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Biology [clear filter]
Tuesday, April 24
 

8:20am PDT

Isolation, Characterization, And Antibiotic Extraction Of Bacterial Strains
Multidrug resistant bacteria pose a threat to human health due to overprescription and misuse of antibiotics, an industry of agriculture reliance, and the decline of novel antibiotic discovery. According to the CDC, at least two million people in the United States each year are infected with multidrug resistant bacteria and more than 23,000 succumb to those infections. Natural product (NP) isolation remains a robust source of novel antibiotics even though rediscovery is an ongoing problem. This research reports the isolation of 101 bacteria from soil samples collected in the Southwestern United States and subsequent antibiotic screening of those bacteria. Bacteria from a library of isolates found to be strong antibiotic producers were then identified by 16S rDNA analysis and subjected to scale up and extraction to isolate the produced antibiotic. Currently, antibiotics produced by three bacteria, a Bacillus strain (SS729), a Microbacterium strain (SS452B) and a Pseudomonas strain (SS827B) are being characterized. SS452B was found to have antibiotic activity against Gram-positive Staphylococcus aureus while the other two, SS729 and SS827B were found to have antibiotic activity against both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli in liquid inhibition assays.

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Tuesday April 24, 2018 8:20am - 8:40am PDT
014 Zeis Hall

8:40am PDT

Development And Evaluation Of A High-Throughput Screening Method For Bacterial Antibiotic Production
Due to the misuse and overuse of antibiotics, the number of multi-drug resistant bacteria continues to rise. To fight this growing threat, new methods for discovering novel antibiotics must be developed. Natural products produced by microorganisms continue to be a robust source of novel antibiotics. Unto today, a rudimentary method that can screen natural sources accurately and efficiently has yet to be established. Current methods for screening bacterial libraries for antibiotic production are either highly complex or inefficient and prone to error. Herein, a rapid and robust high-throughput liquid culture screening method for antibiotic production by bacteria is described, which has the ability to screen both single and multicultural mixtures of bacteria in vitro. Over 300 bacteria were screened in monoculture, and 12% and 15% were found to produce antibiotics capable of ≥30% inhibition of Staphylococcus aureus or Escherichia coli respectively.

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Tuesday April 24, 2018 8:40am - 9:00am PDT
014 Zeis Hall

9:00am PDT

Inducing Gene Expression Of Major Urinary Proteins In The Female Murine Liver Cell Line Hepa1-6
Mice, Mus musculus, are a primarily nocturnal species that rely heavily on their olfactory system to detect changes in their environment. Specifically, mice rely on protein pheromones, the Major Urinary Proteins (MUPs), non-volatile molecules which are detected by the vomeronasal organ (VNO). MUPs are synthesized in the liver, excreted in the urine, and serve as genetically encoded pheromones which direct social behaviors such as countermarking, aggression, or mate preference. Mice can also use MUPs as a way to detect sex, status, and identity of the emitting individual. MUP expression is thought to be controlled by a set of hormonal axes consisting of testosterone, growth hormone, and thyroxine. The mouse genome encodes 21 MUPs, yet, each adult male mouse will express a unique set of 4-12 MUPs. The mechanism by which MUPs are chosen for expression is non-random but not well understood. This study looks to understand how individual MUPs are chosen for expression by utilizing a cell culture model system. The female murine liver cell line Hepa1-6, is being used because it does not endogenously show expression of any MUPs, but previous studies have shown that female mice are capable of producing MUPs at male levels if they are exposed to testosterone. A combination of hormonal and drug treatments consisting of methylation inhibitors and deacetylation inhibitors are being used to induce MUP expression in these cultured cells. Following treatment of cells, they are harvested for RNA isolation, and the resulting cDNA library is examined for MUP expression. The results of this study using the chosen concentrations and treatment periods are not sufficient to induce MUP expression. As such, a working protocol for the induction of MUP expression is yet to be established. The creation of a working protocol will in the future contribute to the greater understanding of gene expression.

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Tuesday April 24, 2018 9:00am - 9:20am PDT
014 Zeis Hall

9:20am PDT

Cloning Of Vomeronasal Type-2 Receptor For Deorphanization
The vomeronasal organ (VNO) is a chemosensory organ present in amphibians, reptiles, and non-primate mammals. In mice, vomeronasal neurons express vomeronasal-1 receptors (V1R) or vomeronasal-2 receptors (V2R), both of which are G protein-coupled receptors involved in pheromone detection. V2Rs are expressed by the basal neurons of the VNO. They are of special interest because they are used to detect protein pheromones, the Major Urinary Proteins (MUPs), which induce intermale aggression, female responsiveness to mating, and territory marking behaviors. Because V2Rs use combinatorial coding instead of a labelled line coding strategy, linking pheromone responses to the correct V2R has so far been difficult. We designed primers for each V2R to amplify separate individual sequences from a cDNA library, which could then be transfected into cells using vectors. This cell culture method would allow for deorphanization of V2Rs by creating entire cell populations which only express a single V2R. With V2Rs deorphanized, mapping of pathways can begin from a bottom-up method, instead of the more difficult top-down approach. Here we show how to isolate and clone V2Rs for individual eventual expression in mammalian cells using DNA purification, Zero Blunt TOPO cloning PCR kit and ligation into mammalian vector. V2R 121 was successfully cloned into a bacterial vector with a complete sequence. The blunt end cloning did result in correct directionality when the V2R was transferred into a mammalian vector. These results demonstrate progress towards setting up a cell culture based V2Rs expression system. This system will allow for further research to deorphanize individual V2Rs by matching them to their corresponding ligands. Stimulation of V2Rs reproducibly activate specific neural circuits in mouse brains, allowing for reliable study of how external environmental cues can direct behaviors. Cloning V2Rs is the first step to identify receptor-ligand interactions, which can be utilized for future research.

