Differential attractiveness of humans to the African malaria vector Anopheles gambiae Giles : effects of host characteristics and parasite infection

The results of a series of studies designed to understand the principal factors that determine the differential attractiveness of humans to the malaria vector Anopheles gambiae are described in this thesis. Specific attention was paid to the role of body emanations and infection (of humans) with the malaria parasite Plasmodium falciparum . The main findings of these studies are summarised in the following sections.Differential attractiveness of humans to Anopheles gambiae ( Chapter 1 )Although it has frequently been reported that human beings differ in their degree of attractiveness to mosquitoes, the principal causes that make certain individuals to be preferred more than others are not well known. This gap in knowledge has hindered the understanding of the transmission dynamics of malaria and other mosquito-borne diseases. From an epidemiological point of view, high malaria transmission rates are expected if mosquito vectors preferentially select infective humans for a blood meal, become infected, survive long enough for the parasites to develop to infective stages and proceed to bite uninfected individuals selectively. In terms of fitness, mosquito vectors would be better off if they select hosts that (1) are docile and less defensive so as to minimise feeding-associated risks of mortality, (2) have blood of a high nutritional value, (3) are free of (gametocyte) parasites, healthy and (4) have no anti-mosquito immunity. The answers to these epidemiological and fitness factors are still lacking. Furthermore, since host seeking is odour-mediated, the identification of chemical compounds responsible for attracting mosquitoes to their vertebrate hosts would help in developing traps that are useful for vector surveillance and control. Research in this area is rapidly advancing but has not produced tools for field application. It is felt that more research effort is needed so that new approaches towards understanding and combating disease vectors can be developed.Characterisation of mosquito blood meals using DNA markers (Chapters 2 & 3)The analysis of arthropod blood meals using molecular genetic markers was reviewed and forensic techniques borrowed from the Federal Bureau of Investigation (FBI), USA, used to evaluate the effect of blood meal size and extent of digestion on the ability to identify human DNA extracted from blood meals of Anopheles gambiae . The review recommended that proper and appropriate storage, determination of the concentration of host DNA and collection within few hours after ingestion are important parameters for improving the success of identifying blood meal sources of field-collected mosquitoes. Further, microsatellite markers were highlighted as being more appropriate than minisatellites in analysing blood meals that have been highly degraded e.g. through prolonged digestion. Also, mitochondrial DNA targets were recommended to be better than nuclear DNA targets for analysing blood meals that have been highly degraded. Blood meal size and (microsatellite) locus (analysed) were shown not to affect the success of amplifying human DNA extracted from blood meals of An. gambiae after having been digested for 0, 8, 16, 24 and 32 hours. However, a significant negative relationship between the time since ingestion and the success probability of obtaining positive PCR reactions among blood meals digested for between eight and 32 hours was demonstrated. There was no significant difference in the success probability of amplifying human DNA from blood meals of mosquitoes killed at zero and 8 hours after ingestion. The research demonstrated that not the quality of ingested blood, but the time since ingestion determined the success of blood meal analysis.Host characteristics and differential attractiveness of humans to An. gambiae (Chapter 4, 5 & 6) A tent olfactometer that accommodates complete humans as sources of host-seeking stimuli was designed, developed and tested. The olfactometer was used to study (1) differential attractiveness of humans to host-seeking An. gambiae and (2) how the differences, so elicited, are affected