In order to perceive attack by herbivores and pathogens and subsequently mount appropriate defensive regimes, plants rely on a variety of protein receptors that recognize and initiate responses to damage, pathogen, and herbivory associated molecular patterns (DAMPs, PAMPs, and HAMPs, respectively). Although DAMPs and PAMPs have been studied extensively, the first HAMP-receptor pair was only recently discovered. The inceptin receptor (INR) discovered in cowpeas (Vigna unguiculata) binds to inceptin, a small peptide and potent HAMP found in the oral secretions of Lepidopteran caterpillars, promptly eliciting an immune response leading to enhanced resistance against herbivory. In order to further characterize the structure and function of INR, I am developing a fluorescent reporter for use in Nicotiana benthamiana, a model organism that normally lacks INR. Expression of the fluorescent protein mScarlet will be driven by putative promoters of genes found to be upregulated in response to inceptin binding to heterologously expressed INR in N. benthamiana. Transfer of this reporter system into N. benthamiana via Agrobacterium-mediated transformation will allow fluorescence to act as a reporter of INR function by generating quantifiable fluorescence in the presence of inceptin binding functional INR. By simultaneously transforming wild-type N. benthamiana plants with the reporter and mutagenized variants of INR, key domains and residues for the recognition of inceptin by INR and subsequent activation of plant defense will be elucidated. This will identify key structural and functional aspects of INR that will inform engineering practices for enhancing crop resistance to herbivory.

Genetic data provide important insights into evolutionary relationships within and between species, and are especially useful for studying morphologically cryptic species (species that are visually indistinguishable from one another). I investigated the identity of an unstudied population of Sceloporus lizards from the Laramie mountain range of Wyoming. This population occurs at a boundary between the recorded distributions of S. tristichus and S. consobrinus. These species are morphologically cryptic, so molecular methods are necessary for accurate species determination. In collaboration with the Wyoming Fish and Game Department (WFGD), I conducted molecular genetics work on 10 samples, including extraction of DNA and PCR amplification of the ND1 mitochondrial DNA (mtDNA). Genetic divergence between populations of S. tristichus in western Wyoming (Sweetwater) and the Laramie Range is over 8%. Genetic diversity between a population of S. consobrinus from the Rocky Mountains in Colorado and the Laramie population is low (0.15%). I performed a phylogenetic analysis using maximum likelihood in the program RAxML and the population from the Laramie range is placed in a clade with S. consobrinus. Sceloporus consobrinus is currently not in the State Species Index for Wyoming, yet this result provides support for their addition. Before confirming that this population is S. consobrinus, additional phylogenetic analyses must be done. Previous studies of lizards in this genus have revealed discordance between gene trees made from mtDNA and nuclear DNA (nDNA) for populations occupying boundary zones between species. Therefore, current research aims to check for discordance or agreement between the nuclear genome and mtDNA gene tree.

Ceratophora is an endangered genus of lizards endemic to Sri Lanka containing five species. Of them, Ceratophora aspera has the widest distribution spanning the lowland and montane rainforests of Southwestern Sri Lanka. Very little is known about the ecology, population biologyand natural history of C. aspera, although this rainforest dwelling species can serve as a precedent for many more conservation and management projects. The goal of this project is to assess the genetic diversity within and among populations, and determine the phylogenetic relationships among the populations. During the 2018 and 2019 field seasons, tail clippings were collected from lizards that were captured in the wild. In the University of Washington's Biology Department, DNA sequencing of mitochondrial DNA is being used for an initial screening of genetic diversity, followed by the acquisition of genomic information using Single Nucleotide Polymorphism (SNP) data. Together these data are enabling us to model the relationships between geography and genetics to visualize where populations are connected and fragmented. Preliminary analysis suggests that there is low intrapopulation diversity (<0.1%), while the highest interpopulation diversity is 3.8%. Furthermore, the phylogeny supports two main clades indicating that population structuring corresponds to the two main forest complexes, the Sinharaja Forest Complex and the KDN (Kanneliya-Dediyagala-Nakiyadeniya) complex. Each of these forest complexes were historically entirely forested but are now composed of forest patches. Restoring forest landscapes and reestablishing metapopulation and genetic diversity across the landscape and locations of migration barriers and corridors, provides necessary information for effective conservation management of this species and others like it. 

