The Dengue vector Aedes aegypti: Microbiota and the Chemical Ecology of Oviposition sites

Lead Research Organisation: University of Greenwich
Department Name: Natural Resources Institute, FES

Abstract

Dengue is the most important arbovirus; there are an estimated 2.5 billion people in over 100 countries living at risk of infection. There are an estimated 100 million new cases of the disease each year, and 20 000 deaths. In Brazil, annually, there are more than 800,000 cases and over 200 deaths. There are a number of different vectors of Dengue, but in Brazil, the virus is largely transmitted by the Aedes aegypti mosquito. Aedes aegypti is commonly found in urban areas, where it has adapted superbly well and is able to reproduce in a wide range of man-made containers, such as tyres, cups, tin cans and plant pots. Controlling vector mosquitoes is a key way of limiting the transmission of vector-borne diseases such as Dengue, and to do this it is important to have accurate data on the distribution and dispersal activity levels of the mosquito species in different environments, and to understand the factors which influence the reproductive success of the species.
All animals pick up micro-organisms from their environment. It is well known that mosquitoes acquire their microbiota from plants as well as larval breeding sites. This microbiota is not only important to the mosquito physiology (e.g., bacteria provide nutrients to the insect) but it also affects how the mosquito responds to pathogens (becoming more susceptible or resistant to the infection). Micro-organisms can be particularly important for mosquitoes, because they are filter feeders as larvae and the make-up of the micro-organisms in their breeding water has a significant effect on the quality of food the larvae have access to. Thus, the microbial make-up of the water in which the females lay their eggs is ultimately crucial to their survival. Here we will study how the microbiome environment influences mosquito breeding capacity and how we can manipulate these associations to provide novel means of controlling this vector of deadly diseases such as dengue, yellow fever, chikungunya and zika.
In addition, analysis of the micro-organisms picked up by individual mosquitoes can indicate where it has been. In this study, we aim to use the identity of micro-organisms found on individual mosquitoes to track their movement through the environment. Knowing how far a mosquito moves can be critical for modelling/predicting the range and rate of spread of a vector borne disease.
Finally, if we can identify the odours that draw a female to an egg-laying site, we can synthesize the odour and use it as a bait for trapping systems to monitor mosquito populations, or to kill females whilst they attempt to locate egg-laying sites. These control tools could contribute to an integrated control strategy for the vector, combining traps for the adult females with other measures aimed at controlling the immature stages to reduce populations of vectors, and thus the spread of diseases.
Dengue is imposing a heavy burden on both global health and economics. A better understanding of the transmission of Dengue by Ae. aegypti can contribute to reducing the incidence of a disease that takes a heavy toll in densely populated, often poor urban environments.

Technical Summary

Estimates of population boundaries and mosquito dispersal have previously required mark-release-recapture experiments, where thousands of mosquitoes are radioactively labelled or marked with fluorescent dye before release. Often only a handful of mosquitoes are recaptured and this technique is plagued by a plethora of technical problems, and massive releases of vectors of disease bring ethical issues, often producing poor data. In contrast, from preliminary data obtained in a study of malaria vectors in Burkina Faso, it has been demonstrated that we can clearly assess the spatial dynamics in population distributions of An. gambiae mosquitoes. On the basis of the bacterial flora obtained from 30 mosquitoes from 3 villages within flight distance (2 km) we found clear patterns of movement of individuals in local populations within a meta-population network. We now want to expand and verify the technology developed in one country by performing a follow-up study in another country. Knowing the movement patterns of vectors will provide new knowledge which better allows control measures to be focused and applied.
The development of ovitraps has been an area of interest in the control of malaria vector mosquitoes. The techniques of headspace collection and the use of odour dispensers are well developed, particularly within NRI labs. The tight spatial arrangement of hosts, breeding sites and resting sites for Dengue vectors presents a different scenario to the pattern for Anopheles species, which move through much larger areas. It is likely that the patterns of movement of Ae aegypti may be even more amenable to control than An. gambiae. Also, the successful application of techniques to identify malaria mosquito oviposition stimuli to Dengue vectors is highly attractive, and the urban setting of these species makes ovitraps a realistic tool to deploy within integrated vector control systems. These are techniques that are mature and are ripe for wider application.

