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

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.


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

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.


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

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 (Rand variation in secondary infections). We found that outbreak size is nonlinearly associated with Rand 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 theoryabout 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.”Nature438(7066): 355.


Extreme heat reduces fitness of monarchs and their parasites

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.


Vive la resistance: the impact of antibiotic use in US livestock on emerging antibiotic resistance

Sydney Rentsch, a junior from Connecticut College, worked with Dr. JP Schmidt to examine the relationship between antibiotic use and resistance in US livestock.

Abstract:  The potential for livestock to spread antibiotic resistant pathogens to human populations is a cause for concern. This research focused on finding trends in data on US livestock antibiotic resistance, US livestock inventory and US livestock antibiotic consumption. Data was compiled from CDC, USDA and FDA reports and publicly available datasets. Data was analyzed in R and generalized additive models (GAMs) were used to test for increasing resistance as a function of time. We found that the tetracycline class of antibiotics had consistently high resistance over time. The antibiotic class lincosamides, had a sharp increase in resistance which was positively associated with the data from turkeys and chickens. Analyzes also found that poultry had the highest burden of antibiotic resistant pathogens. These results may lead future studies focused on antibiotic resistance in poultry and provide framework for future data analysis.


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Behavioral and environmental determinants of parasite transmission in a butterfly host

Chastity Ward, a senior from Fayetteville State University, worked on a project with Dr. Sonia Altizer, Dr. Richard Hall and Dr. Paola Barriga to examine how parasites of the Monarch butterfly are transmitted.

Abstract:  Many pathogens can be transmitted when infectious stages shed into the environment are later encountered by susceptible hosts. Environmental transmission is common among insect parasites, and also occurs for human diseases such as cholera and polio. Understanding how host behavior and environmental variables affect the shedding of infectious stages is crucial for predicting patterns of infection risk. Monarch butterflies Danaus plexippus are commonly infected by the protozoan Ophryocystis elektroscirrha (OE); this parasite is transmitted environmentally when infected adults deposit spores onto host plants (milkweed) that are consumed by monarch larvae. To quantify host contact with milkweeds as an estimate of parasite transmission, we set up outdoor flight cages with adult monarchs and milkweed plants. Cages varied in the number of adult monarchs and milkweed plants, and were assigned to one of two milkweed species. We used captive-raised monarchs from several genetic lineages, and marked the monarchs with unique number and color codes to track activity. We observed cages for replicate intervals over a week-long period, during which we noted observed monarch contacts with plants, and recorded monarch and plant identity, activity type, temperature, weather, and time of day. Our results showed strong heterogeneity in plant visitation rates among monarchs that was best explained by monarch sex (females had 4.7 times higher visitation rates than males, owing to frequent oviposition on milkweeds).  We also found wide variation among individual plants in the number of visits by monarchs. Milkweed species, plant flowering status and plant leaf number did not affect visitation rates, but plants in cages with a higher number of monarchs were visited more frequently. In sum, our findings provided evidence for individual monarch’s serving as superspreaders of infection, and for some milkweed plants serving as hotspots of infection. This study provides a starting point for estimating environmental parasite transmission in wild milkweed patches, and suggests that individual-level heterogeneity might be more important than environmental variation in driving parasite transmission in this system.


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Influenza inoculum dose and disease outcome

Wei-En Lu, a junior from Grove City College, worked with Dr. Brian McKay and Dr. Andreas Handel to examine the relationship between incolum dose and disease outcome in influenza.

Abstract:  The purpose of this study is to determine the relationship between influenza inoculum dose and disease outcomes.  A systematic review to identify and abstract data from all influenza challenge studies were conducted. Exponential and linear models were used to assess the impact of inoculum dose on disease outcomes. This study found that inoculum dose has a positive relationship on the proportion infected. However, there was a negative trend between inoculum dose and proportion of fever or systemic symptoms and between inoculum dose and the mean peak viral titers. There was also a rise of inoculum dose given to individuals and a decrease in the proportion of individuals with disease outcome over time. In conclusion, inoculum dose has a definite impact on disease outcomes.


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Using SpatialDE to characterize spatiotemporal changes in mitochondrial morphology

Brittany Dorsey, a sophomore from Mercer University, worked with Dr. Shannon Quinn and Dr. Fred Quinn to test the use of a new method to detect changes in organelle morphology.

Abstract:   Intracellular bacterial pathogens have the capacity to greatly alter target organelles’ morphology, which can easily be visualized through fluorescence microscopy, but is more difficult to quantify succinctly. Work is being done to consider Gaussian Mixture Models as a viable solution by viewing mitochondria as social networks, but there are difficulties with this method. Therefore, the goal of the current project is to explore the feasibility of SpatialDE as an alternative way to quantify the spatiotemporal changes in organelle morphology. Using time series footage of mitochondria, three morphological phenotypes were analyzed: control, fragmented, and fused. The raw video was converted to a three-dimensional matrix of pixel values, which was then raster scanned into a two-dimensional matrix. This matrix was normalized, then input into the SpatialDE framework using the programming language Python. The data frame output gave 18 different variable values for each pixel location throughout the footage, which was converted back into “image” format in order to be analyzed. The results showed little to no discernable patterns between treatments. In comparison, the Gaussian Mixture Model output shows clear similarities among phenotypes. Therefore, it was determined that Gaussian Mixture Models continue to be the best option to model spatiotemporal changes in diffuse organelles. Once this method is fully developed, the mechanisms by which bacterial virulence factors transform mitochondrial structure in host cells will be better understood, which will have crucial implications for structural biology and biomedicine.


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