Category Archives: 2019 Projects

Is relative viral load an important metric for treatment and prognosis of influenza A?

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Zane Billings, a student at Western Carolina University, worked with Dr. Andreas Handel and graduate student Brian McKay in the UGA College of Public Health.

Abstract
Influenza-like illnesses (ILIs) present with several of the same symptoms, including cough, fatigue, and weakness. However, ILIs can be caused by a range of different pathogens with vastly different treatments. Quantitative PCR is an incredibly specific and sensitive method to detect several ILIs, but until recently, qPCR methods were prohibitively expensive and required special training and equipment. Recent advances in qPCR technology have allowed for machines such as the Roche cobas Liat system to become available to point-of-care physicians. Using data collected from the University of Georgia Student Health Center, qPCR data was examined relative to patient and physician reported symptoms, as well as impacts and recovery from disease to determine if quantitative estimates of relative viral load are important for physicians to make informed decisions. While relative viral load estimates were found to be correlated to days since the onset of illness and patient temperature at diagnosis, no correlations were found between recovery or severity of illness and relative viral load. However, the study sample was very limited and more research should be performed on broader study populations.

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Understanding the dynamics of viral shedding within Norovirus infected subjects

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Simran Budhwar from the University of Virginia, worked with Rachel Mercaldo, Brian McKay, and Dr. Andreas Handel to study shedding of Norovirus.

Abstract: Norovirus (NoV) is a common cause of acute gastroenteritis. Symptoms include vomiting and diarrhea, which can lead to complications such as dehydration and also serve to spread viral particles through bodily fluids. While some infections are asymptomatic, infected individuals shed the virus regardless of disease severity, primarily through stool. To understand the dynamics of viral shedding, previous studies measured viral load in healthy human subjects challenged with various doses of the virus. In the present analysis, data from these studies was combined to better describe NoV shedding over time. We calculated key variables such as peak viral titer, time to peak viral titer, and duration of shedding, in addition to estimating total shedding through the area under the curve (AUC) value of each participant’s total shedding time-series curve. On average, patients shed the virus for 22 days, with the peak viral titer appearing on day 5 following challenge. Peak viral titers were 10.551 (log10) genomic equivalence copies per gram of stool, while AUC averaged at 11.58 (log10) genomic equivalence copies per gram stool. Though these are key variables that are necessary to understand viral shedding, future work should focus on exploring the drivers of variation in viral load and shedding, such as symptoms or other patient-specific factors.

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Effects of parasites and predators on heart rates of Daphnia laevis using an innovative electronic stethoscope

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Lutchie M. Carrasquillo, a student at the University of Puerto Rico at Arecibo, worked with Christian Hurd and Dr. Andy Davis to study the effects of parasitism on Daphnia heart rate using a new methodology.

Abstract: Daphnia are a model organism often used in investigations of chemical toxicity, and for biology classes. Measuring changes in heart rate is a commonly-used approach to assess responses to toxins. However, these assessments are usually done manually, which is time consuming and tedious. We developed a novel apparatus for monitoring changes in Daphia heart rates in real-time, without harming the animals. We used this approach to investigate how heart rate changes in response to naturally-occurring parasites (epibionts) and a natural Daphnia predator (glassworms). Our results showed Daphnia heart rates were not greatly affected by these, but we did discover an unusual diurnal effect, where the heart rate response differed between the morning trials and the evening trials.

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Wolbachia and its effects on mating preference in two Drosophila species

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Kareena Collins, a students at the University of Maryland Eastern Shore, worked with Paul Ginsberg and Dr. Kelly Dyer.

Abstract: Wolbachia is a maternally inherited intracellular endosymbiont that can manipulate reproduction in many different species of arthropod hosts, enabling its invasion into novel host populations. The most common types of reproductive manipulation is cytoplasmic incompatibility (CI), where mating between an uninfected females and infected males results in embryonic mortality.  Two Drosophila species, D. recens and D. subquinaria, were used to investigate whether Wolbachia can affect mating preference in a native versus non-native host species. D. recens is the infected species of Wolbachia with a frequency ~ 98%, while D. subquinaria is the uninfected host of Wolbachia. In the geographic region where both species overlap there is gene flow between species. We introgressed Wolbachia from D. recens into D. subquinaria in the laboratory. Both species show the CI phenotype in the lab when there is a cross with an uninfected female and an infected male. We conducted no choice mate trials for all crosses among infected and uninfected individuals for each species (all intraspecific crosses), and watched for mating for a three hour observation period. We found that Wolbachia had no effect on mating preference in the native host, D. recens. However, in the non-native host, D. subquinaria, Wolbachia had a huge effect on mating preference, with a significant reduction of mating rate in the cross between an uninfected female and an infected male ( the “incompatible” cross). Because Wolbachia had such a significant effect on mating preference only in the non-native host of D. subquinaria, it has potential implications for Wolbachia’s inability to become established as a native host in the population and/or species.