The spread of grasslands 26-22 million years ago has been linked to global climate changes in the late Oligocene to early Miocene. The pattern of vegetation change was established analyzing assemblages of microscopic plant silica (phytoliths) extracted from sediment samples from the Central Great Plains of North America. It is often presumed that as open-habitat grasses became abundant, vegetation structure concurrently transitioned from closed forests to open landscapes. However, recent work in the Cenozoic of Patagonia has pointed to a decoupling of grass dominance and habitat openness, each independently driven by climatic conditions. We set out to test if a similar decoupling occurred in the Central Great Plains by means of an a-taxonomic phytolith proxy using phytoliths produced in non-grass epidermal cells. Work in modern plants and soil assemblages has shown that the size and degree of undulation in these phytoliths (quantified by, respectively, Phytolith Area, PA, and the Phytolith Undulation Index, PUI) is correlated with the amount of light in the environment, reflecting habitat openness (measured as Leaf Area Index, LAI). We measure the PA and PUI of phytolith samples from Nebraska, dating 35 to 17 Ma, to reconstruct the regional LAI over time and place time constraints on the opening of habitats. By comparing this timeline to that of the rise to dominance of grasses, we hope to better understand changing vegetation and linked climatic conditions in Cenozoic North America.

Ever since they first appeared during the Late Cretaceous, members of the palm family (Arecaceae) have been ubiquitous in the fossil record. Traditionally, palms have been considered a key indicator of warm climates. In addition to leaf macrofossils, fruit, and pollen, palm phytoliths have gained utility as paleoecological indicators. Phytoliths are microscopic silica bodies accumulated in the tissues of many plants. Different plant taxa have unique phytolith morphologies, making them useful diagnostic tools. However, palm phytoliths currently lack diagnostic resolution below the family level, limiting our ability to fully utilize these powerful tools. The goal of our project was to increase this resolution by analyzing the morphology of phytoliths from across the entire Arecaceae family in more detail than has been possible before. We used confocal microscopy to take sharp, high-resolution images of palm phytoliths. Using these images, we took several key measurements, to which we applied multivariate ordination methods. Our analysis allowed us to test how well we can differentiate palm subclades within Arecaceae based on phytolith morphology. Ultimately, we hope to use this information to determine when and where specific clades of palms appeared in the fossil record, increasing our understanding of the evolution of the palm family. This will also allow us to describe past environments in more detail based on palm phytoliths, including estimating more specific climate parameter ranges, and characterizing particular biomes and habitats.

Microplastics (plastic < 5mm) has become a prominent research topic over the last 15 years. Microplastic research papers are published around the world, using a variety of organisms, environments, and interactions as study systems. Findings from these papers suggest that microplastics have negative effects, like behavior disruption and cell death, on marine organisms. This research project uses scientometrics to analyze if trends in regional plastic policies are correlated with marine microplastic research papers. Utilizing spatial analysis, we compared the rate and spread of microplastic research and plastic policies across the globe to identify a statistical relationship between them. We then further explored this relationship by determining if the distribution of study systems in a region’s research affected the quantities of policies in that region. We used the Web of Sciences database to obtain data on microplastics papers from 2006 to 2018. Our data has revealed that specific species are being used in research more than others and that there are large concentrations of microplastics papers in areas like Europe and China. Countries with large amounts of plastic research also have the most plastic policies. The results of this project help decipher how marine microplastics research can have an impact on plastic policy.

