8 undergraduate students awarded GCC research grants for 2020

Project Title: Effects of temperature and humidity on the metabolism of nectar in Aedes albopictus and Ae. aegypti mosquitoes

• GCC Faculty mentor: Dr. Chloé Lahondère

Mosquitoes transmit several pathogens to humans and other animals killing about a million people every year. If females need blood to produce eggs, both females and males feed on plant nectar which enhances their survival and longevity. For this project, we will focus on two invasive mosquito species, Aedes aegypti and Ae. albopictus. These mosquitoes are originally indigenous to the tropics but have now spread around the world (including the US). Global warming has been shown to increase the habitable range of these mosquitoes in part due to raising temperatures. As their geographical range expands it increases their threat to public health. Surprisingly, how environmental factors such as temperature and humidity, in combination with access to nectar sources, affect mosquito population dynamics remain unknown. To fill this knowledge gap, we will first conduct studies on laboratory strains of mosquitoes and quantify their metabolism and digestion on nectar under different environmental conditions using calorimetric assays. Then field work will be conducted in Virginia to assess sugar feeding prevalence in these invasive species. Altogether, these data will shed light on how mosquitoes are utilizing nectar in the field and will inform on the development of new traps to control mosquito populations.

Project Title: From water chemistry to growth: making sense of the abiotic world of frogs

• GCC Faculty mentor: Dr. Meryl Mims

Many organisms rely on environmental cues to signal when to grow and develop to maximize chances of survival. However, anthropogenic threats, such as climate change, habitat alteration, and water pollution, pose a challenge to many species as it alters their environment. In Spring 2019, we conducted an artificial pond experiment to explore the effects of water temperatures, increased drying rates, and the combination of both on wood frog, ​Lithobates sylvaticus, a​ nd spring peeper, ​Pseudacris crucifer ​tadpoles. We examined how these environmental changes – anticipated to be an effect of climate change – influenced tadpole growth and survival. Analyzing how temperature and drying influence water quality parameters such as pH, dissolved oxygen, temperature, and conductivity may also lead us to a deeper understanding of the mechanistic pathways through which these environmental changes influence tadpole growth and overall survival. This past semester, I examined the significant differences of these parameters between treatments; however, we have yet to analyze chlorophyll-a to determine how algae, the tadpoles food source, is affected. I propose to expand this project by extracting chlorophyll-a from algae samples collected during the experiment. Analyzing this information along with the other water chemistry parameters will provide a detailed understanding of the tadpoles’ abiotic environment under warming and drying conditions and how this ultimately affects their growth and survival.

Project Title: Wetlands in a Warming World: The Importance of Wetlands in Headwater Carbon Cycling

• GCC Faculty mentor: Dr. Erin Hotchkiss

Wetlands are productive ecosystems that play an important role in carbon cycling. However, wetland contributions to landscape CO2 and CH4 emissions are often overlooked and the role of wetlands in producing carbon emissions remains a critical gap in carbon budgets. To estimate current and future emissions from wetlands at Coweeta, NC and Jefferson National Forest, VA, I will measure CH4 and CO2 emitted from each wetland to the atmosphere and conduct laboratory warming experiments to calculate microbial organic carbon (OC) uptake and CO2 production. I will use a flux chamber attached to a portable greenhouse gas analyzer to get real-time estimates of CO2 and CH4 fluxes across each wetland. I will develop and test an improved flux chamber design to allow for more accurate and abundant flux measurements. To test how microbial uptake of OC (and subsequent CO2 and CH4 production) may change with increasing temperature, I will incubate filtered and microbially-inoculated wetland water at ambient and +3°C in triplicate bioassays with ambient and amended (+CNP) nutrients. I predict that increased temperature increases OC uptake and CH4 and CO2 production. This experiment will provide insight into the current and future role of wetlands in carbon cycling with changes in temperature.

Project Title: Development of Poison Ivy Clonal Lines Differentiated for High or Low Urushiol Levels: Genetic Resources for Ecological Studies in Urushiol Chemical Ecology

• GCC Faculty mentor: Dr. John Jelesko

Poison ivy seedlings grown in vitro produce markedly different steady state urushiol accumulation levels, apparently due to underlying genetic factors. I will leverage these findings to develop poison ivy clonal lines with varying urushiol levels. I have developed an experiment to germinate 50 poison ivy seeds collected from each of four states: Michigan, Iowa, Virginia, and Texas. I will harvest the first true leaf pair from each seedling and assay total-urushiol levels by Gas Chromatograph-Mass Spectrometry. From these results, I will choose from each accession the four plants with the highest urushiol levels and the four with the lowest urushiol levels. This subset of plants from each state will be transplanted to pots with potting mix and grown in a Washington Street greenhouse with supplemental lighting and automated watering. There, they will produce stolons with genetically-identical daughter plants. These clones will again be assayed for total urushiol levels to confirm the stability of the high and low urushiol accumulation traits. The long term goal of this experiment is to transfer these plants to Kentland Farm this summer and quantify whether urushiol actually reduces herbivory by extant native fauna.

