Most procedures for DNA transformation of chloroplasts involve the use of circular DNA as vectors and green leaves as the target tissue. Although such procedures are successful with some plants, they are inefficient and do not work with other plants, especially monocots. In maize, plastid DNA (ptDNA) is comprised largely of linear molecules. Furthermore, ptDNA is rapidly degraded as the cells become green. In consequence, the introduction of transgenes into maize chloroplasts has been problematic. In order to create vectors for monocot transformation, the plastid chromosome is most efficiently targeted in its natural form. Previous work suggests that the plastid chromosome is found primarily in a complex, branched linear form with defined ends. We chose one of these ends, “End 1,” which is near an origin of replication, as our target for transgene integration. We constructed vectors with the “End 1” gene sequences and a mannose-metabolizing transgene, PMI, as the selectable marker. Plastid transformation was performed by particle bombardment using gold microcarriers coated with either the circular or linear vector. The successful introduction of a transgene using the “End 1” DNA sequence indicates that the transgene integrates either by end joining of the linear vector to the linear ptDNA or by homologous strand invasion. Thus, using a linear vector with an end sequence produces optimized results when engineering monocot plastid transformants.

Active learning generally increases student achievement, but not all instructors experience the same magnitude of gains. This variation in student achievement is likely due to differences in how active learning is implemented in the classroom as well as differences in total classroom time devoted to active learning. Although multiple tools exist for characterizing active learning in the classroom, none have been used to explain variation in student achievement. We are developing a new type of Classroom Observation Tool (COT) that characterizes how closely an instructor’s use of active learning matches the best practices suggested in education literature and can be correlated with observed variation in student exam achievement. Currently, we are using the COT to analyze 3 randomly selected classroom sessions from 27 different introductory biology instructors using archived footage. We will create a linear model that combines instructor’s COT scores with controls for variability in student ability and exam challenge between instructors to predict student exam performance. This model will allow us to identify which elements measured by the COT are most strongly correlated with variation in student performance. With this baseline data, we will be able to recommend the use of the COT to help Faculty assess the effectiveness of their instruction, thereby enhancing teaching and student learning not only within Biology education at UW, but also across all postsecondary education.

Cortinarius is the largest genus of ectomycorrhizal fungi. Ectomycorrhizal fungi are extremely important to the function of forest ecosystems and plant communities as they form symbiotic relationships with over eighty percent of vascular plants by colonizing the feeder roots and increasing nutrient and water uptake. My research will focus on the identification and phylogenetic relationships of a subset of Cortinarius species associated with the Oregon oak in the Columbia George of southwest Washington. Identification of the species associated with Oregon oak will allow me to better understand their ecology and relationships to other Cortinarius species. I am extracting DNA from samples collected in the field over the past four years. Using PCR, the nuclear ribosomal DNA (rDNA) internal transcribed spacer region (ITS1-5.8S-ITS2) will be amplified with primers ITS1F and ITS4. DNA sequencing and analysis will be done using Finch TV and Basic Local Alignment Search Tool (BLAST) software. We expect that most of the species will be new to science, and that these analyses will provide insight into their ecology and relationships to other oak associated species in western North America and Europe.

Isochrysis galbana is a marine alga that produces large quantities of lipid per cell. Lipids contain extensive amounts of potential energy. Therefore, this haptophyte may be considered for use in biofuel production. The lipid body organelle—the site of neutral lipid storage—is involved in many cellular processes. To date, little is known about this organelle in I. galbana. Chloroplasts contain lipids stored within smaller structures, termed plastoglobuli. For the purpose of the study, lipid body and chloroplast isolation techniques have been developed for I. galbana.  While light intensity has shown no direct effect on I. galbana’s growth rate, we hypothesize different qualities of light will affect growth by altering cellular protein, carbohydrate and lipid content. We will culture I. galbana under red and blue wavelengths of light. Using chloroplast isolation techniques, as well as developed lipid body isolation procedures, we will analyze the lipids contained within I. galbana lipid bodies and chloroplasts when grown under different light qualities. We will also determine if these wavelengths of light induce a stress response concurrent with lipid production. Preliminary data shows that heat shock proteins 70 and 90 –proteins that maintain homeostasis within the cell—are present in I. galbana whole cell protein extracts as well as isolated lipid body proteins. Using Western blot techniques, the abundance of these stress proteins will be measured under different light conditions. These studies will provide information on growth and lipid production under different light qualities, and whether these wavelengths of light elicit a stress response in I. galbana, an alga with potential for applied biomass production.

The evolution of multicellular animals required the development of epithelial tissues that function in controlling the transport of molecules from environment to organism. Collagen proteins are crucial to the formation of epithelial tissues, and are therefore critical in understanding the origins of multicellularity and Metazoan evolution. We characterized the collagen complement from the sequenced genome of the ctenophore Pleurobrachia bachei. 1) We discovered that P.bachei has 7 distinct type IV collagen genes, an expansion unseen in any organism sequenced to date. These genes show unique distribution across the genome: four were in an in-line pattern (αAand αB, αD and αE); two were found independent of other genes (αF, αG), while another two were aligned in a head-to-head fashion (αB,αC). This exceptional arrangement suggests both traditional and inverted gene duplication events in the ctenophore lineage. Ctenophore collagen intron/exon arrangements were also uniquely diverse, with a range of 14-40 exons, depending on the gene. 2) Phylogenetic analysis revealed two distinct collagen groups, the α1-like and α2-like sub-families that are common in bilaterians. Yet, gene to gene comparisons reveal a more diverse type IV collagen complement than found in chordates, including humans. 3) We found widespread expression of type IV collagen. Our findings imply that the common ancestor to all Metazoa might have contained a much more developed collagen complement than was previously appreciated. At the same time, there is extensive parallel evolution of ancestral collagens with remarkable functional specification and diversity of body plans in ctenophores.

Possession Sound is a complex estuary system home to diverse marine life that is impacted by local anthropogenic activity such as surface point and non-point source runoff. Introduction of nutrients with runoff as well as from the Snohomish River and upwelling can lead to increase nutrient concentrations in the surface water, which can cause algal blooms. Phosphate, one of the nutrients, is known to act as a fertilizer for phytoplankton while preventing the growth of zooplankton populations (Phosphates in Water Pollution). We hypothesized that Coscinodiscus spp. will vary directly with phosphate concentrations. To test our hypothesis, we reviewed historical data collected on State of Possession Sound (SOPS) cruises collected in the fall by Ocean Research College Academy (ORCA) students. Nutrient and plankton data in October, November, and December of 2010 as well as November of 2011 were compared. We found that during plankton blooms, phosphate levels are extremely low, possibly due to increased absorption of the micronutrient for photosynthesis. Similarly, when phytoplankton populations were low, phosphate levels increased, likely because of decreased absorption. Conversely, an analysis of tides and seasonal anthropogenic activities are likely to have affected the results of our study and could lead us to further questions.

Possession Sound is located at the Snohomish River estuary system between Everett and Whidbey Island in the greater Puget Sound. Mixing between fresh and saline water occurs as a result of the river’s influence and varying tidal exchange. It was hypothesized that more plankton will be present at North Jetty, a site near the mouth of the Snohomish River compared to Buoy, a site located on the opposite side of the jetty, farther from the river. Therefore, it is expected that the plankton biodiversity of North Jetty will be greater when compared to Buoy. Plankton samples were collected at flood and ebb tide at North Jetty to observe the effects of both location and tide height on plankton biodiversity. At North Jetty, there was a wider variety of phytoplankton and zooplankton species during flood tide while ebb tide demonstrated less biodiversity in a smaller population according to data collected in February of 2013. The data from North Jetty depicts that at flood tide there is a greater influence of salt water flow moving planktonic organisms up river.  Gaining an understanding of the effect of flow on plankton in this local area will help strengthen students' understanding of the Possession Sound estuary system.

Rhododendrons are plants native to the Northern hemisphere, comprising over 1000 species, most of which have eye-catching flowers. The native Rhododendron of the Pacific NW, R. macrophyllum, harbors a degree of DNA sequence variation exceptional for a single species. Our study focuses on RPB2d, the gene encoding the second largest subunit of RNA polymerase II. Within intron 4 of R. macrophyllum RPB2d, there exist four conserved haplotypes, which have a structured geographical distribution. (Puget Sound differs from Oregon Cascades differs from Oregon Coast.) This surprising pattern of intraspecies variation inspired my project of looking into the degree and pattern of genetic homogenization in these populations by recombination events within and near the RPB2d gene. Thus far, I have sequenced most introns throughout RPB2d and some of the non-coding region upstream. The results have shown that the intron 4 haplotypes are linked to specific variants in intron 1. However, the later introns (6-13, 13-15, and 23-24) are uncoupled from the intron 4 haplotypes and show increasing recombination. About 1 kb upstream, there is a 341 bp DNA sequence inversion in certain plants. An inversion is a segment of a chromosome that has been reattached on the same chromosome in the same location, but in the opposite direction. The inversion found in most RPB2d genes of R. macrophyllum is not found in other species or in haplotypes 1 and 4, the ones most similar to other species. The data continues to show that fewer recombination events occur in introns closer to the inversion. My research is now focused on how the inversion limits the rate of recombination. The next step will be to sample heavily from homozygous populations to see if there is a normal rate of recombination. This regulation could help explain the persistence of varied haplotypes in intron 4.

Drilled oil is becoming increasingly expensive because it is a non-renewable resource that the planet depends on for energy and transportation. Algae may become the next source of global fuel. Chrysochromulina sp. is an ideal model for algal biofuel production because it has a high growth rate in culture, and the two lipid bodies (the organelles that store potential biofuel precursors) are easily detectable using cell biological techniques. The ability of Chrysochromulina sp. to change its lipid metabolism quickly in response to replicated environmental stress conditions is a crucial factor to better understanding its growth optimization and survival. Based on our preliminary data, this microalgae has the potential for production and commercialization of its lipid-based compounds. Guschina and Hardwood’s 2006 studies have shown that lipid production increases in algae under various environmental stress conditions, such as nitrogen limitation, salinity, or increased temperature. Thus, we have investigated the growth dynamics of Chrysochromulina sp. under the following stress conditions: increased salinity, depleted nitrate levels, depleted ammonium levels, and depleted phosphorous levels. For each of these experiments, flow cytometry was used to obtain cell counts and determine growth rate (K’). Neutral lipid content and lipid body size are essential measurements because they represent how efficiently Chrysochromulina sp. produces lipids (i.e. biofuel) and the characteristics of the different lipids being produced. Using Gas Chromatography/Mass Spectrometry (GC/MS), we have elucidated that the lipid profiles change under different growth conditions. The results attained from these studies provide relevant information about the physiology and ideal growth of lipid producing conditions of Chrysochromulina sp. Using this information, we will be able to determine the proteins involved in lipid biogenesis and apply genetic modification techniques to optimize lipid production for commercial use.

Thermostability is the capacity of an organism to survive and thrive at high temperatures and is a characteristic with many biotechnological applications, such as food processing. We are seeking to understand the mechanisms of thermostability in viruses by using an RNA virus, phi-6 Cystovirus. We are evolving the phi-6 virus to become thermostable by exposing it to a target temperature of 61°C over many generations (wild type viruses grow at 25°C). In order to see if exposure to different intermediate temperatures affects the genetic pathway to thermostability, we will expose the phi-6 virus to different temperature regimes prior to reaching the target temperature. The temperature treatments are sudden, where the virus is exposed to the target temperature throughout the entire course of the experiment, moderate, where the virus is exposed to the target temperature halfway through the experiment, and gradual, where the virus is exposed to the target temperature on the final day of the experiment. The genome of phi-6 has already been characterized, which enables us to compare the genome of the evolved, thermostable phi-6 viruses to the ancestor and allows us to study the genetic basis of thermostability. In future studies, we hope to use these thermostable viruses to learn about costs and benefits of maintaining thermostability.