Chondricthyes such as sharks, skates, rays, and chimeras share the slow maturation and low fecundity traits, thus making them susceptible to overexploitation. To ensure the survival of these vulnerable species it is necessary to protect the habitat important to their recruitment. The purpose of the present study was to determine the sediment preference of juvenile Beringraja binoculata to gain evidence as to where their nursery grounds may be located in the Salish Sea. To do this we conducted one-hour filming trials of three four-month old skates in a tank sectioned off into four different sediment size classes. Skate 1 died one-third of the way through the trials so was removed from statistical analysis. However, we found that Skate 2 and 3 preferred to bury and rest in the smallest sediment size of 0.125-0.3 mm grain size with p-values of 0.00126 and 0.0814 respectively. This is consistent with literature on different species of skates around the world. Ongoing research would be valuable to determining the reason behind this preference and to use the information to locate the Salish Sea nurseries.

Microplastic (plastic < 5mm) pollution is increasing at an exponential rate in marine environments, leading to increased contamination of marine organisms. Previously, it was thought that the majority of microplastics were found in surface waters due to their positive buoyancy; however, recent studies show pollution exists throughout the water column. Mussels, suspension feeding bivalves, can be catalysts to the spread of microplastic pollution to different trophic levels through filtration, ingestion, and egestion of microplastics. Mussels are prominent benthic-pelagic coupling organisms in marine ecosystems, moving particles and nutrients between habitats through different types of biodeposits. Mussels produce feces and pseudofeces, where feces is the digested biodeposit and the pseudofeces are the rejected materials. Accumulation of microplastics in their biodeposits may interfere with this coupling by altering sinking rates, giving other organisms more opportunity to feed on deposits and promote the spread of microplastics through the food chain.The purpose of our project is to test how different types of microplastics affect sinking rates of mussel biodeposits. Our hypothesis is that mussels filter microplastic particles differently.The more readily ingested microplastic will lower the sinking rate of the feces more drastically. In our experiment we fed mussels either polystyrene or polyethylene microspheres along with algae, collected their biodeposits, and measured sinking rate. Further, we quantified microplastic and algae found in each type of biodeposit. The selectivity of mussels toward a particular microplastic was determined by the amount of a microplastic present in the feces vs the pseudofeces. The results from this study can help us understand the impact of microplastic pollution and how mussels play a major role in the spread of microplastics. They may also provide insight into which types of microplastic are more readily spread, potentially providing information on how to distribute microplastic waste.

Mosquitoes primarily navigate using their olfactory system and can use this system to form “memories” that influence their choice in hosts. When a mosquito encounters an odor, information is sent through the antennal lobes in the brain to the mushroom bodies, which are structures responsible for learning and memory consolidation. This odor-learning pathway is mediated by neurotransmitters like dopamine and serotonin. Recent research has shown that the mosquito Culex quinquefasciatus has extremely low levels of dopamine in the antennal lobes compared to other species, and is unable to learn to avoid odors associated with a negative response. This led us to predict that dopamine is essential for aversive learning in mosquitoes. We hypothesized that Cx. quinquefasciatus differed from other mosquitoes in learning ability because they were previously tested in the light and they are the most nocturnal of the originally tested species. To test this hypothesis, we conditioned the mosquitoes in the absence of light in an aversive learning paradigm to measure how frequently they chose to avoid the conditioned odor. An inability to learn regardless of light condition would indicate that the role of dopamine as a neuromodulator in the antennal lobes evolved partly to allow diurnal mosquitoes to avoid defensive hosts. Next, specific neurotransmitters in the antennal lobe were mapped using confocal microscopy, revealing their concentrations which may explain behavioral differences from other mosquitoes. Most mosquito species show some plasticity in host selection, which can lead to the transmission of animal diseases, like West Nile Virus, to humans. Mosquitoes are the world’s deadliest disease vector, killing over 700,000 people globally each year, so understanding how and why this adaptation occurs can help us understand the framework that underlies the spread of mosquito borne diseases and bring us one step closer to solving this global issue.

