Exploring links between mosquitoes, the environment, and disease transmission

The Asian tiger mosquito, Aedes albopictus is one of the most highly invasive mosquito species seen to date.  The physiological and ecological plasticity of Ae. albopictus has led to its rapid global expansion.  Additionally, its ability to vector a wide-range of recently emerging arboviruses, such as dengue and Chikungunya, make it a significant public health threat.  The transmission of many mosquito-borne pathogens is strongly influenced by environmental temperature due to effects on the physiology of the insect vector and the pathogen.  Therefore, changes in local environmental conditions could significantly impact the distributions and dynamics of a range of mosquito-borne diseases.  Predicting the extent of possible changes in disease dynamics will require a detailed understanding of how a suite of mosquito-pathogen traits respond to variation in environmental temperature and other biotic factors. Projects can explore the following potential questions: 1) what are the microclimate conditions mosquitoes experience in the larval environment and relevant transmission settings?, 2) how does thermal variation influence mosquito life history traits relevant for transmission (e.g. larval development rates, larval survival, adult longevity)?, 3) can we use remotely sensed data to predict relevant mosquito microclimate?, or 4) what factors contribute to Ae. albopictus oviposition behavior and density-dependence in the larval environment. We are looking for two REU students that are interested in combining field work with computational approaches to carry out projects in the Athen’s system this summer mentored by Drs. Courtney Murdock (Infectious Diseases & Odum School of Ecology) and Craig Osenberg (Odum School of Ecology).

Host Laboratories: Courtney Murdock and Craig Osenberg
Type of Project: Combination of Empirical/Field-based, and Computational/Computer-based

Declining water quality associated with failing water infrastructure

Globally, failing water infrastructure has been linked to declining water quality and increased exposure to contaminants, and potentially harmful bacteria infections including, but not limited to Escherichia coli. To assess temporal and spatial changes in the chemical and bacterial composition of water associated with failing water infrastructure in watersheds in Atlanta, members of the Capps Lab will conduct a field- and lab-based empirical study. Field activities will be conducted in Atlanta, GA in stream reaches that have been sampled sporadically since the 1970’s. In conjunction with the Principal Investigator and a UGA-based graduate student, the participating student will be trained to collect and analyze water quality samples to begin relating in-stream environmental conditions with water infrastructure. Results will be applied to ongoing and future research projects in the Capps Lab. Applicants should have some previous experience conducting field research in aquatic ecology and an interest in learning techniques in analytical chemistry. Successful candidates will be expected to work effectively individually in the lab and within a team environment in urban environments in the southeast during the summer. Though hiking will be limited, in order to access the field sites, candidates should be able to walk/wade through streams for at least 1 mile while carrying up to 30lbs.

Host laboratory: Krista Capps
Type of project: Empirical/ Field and laboratory-based

Do parasites impair the fight-or-flight reactions of their hosts? Experimental investigations of beetles infected with nematodes

Temporary stressors are a part of life in the animal kingdom, whether they be encounters with predators, transient anthropogenic disturbances or severe weather events. All animals must therefore be capable of dealing with such stressors to ensure their survival, which is the ‘fight-or-flight reaction’. A number of recent research studies, across a range of animal taxa, have found that certain parasites can affect how their hosts deal with these stressors.

In the Davis lab, students have recently conducted a variety of experiments using a common beetle species, the horned passalus (pictured), which is host to a nematode called Chondronema passali. Recent work has shown parasitized beetles have reduced physical strength, cannot fight as well as non-parasitized individuals, and importantly, their stress reactions appear to be affected.

In summer 2019, a project is planned where an REU student will conduct a series of benchtop lab experiments that will all attempt to identify how parasites influence the ability of their hosts to deal with an acute stressor. This will involve field-collection of beetles (from the surrounding area), bringing them to the lab and performing behavioral experiments with them over the summer. The experiments will primarily focus on monitoring changes in stress levels of beetles before and after application of non-lethal stressors.

The ideal student for these projects will be someone who is comfortable handling insects and performing icky dissections, and who can work well in the tick- and chigger-infected field (forest habitats).