Visual-Motor processing provides a mechanism for animals to translate visual cues into useful information for locomotion. My research aims to investigate this mechanism by analyzing the responses made by fruit flies to certain visual stimuli. I accomplish this by first tracking and quantifying fruit fly locomotion when two flies are present. By quantifying this behavior I can create a behavioral map of fly responses in relation to other fruit flies. I can then replace one of the flies with a cylindrical, fly-sized magnet that is readily controlled by me. This step is important because by controlling the magnet it is possible to reproduce large amounts of a specific behavior, instead of watching two flies interacting randomly and waiting for relevant interactions. It also will allow me to investigate the affects of object speed and angle of approach and create a behavioral map of the fly's locomotor response. Once this is accomplished it will then be possible to test fruit fly responses to visual stimuli presented on an LED screen surrounding the fly and to measure brain activity in the flies while these stimuli are displayed. Ultimately we hope to identify the neurons that are responsible for visual-motor processing and how they accomplish the transition from visual input to locomotor response.

 Flying organisms achieve flight stability by employing a multitude of control surfaces, most notably the wings. However, airframe deformations, such as abdominal motions in the hawk moth Manduca sexta, have recently been shown to play a significant role in stabilizing flight. In my research I have studied the control potential of abdominal deflections using a closed-loop flight arena. Tethered moths controlled the velocity of a projected black bar with their abdominal angle. Image velocity varied according to the difference between the abdominal angle and the set point (relative to the average abdominal angle), scaled by a gain factor. Experimental trials were performed for a ten-fold range of gains at three different set points and consisted of 60s periods during which the moths attempted to stabilize the drifting bar. The moths were capable of stabilizing the image for all experimental conditions, with the highest average performance (50%) occurring at the medium gain and the set point corresponding to the average abdominal angle. Poor performance (<50%) during low gain trials was characterized by steady state error, likely the result of the relatively low image velocities. For high gain trials, the decreased performance (<35%) was characterized by large abdominal oscillations. These results support an active and plastic role for the abdomen in flight control, but also tested the limits of the abdominal control circuit. To continue exploring the role of the abdominal motion I have been working on performing EMG recordings from the abdominal muscles during free flight. For this research the virtual arena serves as a controlled environment to collect preliminary data and to refine the experimental procedure and animal preparation. Finally, I will present my work developing a biometric controller for the Parrot AR.Drone quadrotor. This project utilized a Kinect and custom software to control the quadrotor with body postures.

Hawkmoths, Manduca sexta, feed mainly during early morning and late evening in low light conditions by hovering and tracking moving flowers. The variable lighting conditions in which the hawkmoths feed in nature allow for the perfect setting to examine how visual signal acquisition can affect the performance of motor controlled tasks. By varying the luminance levels, it could result in a change in the amount of time it takes for hawkmoths to react to visual stimuli. In other words, motion-sensing tasks, like tracking a moving flower while feeding, may vary with the background sensory environment. We tested our hypothesis with freely flying moths feeding from a robotically actuated artificial flower at a low luminance level of .3 lux and a high luminance level of 300 lux. Because the flower motion was composed of the superposition of multiple sine waves (0.2-20 Hz), we were able to examine how moths responded at different frequency levels, making it possible to reconstruct a performance pattern. The flower's movement was done in both the vertical and horizontal axes. By calculating the coherence at each frequency, gain, and phase delay, we discovered that moths reliably track at frequencies exceeding 5 Hz. As predicted, we perceived much larger processing delays from the moth's response to the flower's movement at lower luminance levels than higher. This processing delay corresponds to moths being able to perceive and react to visual stimuli 16ms faster at high luminance levels than low luminance levels. At low luminance levels, moths actually overcorrected by overshooting the flower's position at peak tracking frequencies (1-2Hz), possibly due to longer integration delays. Future experiments involve integrating two degrees of freedom by combining multiple axes. Background sensory environment significantly alters the performance of an ecologically-relevant tracking behavior as predicted from sensory neurophysiological mechanisms.

Building a reference library of life on earth is an enormous task of paramount importance given the current threatened state of species across the planet. Collecting DNA barcodes - essentially genetic fingerprints - for every species on the planet has been proposed as a means of rapid species identification. In 2004, the Barcode of Life Database (BOLD) was initiated and is compiling DNA barcodes for every species using a small fraction of the genome called the, "CO1" gene. Unfortunately, the universal primers used to sequence the CO1 gene have a low success rate at producing DNA sequences for amphibians, and a relatively high error rate for species identification. My research investigates the success and accuracy of new amphibian-specific CO1 primers in comparison to an alternative DNA barcode, 16s rRNA, which is widely used in amphibian systematics and taxonomy, but is presently not accepted by the DNA barcoding community. I analyze and compare both CO1 and 16s genes for their utility as DNA barcodes by building phylogenetic trees using maximum likelihood and Bayesian inference to quantify their species identification accuracy. In collaboration with the UW Burke Museum of Natural History and Culture, I sequenced DNA for 162 frogs (representing 22 species) collected from the rainforest in Southern Ghana. The sub-Saharan rainforests of West Africa host a high number of endemic amphibian species. However, increasing habitat threat are heavily impacting species in these forest, and an estimated one third of amphibians are considered threatened. All DNA sequences obtained for this study are being submitted to the BOLD and GenBank databases and are linked to voucher specimens at the UW Burke Museum, which will inform future biodiversity studies, assist monitoring, and aid conservation efforts.

Fatty acids (FA) are commonly used as biomarkers to infer contributions of different basal energy sources to consumers. However, modification, catabolism, and storage of dietary FA are poorly understood for most animals. Controlled feeding experiments are critically needed to determine the appropriate use of FA as dietary tracers. We conducted two such experiments with juvenile isopods to investigate their use as a model organism for FA signature analysis in a direct herbivore. Newly hatched Idotea wosnesenskii were raised for 10 weeks on five different macroalgal diets at two different temperatures. Broods were raised from hatching on a single food source. Highest growth rates were on Ulva spp., fresh Nereocystis luetkeana, and aged N. luetkeana. Animals grew significantly slower on diets with chemical (Fucus distichus) and structural (Mazzaella splendens) anti-herbivore defenses. Temperature did not substantially affect growth rates. Preliminary analyses indicate that FA in Idotea tissues reflect those of their diets, and that FA driving the patterns include common biomarkers for the algae consumed. Juvenile Idotea are a promising model organism for FA trophic ecology because they generally thrive in a laboratory setting and can be hatched and quickly grown on a variety of diets.

Drift algal material is expected to play a significant trophic role in ecosystems adjacent to kelp beds. The contribution of drift algae to the overall nutritional state of various invertebrates can be assessed using phenotypic traits such as gonad index (larger gonad mass with better nutrition) and for sea urchins, jaw diameter (larger jaws with inconsistent food supplies). In this study, we collected green urchins (Strongylocentrotus droebachiensis) within kelp beds (5m depth) in the shallow subtidal photic environment (SSPE) and outside of kelp beds in nearby deep subtidal environments (DSE; 100m depth) during spring and winter seasons. SSPE and DSE sampling stations were less than 1 km apart. In the initial spring analyses, urchins in the DSE had significantly larger gonads but also larger jaws than SSPE animals. Winter gonad indices showed a reverse pattern, in which SSPE urchins had larger gonad indices, but still had smaller jaw indices than the DSE urchins; gonads in all urchins on this sampling date were much larger than those in the previous spring. These results suggest that food supplies may vary among seasons, especially in deep habitats, where the subsidy of algal material exported from shallow water may be inconsistent; the jaws from DSE urchins concur with this hypothesis. Gut content analyses from winter samples will be used to evaluate the types of foods available to deep vs. shallow-water animals.

Since neuronal morphology is linked to neuronal function, the nervous system goes to great lengths to ensure that neurons achieve their proper shape, and failure to do so is associated with many cognitive disorders. However, the precise regulatory mechanisms underlying the coordinated expression of certain genes in a given cell type, at the right amount, and at the exact developmental time is one of the longstanding questions in developmental biology. MicroRNA biogenesis is a common mechanism by which cells regulate gene expression, often by “tuning” levels of target gene products, allowing for fine regulation of protein levels over a broad dynamic range. From a genetic screen for mutations in fruit flies that affect dendrite morphology in sensory neurons, we identified a novel Dicer-1 allele that causes stereotyped dendrite defects, including deregulated growth and defects in dendrite self-avoidance. Dicer-1 encodes a type III RNA endonuclease that is required for processing many types of small regulatory RNAs, including microRNAs. Time-lapse imaging and quantitative analysis of the dendrite patterning in Dicer-1 mutant sensory neurons reveals that there is a significant increase in the number and overall length of dendrite branches as well as an increase in dendrite-dendrite crossing-over, especially at late stages of larval development. Interestingly, mutation in a gene (pasha) that functions upstream of Dicer-1 to process a subset of miRNAs recapitulates the exuberant branching, but not the dendrite crossing phenotype of Dicer-1 mutants. We hypothesize that different classes of miRNAs regulate dendrite branching and dendrite self-avoidance and we plan to use microarray analysis of Dicer-1 and pasha mutants to identify the pathways regulated by these different classes of miRNAs.

During embryonic neural development mouse hindbrains exhibit spontaneous depolarizations that propagate from cell to cell across the tissue in waves. There is a discrete, anatomically identifiable population of cells responsible for generating these waves: the cells of the initiation zone (InZ). Spontaneous waves of activity have been shown to affect neuronal migration, pathfinding, and differentiation, among other developmental sequences. Spontaneous depolarizing events, in general, allow passage of ions into and out of the cell; of developmental importance are calcium ions, which are involved with regulating gene expression. During critical windows of development these calcium fluxes help to determine the adult fate of a neuron. The spontaneous and variable nature of the propagating neuronal events makes them difficult to manipulate experimentally. Were these propagating events able to be elicited experimentally and discretely that would provide both a vehicle for further study and an indication of what properties of the initiator population allows the cells to generate propagating events. I have used a combination of several techniques, including calcium-dye imaging, whole-cell patch clamp, and extracellular electrical stimulation, to probe the details of the cells’ activity. These experiments have identified separate populations of cells that are capable of initiating propagating waves, both spontaneously and elicited by external electrical stimulation. One population includes the previously identified cells of the InZ, and another population exists at the isthmus between the midbrain and hindbrain. Cells in these populations respond differentially to a variety of electrical stimuli. Within each of these initiator populations may exist single, highly-networked neurons capable of initiating propagating electrical activity evoked by intracellular stimulation, which is the subject of further investigation.

During early development, spontaneous waves of electrical activity propagate across many structures in the central nervous system. These waves are believed to regulate neuronal migration, physiological maturation, and synaptic connectivity. In the developing mouse cerebral cortex such waves manifest as increases in intracellular calcium and bursts of actions potentials that occur simultaneously in the majority of neurons in the cortex. We have developed a microfluidic multi-electrode array for use in the study of these waves of activity. This array records extracellular signals from twelve 50 micron microfluidic apertures that can also be used for focal electrical or chemical stimulation. The array is transparent and is compatible with simultaneous fluorescent imaging of intracellular calcium signals at single-cell resolution. We have used this array to study propagation patterns and mechanisms of cortical waves in postnatal (P) day 1 to day 5 brain slices. Waves initiate in the ventral (piriform) cortex and are detected by the array sequentially at each of the apertures placed along the ventral-dorsal axis of the slice, and by simultaneous calcium imaging as the spread of a fluorescence signal along the same propagation axis. The calcium signals outlast the electrical events by several seconds. We used a power spectral density analysis to identify the unique frequency components of different types of cortical waves. The signals from the dorsal regions show a prominent low-frequency oscillation late in the signal that is not present in ventral initiator regions. We have also used this device to study the contributions of the neurotransmitter GABA to network development by investigating a transgenic mouse missing the primary enzyme that creates GABA. Use of this device will lead to a greater understanding of the development of neural networks in the mouse cortex.