Hydra vulgaris are some of the simplest animals with neurons. They only have two thin, near transparent layers of tissue: myo-endodermal and myo-ectodermal layers. Interspersed in each layer is a network of neurons known as the nerve net. All cells in the animal are constantly renewed, which allows Hydra to regenerate after being cut in pieces or dissociated into single cells, though how the nerve net regenerates has not been well studied. Each cell in the animal can be examined simultaneously due to the animals’ small size and simple, translucent body pattern. Hydra also exhibit stereotypical (regular and defined) behaviors. This makes Hydra great models for examining simple signaling pathways from which the complex pathways in vertebrates derive. The Hydra nerve net is composed of circuits that coordinate the behavior of the animal. The most obvious are the contractile burst (CB) and rhythmic potential (RP) circuits. I selectively blocked these circuits to understand how they drive behavior. It has been found that N-[1-(2-phenylethyl)-3-piperidinyl]-1-benzofuran-2-carboxamide (E9), affects Hydra behavior, appearing to block the CB circuit but leaving others, including the RP circuit, uninhibited (unpublished data, Woods Hole MA). I worked to understand the affinity and response rate of this molecule to Hydra by establishing a dose response curve for E9 on Hydra. To determine effective concentrations of E9, I imaged animals in serial concentrations ranging from 3uM-300uM. I then identified the response of neural circuit firing patterns to varying concentrations of E9 by applying this technique to animals that express GCaMP, a protein that fluoresces when bound to calcium, in neurons. I found that 30uM is the lowest concentration of E9 sufficient to block the CB circuit. This research provides a tool for studying the link between circuits and behavior, and allows us to characterize how behaviors depend on identified circuits.

The Cretaceous-Paleogene boundary is one of the most well-studied mass extinction events in history. However, there are still many questions left about what was occurring in terrestrial ecosystems in the 10-15 million years prior to the event. One major question is if theropod dinosaurs were declining in diversity and ecological stability prior to the event, or if they all died off suddenly. This stability is partially interpreted in this project through a geometric morphometric approach comparing Judithian and Lancian theropod teeth. Geometric morphometrics quantitatively analyzes tooth shape based on points called landmarks, that are universal across theropod teeth so variance can be measured and compared. Teeth are digitally imaged and processed with the landmarks to compute and then interpret their shapes in specialized programs. The landmarks measured are based on established standard literature of mesial and distal points of the tooth, the apex, and the mesial and distal terminal denticle. If the finalized analysis of the remaining confirms theropod dinosaurs are shown to have consistent morphological disparity through this time, this will be an indication that this group of organisms were not declining and were settled in their niches prior to the K-Pg boundary. Given that theropods were a diverse group that occupied many niches, as well as being the top carnivores that would have curbed other taxonomic populations, studying their economic stability through the window of deep time lasting from the Judithian to the Lancian immediately before the K-Pg boundary is leading to an invaluable understanding of how this environment was changing during this time.

Today there are more than 5,000 species of extant mammals that are categorized into three major clades: placentals, marsupials, and monotremes. The deep evolutionary history of marsupials is poorly known due to a fragmentary fossil record. In particular, cranial fossils of the ancient relatives of marsupials (stem marsupials) are extremely rare. The few cranial elements of these taxa that have been found are fragmentary, crushed, or missing major elements. Recently, some more complete cranial fossils of stem marsupials have been discovered at the Late Cretaceous (75 million years ago) Egg Mountain fossil locality in northwestern Montana. My research will describe the morphology of a partial skull of a stem marsupial from Egg Mountain. This delicate skull is encased in a hard siltstone, making it difficult to mechanically remove without damage to the fossil. Instead, we used micro-computed tomography (µCT) to scan the specimen block. Then I used Avizo software to virtually remove the rock and expose the details of the encased fossil. From the resulting files, I will study and describe the cranial morphology in detail. Thus far, we have identified the stem marsupial as Alphadon halleyi. With further study, I hope to (1) expand upon current knowledge regarding the morphology of Alphadon halleyi, (2) make comparisons with other stem marsupials and extant marsupials, and (3) more broadly, incorporate our findings into a phylogenic analysis of early metatherians (the clade that includes stem marsupials and marsupials) that will further elucidate the evolutionary history of marsupials.