Project Title: In Cold Blood: Understanding The Role Mosquitoes Play in Pathogen Transmission to Frogs

• GCC Faculty mentor: Dr. Chloé Lahondère

Amphibian extinction is now happening at an unprecedented rate in part due to alteration of local ecosystems, climate change and diseases. Among diseases, emerging viral pathogens, Ranaviruses, as well as Batrachochytrium dendrobatidis, a fungus that infects the skin, are greatly affecting amphibian populations, most notably frogs. This combination of habitat loss, rising temperatures and diseases has made this issue complicated and multifaceted. This project focuses on the role that Culex territans, a mosquito that feeds primarily on cold-blooded animals including frogs and snakes, plays in disease transmission in amphibians in Virginia. Cx. territans is a known pathogenic transmission vector, capable of spreading parasitic trypanosomes to various species of amphibians. We hypothesized that Cx. territans may also be capable of spreading the aforementioned viral and fungal diseases. To test for this hypothesis, we will screen blood fed Cx. territans from local areas for pathogens during plaque assays and PCR with specific primers. Data emerging from this project will help us better understand the complex dynamics seen between amphibian extinction, transmission of pathogens, and host-vector relationships.

Project Title: CRISPR Genome Editing of Hairy Roots to Identify and Confirm Poison Ivy Urushiol Biosynthetic Genes

• GCC Faculty mentor: Dr. John Jelesko

Urushiol is produced in poison ivy and causes allergic dermatitis in humans. Poison ivy is projected to become more allergenic with higher atmospheric CO2 levels associated with climate change. Urushiol’s primary ecological purpose is currently unknown because it is uninvestigated. Moreover, none of the urushiol biosynthetic genes and enzymes have been previously validated. A previous Jelesko Lab GCC UG Research Awardee Nye Lott successfully developed an Agrobacterium rhizogenes-based poison ivy stable transformation system to produce transgenic poison ivy hairy roots containing CRISPR-Cas9 gene editing plasmids that target poison ivy PolyKetide Sythase-like (PKS-like) genes proposed to be involved in the first step in urushiol biosynthesis. My research project will follow on Nye’s A. rhizogenes strains containing PKS-specific CRISPR-Cas9 T-DNA binary plasmids to produce transgenic poison ivy hairy root lines with mutated PKS genes. The resulting transgenic hairy root lines will be evaluated for urushiol levels using GC-MS. If they show a decrease in urushiol levels, then the hairy roots lines will be sequenced for mutations in the targeted PKS genes. Hairy root lines with mutated specific PKS genes that result in dramatically less urushiol levels will be strong evidence that a specific PKS gene is necessary for urushiol biosynthesis.

Project Title: Identification of Urushiol Biosynthetic Genes by Differential Gene Expression in Tissues with Different Urushiol Accumulation Levels

• GCC Faculty mentor: Dr. John Jelesko

Poison Ivy plants produce urushiol, the compound that causes the characteristic allergic contact dermatitis symptoms in humans. The main goal of this project is to identify genes that are likely responsible for producing urushiol in poison ivy. In order to accomplish this goal, we will use a comparative transcriptomics approach between poison ivy tissues that accumulate markedly different amounts of urushiol. Preliminary work by a former GCC Undergraduate Awardee Nye Lott, suggested that poison ivy drupes accumulated between 8 to 45-fold more urushiol than the adjacent leaves. The lab has 20 matched pairs of poison ivy drupes with adjacent leaf material available for my experiments. I will extract total RNA from five pairs of matched poison ivy drupes and their corresponding adjacent leaves showing maximum differential urushiol accumulation levels. These 10 total RNA samples will be sent to NovoGene for library preparation and Illumina NextGen sequencing (RNA-seq). The collaborating Haak laboratory will perform the RNAseq analyses using their unpublished draft poison ivy whole genome to quantify and identify differentially expressed genes. I will subsequently sort through the differentially expressed poison ivy genes to identify specific predicted enzymatic activities involved in urushiol biosynthesis. This informatic resource will be used for many studies.

Project Title: The Effect of pH and symbiont density on outcomes in a cleaning symbiosis

• GCC Faculty mentor: Dr. Bryan Brown

For over 20 years, the Brown Lab has studied the context-dependent cleaning symbiosis between crayfish and ecosymbiotic branchiobdellidan annelids. These worms act as cleaners for the crayfish, increasing the hosts’ fitness. However, the benefits from the symbiosis for the host are context dependent, and can shift from mutualism to parasitism under some conditions including high symbiont densities. However, what isn’t currently known is whether the symbiotic outcome will change under conditions of host stress. Given that changes in pH are common stressors in aquatic systems, it makes sense to examine how the outcomes of the symbiosis will change with this change in context. I will use varying levels of the branchiobdellidan, Cambarincola ingens crossed with a pH gradient in an aquarium experiment using previously successful methodologies. Response variables will be growth and survivorship of the host crayfish. We have already run preliminary versions of this experiment and produced results that suggest that intermediate densities of C. ingens will increase growth and survivorship at pH levels that depart from known norms. Lessons learned from that preliminary experiment will increase our probability of success with the proposed experiment.