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Reproduction and immunity trade-offs in Aedes aegypti mosquitoes

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Jillian Dunbar, a student at the University of Alabama, worked with Ellen Martinson and Vincent Martinson in the lab of Dr. Michael Strand.

Reproduction and immunity are metabolically expensive systems; therefore, organisms with a limited amount of resources have to invest carefully (Schwenke et al. 2016). With the goal of producing offspring, organisms must invest resources into reproduction, yet also reserve resources for protecting themselves. In many cases reproduction and immunity are not directly linked. However, it has been shown in Aedes aegypti mosquitoes that, in addition to inducing egg production, a blood meal also increases the number of circulating immune cells called hemocytes (Castillo et all. 2011; Castillo et al. 2006). These findings elicit the question, does reproduction (blood-feeding) result in lower immunity for the mosquito? Through a variety of bacterial injections into blood-fed and non-blood-fed mosquitoes, this project worked to understand the possible tradeoffs between immunity and reproduction. We found an inverse relationship between immunity and reproduction, in that mosquitoes laid fewer and smaller eggs when injected with both live and heat-killed bacteria, but only for the more virulent species and higher doses. Interestingly, the results also showed that blood-fed mosquitoes were more successful in clearing or tolerating less virulent bacterial infections, suggesting resources gained from a blood meal are used to produce an anticipatory immune response. These preliminary findings are essential for continuing research and strengthening our understanding of the A. aegypti immune system with hopes of controlling or preventing diseases propagated by A. aegypti in the future.

References

Castillo, J, Brown, MR and Strand, MR (2011) Blood feeding and insulin-like peptide 3 stimulate proliferation of hemocytes in the mosquito Aedes aegypti. PLoS pathogens 7: e1002274.

Castillo, J, Robertson, A and Strand, M (2006) Characterization of hemocytes from the mosquitoes Anopheles gambiae and Aedes aegypti. Insect biochemistry and molecular biology 36: 891-903.

Schwenke, RA, Lazzaro, BP and Wolfner, MF (2016) Reproduction–immunity trade-offs in insects. Annual Review of Entomology 61: 239-256.

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Virulence-related characteristics of Bordetella pertussis mutants deficient in intracellular survival

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Callie Effler, a student at Lee University, worked in the lab of Dr. Eric Harvill.

Abstract: Intracellular survival is a common trait among human pathogenic bacteria that has advantages for the bacteria’s protection from the host immune response, persistence, and dissemination within the host. Bordetella pertussis, the gram-negative bacteria that causes whooping cough in humans, is commonly regarded as an extracellular pathogen. However, it has been recovered from macrophages in in vitro experiments, and reported anecdotally in clinical samples. It is unknown what contribution to pathogenicity the intracellular population has, if any, on the host. In this work, our broad objective was to evaluate the impact of B. pertussis’ intracellular survival and its role in pathogenicity. To do so, we planned to identify mutants similar to the wild type in general measured aspects of virulence, but that failed to survive inside of macrophages. A transposon library of B. pertussis UT25 was screened, resulting in the identification of several putative mutants that were deficient in intracellular survival. These strains were further screened for intracellular deficiency as a confirmatory measure, and went through further in vitro assays screening for cytotoxicity, hemolytic activity, resistance to serum complementation, and general fitness (growth). Based upon these assays, mutant strain G4 was the best candidate among those tested for an intracellularly-deficient mutant with similar virulence-related characteristics to the wild type. Preliminary C57 mouse infection studies suggest that the mutant strain behaves similarly to the wild type in vivo, indicating that intracellular survival may not be contributing to virulence. It is hypothesized that intracellular survival may be a phenotypic remnant of an ancestral strain of B. pertussis that transitioned from the environment to a become a human pathogen using this trait.