Previous studies on plant phenology have found that shifts in the timing of life cycle events are connected to changes in the surrounding climate. Alpine and subalpine wildflowers are of particular interest, considering that the initiation of flowering often depends on the timing of snowmelt, which has been occurring earlier as temperatures warm. However, we often lack the information needed to predict exactly how much wildflower phenology will shift in response to warming. Herbaria, collections of plant specimens collected over the last 100-200 years, have recently been paired with climate data (from the geographic locations and dates of specimens) to examine the relationship between climate and plant phenology. I aim to answer 1) How does climate influence the timing of phenological stages in wildflowers? and 2) Do species vary in their responses according to their average bloom time after snowmelt (e.g. early season vs. late season bloomers)? This study involves five alpine/subalpine species that very in the timing of blooming relative to snowmelt date: Western pasqueflower (Anemone occidentalis), Sitka valerian (Valeriana sitchensis), Sickletop lousewort (Pedicularis racemosa), Rainier pleated gentian (Gentiana calycosa), and Glacier lily (Erythronium grandiflorum). I will access specimen data from the University of Washington Herbarium and the Consortium of Pacific Northwest Herbaria. Phenological stage will be recorded with the date of collection, geographic locations will be determined using GEOLocate coordinate and elevation data, and climate conditions will be spatially modeled using ClimateWNA historical records. I predict that there will be an overall trend of earlier flowering for all four species over time, but that species with later bloom times will experience less deviation from their historical average than those with earlier bloom times. This study is significant in providing information that will help the preservation of wildflower meadows in the high mountains of the Northwest.

Honeybees, Apis mellifera, demonstrate an ability to perform visual learning to such an extraordinary extent that it is not typically associated with smaller brains. Those learning abilities may be linked to their degree of sociality as they aggregate into colonies and synergistically work together on tasks. The Social Brain Hypothesis suggests that an organism's level of intelligence is correlated to how social their environment is. Some bees like the leaf cutter bee, Megachile rotundata, are solitary bees who do not live in a hive but instead reside in individual nests. The purpose of this research is to explore the visual learning capability of leaf cutter bees and compare them to the learning capability of the well documented honeybee. The methods used for this study include placing a tethered bee on a free rotating ball that is placed in front of a screen. Two colors are projected onto the screen and the bee controls the movement of the shapes. A positive and negative reinforcement are assigned to each shape. By analyzing how many times the bee picked each color, how fast it responded to the shapes, and how far from a distance it walked to fixate on that color, we can make an informed statement about the solitary bee’s ability to visually learn. We anticipate one of three outcomes, the leaf cutter bee will either have a greater learning rate, less learning rate, or equal learning rate to that of the honeybee. These results will help supplement more data for the Social Brain Hypothesis and allow a deeper understanding of how sociality is related to cognition. This study also has major relevance to pollination practices as leaf cutter bees are used in agriculture, therefore understanding the neural basis of cognition can open the door for new reforms in crop pollination.

It’s known that plant-pollinator relationships are central to the proper functioning of agricultural and ecological systems. Of the many navigation pathways pollinators use, floral scent signaling for insects is the most complex yet also the most at-risk from atmospheric human activity. Oenothera pallida, a primrose, interacts with the pollinators Hyles lineata, a hawk moth, and Megachile rotundata, a leaf-cutter bee, via this scent pathway. Because of their reactivity with floral scent, human-released ozone and NO₂ are the main perpetrators of scent degradation. To understand this relationship being damaged, I exposed the moth and bee species to a normal Oenothera scent versus a degraded one, recording the antennal response as well as the behavioral, expecting a poorer response to the degraded scent. Moth antennae act as the site of odor reception, bearing sensory hairs that detect odors, allowing the moths to navigate to scent sources. I conducted electroantennographic experiments (EAG) to record the electric signal from the insect antennae in response to each scent blend, with the degraded scent representing the impact of NO_x interactions. Following the EAG, I conducted Proboscis Extension Reflex (PER) experiments with Megachile to show the relationship between insect behavior and antennal physiology when in the presence of the scent blends. I expect that the EAG experiments show that the antennae respond worse to NOx degraded scents in comparison to the normal, unaltered scent blend. Likewise, Megachile has a worse PER when exposed to the degraded scent, linking the chemical biology of the scent interaction to the feeding and pollination behavior. This work has broader implications regarding the importance of plant-pollinator relationships, especially when considering environmental and agricultural health as well as the issue of food security in our changing climate.