Geomyces destructans is a psychrophilic (cold-loving) fungus that causes cutaneous infections in cave dwelling bats and high mortality in North American populations. Geomyces pannorum is a closely related psychrotrophic (cold-tolerant) species that is a rare skin pathogen of vertebrates. Cold-adapted organisms adjust lipid synthesis to lower membrane viscosity and thus survive unfavorable habitats. Lipid profiles, or lipid class composition, may partially explain ecological niche and G. destructans pathogenicity to bats. Additionally, profiles are species specific and may be utilized to differentiate closely related species and detect disease. We incubated Geomyces at 5°, 8°, 15°, 18°, and 22° C and isolated fungal lipid content. Broad lipid classes were determined to be primarily sterols, free fatty acyls (FFAs), and triacylglycerides (TAGs). Geomyces destructans produced higher proportions of unsaturated 18 carbon TAGs than G. pannorum. Geomyces produced more 18:3 (18 carbon, 3 double bonds) TAGs at five degrees than at higher temperatures. Geomyces destructans made higher proportions of TAGs at its growth limits, suggesting alterations in lipid synthesis to decrease cellular toxicity and reproductive effort. Furthermore, these results indicate Geomyces alter lipid structure to survive cold temperatures by increasing lipid unsaturation. Future studies should focus on temperature optima of enzymes involved in TAG synthesis and disruption of lipogenic metabolic processes. Lipid profiles among multiple Geomyces species should be further investigated as a method of disease detection.

White-nose Syndrome (WNS) is a wildlife disease caused by the pathogenic fungus Geomyces destructans that has resulted in the mass mortalities of North American cave bats. One clinical sign of WNS is wing necrosis.  Geomyces destructans may secrete proteases that degrade tissue, thus reducing wing strength and elasticity. We isolated Geomyces destructans extracellular enzymes from an in vitro system and applied enzyme solution to bat wing tissue. The toughness, strength, and elasticity of tissues was assessed with tensile testing. Protease activity was assessed with SDS-PAGE and peptide mass fingerprinting by MALDI-TOF MS. Protein profiles generated by SDS-PAGE indicate higher solubilized protein in treated samples. Major bands were identified as integumentary proteins by MS. Tensile testing did not detect damage, but Geomyces destructans proteases may cleave host integument.

In this project, we explore conditions that are critical for de novo evolution of cooperation and altruism in a bacterial system. Previous theoretical work has shown that there is a competitive advantage to defection and selfishness, but the ubiquity of cooperation in nature suggests that cooperation may be adaptive. Here, we aim to uncover environments and genetic conditions that actually promote greater cooperation. We use an engineered cooperative strain of Escherichia coli. Through a bistable genetic switch, single cells have the capability to be either a producer cell that makes the cellulase enzyme to break down cellulose, or a consumer cell that eats the byproducts of cellulose breakdown. Our first aim is to characterize the social dynamics of this synthetic system and determine if a social dilemma is in fact occurring—i.e., is there a cost to producing cellulase, and can cheaters that do not produce cellulase displace the cooperators that do? Our second aim is to evolve the system over many generations and analyze how it changes. Uncovering the conditions favoring higher cooperation (e.g., greater production of cellulase) provides insight into how cooperation can evolve and how populations circumvent social dilemmas. We hope to use evolution as a tool to tune the synthetic genetic circuit for maximal cellulose breakdown. On a broader scale, these conditions that favor cooperation may have practical applications in waste degradation and biofuel production.

The warmest climatic event of the last 34 million years occurred during the middle Miocene, culminating in the Middle Miocene Climatic Optimum, (MMCO; 17-15 Ma) which was a period of substantial warming. The climate change likely affected plant community composition as grasses spread across North America, but because plant macrofossils from the MMCO in North America are rare, the response of vegetation to this warm interval is poorly understood. We combine analysis of plant silica (phytolith) assemblages and study of stable carbon isotopes from the same paleosols to document vegetation changes during the MMCO in southwestern Montana. Phytolith assemblage analysis provides information about vegetation structure, as well as the composition of grass communities, whereas isotopic data allows determination of the proportion C3 and C4 plants. The site near Drummond, Montana, has previously produced Barstovian-aged mammals and is productive for phytoliths. Our results show that grass silica short cells, diagnostic of grasses, in particular C3 pooid grasses, dominate assemblages (up to 75%). The remainder of the assemblages is made up of phytoliths typical of forest indicators, including palms, woody and herbaceous dicotyledons, and conifers. This suggests a fairly open habitat, with grass communities dominated by C3 grasses. Preliminary stable carbon isotope data are consistent with the phytolith results, indicating dominantly C3 plants (90% or above) in the samples. Lateral and vertical sampling from the site shows very little variation in phytolith composition, indicating low spatial and temporal variability.

Every aspect of nature is governed by the laws of physics. These laws include the basic principle of the center of gravity which is defined as a point in an object’s body at which the total net torque due to gravity equals zero. Understanding and applying this basic principle is crucial for dancers if they are to be successful movers. Yet the popular form of dance known as breaking seems to defy the laws of physics, particularly static equilibrium – a state where an object will not rotate unless an external force is applied because the center of gravity is located above an object’s base of support. Many breakers are able to perform gravity defying movements as they invert their bodies and hold various poses known as “freezes.” Using a series of cameras and a three-dimensional (3D) modeling program, I am able to locate the center of gravity in individual breakers and reveal that their movements do not violate the laws of physics. Using the information collected from this research, I can then propose and create new possibilities for freezes based on the concepts of static equilibrium. This research will be a stepping stone for further biomechanical researches in breaking and other forms of dance. Similar experimental techniques can be used to analyze more complex principles such as dynamic equilibrium – balance while in motion.

The PIRL genes are a family of nine genes discovered during the sequencing of the Arabidopsis thaliana genome, the first fully-sequenced plant genome. PIRLs are a previously uncharacterized class of plant leucine-rich repeat (LRR) proteins that are hypothesized to be important to plant development. As part of an ongoing systematic study to gain insight into PIRL functions, I examined Arabidopsis plants engineered to overexpress the PIRL9 gene. Plants containing an extra copy of PIRL9 designed to drive overexpression of the gene were identified and observed as they grew. Abnormal leaves were observed in most of these plants and stunted growth was quantified through measurements of leaf length and width, as well as primary stem height. Using qPCR, the amount of PIRL9 mRNA present in the different plants was quantified and linked to previous plant measurements. Data showed a correlation between severely stunted plant growth and overexpression of the PIRL9 gene. Significance of these results for PIRL9 function will be discussed, but further genetic and phenotypic analysis is needed to confirm and assess the effects of PIRL9 overexpression. 

MicroRNAs (miRNAs) are crucial regulators of gene expression in plants and animals and play a critical role in many biological processes, including nervous system development. From our previous study of Drosophila melanogaster peripheral sensory neurons, we found that miRNA bantam is both necessary and sufficient in epithelial cells to initiate an epithelia-derived signaling pathway that regulates dendrite growth. Using miRNA sensors, which report the location on miRNA activity, we found that bantam activity is developmentally regulated in epithelial cells. We hypothesize that this developmental regulation of bantam activity is the result of transcriptional regulation of bantam, and we aim to investigate this using bantam transcriptional reporters. To this end, we are developing bantam transcriptional reporter constructs in which the bantam transcript has been replaced by different reporter genes, such as GFP or Luciferase, while preserving the cis-regulatory landscape. We will use these reporters to monitor bantam transcript levels over developmental  time and, pending the outcome of these studies, we will use the reporters to identify trans-acting factors that regulate bantam transcription.

Dendrites are branched extensions of the cell body of a neuron that act to receive and process synaptic or sensory inputs. The elaborate branching pattern of the dendrite is a hallmark of neural type, and has a considerable impact in determining what signals a neuron receives, and how these signals are integrated. Alterations of dendrite morphology can lead to abnormalities in neuron function--dendritic defects are associated with neurological disorders such as Ret syndrome, Fragile-X syndrome, and Down syndrome, and in many cases the dendrite pathologies are progressive. Therefore we set out to identify mutations that result in progressive defects in dendrite growth and stability with the hope that the genes affected by these mutations and the processes in which they regulate will provide new insight into human disease. A series of complementation tests using chromosomal deficiencies, as well as meiotic mapping have been used to map one such mutant (cc215) to a small chromosomal interval. In addition, we have performed a non-complementation screen to determine other alleles that affect the same gene mutated in cc215. Complementation tests and sequence analysis are currently being performed to classify the gene affected by this allelic series, which will allow us to investigate the underlying cause(s) of the dendrite defects in the mutants.

The first floral mutations ever documented, double flowers have been prized for centuries for their beauty. Double mutants known as ‘Shoaf’s Double’ and ‘Cameo’ are present in the species Thalictrum thalictroides, a member of the buttercup family. The purpose of this research is to determine the genetic mutation causing these phenotypes. We predicted that a mutation in a C-class floral organ identity gene would cause the homeotic transformation of stamens and carpels to petalloid sepals in this species. C-class genes are also involved in floral determinacy, the termination of growth in the flower meristem, which is why double flowers appear to have a second flower nested inside the first. As expected, the expression of the C-class gene AGAMOUS1 (AG1) was greatly reduced in our mutants. To pinpoint the mutation we first sequenced the coding region, the part of a gene that becomes a protein. Finding no changes from wild type I further sequenced the entire genomic locus. In AG1, the first intron is known to have regulatory function. I found evidence of the action of a DNA transposable element in both mutants. In “Shoaf’s Double” a Mutator element has inserted itself into the first intron of AG1, potentially disrupting its function. In “Cameo” there is a ‘footprint’ present in the same site, which indicates the same element may have jumped out, deleting a necessary section of the gene. These results show the presence of at least one active DNA transposon in this species, capable of causing a phenotype due to disruption of a regulatory region. This transposable element could potentially be induced to jump and create new mutant lines. Similar mutations could potentially underlie other popular double flower cultivars, such as those found in roses, camellias and carnations.

Neurons are specialized cells that receive and relay information throughout an organism, and the compartment that inputs the signal has multi-complex structures known as dendrites. The form of the dendrite is tightly regulated to neuron function; therefore, any abnormality that alters the structure of the dendritic arbor can lead to severe defects in neuronal function. However, the mechanisms of how dendrites develop and maintain their arborization are poorly understood. From a forward genetic screen for mutant alleles with progressive defects on dendrite patterning we identified one mutant allele that leads to a complete loss of terminal dendrites. We mapped this allele to a small genetic interval, and using complementation tests and DNA sequence analysis we identified Acsl (Acyl-CoA synthetase long-chain) as the gene affected by this mutation. Interestingly, Ascl is also associated with human X-linked mental retardation (XMR), but to date the role that Ascl plays in dendrite development has not been investigated. Here we report the phenotypic analysis of the Acsl mutant, effects of Acsl knock down in neurons, and our plans for functional analysis of Acsl in dendrite development.

The basic functional unit of the nervous system is the neuron, which contains two highly specialized compartments, the axon and dendrite. Dendrites received synaptic/sensory inputs, and the dendrite arborization pattern has a profound influence on the type and number of inputs a dendrite can receive. We are studying dendrite patterning in the Drosophila peripheral nervous system (PNS), with a particular focus on the role that non-neuronal signals play in shaping dendrite development. In the Drosophila PNS, sensory neurons grow on top of a monolayer of epithelial cells, which provide instructive cues to shape dendrite patterning. To date, there are plenty of tools available to genetically label and manipulate neurons, but few tools are available for genetic manipulation of epithelial cells. My goal is to design a reporter construct that will specifically label and facilitate targeted expression of transgenes in epithelial cells. To this end, I am assembling a modular plasmid construct which contains a minimal promoter, an enhancer sequence which we have found to be sufficient to direct epithelial expression, and the coding sequence of green fluorescent protein or other genes of interest. After assembling the construct, I will use germline transformation to generate transgenic flies and test the efficacy of the construct. If successful, this will represent the first epithelia-specific transcriptional reporter, and will provide a template for assembly of additional tools for studying dendrite-epithelia interactions.