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Genomics of bacterial symbionts to determine nutritional roles in plant-sap feeding insects

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Michael Lansford, a student at the University of Rochester, worked with Dustin Dial and Dr. Gaelen Burke

Abstract: Adelgids are sap-sucking insects that contain bacterial endosymbionts to help them synthesize essential amino acids. The adelgid life cycle alternates between sexual generations that parasitize spruce as a primary host and asexual generations that parasitize a secondary host plant species. The adelgid family consists of five lineages each with a different secondary host plant: Douglas fir, fir, hemlock, larch, and pine. Each adelgid lineage has a different pair of symbionts, a primary symbiont that was acquired by the adelgid first and a secondary symbiont that was acquired second. Vallotia is a symbiont shared between the Douglas fir lineage, where it is the secondary symbiont, and the larch lineage, where it is the primary symbiont. To determine the nutritional roles of Vallotia in different species, genomic data were searched for genes involved in amino acid synthesis. FastQC was used to evaluate the quality of raw adelgid read data. The Georgia Advanced Computing Research Center (GACRC) cluster was used to assemble and annotate genomes from the raw reads. After running scripts to assemble raw reads into scaffolds, BLAST was used to identify which scaffolds were from symbionts. Symbiont genes were annotated using PROKKA and Geneious Prime and biochemical pathways were reconstructed with help from BioCyc. The results showed that Vallotia is primarily responsible for synthesis of all essential amino acids except cysteine in the Douglas fir lineage species A. cooleyi. Gillettellia, the primary symbiont in the Douglas fir lineage, works together with Vallotia in lysine and aromatic amino acid synthesis. In both larch lineage species, Vallotia is only responsible for the final steps in tryptophan synthesis and depends on the secondary symbiont Profftia in A. lariciatus and probably A. abeitis for most steps in aromatic synthesis. These results suggest that Vallotia was acquired by the Douglas fir lineage to account for the loss of most synthesis genes in Gillettellia and Profftia was acquired by the larch lineage to account for the loss of aromatic synthesis genes in Vallotia.

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Effects of larval density on the fitness of the Asian tiger mosquito (Aedes albopictus)

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Courtney Schreiner, a student at the University of Idaho, collaborated with fellow REU student Taryn Waite, along with Nicole Solano, Dr. Courtney Murdock, and Dr. Craig Osenberg.

Abstract: We were interested in studying the effects of larval density on the fitness of the Asian tiger mosquito (Aedes albopictus)­­. Aedes albopictus live all around Georgia and in various types of habitats that can support varying amounts of larvae. This creates competition which can effect downstream traits like fitness and disease transmission. Our experiment took place in a semi-field enclosure. We had 7 different larval density treatments that ranged from 5 to 240 larvae in each jar, for a total of 92 jars. Daily emergence, sex ratio and wing size were all recorded for all emerged mosquitoes. Overall we found that the proportion that survived, proportion female, fecundity, and wing size all decreased as density increased. We calculated the intrinsic growth rate using these estimates and found that lower densities have a higher intrinsic growth rate than those at lower densities. This told us that mosquitoes have a higher fitness at lower densities. Which also means that higher disease transmission would also be found in lower densities.

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Spiders as a gauge for change: linking spider density to riparian habitat structure

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Lily Tanner, a student at New College of Florida, worked with Denzell Cross and Dr. Krista Capps.

Abstract: Urbanization often compromises the diversity and abundance of native species living in watersheds (Rodrigues et al., 2015). Research has shown that riparian spiders are susceptible to land use change associated with urbanization (Sanchez-Ruiz et al., 2017).  We conducted  a spider density survey in riparian habitats in and around Atlanta as a metric to assess the impact of urbanization on regional spider populations. We hypothesized that if overhanging vegetation was important web-building substrate for some taxa  and vegetation provided cover for ground-dwelling species, then the highest density of spiders would be found in transects with vegetation hanging over the stream. Our results indicate that overhanging vegetation was related to higher densities of web-weaving species. Increasing amounts of overhanging vegetation was also related to increasing bank slope, suggesting there may be potentially important interactions between physical and biological characteristics of riparian areas that mediate spider population structure.

