Pain and unpleasant stimuli carry a negative hedonic valence, indicating an intrinsic aversiveness useful for avoiding harm. There are instances, however, of unpleasant stimuli carrying positive valence – such as the pleasure from spicy food, suggesting that pain and aversion can be decoupled. The Dhaka lab has discovered a small molecule Analgesic Screen 1 (AS1) which reverses the valence of a number of nociceptive and other aversive stimuli, whereby animals prefer normally aversive stimuli. Behavioral studies with zebrafish indicate that AS1 induces preference for noxious heat, painful chemical (AITC) and normally aversive dark environments. As positive valence or reward is often mediated by the neurotransmitter dopamine, we tested for the affects of dopamine antagonism on AS1-evoked behavior and found that the effects of AS1 are reversed by a D1 dopamine receptor antagonist. We currently propose that AS1 potentiates activity in the dopamine reward system in the presence of nociceptive and other aversive stimuli via D1R activation, thereby creating “pleasure from pain.” Understanding these pathways and the mechanisms underlying AS1 action, could provide a path forward for the development of novel therapeutics to treat debilitating pain disorders.

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.

Sex determination is a crucial process in many organisms and there are many ways in which it is regulated, including through genetic and environmental mechanisms. Despite its popularity as a model for development and disease research, the precise mechanisms of zebrafish (Danio rerio) sex determination remain uncertain. Zebrafish lack the typical heteromorphic sex chromosomes present in many other species, and no specific chromosome or gene that determine sex has been conclusively identified. In a preliminary experiment, a genetic analysis found sex was associated with inheritance of single nucleotide polymorphisms (SNPs) from various regions of the genome. To test the reproducibility of this observation, we used PCR and Sanger sequencing to identify SNPs in candidate causal regions. We tested these to obtain genotypes of individual zebrafish. These fish are being crossed to assess whether significant deviations from genotype ratios are found in males and females, which would suggest that loci linked to these SNPs are in fact involved in sex determination. This work attempts to further elucidate the genetic contribution to sex determination in zebrafish.

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.

 The northern side of Mount St. Helens violently erupted in 1980 providing a unique opportunity to study the ecological theories of primary succession. The previous sub-alpine environment was destroyed by the pyroclastic blast where new plant communities can be observed or manipulated. We seek to know which factors in the current alpine meadow influence the most change as the environment transitions to a woody forest. To test which facilitative factors influence plant communities associated with pioneering Abies procera (Noble Fir), forty experimental plots were established across an elevational gradient. The sites consisted of a control and four equidistant plots; a naturally established Abies procera specimen, while the other three plots had carbon, nitrogen, and shade additions. For carbon addition, Abies procera needles were collected, dried, weighed and dispersed to each plot. For shade addition, a shade netting was applied early in the growing season. For nitrogen addition, the soil will be treated with a nitrogen supplement. Plant species abundance and coverage was assessed at peak growth. R statistical program was used to calculate the species richness, evenness, and non-metric multidimensional scaling (NMDS) similarities. Species richness was found to be higher in sites of lower elevation, with little to no difference in treatments within the forty macro-plots. Plots treated with shade proved to have no significant species evenness when compared to the control, despite observations that the vegetation appeared healthier and more robust. NMDS results showed a strong similarity between experimentally manipulated plots and a large dissimilarity between those and the established tree. Although these preliminary results suggest little significant differences, they will continue to be used in a long-term ecological study to document the presence of woody trees as they catalyze the shift of plant communities towards a sub-alpine forest.

The direction that plants grow in is dictated by the directions of gravity and light. A mutation in the LAZY-2 (LZ-2) gene in tomato (Solanum lycoperiscum) causes the plant to actively grow downwards, in the direction of gravity. It has been shown that the lz-2 phenotype is dependent on the phytochrome (Phy) protein family; a group of plant proteins that is responsive to red light. In a plant with no functional phytochromes, the lz-2 mutation is restored to wild-type, and the plant will grow upwards as normal. In this project, we discern the roles of three of the five tomato phytochromes, A, B1, and B2, to see if they work together or alone to provide the light cue that initiates the mutant function of lz-2. By gravistimulating combinations of lz-2 and Phy tomato seedlings under red light, we have detetermined that PhyA delays the lz-2 response, while PhyB2 exhibits an additive effect with PhyA and PhyB1 to effectively reverse the lz-2 phenotype. These data expand on previous work to further our knowledge on the relationship between light and gravity sensing in higher plants. 

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.