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

Design of trials for interrupting the transmission of endemic pathogens

Many interventions against infectious diseases have geographically diffuse effects. This leads to contamination between arms in cluster-randomized trials (CRTs). Pathogen elimination is the goal of many intervention programs against infectious agents, but contamination means that standard CRT designs and analyses do not provide inferences about the potential of interventions to interrupt pathogen transmission at maximum scale-up.; A generic model of disease transmission was used to simulate infections in stepped wedge cluster-randomized trials (SWCRTs) of a transmission-reducing intervention, where the intervention has spatially diffuse effects. Simulations of such trials were then used to examine the potential of such designs for providing generalizable causal inferences about the impact of such interventions, including measurements of the contamination effects. The simulations were applied to the geography of Rusinga Island, Lake Victoria, Kenya, the site of the SolarMal trial on the use of odor-baited mosquito traps to eliminate Plasmodium falciparum malaria. These were used to compare variants in the proposed SWCRT designs for the SolarMal trial.; Measures of contamination effects were found that could be assessed in the simulated trials. Inspired by analyses of trials of insecticide-treated nets against malaria when applied to the geography of the SolarMal trial, these measures were found to be robust to different variants of SWCRT design. Analyses of the likely extent of contamination effects supported the choice of cluster size for the trial.; The SWCRT is an appropriate design for trials that assess the feasibility of local elimination of a pathogen. The effects of incomplete coverage can be estimated by analyzing the extent of contamination between arms in such trials, and the estimates also support inferences about causality. The SolarMal example illustrates how generic transmission models incorporating spatial smoothing can be used to simulate such trials for a power calculation and optimization of cluster size and randomization strategies. The approach is applicable to a range of infectious diseases transmitted via environmental reservoirs or via arthropod vectors

Numerical simulation of the influence of the large-scale monsoon flow on the diurnal weather patterns over Kenya

August 1992.Bibliography: pages 204-211.Sponsored by NSF ATM-8915265

Evaluation of textile substrates for dispensing synthetic attractants for malaria mosquitoes

Background: The full-scale impact of odour-baited technology on the surveillance, sampling and control of vectors of infectious diseases is partly limited by the lack of methods for the efficient and sustainable dispensing of attractants. In this study we investigated whether locally-available and commonly used textiles are efficient substrates for the release of synthetic odorant blends attracting malaria mosquitoes. Methods: The relative efficacy of (a) polyester, (b) cotton, (c) cellulose + polyacrylate, and (d) nylon textiles as substrates for dispensing a synthetic odour blend (Ifakara blend 1(IB1)) that attracts malaria mosquitoes was evaluated in western Kenya. The study was conducted through completely randomized Latin square experimental designs under semi-field and field conditions. Results: Traps charged with IB1-impregnated polyester, cotton and cellulose + polyacrylate materials caught significantly more female Anopheles gambiae sensu stricto (semi-field conditions) and An. gambiae sensu lato (field conditions) mosquitoes than IB1-treated nylon (P = 0.001). The IB1-impregnated cellulose + polyacrylate material was the most attractive to female An. funestus mosquitoes compared to all other dispensing textile substrates (P < 0.001). The responses of female An. funestus mosquitoes to IB1-treated cotton and polyester were equal (P = 0.45). Significantly more female Culex mosquitoes were attracted to IB1-treated cotton than to the other treatments (P < 0.001). Whereas IB1-impregnated cotton and cellulose + polyacrylate material attracted equal numbers of female Mansonia mosquitoes (P = 0.44), the catches due to these two substrates were significantly higher than those associated with the other substrates (P < 0.001). Conclusion: The number and species of mosquitoes attracted to a synthetic odour blend is influenced by the type of odour-dispensing material used. Thus, surveillance and intervention programmes for malaria and other mosquito vectors using attractive odour baits should select an odour-release material that optimizes the odour blend

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

The Silver Cyprinid Rastrineobola Argentea as the main diet source for rearing Anopheles arabiensis mosquitoes