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To skip or not to skip: exploring the connections between orviposition behavior and density –dependence in Aedes albopictus mosquitoes

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Taryn Waite, a student at Colby College, collaborated with REU student Courtney Schreiner, Nicole Solano, Dr. Craig Osenberg, and Dr. Courtney Murdock.

Abstract: Conspecific density in larval habitats is an important factor affecting adult fitness in Aedes albopictus mosquitoes, as it drives competition for food and space. We conducted a larval density experiment wherein mason jars containing leaf infusion and varying numbers of larvae were placed in a field enclosure, developmental stage was recorded daily, and emerged adults were collected. Nonlinear regressions were performed on the data for survival to adulthood, sex ratio of adults, and wing length of females, and fecundity was inferred from wing length. Using these regressions, an equation was created to predict short-term population dynamics in habitats with varying conspecific densities. What determines the densities that will actually occur in various larval habitats is where females choose to lay their eggs. Female mosquitoes have the ability to skip-oviposit, which entails spreading their eggs out among multiple habitats instead of dumping them all in one habitat. The population dynamics equation was used to evaluate the theoretical consequences of skip- versus non-skip- oviposition, using scenarios with varying numbers of egg-laying females and a fixed number of available larval habitats. We found that at low densities of ovipositing females, skip-oviposition produces more short-term population growth than non-skip-oviposition. At higher densities, non-skipping becomes more productive than skipping, though there is less divergence between the outcomes. This simulation demonstrates a way in which patterns of density-dependence could act as a link between oviposition behavior and population dynamics. Due to the effects that we found of density dependence in larval habitats, individual females’ oviposition behavior could have consequences for short-term population dynamics.

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Who infected whom? Creating a database of transmission trees for comparative outbreak analysis

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Juliana Taube, a student at Bowdoin College, worked with Paige Miller and Dr. John Drake.

Abstract: Transmission trees contain valuable details about who infected whom in infectious disease outbreaks. We created a database with 81 published, standardized transmission trees consisting of 12 directly-transmitted pathogens (mostly viruses). We also demonstrated how the database could be used to help answer research questions in infectious disease epidemiology. First, we analyzed overall and pathogen-specific patterns between tree parameters (R0 and variation in secondary infections). We found that outbreak size is nonlinearly associated with R0 and the dispersion parameter, but emphasize that pathogen-specific patterns and intervention efforts may alter theoretical relationships between these variables. Second, we examined how superspreader contribution to onward transmission, either directly or through their tree descendants, varies across pathogens. Superspreaders were responsible for most cases via their descendants and the number of superspreaders varied across pathogens. Additional database exploration matched theory1 about how the proportion of superspreaders increases at intermediate levels of dispersion, an idea that should be further explored. We hope that our database will assist both theoretical and applied infectious disease epidemiology research in the future. 


1. Lloyd-Smith, JO, Schreiber, SJ, Kopp, PE, & Getz, WM (2005) “Superspreading and the effect of individual variation on disease emergence.”Nature, 438(7066): 355.

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Extreme heat reduces fitness of monarchs and their parasites

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Maya Sarkar, a student at the University of Minnesota, worked with Isabella Ragonese, Dr. Sonia Altizer and Dr. Richard Hall.

Abstract: It is important to understand the consequences of a warming climate, especially in organisms that are more sensitive to temperature changes and where the outcome of warming may not be intuitive. This project used the Monarch-OE system to study how temperature may affect host-parasite interactions. The monarch butterfly (Danaus plexippus) is an iconic North American migratory species and the specialist protozoan parasite OE (Ophryocystis elektroscirrha) is present in all monarch populations. It has been shown that monarch development proceeds faster with increasing temperatures and that increased temperature exposure lowers OE spore infectivity over time. However, the effect of temperature on the host and parasite during active infection is not known. This project examined how temperature affects the monarch-OE system, focusing on the interaction between monarch immune function and parasite replication. Monarchs were inoculated with strains of OE parasite and placed in different temperature treatments. Three lineages (B,F, and D) of migratory monarch were used to test genetic effects, while 2 spore lines (E3 and E10) were used to study virulence effects within 5 different temperature treatments (18, 22, 26, 30, and 34°C). The results of this study provide novel insight to how extreme temperatures affect the fitness of a host and its parasite.

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