongrass, essential oil at 25% was used as a skin repellent, and it provided 74% protection for 2.5 h against predominantly An. darlingi and 95% protection for 2.5 h against Mansonia spp. Attalea princeps (name not verified) husks, burned on charcoal in the traditional way provided 35 and 51% protection against An. darlingi and Mansonia spp., respectively. Kerosene lamps, often used to light rural homes, were used as a heat source to volatilize 100% Mentha arvensis (Malinv ex. Bailey) essential oil, and they reduced biting by 41% inside traditional homes against Mansonia spp., although they were ineffective outdoors against An. darlingi. All three plant-based repellents provided significant protection compared with controls. Plant-based repellents, although less effective than synthetic alternatives, were shown by focus groups to be more culturally acceptable in this setting, in particular para-menthane-3, 8, idol derived from lemon eucalyptus, Corymbia citriodora (Hook). Plant-based repellents have the potential to be produced locally and therefore sold more cheaply than synthetic commercial repellents. Importantly, their low cost may encourage user compliance among indigenous and marginalized populations

Sugar-fermenting yeast as an organic source of carbon dioxide to attract the malaria mosquito Anopheles gambiae s.s.

<p>Abstract</p> <p>Background</p> <p>Carbon dioxide (CO₂) plays an important role in the host-seeking process of opportunistic, zoophilic and anthropophilic mosquito species and is, therefore, commonly added to mosquito sampling tools. The African malaria vector <it>Anopheles gambiae sensu stricto </it>is attracted to human volatiles augmented by CO₂. This study investigated whether CO₂, usually supplied from gas cylinders acquired from commercial industry, could be replaced by CO₂derived from fermenting yeast (yeast-produced CO₂).</p> <p>Methods</p> <p>Trapping experiments were conducted in the laboratory, semi-field and field, with <it>An. gambiae s.s</it>. as the target species. MM-X traps were baited with volatiles produced by mixtures of yeast, sugar and water, prepared in 1.5, 5 or 25 L bottles. Catches were compared with traps baited with industrial CO₂. The additional effect of human odours was also examined. In the laboratory and semi-field facility dual-choice experiments were conducted. The effect of traps baited with yeast-produced CO₂on the number of mosquitoes entering an African house was studied in the MalariaSphere. Carbon dioxide baited traps, placed outside human dwellings, were also tested in an African village setting. The laboratory and semi-field data were analysed by a χ²-test, the field data by GLM. In addition, CO₂concentrations produced by yeast-sugar solutions were measured over time.</p> <p>Results</p> <p>Traps baited with yeast-produced CO₂caught significantly more mosquitoes than unbaited traps (up to 34 h post mixing the ingredients) and also significantly more than traps baited with industrial CO₂, both in the laboratory and semi-field. Adding yeast-produced CO₂to traps baited with human odour significantly increased trap catches. In the MalariaSphere, outdoor traps baited with yeast-produced or industrial CO₂+ human odour reduced house entry of mosquitoes with a human host sleeping under a bed net indoors. <it>Anopheles gambiae s.s</it>. was not caught during the field trials. However, traps baited with yeast-produced CO₂caught similar numbers of <it>Anopheles arabiensis </it>as traps baited with industrial CO₂. Addition of human odour increased trap catches.</p> <p>Conclusions</p> <p>Yeast-produced CO₂can effectively replace industrial CO₂for sampling of <it>An. gambiae s.s</it>.. This will significantly reduce costs and allow sustainable mass-application of odour-baited devices for mosquito sampling in remote areas.</p

Research agenda for preventing mosquito-transmitted diseases through improving the built environment in sub-Saharan Africa

Mosquito-transmitted diseases are a major threat to health in sub-Saharan Africa, but could be reduced through modifications to the built environment. Here we report findings from a major workshop held to identify the research gaps in this area, namely: (1) evidence of the health benefits to changes to the built environment, (2) understanding how mosquitoes enter buildings, (3) novel methods for reducing mosquito-house entry, (4) sustainable approaches for reducing mosquito habitats, (5) case studies of micro-financing for healthy homes and (6) methods for increasing scale-up. Multidisciplinary research is essential to build out mosquito-transmitted diseases, and not build them in

Keeping track of mosquitoes : A review of tools to track, record and analyse mosquito flight