We are studying the roles of the p120-catenin protein in zebrafish development. p120-Catenin has a critical role in adhesion and migration by associating with E-cadherins and small, Rho family GTPases. The regulatory domain of p120-catenin contains 8 tyrosine and 8 serine or threonine residues that can be phosphorylated. To date, the function of these phosphorylation sites has been controversial. In cell cultures there is evidence that phosphorylation of 6 serine sites cause adhesions to recover quicker as well as form stronger connections. Some studies, however, claim that phosphorylation of these serine residues has no effect on adhesion. Tyrosine phosphorylation has been associated with nascent adhesions suggesting its role in adhesion formation, while its presence in carcinoma cell lines implies a role in cell motility. By selectively blocking phosphorylation at these 16 residues we hope determine the roles for the phosphorylation of serine and tyrosine residues in adhesion and migration of cells during embryogenesis. We mutated mouse p120-catenins at tyrosine residue 228 to phenylalanine, and at serine residues 252 and 268/288 to alanine via site-directed PCR mutagenesis. We inserted the DNA into PCS2+ vectors containing the fluorescent protein mcherry, and translated the mutant DNA into mRNA. We injected one-cell zebrafish embryos with an antisense morpholino RNA, complimentary to a splice-site region of endogenous zebrafish p120-catenin. Injections of this morpholino effectively knockdown the expression of newly translated p120-catenin mRNAs, and inhibit normal somite formation and axis extension during development. Co-injection of a wild-type mouse p120-catenin mRNA with the morpholino showed nearly normal development. Preliminary data suggests that co-injection of Y228F p120-catenin mRNA with the antisense morpholino RNA also produces nearly normal phenotypes. We will be testing the localization and function of the other mutated p120-catenins. This research could have important implications in development, treatment of some cancer types, and wound healing.
 

 As human development continues to encroach on wild places, wetland bird species lose vital nesting habitats.  To combat that loss, many cities nationwide create urban wetlands, in part to protect or replace lost habitat; it is unknown how effective this effort is.  Previous Canadian studies identified four wetland bird species that do not generally nest in urban wetlands in Eastern Canada: American Coots (Fulica americana), Pied-billed Grebes (Podilymbus podiceps), American Bitterns (Botaurus lentiginosus), and Virginia Rails (Rallus limicola).  This project will compare four urban wetlands with four rural wetlands in King and Snohomish Counties to see if the four species show any nesting preference between urban and rural wetlands.  In addition, the project will investigate if those target species that nest in both urban and rural areas exhibit greater habituation to human presence in urban areas.  I will visit each of the eight wetlands weekly during the morning or early evening and note the presence of courtship or nest-building activity and the number of individuals of each species exhibiting those behaviors.  Using a laser distance device, I will measure the distance between the birds or their nest and the closest human access point to determine relative habituation.  If urban wetlands birds are more habituated to humans, they will nest, on average, more closely to human access points than their counterparts in the rural wetlands.  If urban wetlands are effective as nesting habitat, then mated pairs of the target species will utilize them, demonstrating that small created wetlands can serve as a buffer to habitat loss for wetland birds.

The evolution of multicellularity is one of the most important yet least understood biological phenomena. What selective pressures drive this major evolutionary transition? To address this question, we use a theoretical approach to model the spatial properties of cell groups in a simulation entitled Tree-Multicellularity (TreeMu). In TreeMu a simple multicellular organism is represented as a graph, with nodes representing cells, and edges representing physical connections between cells. Like cells, nodes carry intrinsic attributes, such as reproduction, mutation and death rates; these dictate life processes and define an individual's fitness. Like multicellular groups, graphs have similar properties that emerge from individual contributions. When a node dies it splits the graph in two, analogous to group level reproduction. We explore how the evolution of a node's death rate affects multicellular growth. In the simulation we implement an evolutionary algorithm that selects for 'multicellular' graphs of large sizes by removing small groups from the population. Preliminary data supports a tradeoff between group size and reproduction. TreeMu provides a computational framework to explore the dynamics of multicellular evolution.

The ABC model of flower development explains the patterning of organs along the flower axis. Involving mostly MADS box genes, that partake in determining flora organ identity, this model has been the botanical counterpart to the Homeobox genes which participate in the morphogenesis of animals. The model predicts the production of the four floral organs sepals, petals, stamen and carpels, through various combination of the A, B, and C-class genes. The flowering plant species Thalictrum thalictroides, in the buttercup family Ranunculaceae, belongs to an interesting lineage that is sister to all other eudicots, including the model system Arabidopsis thaliana. T. thalictroides flowers consist of three types of organs: sepals, stamens and carpels, lacking petals. However, the sepals express a petaloid morphology, being large and white or pink. I will be using a forward genetics approach to study a horticultural mutant of T. thalictroides, ‘Betty Blake.’ Its phenotype consists of multiple whorls of non-petaloid (green) sepals and multiple whorls of carpels, resembling B-class gene mutants. We hypothesize that the reason for the lack of stamens, the extra carpels and non-petaloid sepals in ‘Betty Blake’ is a mutation in one or more of the B-class genes. The four B-class candidate genes belong to the MADS box gene family and function in petal and stamen identity. I will be sequencing and analyzing the entire genomic locus of 8 candidate genes, by means of basic molecular biology techniques. A mutation could cause a loss of B-class function potentially, resulting in a homeotic conversion from stamen to carpels and sepals. Molecular dissection of this mutant will contribute to understanding the degree of conservation of stamen identity genes across divergent flowering plants, and to test whether sub-functionalization in the B class gene lineage contributes to petaloidy of sepals.

 Vesicular Glutamate Transporters (vGluts) are proteins that package glutamate into vesicles prior to synaptic transmission. Three isoforms have been identified in mammals (vGlut-1-3), with 1 and 2 showing a mostly complementary pattern of expression, and 3 co-localizing in a small number of cells with both 1 and 2. Knockout mice for the vGlut1 gene die shortly after weaning, indicating a crucial role for vGlut1 in development. Intriguingly, avian genomes contain no vGlut-1 orthologs, despite the fact that lizards, a common ancestor of both mammals and birds, have orthologs of both vGlut-1 and -2. The fact that birds function without vGlut1 suggests there may be an increased functional role for vGlut2 in the avian brain. To further demonstrate the presence and potential role of vGlut2 in avian pallial circuitry, we used immunocytochemical methods to study the brain distribution of vGlut2 protein in zebra finch. To confirm the specificity of antibodies to vglut2, we first performed co-localization of anterograde tracer with vGlut-2 in known glutamatergic terminals in RA that originated from pallial song nucleus HVC. Electron microscopy further confirmed that labeling was restricted to synaptic terminals containing vesicles, and in situ hybridization was used to confirm the presence of mRNA in somata. We found that a large number of vGlut2-positive cells are present throughout the zebra finch pallium, and also found evidence for vGlut-2 positive synapses in three different song nuclei (nucleus HVC, Area X, and robust nucleus of the arcopallium), which were imaged and analyzed for size, and the potential identity of post-synaptic elements. Our results are consistent with the notion that avian vGlut2 has largely taken over vGlut1 functions. We believe our study contributes important facts to the long disputed evolutionary relationship between the avian pallium and the mammalian cortex.

The circadian system controls daily rhythms of behavior and physiology, including locomotor activity and motor-task performance. The master regulation of these rhythms is achieved by a circadian clock located in the suprachiasmatic nucleus of the hypothalamus; however, it is not clear how motor tasks are programmed by the motor cortex at different times of the day. Our hypothesis is that similar motor tasks executed at different times of the day may require different motor programs to account for the daily variance introduced by the circadian system. How and where in the brain this variance in motor control manifests has not been established. We propose to identify specific primary motor cortex activity patterns associated with specific motor outputs across the 24-hour day. We implant electrocorticographic (ECoG) electrodes onto the motor cortex of mice and record brain wave activity during wheel running and rest. We first test whether the motor cortex may show a brain-wave signature for wheel running, and second whether this signature may change predictably across the 24-hour day. Initial results indicate a broad-spectrum power increase in brain-wave output from the motor cortex associated with wheel running. Furthermore, there appears to be a circadian modulation of the power of specific ECoG frequencies. The decoding of motor cortex signals is at the core of the design of brain-machine interfaces (BMIs). These devices decode signals from the conscious brain to drive the execution of specific tasks by a machine, such as an artificial limb. Our work will directly contribute to our understanding of how the motor cortex decodes circadian time. This knowledge is essential to create BMIs that can operate effectively throughout the 24-hour day to execute tasks by brain-operated artificial devices.

Cooperative interactions are observed at all levels of biological organization. Genes cooperate to form genomes, bacteria aggregate to form biofilms, and individuals organize into societies. The formation of cooperation facilitates the transition from simple forms of life into higher levels of biological complexity. In these systems a cooperator enhances the fitness of its partner at its own expense. Yet Darwinian evolution favors selfish acts which promote individual fitness, rather than selfless acts which incur a cost to the altruist. How could cooperation evolve under this paradigm which, seemingly, is its antithesis? To address this question, I have used a synthetic cooperative system, amenable to genetic and quantitative analysis, termed CoSMO (Cooperation that is Synthetic and Mutually Obligatory), as a model for incipient cooperation. CoSMO is an engineered system consisting of two reproductively isolated Saccharomyces cerevisiae strains: a red-fluorescent strain that overproduces adenine but requires lysine, and a green-fluorescent strain that overproduces lysine but requires adenine. Evolved CoSMO cocultures are able to survive significant reductions in population density. We have determined that adaptations in the lysine-requiring, adenine-overproducing cooperator are sufficient for this system level improvement. Phenotypic analyses revealed that evolved CoSMO cooperators exhibit increased tolerance to limited lysine, and increased adenine release. These evolved phenotypes are attributed to mutations in ECM21 and RSP5, genes controlling ubiquitin-mediated endocytosis of membrane transporters. These mutations facilitated adaptation to nutrient limitation by stabilizing the high-affinity lysine transporter LYP1 on the plasma membrane. Simultaneously, they increased adenine release to the partner via membrane stabilization of the purine-cytosine permease FCY2. Thus, pleiotropy, the influence of one gene over multiple phenotypes, can serve as a mechanism to stabilize incipient cooperation. Through mutations in pleiotropic genes, genetically optimizing self-interest during the cooperative interaction still allowed individuals to directly benefit their partner, thus allowing cooperation to persist under Darwinian selection.

Darwinian natural selection produces an organism that is adapted to its environment, an organism whose traits (phenotype) are tuned in critical ways to its habitat. If organisms and their phenotypes are so finely tuned to their environment, how can they deal with changes to that environment? One strategy is to evolve the ability to change phenotype in response to a change in the environment, or phenotypic plasticity. Theory predicts phenotypic plasticity to be adaptive when (1) organisms experience different environments either spatially or temporally and (2) different environments favor different phenotypes. In some cases, changes may be accompanied by cues that provide reliable information about future selection. Previous studies modeling adaptive plasticity suggest plasticity to be favored when the environmental cue always predicts the correct selection and not when the environmental cue is unreliable. We experimentally tested theoretical predictions about the de novo evolution of adaptive phenotypic plasticity with a clustering phenotype of baker's yeast by selecting alternately for large or small clusters. Selection for size was cued by alternate forms of growth media and this cue was either a reliable predictor of future selection or an unreliable predictor. After a preliminary run of the experiment, we have found indications of an emergent plastic response in one of our replicates from the reliable cue treatment. This provides a biological example that is consistent with accepted theoretical predictions.