Host laboratory: Andy Davis
Type of project: Empirical/Laboratory-based

Intracellular stage of Bordetella spp.: A path to escape immune recognition

Data published by our group and others, show Bordetella pertussis, B. bronchiseptica and B. parapertussis, can survive and grow intracellular in macrophages and lung epithelial cells. These two aspects have great clinical implications. Intracellular survival could explain vaccine failure (evasion of immune recognition) as well as the long persistence period of clinical disease (disease is reported in adolescent and young adults). This project involves an exceptional opportunity to study all the molecular basis and mechanisms involved in the intracellular survival and replication of Bordetella spp. that can be responsible of the re-emergence, dissemination, lethargy periods, evasion of the immune system, vaccine failure and even transmission.

To understand intracellular survival/growth of Bordetella spp., we propose the following aims:
Aim 1: Identify genes required for specific stages of intracellular survival and replication (intracellular stage).

We will create and screen a transposon library to identify all genes required for survival and replication within macrophages.
Student role: The student will do the transposon library for Bordetella pertussis. Also, the student role in this aim it will be to screen the transposon library of Bordetella bronchiseptica and Bordetella pertussis, using the gentamicin assay and variations of it in order to identify mutations that promote survival / growth within mcarophages.

Aim 2: Define the mechanisms involved during in vitro Bordetella spp. intracellular survival and replication.

Intracellular survival and replication requires to perform several steps; a) adhere, b) invade, c) survive intracellularly, d) obtain nutrients to replicate, and e) avoid killing the host cell (i.e. inducing its apoptosis or targeting it for killing by immune cells). In this specific aim we will examine which of these aspects each mutation affects.
Student role: The student will do immunofluorescence and western blot in order to identify the localization of Bordetella spp. within macrophages, in order to determine if Bordetella can growth within macrophages and to reveal how many of the Bordetella that are engulf are death or alive within macrophages  (this techniques will require microscopy and/or flow cytometer). The student also will perform and develop western blot techniques that will identify the mechanism that Bordetella uses in order to kill macrophages.

Understanding how Bordetella species survive and grow within host cells is likely to shed light on the current problem of the high rates of vaccine failure as well as its re-emergence, as current vaccines do not induce Th1 immunity and are not effective against intracellular bacteria. This work will also reveal key genes contributing to intracellular growth/survival that can provide targets for the next generation of vaccines and therapeutics.

Host Lab: Eric Harvill
Type of project: Empirical/Laboratory-based\

Temperature impacts on an insect-parasite interaction

For insects, environmental temperature can influence their physiology, survival, activity patterns, and large-scale distribution. Shifts in temperature will have pervasive effects, not just on individual insect species, but also on their interactions with other organisms, notably, their parasites. Given that insects are important pollinators and vectors of disease, it is vital that we explore how temperature impacts insect-parasite interactions. Monarch butterflies are parasitized by a protozoan parasite, Ophryocystis elektroscirrha (OE), that can have negative effects on the insect’s survival, reproduction, and flight ability. Their iconic migration exposes the butterflies to a range of environmental temperatures over the course of several generations, making the monarch-OE system quite suitable for investigating how temperature impacts host susceptibility and parasite virulence. This project, aimed at quantifying elements of host and parasite fitness at different temperatures, will include controlled lab experiments and the development of a mathematical model of infection. The student will participate in experimental data collection/analysis, model design, and model parameterization. This project is suitable for students with interests in infectious disease ecology and conservation hoping to integrate experimental and modeling techniques.

Host laboratory: Sonia Altizer & Richard Hall; co-mentored by Isabella Ragonese (PhD student)
Type of project: A combination of Quantitative (computer-based) and Empirical (lab-based)

Copepod survival in water bowls exposed to typical Chadian ambient temperatures

Zavier Eure, a junior from North Carolina A&T, worked with the lab of Dr. Michael Yabsley to study copepod survival in different types of water bowls.