Almost all of our tangible understanding of the world is mediated by the skin, a highly innervated sensory organ. However, many injuries to the skin result in the severance of somatosensory axons, causing temporary or permanent loss of feeling. To reestablish innervation, epidermal and neuronal cells launch wound healing responses. Neuronal cells undergo a transition to a more juvenile state to promote new axon growth for reinnervation. Surrounding keratinocytes and immune cells migrate to the affected area to clear cellular debris and promote axon regeneration. Although we know these cellular processes occur, it is unclear how changes in gene expression direct these responses. My project aims to transcriptomically characterize healing responses using zebrafish (Danio rerio) as a model organism. Zebrafish are widely used in regeneration studies and have a fully sequenced genome available for reference. Unlike in human skin where complete regeneration is not observed, zebrafish have almost perfect regenerative abilities. To address our question, we plucked fish scales to induce a rapid regenerative response of both somatosensory neurons and skin. We then collected and purified RNA from somatosensory neurons and skin at zero, one, and three days post pluck for sequencing to characterize gene expression over time. With high-throughput RNA sequencing, we can uncover expression patterns and ontologies of differentially expressed genes in neurons and skin after injury. Thus far, we have found that several previously characterized regeneration associated genes (RAGs) are transiently upregulated in neurons following injury. Transcription factors involved in somatosensory development show a drop in gene expression following injury and are upregulated as the regenerating axon develops. We hope that by understanding skin reinnervation in successfully regenerating organisms, we can develop strategies to improve tissue recovery in humans through manipulation of conserved mechanisms.

Skin is an organ system with a diverse cell ecosystem that plays important barrier and sensory functions, which are critical to organism survival. Because of its superficial location, the skin, and its composite cells, are easily damaged and need to be repaired to maintain homeostasis. Zebrafish are an excellent model to study skin repair because they regenerate tissue efficiently and share conserved skin architecture with other vertebrates. I am addressing two related questions that examine how the sensory axons that innervate the skin and resident skin cells interact during development and tissue repair. First, how do skin-resident immune cells called Langerhans cells contribute to skin repair? Previous work in the lab found that Langerhans cells phagocytose degenerating cutaneous sensory axons, leading me to hypothesize that Langerhans cells play a broad, and previously unappreciated role, in skin repair. As a first step, I have been examining the effects of genetic mutations on Langerhans cell development. I have compiled a data set from several mutants proposed to regulate Langerhans cell development using image analysis of transgenic zebrafish. Second, how do peripheral nerves in the skin influence tissue regeneration? To answer this, I have created week-long time-lapses of skin regeneration in mutants lacking peripheral neurons compared with wild type sibling fish. To induce regeneration, I removed a concentrated patch of scales, a type of skin appendage, on the lateral side centered above the pelvic fin. I then visualize and quantify scale regeneration using a fluorescent imaging of a stain that labels scales. By comparing the genetic groups, I hope to determine whether nerves influence the rate and amount of regrown scales. Together these projects will provide insights into how interactions between the complex cell types of the skin regulate tissue repair, which we hope will ultimately have implications for wound repair in humans.

