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.

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.

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.

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.

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.

Cantharidin is a terpenoid produced by blister beetles (Meloidae) and false blister beetles (Oedemeridae) for defensive purposes. Despite its toxicity, a diverse range of organisms (mainly insects) are attracted to this compound in nature, but the specific role of cantharidin in the biology of insects is unknown. In summer 2011, a faunal survey was initiated in Northern Idaho and Eastern Washington to determine and observe the cantharidin orienting behavior of various arthropod species. Cantharidin-baited and control pitfall traps were placed in three localities to collect specimens. Through the survey, we discovered a new canthariphilous harvestmen species, Togwoteeus biceps (Thorell) (Arachnida, Opiliones, Sclerosomatidae). Distribution and phenological sex ratio patterns were analyzed to further understand what role sex might have with cantharidin-orienting behavior in this harvestmen species. A total of 281 T. biceps specimens were collected, and approximately 98% of the specimens were collected from the cantharidin traps. About 80% of the cantharidin-orienting T. biceps were female, suggesting a female bias in the canthariphilous behavior of this species. Through our study, we are providing the first documented North American harvestmen species and the second documented species in the world exhibiting the attraction to this compound. Historically, these species of arthropods are understudied despite their abundance, and our research sheds some light on their contributions to arthropod biodiversity.