In evolutionary history, distantly related taxa have often independently evolved the same or similar complex structure as a solution to a common problem presented by their environment. One such example is the plagiaulacoid tooth—a laterally compressed shearing blade that convergently evolved in four clades of mammals, some extant and others extinct. To better understand the selective forces that led to the convergent evolution of this specialized trait in multiple lineages, I investigated how this tooth form correlates with diet in a sample of extant taxa. Specifically, I measured complexity of lower cheek tooth rows of 15 species from five families of extant marsupials with plagiaulacoid teeth. To quantify dental complexity I created digital 3-D models from microCT scans of these specimens and used GIS software to generate orientation maps of the tooth surfaces. Contiguous pixels with the same orientation are considered a patch on the tooth surface, and the number of patches approximates the number of shearing surfaces available to mechanically process food. Previous studies of placental mammals have shown that increases in Orientation Patch Count(OPC) (i.e., increases in tooth surface complexity) correlate with increased consumption of plant material. However, I found no correlation between OPC value and diet as recorded in the literature for these taxa. I then compared complexity of the blade to complexity of the molars to investigate the functional contribution of the plagiaulacoid tooth and again found no correlation with diet. Broader sampling will help determine the pattern of tooth complexity and diet in marsupials, but the range of OPC values generated in this study suggests that the convergent evolution of the plagiaulacoid phenotype is not a simple case of one morphological adaptation to a particular environmental problem.

Wnt signaling is known to be critical for proper embryonic development in most animals studied to date. But key evolutionary questions on the origin and evolution of this pathway in the metazoan common ancestor are still unresolved. Recently, the genome of Pleurobrachia bachei, a member of the early branching metazoan linage ctenophora, has been sequenced. Insights into the function of the Wnt pathway in P. bachei will provide information on early evolution of this key pathway. Three Wnt ligand genes were identified in P. bachei and cloned for in situ hybridization. These genes showed expression in the combs, tentacles, mouth, ciliated grooves and polar fields of the adult P. bachei. In a genomic search for other members of the canonical Wnt pathway, components of the destruction complex and antagonists were incomplete or missing from the genome. High levels of Wnt expression in the adult P. bachei indicates that Wnt could also be playing a role in signaling in the adult.

In typical ascidian development, a tailed larva hatches, swims, becomes competent, and then responds to settlement cues, that initiates metamorphosis. Metamorphosis triggers apoptosis, which is required for tail loss. At this time, the notochord is lost as the tail reabsorbs and the juvenile settles to a substrate. In the ascidian family Molgulidae, species have evolved tailless larval development at least four times independently from a tailed ancestor. We hypothesized that a heterochrony in metamorphosis is responsible for the evolution of tail loss in the tailless ascidian larvae. We show that there is early expression of metamorphosis genes in Molgulidae, including genes involved in the Mannose Binding Lectin (MBL) Complement pathway of innate immunity. We hypothesize that the maturation of the immune system early in tailless larvae could be a progression to competency and trigger to metamorphosis. Jeffery (2002) showed programmed cell death happens in cells that form a tail early in development. We further hypothesize that a heterochrony in metamorphosis promotes the early activation of programmed cell death in the tailless ascidian larvae. Using next generation sequencing techniques, we sequenced transcriptomes of a tailed and tailless species and their hybrid through development. We examined genes that have been shown to be expressed during metamorphosis in other ascidians and saw earlier expression of genes critical for metamorphosis in both species. Our ultimate goal is to elucidate the ascidian molecular trigger for metamorphosis and uncover whether heterochrony of metamorphosis leads to tailless ascidians.

Flowering in plants is induced and tightly regulated through the use of internal circadian clocks that respond to changes in day length and light quality.  In the model plant, Arabidopsis thaliana, the molecular mechanism behind flowering induction relies upon expression of the CONSTANS (CO) gene and subsequent CO protein stabilization.  Daytime CO expression occurs strictly under long day (LD) conditions and is reliant upon removal of the transcriptional repressor CYCLING DOF FACTOR 1 (CDF1) from the CO promoter by a complex consisting of FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) and GIGANTEA (GI).  Following FKF1/GI mediated induction of CO expression, CO protein is stabilized in the LD afternoon through direct binding to FKF1.  Our most recent unpublished data show CO stabilization is also crucially dependent on the presence of GI protein.  However, the mechanism by which GI interacts with CO has yet to be determined.  We hypothesize that GI is involved in CO stabilization through either binding to CO independently of FKF1 or via an FKF1/GI complex.  Through the use of molecular genetics and nuclei-specific protein analysis, we aim to elucidate the mechanism by which GI interacts with CO, and furthermore, establish GI as a regulator not only in expression of CO mRNA, but also in stabilization of CO protein.  

The presence of fungal symbionts residing in the root tissue of plants is a well-documented occurrence, yet questions regarding the identification and comparison of fungal partners in mycorrhizal relationships have been largely unanswered. Ericoid mycorrhizae, an example of a mycorrhizal relationship, are found in host plants within the order Ericales. Ericales, which contains such familiar species as persimmon, blueberry and Rhododendron, are able to persist in edaphic conditions due to their fungal symbionts. These symbionts form hyphal coils inside plant cell membranes, and thereby exchange crucial nutrients with the host plant. This project aims to evaluate the specificity between fungal communities and their host Rhododendron species, with the expectation that differing communities may exist, even in closely related hosts. Using known techniques to extract fungal DNA from the root systems of Rhododendron species in varying conditions and proximity, this DNA is then used to generate species based communities within specific Rhododendron hosts. By using type-cultures and genomic sequencing, comparisons of the presence or absence of fungal species within host roots can be made. This will shed light on infection intensity, and specificity between roots and fungal symbionts. Anticipated results of high levels of specificity between host plant and fungus could prompt questions regarding the importance of fungal symbionts in genus Rhododendron, especially with regards to the speciation between individual host plants.

Phylogenetics is the study of evolutionary relationships and in order to infer these relationships in plants, various loci that are phylogenetically useful in nuclear as well as chloroplast DNAs should be utilized. Presently, the use of chloroplast loci is widespread in phylogenetic studies whereas the nuclear genome is still somewhat under-utilized. The pentatricopeptide repeat (PPR) gene family is a large group of protein coding nuclear genes that has been shown to be highly informative in the inference of evolutionary relationships among closely related species. There are multiple reasons that make these genes useful to these types of studies. The PPR gene family is very large which offers researchers multiple loci that are available for phylogenetic analyses. This is important because having multiple loci is imperative to answering important phylogenetic questions. A large portion of PPR genes are intronless making it possible to sequence and align data with little or no difficulty. They also have a high rate of evolution, and are single-copy in most plant genomes. 127 of these loci have been identified as phylogenetically useful but of these only 5 have been developed. This research looks to develop 10 more loci that will specifically target the Lamiales. To do this, 10 primer sets were tested using a sampling of species across the Lamiales. The primers were used to amplify these loci in order to ascertain their usefulness for a wide range of species and to obtain sequence data for each species. The sequences obtained were aligned and the alignments were used to design new primers. These new primer sets will allow for new data to be more easily collected that has been either difficult to obtain or previously unavailable and will increase the number of nuclear loci available for phylogenetic studies.

 The diatoms are a prolific and productive group of phytoplankton belonging to the phylum Heterokontophyta. They are responsible for ~30% of oceanic primary production and their high photosynthetic capacity depends on their abilities to synthesize chlorophyll. The penultimate step in the biosynthesis of chlorophyll is catalyzed by the enzyme ‘light-dependent protochlorophyllide oxidoreductase’. To understand the evolutionary history of the gene (por) encoding this enzyme (POR) we have constructed an algal por gene tree using diatom por genes as well as those from sister taxa and distantly related taxa. This process involved the cultivation of various strains of algae, DNA extraction, PCR, gene sequencing, data mining from public databases, and bioinformatic analysis. We found two distinct por genes (por1 and por2) within the diatoms and a single haptophyte alga. We propose three hypotheses as to the origin of dual por genes in diatoms: (1) a por gene duplication event occurred before the divergence of the Heterokonts and Haptophytes; (2) a por gene was laterally transferred to a common ancestor of these two algal taxa; or (3) reciprocal lateral gene transfers occurred between these two algal taxa. Whereas the first two hypotheses are more parsimonious, we are unable to determine which scenario is correct with the current dataset. Nonetheless, these data provide evidence for a shared common ancestor between the heterokonts and haptophytes, a hypothesis that is currently hotly debated. Furthermore, the presence of these two por genes in various algal taxa presents a rich opportunity to study the evolutionary forces that either modify or maintain cellular gene function after gene duplication or lateral gene transfer. Thus, the results of this study will soon be complemented with research regarding the enzymatic function and regulation of the two por genes in diatoms.

Lipid bodies are cellular organelles involved in lipid production and storage within cells. Until recently, lipid bodies were thought of as inert storage units rather than complex organelles that play a large role in lipid biogenesis. The freshwater haptophyte Chrysochromulina sp. has served as an excellent model organism for research on lipid bodies because of its high growth rate, fully sequenced genome and two large, distinct lipid bodies. In algae and other eukaryotes, heat shock proteins can be investigated to detect when the cell is stressed. In many species of algae, when the cell population is placed under stress, the cells produce large lipid bodies. A classic stress response has also been detected in Chrysochromulina sp. after discovering genes coding for heat shock proteins (HSP 70 and HSP 90). Previous studies show that HSPs appear to be induced under high salinity stress in Chrysochromulina sp. The current study examines two important aspects of the correlation between salinity stress and lipid body production: what concentration of salt and what time frame is needed to cause haptophyte cells to become stressed enough to produce large lipid bodies. Previous experiments show that adding salt to algal media during log phase of growth fails to produce large lipid bodies while adding salt initially to media does not produce significant biomass to allow protein assays. Since HSPs are induced under high salinity concentrations, the second part of this study aims to determine a correlation between lipid body size, lipid production and how/when HSP proteins are induced. This study uses flow cytometry, the neutral lipid stain BODIPY 505/515 and Western Blotting to examine the relationship between lipid bodies and HSPs. Future research projects may include manipulation of stress response pathways that are relevant for biofuel applications.

Darwin and Wallace recognized natural selection as an essential force in evolution over 150 years ago. Today, we still investigate the processes that drive evolution and the origin of new species. Two sister species of wildflower in the genus Mimulus (monkeyflowers) present an ideal case for studying the genetic and ecological basis of speciation. M. lewisii and M. cardinalis coexist in overlapping ranges in the Sierra Nevada Mountains, and although they successfully interbreed in the laboratory, hybrids are extremely rare in the wild. Each flower species is visited by a unique pollinator – M. cardinalis by hummingbirds, M. lewisii by bumblebees. This indicates that traits influencing pollinator choice and pollination success account for reproductive isolation. We hypothesize that these interspecific differences can be traced to single genes with large effects on flower traits, such as color, that pollinators use in making the choice of which species to visit in the field. If our hypothesis is supported, this would demonstrate that single genes of large phenotypic effect are directly responsible for the origin of new species. Field work indicated that hummingbirds prefer red flowers (high amounts of purple and yellow pigment) while bumblebees prefer pale pink (small amounts of purple pigment). Using a genetic technique called positional cloning, my main contribution to the project was to locate a novel gene that regulates the amount of purple pigment (anthocyanin) in Mimulus flowers. We named this gene ROSE INTENSITY1 (ROI1), verified its identity and function by creating transgenic plants carrying the alternative M. lewisii and M. cardinalis alleles, and showed that ROI1 represses transcription of the genes that encode enzymes for anthocyanin biosynthesis. Our findings are being published in the May issue of Genetics. Future directions include quantifying the effect of ROI1 on pollinator visitation in the Sierra Nevada Mountains.