by human breath, body odour, heat and moisture. Nine human subjects were successfully ranked for their attractiveness to the mosquitoes based on (mosquito) responses to their complete body emanations encompassing body odour, heat and moisture. The nine subjects were classified into least (3 persons), medium (4 persons) and most attractive groups (2 persons). Breath was shown to reduce mosquito responses, whereas body odour was highly attractive. Breath was also shown to be an important contributor to between-person differences in relative attractiveness to An. gambiae . Whereas differential attractiveness of two human subjects for the mosquitoes could be demonstrated based on their total body emanations (breath plus body odour), the attractiveness of the two subjects did not differ significantly based on body odour alone. Body odour from either individual was consistently more attractive than total emanations from the other. The same results were obtained with another pair of individuals. It was concluded that breath, although known to contain attractive semiochemicals like carbon dioxide, may also contain compounds that inhibit attraction and may thus serve as an important contributor to between-person differences in relative attractiveness to this important malaria vector. The inhibitory effect of breath was postulated to be allomonal as it benefits the emitter (human being) but does not harm the recipient (mosquito vector). Body heat and moisture were shown to have significant effects on the attraction of An. gambiae to humans. In general, An. gambiae was more attracted to the individual whose body emanations were warmer but less moist than those of an opposing counterpart, in choice experiments. It was concluded that body heat and moisture influence host-selection by An. gambiae at short range and that their effect is probably achieved through interaction with breath components.Parasite infection and differential attractiveness of humans to An. gambiae (Chapter 7 & 8) A rare window of opportunity allowed for the investigation of the effect of clinical symptoms and parasitaemia due to Plasmodium falciparum on variability in human attractiveness to the malaria mosquito An. gambiae using the tent olfactometer previously developed. The relative attractiveness of an individual who was always more attractive than a specific counterpart (when both were uninfected) was suppressed when he (the 'putatively' more attractive individual) displayed clinical symptoms (fever and profuse sweating) of malaria caused by P. falciparum . This finding provided both new and alternative evidence, within the intricate web of Anopheles-Homo-Plasmodium interactions, that the malaria parasite P. falciparum influences the olfactory signals produced by human hosts. Field studies, in which a twin pair of male humans was recruited as a follow-up to the olfactometer study, found that being positive with malaria parasites (trophozoites and possibly gametocytes of P. falciparum ) is associated with higher numbers of attracted mosquitoes ( An. gambiaesensu lato ). This effect was not a consequence of the time of the year and the higher number of mosquitoes present in that period. It was recommended that further investigations be carried out. If these results are confirmed to be true, this work will constitute the first evidence that P. falciparum is capable of enhancing its own transmission by manipulating the physico-chemical characteristics of its vertebrate hosts in such a way that infected individuals are preferentially selected as blood meal sources by host-seeking malaria vectors.What questions remain unanswered (Chapter 9) Much as this thesis has provided some answers to why humans differ in their degrees of attractiveness to mosquitoes ( An. gambiae ), many more questions remain unanswered. The key questions in point include the following: (1) How can we tell whether one is more or less attractive to host-seeking mosquitoes? (2) How do parasites affect the host-seeking behaviour of their insect vectors? and (3) How do evolutionary processes related to host-seeking behaviour affect the fitness of mosquito vectors? The answers to these questions and those to a myriad of others are still waiting to be resolved