The health impact of mosquito-borne diseases causes a huge burden on human societies. Recent vector control campaigns have resulted in promising declines in incidence and prevalence of these diseases, notably malaria, but resistance to insecticides and drugs are on the rise, threatening to overturn these gains. Moreover, several vector-borne diseases have re-emerged, requiring prompt and effective response measures. To improve and properly implement vector control interventions, the behaviour of the vectors must be well understood with detailed examination of mosquito flight being an essential component. Current knowledge on mosquito behaviour across its life history is briefly presented, followed by an overview of recent developments in automated tracking techniques for detailed interpretation of mosquito behaviour. These techniques allow highly accurate recording and observation of mating, feeding and oviposition behaviour. Software programmes built with specific algorithms enable quantification of these behaviours. For example, the crucial role of heat on host landing and the multimodal integration of carbon dioxide (CO2) with other host cues, has been unravelled based on three-dimensional tracking of mosquito flight behaviour. Furthermore, the behavioural processes underlying house entry and subsequent host searching and finding can be better understood by analysis of detailed flight recordings. Further potential of these technologies to solve knowledge gaps is discussed. The use of tracking techniques can support or replace existing monitoring tools and provide insights on mosquito behaviour that can lead to innovative and more effective vector-control measures

Effect of insecticide-treated bed nets on house-entry by malaria mosquitoes : The flight response recorded in a semi-field study in Kenya

Insecticide-treated nets are currently a major tool to reduce malaria transmission. Their level of repellency affects contact of the mosquito with the net, but may also influence the mosquito's entry into the house. The response of host-seeking malaria mosquitoes approaching the eave of an experimental house was recorded within a large screen house. We compared entry- and exit rates in relation to the presence in the house of different insecticide-treated bed nets (ITNs) with an untreated net. Mosquitoes were lured towards the house by dispensing a synthetic host-odour blend from within the net in the house. Complementary WHO bioassays revealed that the treated nets caused high knock-down- and mortality responses to the Anopheles gambiae sensu stricto strain tested. The proportion of mosquitoes that came into view of the cameras and subsequently entered the house did not differ between treated nets and the untreated net. Treated nets did not affect proportions of mosquitoes that exited the house and departed from view around the eave. However, the percentage of house-leaving and re-entering mosquitoes when an insecticide- treated net was present, was lower than in the presence of an untreated net. Our results indicated that there was no spatial repellent effect from pyrethroid-treated nets that influences house-entry at eave level. It is argued that the toxic effect of treated bed nets resulted in a reduced number of mosquitoes re-entering the house, which could thereby affect malaria transmission in neighbouring, unprotected houses

Visualization of house-entry behaviour of malaria mosquitoes

Background: Malaria mosquitoes often blood feed indoors on human hosts. The mosquitoes predominantly enter houses via open eaves. Host-seeking is odour-driven, and finding a host depends on the quality of the odour plume and whether the route towards the host is free of obstructions. Little is known about in-flight behaviour of mosquitoes during house entry. This semi-field study visualizes mosquito house entry in three dimensions (3D) and offers new insights for optimizing vector control interventions. Methods: The approach and house entry of Anopheles gambiae sensu stricto was studied in a semi-field set-up using video-recorded flight tracks and 3D analysis. Behavioural parameters of host-seeking female mosquitoes were visualized with respect to their position relative to the eave as well as whether a mosquito would enter or not. Host odour was standardized using an attractive synthetic blend in addition to CO2. The study was conducted in western Kenya at the Thomas Odhiambo Campus of the International Centre of Insect Physiology and Ecology, Mbita. Results: The majority of host-seeking An. gambiae approached a house with a flight altitude at eave level, arriving within a horizontal arc of 180°. Fifty-five per cent of mosquitoes approaching a house did not enter or made multiple attempts before passing through the eave. During approach, mosquitoes greatly reduced their speed and the flight paths became more convoluted. As a result, mosquitoes that passed through the eave spent more than 80 % of the observed time within 30 cm of the eave. Mosquitoes that exited the eave departed at eave level and followed the edge of the roof (12.5 %) or quickly re-entered after exiting (9.6 %). Conclusions: The study shows that host-seeking mosquitoes, when entering a house, approach the eave in a wide angle to the house at eave level. Less than 25 % of approaching mosquitoes entered the house without interruption, whereas 12.5 % of mosquitoes that had entered left the house again within the time of observation. Advances in tracking techniques open a new array of questions that can now be answered to improve household interventions that combat malaria transmission.</p