Abstract:  Guinea Worm Disease (GWD), caused by the nematode Dracunculus medinensis, has been detected with increasing incidence among dogs in Chad, Africa. Cyclopoid copepods (freshwater crustaceans) are intermediate hosts for D. medinensis. Currently the route(s) of D. medinensis transmission to dogs is still unknown but drinking from unprotected water sources would pose a risk. Dogs have access to water dishes provided for domestic animals and depending on the source of water, these dishes could harbor infected copepods, thereby acting as a source of transmission. To determine how long copepods survive in water dishes when exposed to Chadian ambient temperatures (41.1ᵒC), copepods were placed in three different container types (plastic, glass, and metal) and heated to 40ᵒC. Our results indicate that under simulated Chadian temperatures, metal dishes result in the highest rate of copepods death in the shortest period of time (2hrs) and were the only container to reach 100% copepod mortality. Conversely, plastic dishes exhibited the lowest mortality of copepods. These results indicate that the type of dish used when supplying water for animals in Chad is an important consideration in terms of preventing or interrupting transmission of D. medinensis among dogs.



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Using long-term science data to examine relationships of wastewater infrastructure and water quality

Sarah Williamson, a junior from Baylor University, worked with Denzell Cross in the lab of Dr. Krista Capps.  They used community-collected data to examine spatial and temporal changes in water quality in a local watershed.

Abstract:  The integrity of freshwater systems throughout the world is threatened by increasing concentrations of gut bacteria, such as E. coli. In urban watersheds, increases in bacterial concentrations are associated with intentional municipal wastewater discharge and aging and obsolete wastewater infrastructure (e.g., sewage leaks and failing septic systems). Tracing sources of bacterial contamination in surface waters is often difficult due to the need for long-term monitoring. Moreover, long-term monitoring can be time intensive and costly.
Community science organizations can be a cost-effective way to collect large amounts of environmental data across broad spatial and temporal scales. However, data collected by community scientists are often criticized due to concerns about scientific rigor, data fragmentation, and inaccuracy. Other work has demonstrated that groups employing robust protocols can produce data that align with data collected by professionals, and can be used reliably in decision-making processes pertaining to environmental health and watershed management. The purpose of this study was to examine spatial and temporal changes in water quality using data collected by community scientists of the Upper Oconee Watershed Network, and use community data to investigate relationships between water quality metrics and wastewater infrastructure in Athens-Clarke County (ACC). Specifically, we wanted to ask if water quality metrics (i.e., conductivity and turbidity) correlated to fecal bacteria concentrations in ACC surface waters and if there are relationships between site- and watershed-specific concentrations of E. coli and the proximity and density of wastewater infrastructure. We found that turbidity may be a strong predictor in bacteria concentrations however, the relationship between bacteria and conductivity was not clear. Our data also suggests that there may be important links between wastewater infrastructure and reduced water quality.

What’s the buzz? An investigation on how urbanization impacts mosquito species distribution

Lilith South, a junior from the University of Georgia, worked with Mike Newberry in the lab of Dr. Courtney Murdock to study the relationship between urbanization and distribution of mosquito species.

Abstract:   Impervious surfaces, mainly paved roads and buildings, significantly impact microclimate by making an area hotter and less humid. For this reason, urban areas are warmer than less developed rural areas. Heat associated with high impervious surface coverage impacts mosquito development and decreases larval survival in Aedes albopictus. Although many species of mosquitoes are present in Athens, Georgia, the most prominent species and most important species for human health, Ae. albopictus, is one of few species that dominate the area. Ae. albopictus has shown potential vectoral capacity for diseases such as Zika, Dengue, and Chikungunya. Fortunately, it does not yet transmit these diseases in the south eastern United States, but with changing climate and urbanization these diseases have potential to spread. The impact that impervious surface coverage has on mosquito community composition was not previously known. To investigate this effect, sites were classified and selected by their impervious surface coverage. Rural sites had impervious surface coverage ranging from 0-5%, suburban had 5-55%, and urban had 55-100% coverage.  Larval samples from each site were identified to species and the proportion of occupied habitats for each species in each site was noted. Overall, species richness decreased in suburban and urban areas with higher impervious surface coverage. Diversity was highest and there was a more even spread of species in rural areas. Contrary to what was expected, the percentage of Ae. albopictus occupied habitats did not significantly change with impervious surface coverage. Although previous studies suggest that Ae. albopictus is sensitive to hotter urban areas, this species may be more resilient than other mosquito species to the effects of urbanization. Knowing how urbanization impacts mosquito community composition can help researchers better understand disease transmittance and develop solutions for potential viral outbreaks.