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Tuesday April 24, 2018 9:20am - 9:40am PDT
014 Zeis Hall

9:40am PDT

Developing a Model System to Establish Electrophysiological Protocols Necessary for the Deorphanization of Vomeronasal Sensory Receptors (VNSRs)
Chemical signaling mediates many complex behavioral interactions such as mating and the establishment of hierarchy for a variety of animal species. A key form of chemical signaling in rodents is facilitated by the production and detection of intraspecific pheromones known as major urinary proteins (MUPs). Pheromones such as MUPs convey specific information which is innately recognized by members of a species. In rodents, MUPs are detected by receptors on specialized sensory neurons in the vomeronasal organ (VNO) known as vomeronasal sensory receptors (VNSRs). Once a MUP binds to a VNSR, the sensory neuron initiates neural circuits extending to other sections of the brain such as the amygdala and surrounding limbic structures leading to a behavioral response. Sensory neurons expressing different VNSRs will activate different neural circuits, thus allowing different MUPs to evoke specific behaviors. The exact behavioral response to a certain MUP is predictable for all members of the species and indicates common neural circuitry associated with MUP communication. This uniformity of neural circuitry in mice allows for MUP communication to serve as a reliable model system for studying how chemical stimuli code for behavioral outputs. This study aims to determine which MUPs activate a given VNSR in order to elucidate the neural circuits responsible for specific behavioral reactions. To achieve this understanding, patch clamp analysis will be used to deorphanize VNSRs by monitoring electrophysiological changes in cells expressing VNSRs upon exposure to specific MUPs. If the MUP being introduced is able to bind the specific VNSRs expressed by the cells, a measurable change in the cells voltage will occur. Currently, a model system using CHO cells transfected to express kir2.2 channels is being established to yield preliminary methodologies that will be used to complete this study.

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Tuesday April 24, 2018 9:40am - 10:00am PDT
014 Zeis Hall

10:15am PDT

Predation Rates On Ambystoma maculatum (Spotted Salamander) Polymorphic Egg Masses
A polymorphism results when a gene has multiple alleles, and polymorphic species can have many different forms. Ambystoma maculatum (spotted salamander) lays polymorphic egg masses that appear either clear or white. This study examined the effects of egg mass morph on predation rates in the natural environment to determine possible advantages of the polymorphism. It was hypothesized that clear egg masses would be predated upon more heavily because white masses have a survivorship advantage under predation. This study examined the relationship between egg mass volume and embryo number for masses of both morphs, surveyed natural predators in the field, and used volume as an indicator of predations rates. Twelve different taxa were surveyed as possible predators in the field, with Ambystoma opacum being most prevalent. Change in egg mass volume was not significant for either morph, but white masses experienced a relatively smaller change in volume than clear masses. The lack of significant difference in the change in volume suggests that morph does not significantly influence predation rates. It is expected that some other fitness advantage maintains the white morph rather than greater survivorship under predation.


Tuesday April 24, 2018 10:15am - 10:35am PDT
014 Zeis Hall

10:35am PDT

Genetic Diversity In Captive Lineages Of Chilabothrus inornatus, Puerto Rican Boa Constrictor
The endemic endangered Puerto Rican boa (Chilabothrus inornatus) has faced a variety of perils in its natural habitat, making it a species of concern in recent decades. While information regarding genetic diversity in wild populations of this species is finally being accrued, an analysis of the viability and relative genetic diversity contained in captive populations is entirely unknown, which hampers efforts to develop a captive species management plan. Here we provide that analysis using an 1100bp fragment of the mitochondrial cytochrome b gene as well as nine microsatellite loci. We obtained 46 individual boas from captive populations in the United States from both private breeders and zoos. We were able to determine relatedness among individuals as well as overall genetic diversity in the captive population. We then compared these data to the same data obtained from wild populations across the island of Puerto Rico to determine genetic diversity in the captive population relative to wild lineages. These analyses provide a starting point for informed breeding decisions in captive individuals by decreasing potential for inbreeding. We hope to provide this information to facilitate stronger conservation decisions regarding this species.