Planned Impact

The intended identification of key microbiota from oviposition sites will allow the manipulation of female Ae. aegypti behaviour in order to recruit gravid females into traps or to expose them to xenobiotics. Consequently, an impact is expected fundamentally in terms of allowing the development of biotechnological tools to promote a reduction in the population of mosquitoes that transmit human disease. Besides, understanding the structure of mosquito populations (and the dispersion patterns) would help the prevention of dengue outbreaks. In principle, the initial beneficiaries of the output of these studies would be public sector agencies responsible for dengue control in endemic countries. The commercial private sector could benefit from the research output because the information gathered would represent the basis for future technologies mentioned above. Therefore, if research goals are met it would be feasible to decrease the number of dengue cases through innovative vector detection and control interventions. From such a perspective, the population would be the final beneficiary of these improvements.
Considering the public sector agencies, these advances would allow them to create programmes based on dispersion detection and the use of biotechnological interventions with a focus on breeding site manipulation. To date, they have failed in eliminating the sites where female mosquitoes lay their eggs in urban landscapes. Instead, the new approach would target this critical mosquito behaviour, intending to manipulate it to control females. In case attractive or repellent compounds or bacteria were successfully identified, they could be exploited to make tools available to the market, and at that stage, private partners would be needed to develop them. This could be initially performed in collaboration with the scientific team.
Dengue represents a heavy burden to Brazilian health programmes and therefore, it impacts directly on the economy of the country. As well, this can be asserted for most countries suffering epidemics of this viral disease. It seems evident that a significant decrease in the number of disease cases would directly impact the economy, promoting savings that could be redirected to other relevant needs. This is especially true when the focus is given to the public sector, and specifically the health agencies responsible for mosquito control, disease diagnosis and treatment. For these improvements to take place a time frame longer than the three years of research would be necessary. Linking this work to the Eliminate Dengue Program and collaboration with the communications team of the Oswaldo Cruz Foundation, will ensure that the results of this study reach the existing programmes for Dengue and translate results and research progress to the organs that can work more directly for the benefit of the public.

Publications

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Description Field cages
Work performed in field cages in Brazil indicated that water dishes containing pot plants were a much more highly utilized oviposition site by Ae. aegypti than any of the other common options (car tyres, drains, bottles or buckets). This was found to be the case even when containers were corrected for volume. Further work was indicative that the water washed through from the plant soil was a strong influence on this and that this was the case even in the absence of the plant itself. The importance of the work is that it demonstrates that whilst the items of urban rubbish that are commonly named may be attractive for the mosquitoes, plants which have been placed to improve home environments may be providing highly attractive egg laying sites.

Understanding egg laying strategy
Work has been performed to separate the key behaviours of number of times oviposition occurred in a water type and the number of eggs laid per ovipositing instance. There was no difference in the number of eggs laid per visit in deionised water or plant water, indicating that larger numbers of eggs were accrued through an increased number of visits to the egg laying sites.
Ae. aegypti are commonly stated to oviposit just above the water line inside of a container, however, they have been observed to oviposit on the water's surface. When Ae. aegypti were presented with deionised water a higher proportion of eggs were placed on the water surface (rather than on the pot wall) compared to plant water, or when conspecific eggs were already on the water surface. This aligns with our hypothesis that when low nutritional content is present in the water, that the mosquito will "seed" the water by ovipositing more eggs on the water surface introducing bacteria in the process which will then multiply to provide food for the growing mosquito larvae.

Larval survival:
Results of bioassays indicate that although water from plant pots was preferred by adults, it was not a good media for larval growth. Experiments also indicated that spreading out the eggs over time in survival assays did not appear to affect the survival of mosquito larvae but could have an effect on the size of the adult that ultimately emerged. This can be of great importance for understanding the population dynamics of Ae. Aegypti.

Spatial arrangement of egg laying sites
Results indicate that both the distance between sites and the spatial arrangement of highly accepted and less accepted egg laying sites can be critical to the distribution of eggs by Ae. aegypti. Indications are that the more closely related the sites are to each other the more it reinforced the usage of those preferred sites. 3D tracking data currently being analysed to indicate the patterns in which the insects used the individual egg laying sites the sites.

Microbiota of the water
The microbiota of the water has been partially investigated from the field in Brazil. Samples were collected as planned and DNA extraction protocols tested. The combined effect of the economic crisis in Brazil leading to funds issues and a key staff member leaving FIOCRUZ has led to this work stopping at this stage.
Exploitation Route Knowledge of the role of plant pots in Aedes reproductive can strongly inform strategies to manage reproductive sites of this key vector.
The insights into the roles of the pots can inform trap design and the development of push-pull systems to impact the reproduction of Aedes in urban environments.
Sectors Communities and Social Services/Policy,Environment,Leisure Activities, including Sports, Recreation and Tourism

 
Description UA-DTA Biomedical, PhD scheme
Amount £50,000 (GBP)
Organisation University of Greenwich 
Sector Academic/University
Country United Kingdom
Start 10/2016 
End 10/2019
 
Description Fiocruz 
Organisation Oswaldo Cruz Foundation (Fiocruz)
Country Brazil 
Sector Public 
PI Contribution This grant has led to the creation of a new partnership between The Oswaldo Cruz Foundation, Brazil and the Natural Resource Institute, University of Greenwich. The primary input from NRI is in Insect Behaviour and Insect Chemical Ecology. NRI provides expertise and facilities for these, and has established a colony of Aedes aegypti from Brazil which allow us to perform work on the same broad biological material as the wild material from Brazil. In addition, we are undertaking chemical analyses of material collected in Brazil, and will proceed to bioassay and GC-EAG investigation of chemical from water in Brazil.
Collaborator Contribution The partners in Brazil provide expertise in the ecology of Aedes aegypti, and have assisted NRI in establishing a colony based on local material. Fiocruz, in addition to the above, has given access to a large size field cage where we have conducted investigations of oviposition site selection in a collaboratively designed experiment. Collaborators in Brazil are performing analyses of the micriobial composition of water collected in Brazil, in parallel to the chemical analyses being performed at NRI.
Impact This is a year 1 report, and the data produced from the first component is currently complete and undergoing statistical analyses. Chemical and microbial investigations are in process.
Start Year 2016