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How does the proportion of sugar fed Aedes albopictus mosquitoes vary across land use?

Alyssa Slicko, a junior from the University of Arkansas Little Rock, worked with Nikki Solano in the lab of Dr. Courtney Murdock to look at sugar-feeding and its relationship to land use in an invasive mosquito.

Abstract:  The Asian tiger mosquito, Aedes albopictus, is a non-native species to North America and is known to be highly invasive with an ability to vector up to 27 different arboviruses. Since female mosquitoes feed on both sugar and blood to survive, understanding the tendencies for sugar feeding could explain the differences in the abundance of invasive mosquito populations. Past studies have shown that temperature plays an important role in the distribution of vector borne diseases, but it has not been discovered whether other environmental factors such as sugar availability is a limiting resource for mosquito populations. Some species have evolutionarily adapted to low sugar resources, meaning they primarily feed on blood. However, little is known about the sugar feeding habits of Aedes albopictus. We collected A. albopictus from nine field sites classified as suburban, urban and rural based on percentage of impervious surface. A backpack aspirator was used to collect mosquitoes that were then frozen and identified by sex and species. A total of 90 female A. albopictus mosquitoes were collected, 30 from each land use type. Using homogenized solution of each individual mosquito, colorimetric sugar assays were performed with serial dilutions to determine relative sugar content per mosquito. The absorbance values of these solution were read through a spectrophotometer. At the 1:4 dilution values, urban sites have the greatest overall amount of sugar followed by rural and suburban land uses. There is evidence that mosquitoes in Aedes albopictus females do sugar-feed and that there are differences between sugar contents across land use types. However, a negative relationship was found between absorbance and concentration values across sites. This could be due to a potential chemical inhibitor formed with highly concentrated mosquito dilutions not allowing complete reading of absorbance values and determination of sugar content.

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The world’s smallest escape artists: manipulation of the host innate immune response by Bordatella bronchiseptica

Margaret Dedloff, a junior at Clarkson University, worked with Dr. Monical Gestal in the lab of Dr. Eric Harvill to study the role of a sigma factor by bacteria in manipulating host immune response.

Abstract:  During the course of infection, some pathogens are able to sense and respond to the host immune system. This can cause prolonged pathogenesis, high transmission rates, and vaccine failure. One group of bacteria known to manipulate the immune response in varied ways is the Bordetella spp. B. pertussis, B. bronchiseptica and B. parapertussis are able to inhibit complement and suppress B and T-cell functions [1].  The BvgAS two-component system is understood to be a regulator of many virulence factors and is known as the master virulence regulon, however this is most likely an oversimplification. We hypothesized that pathogens like bordetellae should be under strong selective pressure to sense and respond to signals in blood and serum, in order to modulate immune defenses. When studying blood and serum responsive genes, we identified a putative sigma factor up-regulated in both conditions, and we hypothesized that this might be a regulator that dictates adaptation to pressure from the immune system. Here we identified the role of this sigma factor, the Bordetella Sigma Factor, or bsr, in manipulating the immune response. Through the use of a B. bronchiseptica bsr knockout and in vitro assays, we have found that bsr interferes with the innate response. bsr inhibits survival in macrophages by changing the dynamics of phagocytosis, causing macrophage death, and causing bacterial death and replication within the macrophage by interfering with cytokine and chemokine expression as well as differentially interacting with TLR receptors. This suggests that subsequent cell recruitment will be different within the host. Our results demonstrate that the bsr gene plays a critical role in Bordetella interaction with the innate immune system. A better understanding of this gene and its function will be valuable in efforts to create successful vaccines and treatments not only for Bordetella spp. but also for other bacterial species.

[1] Gestal M.C., Whitesides L.T., and Harvill E.T. “Integrated Signaling Pathways Mediate Bordetella Immunomodulation, Persistence & Transmission”. In revision, Current Trends in Microbiology.

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