Auxin is the plant hormone, guiding phototropism, gravitropism, cell elongation, cell division, cell determination, and a host of other processes. Plant cells respond to auxin by turning on certain genes. When auxin enters the cell, it binds to a receptor protein; this receptor then binds to a repressor protein which typically prevents expression of auxin genes. This interaction triggers repressor breakdown and gene expression. We are investigating auxin-induced interactions between the receptor and repressor proteins (IAA’s). In our model plant, Arabidopsis, there are 29 IAA repressor proteins and 6 receptor proteins. Some combinations of repressors and receptors are known to result in differential degradation of the repressor; other interactions do not seem to lead to degradation. Our data suggests that receptors AFB4 and AFB5 cannot degrade repressors. This could be because AFB4/5 do not interact with the repressors (hypothesis 1) or because their interaction with the repressors does not trigger degradation (hypothesis 2). We are testing these hypotheses by examining repressor/receptor interactions in Arabidopsis proteins expressed in budding yeast cells. To address hypothesis 1, we are using a yeast two- hybrid assay to investigate the strength of interaction between several IAA repressors and the AFB4/5 receptors in the presence of auxin. To address hypothesis 2, we will determine whether IAA: AFB interactions identified at the yeast two-hybrid assay lead to repressor degradation. We have tagged the IAAs with yellow florescent protein and will co-express them with AFB4/5 in yeast. Flow cytometry will be used to measure changes in florescence levels upon addition of auxin. The next step will be to confirm these findings in plants. Understanding the variation in auxin repressor/receptor interactions brings us a step closer to understanding how auxin drives so many different plant behaviors and responses.

Waves of synchronous spontaneous activity (SSA) propagate throughout the nervous system during early stages of development. These waves consist of simultaneous action potential firing across large cell populations, with correlated increases in cellular calcium levels. Specific types of calcium signals mediate different developmental processes in the brain. While calcium signaling is known to regulate activity-dependent developmental programs that help to finalize the maturation and connectivity of neurons, the particular function of calcium waves in neuronal migration is unknown. The aim of this project is to determine the contribution of calcium waves to the development of cortical inhibitory interneurons. To label inhibitory interneurons in the developing cortex we are using red fluorescent protein (RFP) under control of the dlx5/6 promoter region to drive RFP expression in these cells. We used a fluo4 calcium indicator to measure single cell activity of these interneurons. We have found both cortical interneurons that participate in asynchronous activity mediated by L-type calcium channels, and also cells that participate in synchronous activity mediated by synaptic mechanisms. We seek to determine whether the asynchronous interneuron population becomes synchronous at the time of migration termination and whether this shift in the synchronicity of calcium activity is a mechanism for migration termination.

Red-tailed Hawks (Buteo jamaicensis) and American Kestrels (Falco sparverius) are sit-and-wait predators that rely on perch-sites to forage efficiently. Overwintering Red-tailed Hawks and American Kestrels use available perches (i.e., utility poles and wires, trees, fences, gates, etc.) to hunt for prey items in the agricultural fields in Northeast Arkansas. Observations were made from December 2011 to the present on three representative cover types: short rice stubble, soybean stubble, and fallow areas including roadsides in order to determine which perch-sites were used by Red-tailed Hawks and American Kestrels the most. Prey density and vegetation cover were also estimated in each cover type. Utility pole crossbeams at a height of 6.3 meters are the main perch-site used by Red-tailed Hawks, demonstrating the use of man-made structures as perch-sites. These perches were generally in or near short rice stubble fields, which were found to have the lowest amount of vegetation cover, and low prey density. Conversely, American Kestrels most used utility wires at a height of 4.9 meters from the ground, over fallow roadsides as perch-sites, representing an area with high prey density and vegetation cover. Although there have been documented cases of inter-specific competition between these two species, Red-tailed Hawks and American Kestrels may limit direct interaction via differential uses of perch-sites. The study gained insight into the behavioral ecology of two competing raptors in northeast Arkansas.

Cooperation occurs in the natural world between molecules, cells, individuals, and communities. In cooperative systems, both cooperating partners benefit from each other. To discover how cooperative systems evolved from its incipient state, we utilized an engineered system called CoSMO (Cooperation that is Synthetic and Mutually Obligatory) as a model for incipient cooperation. CoSMO consists of two non-mating yeast strains that are reliant on the other’s metabolite. CoSMO requires a minimum total cell density in order to be viable, i.e. able to grow from low to high cell density in the absence of supplemented metabolites. Evolved CoSMO cocultures are viable at significantly lower starting cell densities compared to their ancestors. What changes facilitated this increase in the system’s viability? We hypothesize that there will be changes in the yeast’s genome that accounts for this change in viability. To address this, we will perform genome-wide re-sequencing on evolved isolates to identify relevant mutations that contribute to increased system viability in evolved CoSMO. Specifically, we will extract genomic DNA of evolved cooperators at early and late time points during their evolution. We anticipate that there will be mutations in the genomic sequences of these strains. We will then compare these genomic sequences to their ancestral sequence and couple the identified mutations with phenotypic changes such as increased affinity for metabolite and starvation tolerance. We will perform similar analysis on replicate lines to elucidate the diversity in evolutionary trajectories of incipient cooperative systems. Through this, we hope to learn how an incipient cooperative system could evolve to an improved state.

The host seeking behavior of disease-vector mosquitoes may be impacted by olfactory and visual learning. By enabling mosquitoes to better find bloodhosts, this behavior may allow mosquitoes to remain longer in the environment, transmitting diseases to more organisms. Recent work has shown that mosquitoes respond to Pavlovian conditioning that pairs visual and olfactory cues with an unconditioned stimulus. However, the function and relative importance of each sensory system has not yet been explored. We investigated both subjects using adult and larval Aedes aegypti. Firstly, we conducted appetitive conditioning experiments on Aedes adults. We found that adults were able to learn associations between novel odors and blood-feeding: they recruited to the conditioned odor consistently and significantly more than the odorless location choice. In contrast, naïve animals showed no innate preference to these odors. Secondly, we conducted aversive conditioning experiments on Aedes larvae. We compared the learning capabilities of wild-type specimens and specimens molecularly (RNAi) or chemically altered (receptor antagonists) to decrease response to dopamine, a critical neuromodulator involved in the aversive learning pathway. Our assays included a conditioned stimulus of indole and multiple unconditioned aversive stimuli (e.g., salt, bitter tastants). We found that wild-type Aedes exhibited a consistent behavioral change after conditioning, whereas transgenic or pharmacologically altered Aedes larvae did not. Finally, we analyzed threat-sensitive larval learning. We tested larval mortality in treatments containing a natural predator (a dragonfly nymph). We found improved survivorship in trained control-injected larvae versus dopamine antagonist injected larva. This is consistent with our data that lowering dopamine sensitivity alters an important pathway in the Aedes larvae olfactory systems to learn and recognize threats. These experiments suggest that both appetitive and aversive learning may be important to mosquito behavior. Our data also supports the conclusion that learning in mosquitoes is at least in part mediated by dopamine.

The goals of this project are to produce transgenic maize and wheat tissue expressing a gfp marker in chloroplasts and to regenerate homoplasmic plants from the transformed tissue. There are several advantages of transgenic chloroplasts, including multiple copies and high expression of the transgene without gene silencing. In addition, plastid DNA (ptDNA) is maternally inherited so that the transgene will not be present in pollen, thus providing containment for the transgene. We used circular and linear vectors that contain ptDNA sequences corresponding to the ends of the plastid chromosome and the gfp transgene. We used particle bombardment to deliver the vector to plastids of maize and wheat. Transgene expression was assessed by fluorescence microscopy and imaging of GFP in plastids. In addition, both standard PCR and quantitative PCR were performed using DNA extracted from the tissue and primers for the gfp transgene. Although we achieved successful plastid transformation for both maize and wheat, the tissues were heteroplasmic, containing both wild type and transgenic ptDNA. We found that a linear vector gave higher transformation efficiency than a circular vector. The next step is to develop procedures for generation of homoplasmic maize and wheat plants.

Select proteins from the HSP70 gene family were studied in Anthopleura artemisia and Anthopleura elegantissima, two intertidal sea anemones found in Puget Sound. A. elegantissima are clonal aggregates that are found on rocks while A. artemisia live buried, solo within sand. HSP70 are a family of heat shock proteins that are present in prokaryotes and eukaryotes that regulate various protein functions such as heat stress or protein folding. PCR techniques were utilized to determine the presence of HSP70 genes. It is predicted that both species have similar or identical genes that produce HSP70 heat shock proteins, but differ in how the HSP70 genes are expressed. In prior studies, A. elegantissima are found to express HSP70 proteins depending on position in a colony (edge versus central positions). A. artemisia, allows for an interesting comparison as to how living conditions would affect the expressions of HSP70 proteins. RT-PCR techniques were utilized to determine HSP70 gene expression in both species. Variations of HSP70 expression can be used to generate models to predict the effect of climate change in the Puget Sound area.

Mosquitoes are the principle disease vector of malaria, yellow fever, and dengue fever, which are responsible for more than a million deaths worldwide annually. This makes controlling transmission a global health priority, and underscores the importance of understanding their behavior. Although it is known that olfaction is the main sensory modality mosquitoes use to select prey, many behavioral patterns, such as changing prey preferences, suggest that associative learning has a role in mosquito behavior. Previous research has established mosquito’s affinity for human scent and characterized their olfactory receptors, but little is known about the neural substrates and molecular underpinnings that modulate olfactory learning. To gain insight into the importance of olfactory learning, I have been introducing controlled perturbations into the larval mosquito nervous system and assaying their behavior using a classical conditioning model. This is achieved through genetic manipulation and injection of drugs with known targets. Initially, I have focused on the role of dopaminergic neurons, because they are known regulators of associative learning in other species. To this end, I have developed microinjection techniques that allow for delivery of silencing RNAs (siRNAs) to knockdown dopamine receptor expression. Using this approach, I have found that Dopamine receptor deficient animals, as well as those injected with Dopamine antagonists, are unable to learn like their untreated counterparts. Knockdown animals suffer a much higher mortality rate in survival experiments that test their ability to avoid predation, underscoring the importance of these receptors in vivo. Currently, we are using cell biological approaches to visualize the relevant neurons. Together these data show that larval mosquitoes are able to learn within days of hatching, and that dopamine plays a crucial role in facilitating aversive learning. In addition to clarifying the mechanistic basis for olfactory learning, these findings may ultimately enhance the development of vector control strategies.

Circadian rhythms like the sleep-wake cycle are governed by a circadian clock located in the suprachiasmatic nucleus of the hypothalamus (SCN). This clock is synchronized by the light-dark (LD) cycle and the cyclic-guanosine monophoshate (cGMP) pathway is known to mediate its phase advances. Phase-advancing light pulses increase the levels of cGMP in the SCN and inhibition of phosphodiesterase-5 (PDE5), the enzyme that degrades cGMP, by artificial drugs such as sildenafil, enhance light-induced phase advances. These inhibitors act by inducing PDE5 phosphorylation and converting it to phosphorylated-PDE5 (phPDE5). This evidence suggests that the inactivation of PDE5 through its phosphorylation could be a mechanism through which light induces phase advances, and that sildenafil utilizes the same mechanism to enhance phase advances upon light stimulation. We are testing this hypothesis by measuring PDE5 and phPDE5 in the SCN of mice treated with a phase-advancing light pulse. Mice are administered either a light or a dark pulse at the time that light causes phase advances. Animals are sacrificed at different times after the presentation of the pulses, their brains are dissected and alternate sections are processed for immunohistochemistry against either PDE5 or phPDE5. Our hypothesis predicts an increase in phPDE5-expressing cells in light-treated animals. Future experiments will determine if sildenafil- induced enhancement of phase advances are mediated by further increasing phPDE5. Our findings may lead to the development of pharmacological treatments to treat circadian rhythm disorders such as insomnia, fatigue, and jet lag, as well as to manipulate circadian rhythms to achieve periods of extended wakefulness and alertness.