Contained semi-field environments for ecological studies on transgenic African malaria vectors: benefits and constraints

New interventions are needed to reduce the burden of vector-borne diseases like malaria and dengué, which are among the most serious and prevalent infectious diseases worldwide. The release of genetically modified (GM) mosquitoes may offer an alternative strategy to do so while circumventing the pitfalls of current vector control methods. Current methodologies are stalling because of drug resistance, absence of vaccines and inadequate mosquito control techniques. GM mosquitoes have been developed that are resistant to pathogen infection and transmission, but the public-health and environmental consequences of releasing such insects are unclear, mainly because of a lack of knowledge of the ecology and population biology of mosquitoes. This book is the reflection of a workshop, held in June 2002, that addressed these issues. Experts on mosquito ecology met for the first time to discuss the current knowledge of mosquito ecology with respect to GM-insect technology. Emphasis of the workshop was on evaluating how human health and natural ecosystems, including target wild-mosquito populations, will respond to the invasion of GM vectors. This volume will stimulate discussion by clearly showing the importance of vector ecology for prevention of vector-borne disease

Variation in malaria transmission dynamics in three different sites in Western Kenya

The main objective was to investigate malaria transmission dynamics in three different sites, two highland villages (Fort Ternan and Lunyerere) and a lowland peri-urban area (Nyalenda) of Kisumu city. Adult mosquitoes were collected using PSC and CDC light trap while malaria parasite incidence data was collected from a cohort of children on monthly basis. Rainfall, humidity and temperature data were collected by automated weather stations. Negative binomial and Poisson generalized additive models were used to examine the risk of being infected, as well as the association with the weather variables. Anopheles gambiae s.s. was most abundant in Lunyerere, An. arabiensis in Nyalenda and An. funestus in Fort Ternan. The CDC light traps caught a higher proportion of mosquitoes (52.3%) than PSC (47.7%), although not significantly different (P = 0.689). The EIR's were 0, 61.79 and 6.91 bites/person/year for Fort Ternan, Lunyerere and Nyalenda. Site, month and core body temperature were all associated with the risk of having malaria parasites (P <0.0001). Rainfall was found to be significantly associated with the occurrence of P. falciparum malaria parasites, but not relative humidity and air temperature. The presence of malaria parasite-infected children in all the study sites provides evidence of local malaria transmission

Extent of digestion affects the success of amplifying human DNA from blood meals of Anopheles gambiae (Diptera: Culicidae)

The success of distinguishing blood meal sources of Anopheles gambiae Giles through deoxyribonucleic acid (DNA) profiling was investigated by polymerase chain reaction (PCR) amplification at the TC-11 and VWA human short tandem repeats (STR) loci. Blood meal size and locus had no significant effect on the success of amplifying human DNA from blood meals digested for 0, 8, 16, 24 and 32 h (P = 0.85 and 0.26 respectively). However, logistic regression found a significant negative relationship between time since ingestion and the success probability of obtaining positive PCR products among meals digested for between 8 and 32 h (P = 0.001). Approximately 80 f fresh blood meals were successfully profiled. After 8 h, the proportion of blood meals that could be successfully profiled decreased slowly with time after ingestion, dropping to below 50 fter approximately 15 h. There was no significant difference in the success of amplifying human DNA from blood meals of mosquitoes killed at time 0 and 8 h after ingestion (P = 0.272)

Evaluation of low density polyethylene and nylon for delivery of synthetic mosquito attractants.

BACKGROUND: Synthetic odour baits present an unexploited potential for sampling, surveillance and control of malaria and other mosquito vectors. However, application of such baits is impeded by the unavailability of robust odour delivery devices that perform reliably under field conditions. In the present study the suitability of low density polyethylene (LDPE) and nylon strips for dispensing synthetic attractants of host-seeking Anopheles gambiae mosquitoes was evaluated. METHODS: Baseline experiments assessed the numbers of An. gambiae mosquitoes caught in response to low density polyethylene (LDPE) sachets filled with attractants, attractant-treated nylon strips, control LDPE sachets, and control nylon strips placed in separate MM-X traps. Residual attraction of An. gambiae to attractant-treated nylon strips was determined subsequently. The effects of sheet thickness and surface area on numbers of mosquitoes caught in MM-X traps containing the synthetic kairomone blend dispensed from LDPE sachets and nylon strips were also evaluated. Various treatments were tested through randomized 4¿×¿4 Latin Square experimental designs under semi-field conditions in western Kenya. RESULTS: Attractant-treated nylon strips collected 5.6 times more An. gambiae mosquitoes than LDPE sachets filled with the same attractants. The attractant-impregnated nylon strips were consistently more attractive (76.95%; n¿=¿9,120) than sachets containing the same attractants (18.59%; n¿=¿2,203), control nylon strips (2.17%; n¿=¿257) and control LDPE sachets (2.29%; n¿=¿271) up to 40¿days post-treatment (P

Spatial heterogeneity of malaria vectors and malaria transmission risk estimated using odour-baited mosquito traps