Numerous interventions and genetic modifications have been shown to extend lifespan across a diversity of species. However, these studies often assume that extended lifespan is synonymous with extended healthspan. Recent research in the nematode worm, Caenorhabditis elegans, has questioned this assumption, and suggests that increasing lifespan can prolong the frailty associated with old age. This is particularly important for humans, as increasing lifespan without a corresponding increase in healthspan could spell disaster. The majority of healthcare costs are associated with aging-related pathologies, and prolonging life without prolonging health could radically inflate these costs. To parse out the genetic relationship between healthspan and lifespan, we have turned to Drosophila melanogaster, a well characterized model organism for studies on the genetics of aging. We have collected lifespan data as well as multiple measures of healthspan, such as negative geotaxis (climbing), intestinal permeability, Cold stress resistance, and metabolomics data across 16 inbred genotypes. We found a strong positive correlation between lifespan and climbing, and no correlation between cold stress resistance and lifespan. This confirms the importance of lifespan as a primary parameter in aging studies, but suggests additional measures of health are needed to accurately assess health. 

Understanding complex population dynamics between species is key for guiding environmental and wildlife management decisions. Accurately identifying the diet of various predator species across northeastern Washington (NEWA) and central Washington (CWA) can provide comprehensive insight into these relationships in terms of predator-predator and predator-prey dynamics. DNA metabarcoding can identify species-specific DNA within a sample and presents an ideal way to perform diet analysis in this context. In a previous NEWA study, the diet profiles of a range of predators were fully resolved, but for the American black bear (Ursus americanus), approximately 80% of its diet composition was undetermined. For increased understanding of the black bear’s diet in Washington, prey species must be identified across a range of geographic areas. This study compares the prey components of the black bear’s diet in both NEWA and CWA in order to provide a comprehensive analysis of its role in the predator-prey community. DNA samples used for analysis were from scat collected by detection dogs during a 2015-2016 NEWA and 2018 CWA field term. Of the 12 bear samples from CWA,9 samples had identified prey and of the 15 bear samples from NEWA, 6 had identified prey. These results add valuable information about prey species composition in a key predator’s diet across a wide geographic region, as well as seasonal shifts in diet composition in relation to other carnivores in the NEWA community. Future research will be conducted on the plant portion of the black bear’s omnivorous seasonal diet. Data collected from this project will provide valuable information that must be considered for further studies on the Washington black bear population and the food groups it consumes.

Over the last decade, wolves have been naturally returning to Washington state. Mapping the population growth and reproductive activity of wolves across Washington is key to understanding their recovery and to assisting wildlife conservation management. Accurate identification of the number of pregnant wolves per pack during the breeding season could help. Progesterone levels excreted in feces provide a reliable index of pregnancy in most mammals; progesterone rises post-ovulation but only remains elevated above a “pregnancy-threshold” among females that become pregnant. Unfortunately, this pregnancy-threshold metric is less reliable in canids because progesterone levels often remain above this threshold during the typical gestation period among all post-ovulatory females, regardless of whether the females become pregnant. Since gut microbiome diversity has also been shown to differ between pregnant and non-pregnant mammals, this study examined whether the combination of progesterone levels and gut microbiome diversity can refine pregnancy diagnosis in free-ranging wolves. Five high progesterone and five low progesterone fecal samples from ten unique female wolves were provided by the Center for Conservation Biology from the 2015-2017 study in Northeast Washington. Gut microbiome profiles were generated by sequencing the V4 16S rRNA gene region in each sample and analyzed using Qiime 2 and R with the Silva reference database for microbial taxonomy classification. Principal coordinates analysis of Bray-Curtis distance between samples at the microbiome phylum level showed separate clusters among high versus low progesterone samples, with one exception. The microbiome community of one high progesterone sample clustered with the low progesterone samples. This sample also had the lowest progesterone concentration among the high progesterone samples and may thus be from a non-pregnant post-ovulatory female. These initial findings suggest that the combination of progesterone levels and microbiome diversity show promise as a pregnancy diagnostic tool that may be able to distinguish pregnant from non-pregnant wolves.