Currently there are three major classes of known viruses: exclusively RNA viruses, which do not require a DNA intermediate in their replication and propagation; DNA viruses, which use DNA dependent processes to perform their genome replication; so-called retroviruses, which go through both an RNA and a DNA phase with the help of a reverse transcriptase enzyme. Until very recently there was no evidence that there was genetic exchange between these distinct virus classes. However, a single stranded DNA (ssDNA) virus genome, recently found using metagenomics in Boiling Springs Lake in Lassen Volcanic National Park, USA (BSL) contains elements homologous to both ssRNA and ssDNA viruses. The genome of this RNA-DNA hybrid virus (BSL-RDHV) contains a gene similar to that of ssDNA circoviruses that infect animals and a capsid protein (CP) gene similar to that of the ssRNA tombusviruses that infect plants. It is currently unclear how the RNA virus capsid gene integrated into the DNA virus genome and what, if any, evolutionary impacts are associated with this recombination. My current work focuses on the characterization of the BSL-RDHV CP using recombinantly expressed protein. The CP gene will be amplified from the BSL viral metagenome using Polymerase Chain Reaction and expressed in bacterial cells. The long term goal is to determine the structure of this protein using X-ray crystallography and use the protein for in vitro assembly reactions. Successful completion of this work will be an important step towards understanding virus evolution and possibly the emergence of novel virus disease.

The development of best practices in instruction, defined by student learning outcomes, requires evaluation of student engagement. How can engagement be measured? Student engagement or disengagement is manifested in body language and behavior. Educational studies that employ behavior to evaluate engagement often subjectively rely on a classical perception of how an engaged student behaves; however, recent work suggests that traditional engaged-behavior patterns can fail to account for student-reported engagement, as attentiveness may have many manifestations. Our research pioneers an objective approach to identifying and evaluating student behaviors that function as accurate indicators of engagement level. Using video recordings of Biology 200 students in lecture, student behaviors were catalogued. These same students were surveyed to determine their perceived engagement. The completed catalogue indicated both the presence and frequency of behaviors unique to individual target students, allowing us to compare students’ self-reported engagement level with their corresponding behaviors. The combination of in-class engagement self-assessment and objective cataloguing of behaviors presents the opportunity for quantitative analysis of each behavior’s ability to serve as a hallmark of engagement. Analyses are in progress to determine the predictive power of classical engaged-behavior patterns, and to identify the behaviors most strongly correlated with student-reported engagement. Our purpose is to provide the educational field with a method for analyzing student behaviors as a means for quantifying engagement, and to lay the groundwork on which a widely applicable engagement evaluation tool can be developed in the future.

During the first week following birth in mice, waves of synchronous spontaneous activity (SSA) initiate in the ventral piriform cortex, and some propagate past the rhinal fissure into the dorsal cortex. These large-scale waves of electrical activity, involving a vast number of neurons firing action potentials simultaneously, are critical for the early postnatal stages of cortical development. By examining what combination of cellular and emergent network properties cause these regional activity differences (the initiation of waves in the ventral cortex and the participation of waves in the dorsal cortex), we have gained insight into the generation of SSA. Cellular activity can be observed by imaging calcium transients, which serve as an indicator of electrical activity. In the present experiments we demonstrate that ventral neurons are more asynchronously active between waves of SSA than are dorsal neurons. In order to determine what portion of the activity is an autonomous property of the neurons (i.e. independent of synaptic communication), we applied synaptic receptor antagonists during calcium imaging to eliminate synaptic transmission between cells. In the presence of synaptic blockers, we observed a greater reduction in activity in the dorsal cortex, whereas many ventral cells remained highly active. Our findings suggest that ventral neurons have a higher probability of being active in the absence of synaptic input, which supports the hypothesis that they may be more likely to initiate SSA if a sufficient number become synchronously active by chance. Since cortical interneurons are believed to play a key role in generating cortical waves of SSA, we are currently performing a similar analysis using a transgenic mouse, in which the interneurons express a red fluorescent protein, TdTomato. By determining if interneurons are more intrinsically active than other neurons in the ventral cortex and if they tend to initiate waves based on their temporal firing patterns, we can gain a greater understanding of the interneurons role in SSA.

Essentially all growth and developmental processes in plants require action of the hormone auxin. Auxin regulates gene expression by mediating the degradation of repressor proteins called IAAs. A subset of IAA proteins are involved in the initiation of new lateral roots, which are important for overall root architecture and function. Members of the Nemhauser lab have shown that proteins within this subset from Arabidopsis thaliana have varying auxin-induced degradation rates, using degradation assays in a heterologous yeast system. I hypothesize that differences observed between the degradation rates of the IAAs contribute to the specificity and diversity of auxin responses. When one of these lateral-root expressed IAA genes, IAA28, is mutated so that it is completely unable to be degraded, the plant does not form lateral roots. I predict that small changes to the degradation rate of IAA28 will lead to differences in plant phenotype, specifically the developmental stages and numbers of lateral roots that develop, as is suggested by preliminary data from work with IAA14. Currently, I am engineering and testing modified versions of IAA28 with the goal of obtaining a range of degradation rates. It is difficult using plant-based system to measure the degradation rates of modified IAA28 proteins due to feedback and the inability to control auxin. To test the degradation rates of modified versions of IAA28, I am expressing them in the yeast, Saccharomyces cerevisiae. Once I have obtained a cohort of modified IAA28 proteins that display a range of degradation rates, I will transform these proteins into plants. I predict that my observations of plant phenotypes will allow me to determine whether the precision of IAA28 degradation rates matters. The results of my research may partially explain the importance of the differences between IAA degradation rates in determining specificity of auxin response phenotypes in the plant.

Pollination syndromes refer to suites of floral traits that influence biotic or abiotic vectors transporting pollen from one flower to another. These traits include flower color, position, shape, size, scent and reward. Scent is a major component of pollination, as the volatile organic compounds (VOCs) that compose “scent” often attract pollinators or deter predators. The genus Thalictrum in the Ranunculaceae (buttercup family) possesses both insect- and wind-pollinated species, enabling comparisons of the role of scent in floral diversification among recently diverged taxa with distinct pollination syndromes. To characterize floral scent, we identified and compared the bouquet of VOCs emitted by insect- and wind-pollination species, as well as by floral vs. vegetative tissue. Focusing on twelve species representative of the two pollination syndromes and spanning the Thalictrum phylogeny, we identified 8 primary compounds of interest. These compounds are known to be chemical communicators for various insects, including bees and syrphid flies, reported to visit Thalictrum. The overall purpose of this project is to determine the differences in scent emissions between species in an evolutionary context, and identify the key volatiles involved in pollination syndrome variation. This will further illuminate our understanding of the biochemical and, ultimately, the genetic basis of these starkly different pollination syndromes. In addition, the variation in distribution of VOCs across species will also be used to test implications of patterns of compound development and reuse during evolution.

It is well documented that low levels of antibiotics are present in both surface water sources and in soil near industrial, large-scale farms. However, very little work has been done exploring the selective pressure for the evolution of resistance at these sub-inhibitory antibiotic concentrations. It is our concern that mutations acquired in a low antibiotic environment may be “potentiating:” that is, the presence of these first mutations opens more mutational paths after a shift to a high antibiotic environment. The converse may also be true in terms of “dooming” mutations that close off evolutionary paths in the new environment. Here we aim to investigate the prevalence of potentiating and dooming mutations and to understand their role in adaptive evolution. We study these phenomena by performing evolution experiments with Escherichia coli grown in a permissive environment (low drug concentration) followed later by a sudden shift to a new environment that imposes a strong selective pressure (high drug concentration). Here the permissive environment serves to generate mutational diversity and the harsh selective environment serves to assess the evolutionary potential of mutant isolates relative to their wild type ancestor. This will increase our knowledge of the evolution of antibiotic resistance and as well as the unintended but potentially dangerous effects of long term selection in the presence of low levels of antibiotics.

Paternal effect mutations provide experimental tools to identify the unique contributions that fathers provide to their offspring. We are studying the ddbt (deadbeat) gene of Drosophila melanogaster, which encodes a chromosomal protein that is incorporated into spermatid nuclei during spermatogenesis and is required for normal embryogenesis. Embryos fathered by ddbt mutant males have extensive chromosome damage and embryogenesis terminates prematurely. This study asks whether ddbt-induced damage is a consequence of improper mitotic chromosome segregation in the embryo, or if the damage is independent of chromosome segregation. To address these alternatives, ddbt males will be mated to females carrying the giant nuclei (gnu) mutation. This maternal effect mutation permits repeated rounds of DNA replication but prevents chromosome segregation in the embryo. If DNA replicates to the same extent in embryos produced by gnu mothers and ddbt fathers compared to those of control fathers, this would suggest that ddbt-induced breaks occur during chromosome segregation. The alternative is that the chromosomes of ddbt fathers have preexisting damage, in which case replication would arrest due to preexisting DNA breaks. We are examining nuclear size and morphology in experimental and control embryos using confocal microscopy and assay for chromosome damage using techniques that detect breaks in DNA. Investigation of ddbt promises to improve our understanding of the paternal contributions to embryonic development.

Auxin is one of the most important plant hormones that regulates almost every developmental process during plant life cycle. Auxin response factors (ARFs), are DNA-binding transcription factors that regulate auxin-mediated transcription by activation or repression. In Arabidopsis, ARFs are encoded by a family of 28 genes, and each member is thought to play a central role in various auxin-mediated developmental processes. ARF2 has shown to be a transcriptional repressor. Nemhauser lab uses Arabidopsis seedling hypocotyl, the embryonic stem, as a model to study growth. Loss of ARF2 alters hypocotyl growth in Arabidopsis seedlings. arf2-9 is a single point mutation in DNA-binding domain that results in loss of ARF2 function. I have analyzed the hypocotyl growth in arf2-9 mutant in presence and absence of exogenously applied auxin. The results indicate that arf2-9 exhibits longer hypocotyl in comparison with wild type seedlings not only in control untreated condition but also in response to auxin application. This data suggests that ARF2 is a negative regulator of hypocotyl elongation. To investigate the mechanisms how ARF2 regulates hypocotyl growth and elongation, I have crossed arf2-9 mutant with a reporter line P_PKS4::GUS that marks actively growing hypocotyl region. I have used dCAP genotyping method for arf2-9 single point mutation and Basta resistance selection for P_PKS4::GUS transgene to isolate double homozygous arf2-9 X P_PKS4::GUS plants. My hypothesis is that long hypocotyl in arf2-9 is a consequence of indeterminate growth. Given that, my prediction is that GUS staining pattern of P_PKS4::GUS will be different in arf2-9 genetic background compared to the original line. This includes possibility of having GUS staining in different regions of hypocotyl or extended staining pattern over the time when in wild type background P_PKS4::GUS marker is no longer active. Those differences will help us understand the function of ARF2 better.

Commercial fish stock populations can be linked to the health of forage fish populations. The Pacific sand lance, Ammodytes hexapterus, is an essential link between zooplankton and higher trophic levels such as birds, marine mammals, and fishes. Understanding the behavior and habitat of Pacific sand lance is key to ensuring the long-term sustainability of sand lance populations. Sand lance spend most of their time either schooling in the water column or buried beneath the substrate. We performed paired preference trials during daylight hours to compare burrowing tendencies between sediment sizes. Sediments were collected on San Juan Island and sorted using a RoTap sediment analyzer. The grain sizes chosen for the trial were medium sand (0.25 – 0.5 mm), coarse sand (0.5 – 1.0 mm), very coarse sand (1.0 – 2.0 mm), and very fine gravel (2.0 – 4.0 mm). The study was conducted on sub adults (8.5-11.5cm total length) to account for any ontogenetic variability in burrowing behavior. We found that sand lance could burrow into all of the provided substrates, but they prefer coarse sand. Observations have shown that Pacific sand lance spend the majority of the night buried under the sediment. We also conducted nocturnal preference experiments during night time hours (2300 – 0300). At night, A. hexapterus displayed no preference between coarse sand and very coarse sand, but did prefer coarse sand over medium sand. At night, when visual clues are eliminated, the preference for substrate type is relaxed. However, preference is also being evaluated by other sensory systems as there is still a notable distinction in the proportion of buried sand lance between substrates.