Background Prior to the commencement of a large-scale malaria intervention study on Rusinga Island, western Kenya, intensive baseline surveillance of the mosquito population was performed using odour-baited traps. The survey aimed to determine the relative abundance and species composition of malaria vectors, and to measure seasonal and spatial heterogeneity in populations. Human malaria prevalence was combined with entomological data to provide information about malaria transmission risk before the intervention began. Materials and methods From September 2012 until June 2013, mosquito monitoring took place over successive six-week sampling periods. MM-X traps baited with attractant lures and carbon dioxide were used to collect mosquitoes from inside and outside houses, and a new random sample of 80 households was drawn for each sampling round. During the baseline period, malaria prevalence was measured twice in a randomly selected 10% of the human population. A QuickBird satellite image and digital elevation map were used to describe environmental features of the island. Mosquitoes were initially identified on the basis of morphology and anophelines were processed by PCR to confirm species identifications. Results Odour-baited MM-X traps proved to be a good tool for monitoring malaria vectors inside and outside houses. Using this tool a marked temporal and spatial heterogeneity was described for the malaria vector species Anopheles gambiae s.s., An. arabiensis and An. funestus. Regions of potentially high malaria transmission intensity were identified after mapping the distributions of malaria mosquitoes and Plasmodium-positive persons. Despite studying a range of environmental and topographical features, no strong associations were found between environmental variables and the presence or absence of adult Anopheles. Conclusions Malaria vectors and malaria prevalence are not homogeneously distributed across Rusinga Island; the risk of malaria transmission is therefore greater in some areas than others. The finding that environmental features were not closely associated with adult malaria vector distribution, indicates that other factors, such as house construction or the presence of livestock, may play a more important role in the decision of a female anopheline to approach the domestic environment of a particular house in search of a blood meal. The findings of this study demonstrate how trans-disciplinary data can be integrated to provide a better understanding of mosquito population dynamics and malaria transmission risk. Intensive mosquito monitoring before the commencement of, as well as during, a large-scale malaria intervention study, contributes valuable information which will be used in describing the eventual impact of the intervention

The effect of essential oils of tagetes minuta and tithonia diversifolia on on-host behaviour of the brown ear tick rhipicephalus appendiculatus

On-host behaviour of Rhipicephalus appendiculatus was studied in the field in Bungoma County in Kenya to evaluate the putative repellent effects of essential oils of Tagetes minuta and Tithonia diversifolia at its predilection feeding site. Oils of both plants caused a disruption of orientation, movement and attachment behaviour of ticks. More ticks dropped off in the treatments with the two essential oils than with the control. Treating the ear pinna with the essential oil of T. minuta caused the highest percentage of ticks to drop off the host body. No tick reached the ear pinna treated with the essential oil of T. minuta and up to 30% of ticks (from the forehead release site) reached the ear base. When the ear pinna was treated with the essential oil of T. diversifolia, one tick reached the ear pinna and up to 40% of ticks (from the dewlap release site) reached the ear base. The results show that T. minuta repels ticks more strongly than T. diversifolia. However, both essential oils offer possibilities for exploitation of potentially effective and environmentally acceptable tools for on-host tick control.</p

Human skin microbiota and their volatiles as odour baits for the malaria mosquito Anopheles gambiae s.s

Host seeking by the malaria mosquito Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) is mainly guided by volatile chemicals present in human odours. The skin microbiota plays an important role in the production of these volatiles, and skin bacteria grown on agar plates attract An. gambiae s.s. in the laboratory. In this study, the attractiveness of volatiles produced by human skin bacteria to An. gambiae s.s. was tested in laboratory, semi-field, and field experiments to assess these effects in increasing environmental complexity. A synthetic blend of 10 compounds identified in the headspace of skin bacteria was also tested for its attractiveness. Carbon dioxide significantly increased mosquito catches of traps baited with microbial volatiles in the semi-field experiments and was therefore added to the field traps. Traps baited with skin bacteria caught significantly more An. gambiae s.s. than control traps, both in the laboratory and semi-field experiments. Traps baited with the synthetic blend caught more mosquitoes than control traps in the laboratory experiments, but not in the semi-field experiments. Although bacterial volatiles increased mosquito catches in the field study, trapping several mosquito vector species, these effects were not significant for An. gambiae s.l. It is concluded that volatiles from skin bacteria affect mosquito behaviour under laboratory and semi-field conditions and, after fine tuning, have the potential to be developed as odour baits for mosquitoes