Lateral roots are branches originating from the main branch of a primary root. They provide stability to the plant, and assist in acquisition of nutrients and water, similar to the primary root. It is well understood from studies in Arabidopsis thaliana that lateral root development is regulated by the plant hormone auxin. A few undifferentiated cells respond to a  pulsatile auxin signal to become ‘specified’ lateral root stem cells, retaining potential to proliferate and ability to differentiate into a lateral root. These specified lateral root stem cells are arrested in G2 phase of cell cycle, respond to auxin signaling and undergo rounds of cell division marking the onset of lateral root development. The focus of my study is to better understand how cell cycle control influences lateral root developmental transitions from undifferentiated to specification to initiation. In particular, I am interested in addressing the question of whether cell cycle arrest in G2 phase crucial for lateral root development, and if so at which step of cellular transitions get altered specification or initiation. An important tool necessary to address this question is the ability to control the length of G2 cell cycle arrest for lateral root stem cells, and we have recently generated transgenic lines for this purpose. I am inducing lateral roots in a number of transgenic plant lines and using fluorescence microscopy to observe the early stage of lateral root development. These experiments allow viewing of both the staging of development and cell cycle stage associated with development over time. The understanding gained from these experiments will help build a framework for how cell cycle contributes to lateral root development, allowing for future genetic modifications to improve root structure in crop plants.

Plants respond to environmental changes by changing their growth patterns. For plants to continue to grow throughout their lives, they are constantly undergoing cell fate determination—how the plant determines the specific fate of new cells they are generating. One example of this is the development of roots that emerge from the primary root, called lateral roots. The number and spacing of lateral roots determine the overall root structure, which determines how well the plant takes advantage of resources in its environment. Auxin is a plant hormone involved in many aspects of growth and development, including the regulation of lateral root production. Using data from an experiment where mRNAs were sequenced from single cells isolated from roots, the Nemhauser Lab identified specific genes that may be active during lateral root development. My research question is: are these genes targets of auxin signaling? To test this, I will use quantitative RT-PCR to measure expression of the candidate genes in wild type and in plants that are deficient in the auxin response pathway. Genes that are targets of auxin should show lower expression levels in the mutants relative to wild type. The more we understand about how cell fate determination occurs in lateral roots, the more we can understand the underlying mechanisms by which plants arrive at their final root structure. Our understanding can then guide engineering or breeding projects, allowing optimal root growth that could drastically increase plant survivability and yield.

Plant hormones are critical to their growth, development, and overall function. Gibberellins (GAs) are a class of plant hormones that are crucial for cell elongation and growth. The GA pathway has been genetically manipulated in many crops to enhance agricultural yields. Through my research, I hope to test whether modulation of GA signaling pathways can make crops that are more productive and better adapted to climate change. These types of interventions are needed because climate change is occurring more quickly than plants are able to adapt or evolve. Rewiring of the GA pathway is a potentially significant solution to this problem because we can modify plants in a manner that is likely transferable across many species. By using a novel genetic tool called a GA-sensitive Hormone Activated Cas9-based Repressor (GA HACR), we can modulate targeted genes in the GA hormone response pathway to turn down their transcriptional activity. The GA HACR targets specific genes through guide RNAs to repress the gene with complementary DNA sequence. The GA HACR is degraded in the presence of GA, which allows them to have a natural response to the activating hormone signal. We hypothesize that by targeting the HACRs to genes involved in GA biosynthesis (GA20 oxidase) and GA response (GID1 genes), we can modulate root growth of these plants. Once we identify plants with modulated growth behaviors, I will grow them in a CO2-enriched growth chamber to simulate future climate conditions. In this way, I will determine whether genetic intervention targeting the GA is a feasible strategy.

Tracing the lineage of individual cells within a multicellular organism has been one of the key struggles of modern developmental biology. The ability to trace the differentiation of individual cells over various timescales would give extensive insight into many fields of biology. Techniques using large scale genomics based on natural DNA mutations have been used in the past. However, in recent years, novel techniques using CRISPR-Cas9, and more recently recombinase, have been developed to study cell lineage in a more precise and dynamic manner. Each of these methods have different specifications in their readout methods, time-dependent resolution, spatial integrity, and accuracy. I have constructed a review summarizing these methods, and will present their impact on dynamic cell lineage tracing. While most of this research has been done in animals, I will also propose a design for cell lineage tracing in plants based on these reviewed methods.