Background & objectives: All organisms that are cultured for laboratory bioassays need food for sustenance and amplification of the colony to such numbers that would not compromise the progress of the research at hand. For effective turnover rate in the population generation, the diet should be such that it is readily available and provide adequate nutrients to the organisms. The aim of this study was to test and evaluate the efficacy of Rastrineobola argentea as a diet source for rearing Anopheles arabiensis Patton mosquitoes for research. Methods: Crushed fish, Rastrineobola argentea and two other commercial larval feeds (Tetramin Baby fish food and baker’s yeast) were compared for their efficacy as diet sources for feeding Anopheles arabiensis mosquito larvae. The effects of these diet types were determined against fecundity, size, longevity and male mating competitiveness. Results: Mosquitoes raised on Tetramin Baby fish food laid larger egg batches (66.45 ± 5.03 mm) as compared to crushed fish (64.86 ± 4.93 mm) or bakers’ yeast (50.49 ± 4.25 mm). However, the number of eggs laid by the mosquitoes irrespective of the diet type did no differ significantly (p < 0.132). Mosquitoes raised on Tetramin Baby fish food were larger in size (3.06 ± 0.02 mm) as compared to those raised on crushed R. argentea (2.93 ± 0.01 mm) or baker’s yeast (3.00 ± 0.02 mm). The choice of diet was found to influence both mosquito size (p < 0.001) and fecundity (p < 0.013). Conclusion: This study found that crushed R. argentea is an effective mosquito larval diet and that it has the potential of being more effective than tetramin if refined further and supplements added. Keywords Rastrineobola argentea; Anopheles arabiensis; Fecundity; Longevity; Eclosio

Trapping of the malaria vector Anopheles gambiae with odour-baited MM-X traps in semi-field conditions in western Kenya

BACKGROUND: The successful development of odour-baited trapping systems for mosquitoes depends on the identification of behaviourally active semiochemicals, besides the design and operating principles of such devices. A large variety of 'attractants' has been identified in laboratory investigations, yet few of these increase trap catches in the field. A contained system, intermediate between the laboratory and open field, is presented and previous reports that human foot odour induces behavioural responses of Anopheles gambiae confirmed. METHODS: The response of 3–5 day old female An. gambiae towards odour-baited counterflow geometry traps (MM-X model; American Biophysics Corp., RI) was studied in semi-field (screen house) conditions in western Kenya. Traps were baited with human foot odour (collected on socks), carbon dioxide (CO(2), 500 ml min(-1)), ammonia (NH(3)), 1-octen-3-ol, or various combinations thereof. Trap catches were log (x+1) transformed and subjected to Latin square analysis of variance procedures. RESULTS: Apart from 1-octen-3-ol, all odour baits caused significant (P < 0.05) increases in trap catches over non-baited traps. Foot odour remained behaviourally active for at least 8 days after collection on nylon or cotton sock fabric. A synergistic response (P < 0.001) was observed towards the combination of foot odour and CO(2), which increased catches of these odours alone by 3.8 and 2.7 times, respectively. CONCLUSION: These results are the first to report behavioural responses of an African malaria vector to human foot odour outside the laboratory, and further investigation of fractions and/or individual chemical components of this odour complex are called for. Semi-field systems offer the prospect of high-throughput screening of candidate kairomones, which may expedite the development of efficient trap-bait systems for this and other African mosquito species

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

Recent successful genetic transformation of disease-transmitting insects has fuelled enthusiasm towards its potential application for disease control in the future. However, advances to date have been confined to laboratory settings and many questions relating to the fitness, behaviour, ecology and phenotypic characteristics of transformed insects remain unanswered. Spread of desired traits, such as refractoriness to Plasmodium infection, will depend on the reproductive fitness and manifestation of life-history behaviours, such as dispersal and mating, by engineered specimens. These should preferably be similar to those displayed by their wild conspecifics but may be compromised by genetic modification and difficult to assess realistically under standard laboratory conditions. Contained semi-field environments that mimic a near-natural environment and are exposed to ambient climatic conditions may serve to verify laboratory findings and yield valuable insights into transgene fixation processes prior to field releases of transgenic specimens into the wild. Here we describe the constraints and benefits of this approach with respect to containment stringency, facility design and operational guidelines for studies involving genetically-engineered malaria vectors. We also report on our initial success with such semi-field systems in West Kenya, using non-transgenic mosquitoes in a variety of behavioural and ecological studies. Successful completion of the Anopheles gambiae life cycle, and thus expression of all major life-history behaviours, occurred in three separate trials. However, our results show that the sustenance of successive and overlapping generations in such systems may be difficult. Considering the frequently expressed and explicit need for contained semi-field trials with engineered insects prior to field releases, this calls for intensified development of improved semifield systems, preferably in field sites earmarked for future release