Pareiasaurs were a group of herbivorous reptiles that lived during the Middle to Late Permian (~265–252 mya) in what is modern day Europe, Asia, and Africa. Fieldwork in the Moradi Formation of Niger has produced three scapulocoracoids of the pareiasaur Bunostegos akokanensis, two of which were identified to taxon by associated skull material, with the third not found in association with a skull. The considerable size disparity and slight morphological variation among the scapulocoracoids suggest that they are from different ontogenetic stages ranging from juvenile to adult. This variation permits the first description of ontogenetic change in the scapulocoracoid in pareiasaurs. An increase in overall size is associated with morphological change in each region of the scapulocoracoid, including the expansion of the anterior coracoid, the reduction of the posterior coracoid, increased robustness of the scapula, increased depth of the glenoid fossa, and a higher angle of the acromion process. To analyze these trends, precise measurements were taken from important physical landmarks in order to quantify ontogenetic shape change. Following a description of the morphology and ontogeny, an analysis of phylogenetic relationships will be conducted based on morphological characters, comparing Bunostegos to other pareiasaurs and their closest relatives. This research will further understanding of ontogenetic change in pareiasaurs, but more importantly, it will form the basis for further hypotheses regarding function, locomotion, and ecology in this important group of Permian reptiles.

Individual bees often show flower constancy, specializing on a floral resource while avoiding switches to newly available resources. The familiar plant species benefits by receiving high rates of visitation even when competing floral resources become available. However, the phenology of some plants is shifting more rapidly than others in response to climate change, resulting in a changed sequence of floral resources bees encounter. To determine whether this affects plant fitness, we asked how pollinator visitation to competing plant species depends on the order in which those species bloom. We used two plant species with overlapping phenology and a common pollinator, Hypochaeris radicata and Campanula rotundifolia. We allowed Bombus impatiens to forage in floral arrays representing three phenological progressions: (1) CR Early, with C. rotundifolia decreasing from 100% to 0% while H. radicata gradually increased from 0% to 100%; (2) HR Early, with H. radicata decreasing from 100% to 0% while C. rotundifolia gradually increased from 0% to 100%; and (3) a Control with both species at 50% throughout. We quantified visitation rate to both species, and seed production by H. radicata. Overall, the number of bee visits received by both species was significantly higher when that species was introduced first than when it was introduced after its competitor. In treatments where the proportion of the early species decreased over time, the visitation rate per flower to that species increased, for both species and across all stages of experimental treatments. Seed set for H. radicata showed a similar pattern to visitation. Our results corroborate other work suggesting that individual bees learn and become specialized on a single floral resource and are slow to switch to a newly emerging resource. As a result, the flower species that blooms first can benefit from higher bee visitation and thus, potentially higher reproductive success.

The late Oligocene-early Miocene fossil record (30-18.8 million years ago) of North America includes an abundant fauna of entoptychine gophers (Rodentia: Geomyidae: Entoptychinae). These burrowers make up as much as 20% of the mammalian individuals of the faunas they are part of, and can be involved in habitat partitioning or competition with other burrowing herbivores. The wide geographic range of these animals and their use in constraining the age of the fossil assemblages of the Cabbage Patch beds (western Montana) and John Day Formation (eastern Oregon) makes them critical to understanding the biogeographic relationships among these fossil faunas. To understand these spatiotemporal patterns, I am evaluating the taxonomic richness of entoptychine gophers. Previous studies have discriminated among these gopher species by using morphological features of skulls and dentaries. However, anatomical elements are rarely preserved in fossil assemblages, and isolated teeth are commonly the only material available for identification. Since the occlusal surfaces of the cheek teeth are usually worn down as a consequence of feeding, they lack discrete features for species identification. Instead, some researchers have hypothesized that the more continuous variation in the enamel outline of select cheek teeth could be used for discrimination. Here, I use semi landmark-based geometric morphometrics (an arithmetical comparison of geometric coordinates) on the upper last premolar and molar. Applying this method to 98 modern geomyine gopher specimens (Geomyidae: Geomyinae), representing 10 taxa, helps quantify intra- and interspecific tooth shape variation and offers guidelines for recognizing fossil gopher species. Preliminary analyses indicate that tooth shape is species-specific in modern gophers, supporting the use of this method to differentiate fossil gopher taxa. Applied to 96 specimens of fossil gophers, I found a strong overlap of fossil taxa in the morphospace, suggesting that previous researchers may have overestimated the species diversity of some fossil gophers.

GCS1 is a protein that plays a crucial role in angiosperm plant fertilization. Surprisingly, comparative studies show that GCS1-like proteins exist within a wide variety of eukaryotic species. In Drosophila, the protein Pskl is predicted to have a GCS1-like domain that affects male gametes, but its exact role in sperm formation and sperm function is not well characterized. Studying Pskl is imperative towards broadening our understanding of sexual reproduction in eukaryotes. Comparisons of Pskl to its homologs in angiosperms, algae, and protists may provide insight into mechanisms of fertilization that are evolutionarily conserved across diverse species. Furthermore, defining how the protein works could contribute to the development of reproductive therapeutics or drugs that inhibit the procreation of illness-causing organisms. The goal of this research is to understand how Pskl regulates fertility by defining its functional domains. Through gene deletions generated by imprecise excisions of a transposable element called MiMIC, analysis of both the molecular and phenotypic consequences can improve our understanding of sequences necessary for normal pskl expression. If male flies with the chromosomal derivative are sterile or have reduced fertility, then removed sequences flanking the MiMIC insertion site are likely crucial for successful fertilization. Alternatively, generated deletions may extend in either or both direction of the insertion site and impact viability, shedding light on whether pskl or its adjacent genes are essential for development. In this report, we describe the nature and consequences of the transposon excisions in pskl, both molecularly and phenotypically.

Climate change is likely to affect the altitudinal range limits of conifers in the Pacific Northwest as population performance declines at lower elevations and populations expand into upper elevations. However, it is possible that populations are locally adapted, thus influencing range shift dynamics. To assess the potential for local adaptation for three conifer species, Abies amabilis, Tsuga heterophylla, and Tsuga mertensiana, we monitored bud set and bud dormancy in seedlings collected from two to three different source elevations at Mt. Rainier National Park. Phenotypic differences in bud set and bud dormancy influence the length of the growing season, and thus, have impacts on fitness. However, source elevations from which we collected seeds differ strongly in climate, meaning that the optimal climatic triggers for bud set and dormancy are likely to differ. Observations on apical (terminal) bud formation were taken for one year in a common garden at the UW Greenhouse, to correct for environmental differences. Our study thus provides a glimpse at whether or not populations of one species in different locations on Mount Rainier are producing seeds with similar or different phenological traits when observed in the same climate. If seeds of the same species from different locations have inherent differences in the timing of key ecological events, a species may experience difficulty shifting its range when between-population interactions from different elevations become necessary because of climate. Dormancy and bud set differences at the seedling level could influence how conifer population dynamics on Mount Rainier and in other places around the Pacific Northwest will change. In preliminary analyses, we found that seedlings differ in budset and dormancy by species, and there is a trend for differences by elevation within species.

Within the last 20 years, global climate change has become well accepted. Research on plant dynamics has documented species shifting their ranges to higher elevations in order to remain within physiologically optimal conditions. This general observation has motivated my investigation of elevation as a driver of life history traits within alpine and subalpine plants. Specifically I’m interested in sex-ratios among the subalpine flower Erythronium montanum. This species of flower has been shown to exhibit a unique trait known as androdioecy, wherein some individuals produce male-only flowers instead of hermaphroditic flowers. In other species this trait has been attributed to sexual dimorphism, the variability between the sexes in terms of morphology and/or phenology. The discovery of this trait in E. montanum has inspired my research project with two distinct goals: To determine whether 1) abiotic factors such as elevation, soil moisture and time of flowering and 2) biotic factors such as canopy cover, and flowering density, influence sex-ratios within E. montanum. Surveys done at Mt. Rainier in 2011 and 2012 by the Hille Ris Lambers lab provide data on multiple factors which should allow me to test a number of possible hypotheses. Preliminary data analysis spanning both elevation and flowering time, show biased sex-ratios where the number of males to hermaphrodites decreases at higher elevations, late in the season. A future look at the extent of sexual dimorphism within the population can help to shed light on the mechanism behind sex-ratio bias and increase our understanding of plant reproductive-ecology over an altitudinal gradient. As many species begin a slow but steady march up the mountain, it’s paramount that we have a better understanding of how and why life-history traits fundamental to reproduction (e.g. sexual dimorphism) vary with elevation. Ultimately, future generations and plant communities rely on what is happening today.

During the postembryonic transformation from larva to adult, zebrafish undergo a dramatic metamorphosis in pigment pattern. New pigment cells differentiate, interact, and migrate to form stripes of black cells (melanophores) and the interstripe regions of yellow cells (xanthophores); this pigment pattern characterizes adult zebrafish. My project investigates the roles of thyroid hormone (TH) in this dramatic transformation. We have produced a stable transgenic line expressing a transgene for nitroreductase exclusively in the thyroid gland. When I expose these transgenic fish to the drug metronidazole, toxic byproducts are produced in the thyroid gland, causing cell death and ablating the gland and preventing the fish from producing any TH. Using these thyroid ablated fish, I have characterized pigment cell differentiation and pattern formation in the absence of TH. I find that these thyroid ablated individuals do not develop xanthophores and show resulting disruptions in stripe boundary formation. Our lab has also found a zebrafish pigment mutant (opallus), which produces an excess of xanthophores. Mapping data from this mutant leads us to hypothesize that the phenotype is caused by a gain-of-function mutation in the receptor that stimulates TH production in the thyroid gland. I test the prediction that the excess xanthophore phenotype is caused by overproduction of TH by blocking TH synthesis using the drug methimazole, and show that these treatments rescue the opallus pigment phenotype, returning them to a wild-type pigment pattern. Moreover, I test the prediction that exposing wild-type zebrafish to exogenous TH produces an opallus-like pigment pattern. Thus, by quantifying the number of melanophores and xanthophores and their distribution across the flank in these hypothyroid and hyperthyroid backgrounds, I show that TH is both necessary and sufficient for xanthophore differentiation, and that both too much and too little thyroid hormone during the adult transformation grossly disrupts the formation of adult stripes.

Asymmetric cell division creates diverse cell-types that are critical for stem-cell maintenance and tissue patterning in multicellular organisms, including ourselves. Therefore, understanding the mechanism of asymmetric divisions can have a huge impact on regenerative medicine. In plants, asymmetric divisions are important for producing proper patterns of stomata, which are microscopic valves that regulate proper exchange of carbon dioxide and oxygen while simultaneously minimizing water loss. A precursor of stomata, called a meristemoid, divides asymmetrically and renews itself, while its daughter cell, called a stomatal-lineage ground cell (SLGC), shows rapid polar expansion and loses stomatal precursor fate. How can a meristemoid create two daughter cells with contrasting characters? Lines of evidence suggests that rapid expansion of SLGC associated with polar localization of novel proteins is essential for cell fate determination. In this study, I will test this hypothesis within Arabidopsis thaliana by expressing a known regulator of cell polarity, Rho-GTPase2 (ROP2), in stomatal cell lineages. I have generated transgenic plants expressing green fluorescent protein (GFP) tagged Rho-GTPases (ROPs) driven by the stomatal-lineage specific POLAR promoter, which is short for POLAR LOCALIZATION DURING ASYMMETRIC DIVISION AND REDISTRIBUTION (POLAR) in order to view potential ROP localizations using confocal microscopy within stomatal cells. I expressed three forms of ROP2: a wild type form, a constitutively active form (CA ROP2) that always binds GTP and causes isotropic cell expansion, and a dominant negative form (DN ROP2) which always binds GDP and in inhibits cell expansion. Visualization of these ROPs may provide evidence indicating their presence is a requirement for meristemoid differentiation. Because ROPs are present in both plants and animals, this study is an important player in understanding stem cell differentiations, which may resolve the modern challenges we face with respect to regenerative medicine, world hunger, and biomechanical operations.