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Tuesday April 24, 2018 10:35am - 10:55am PDT
014 Zeis Hall

10:55am PDT

Elucidating Cryptic Diversity In Burmese Lygosoma Skinks Via Integrative Taxonomy
Supple skinks (Reptilia; Lygosoma), named for their attenuate limbs, like many other Indochinese herpetofaunal groups, have experienced particular taxonomic turbidity over the past few decades. With a widespread geographic breadth (spanning most of Africa to continental and insular Asia), the majority of species within this group are exceptionally poorly known. As is the case with so many understudied cryptic species groups, Southeast Asian Lygosomine species-complexes, such as Lygosoma quadrupes, require comprehensive molecular and morphological assessments to resolve the evolutionary relationships of these cluttered taxa. Here, using comparative morphological and novel multilocus phylogenetic data, we apply targeted species delimitation techniques to the Lygosoma quadrupes complex, focusing in particular on species from Myanmar. In so doing, we reveal multiple cryptic lineages and provide insight on the taxonomic status of this group, as well as additionally biogeographic discussion. Our dataset also provides opportunities to examine the evolution of body elongation and limb attenuation, which has happened independently in several Indochinese skink lineages.

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Tuesday April 24, 2018 10:55am - 11:15am PDT
014 Zeis Hall

11:15am PDT

Population Genetic Structure And Hybridization Within Western North Carolina Sarracenia (Pitcher Plants)
The Sarracenia (pitcher plant) genus, a group of carnivorous perennial herbs, includes many species of conservation concern. Sarracenia species can hybridize when in sympatry, with seemingly few pre-zygotic barriers to cross-fertilization. Two pitcher plant species, S. jonesii (mountain sweet pitcher plant) and S. purpurea var. montana (mountain purple pitcher plant), are native to western North Carolina bogs, and others, including S. flava (yellow pitcher plant) and S. leucophylla (white pitcher plant), have been introduced to the region. In this study, we examined the genetic composition of phenotypically hybrid plants and S. pupurea var. montana individuals from a site in which these four species co-occur. Plants were non-destructively sampled, DNA was extracted, and samples were PCR-amplified at 6 (hybrid) or 8 (S. purpurea var. montana) microsatellite loci; after fragment analysis, microsatellite lengths were quantified in Geneious. Calculations of hybrid indices showed that all individuals contained S. jonesii and S. purpurea var. montana DNA, and that the contribution of S. purpurea var. montana DNA to hybrid plants ranged from 20 - 40%. The latter result is of particular concern, as S. purpurea var. montana is being considered for federal listing. Genetic diversity indices for S. purpurea var. montana individuals showed low levels of allelic and genotypic diversity. Ongoing experiments are investigating genetic diversity within and among S. jonesii and S. purpurea var. montana sites in western North Carolina, to better understand population dynamics and prioritize conservation work.

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Tuesday April 24, 2018 11:15am - 11:35am PDT
014 Zeis Hall

11:35am PDT

Using morphology and Mitochondrial Cytochrome-B gene comparison in determining origins and genetic similarities.
Clinostomus funduloides, commonly known as rosyside dace, are a minnow species found in western North Carolina where they inhabit areas of narrow, rocky, headwater streams. Their range extends into Ohio, South Carolina, Tennessee, and a few parts of Pennsylvania. Within western North Carolina rosyside dace can be found in almost every water basin and recently have been found in the Upper French Broad (UFB) river basin. Past work on the CYT-B gene of C. funduloides revealed a clade between the Catawba and French Broad samples dating back 2.4 million years. This lead to a conclusion of bait-bucket release as the point of introduction of C. funduloides into the French Broad river basin. Sequencing of the 1140 bp mitochondrial cytochrome-B gene (CYT-B) gene have now been done on four populations of C. funduloides (from the Broad River basin, Catawba river basin, and the French Broad River basin) to gain a better understanding of the relationships among the different populations of C. funduloides. Morphology of C. funduloides is also being taken into account by measuring body depth and counting scales along the lateral line. C. funduloides’ presence in the UFB is intriguing because of its separation from other populations by the Eastern Continental Divide. By looking at genetics and morphology, we will be able to determine the origins of the UFB population and how unique the UFB population is compared to others.


Tuesday April 24, 2018 11:35am - 11:55am PDT
014 Zeis Hall

12:00pm PDT

A New Species Of Dwarf Gecko (Sphaerodactylus) From The Virgin Islands
West Indian dwarf geckos, belonging to the genus Sphaerodactylus, are found throughout the Caribbean, with eleven species found on the Puerto Rican islands alone. Despite huge diversity and widespread abundance, comprehensive multi-locus phylogenetic studies of this group are largely lacking, or are based on older methods such as allozyme data. As part of a larger study on the phylogenetics of Puerto Rican herpetofauna, a molecular phylogeny of 16S RNA mitochondrial sequence data suggested that a cryptic and potentially new species of the Sphaerodactylus macrolepis complex might inhabit the Virgin Islands to the east of Puerto Rico. To confirm the relationship of this new species to Sphaerodactylus macrolepis sensu lato, we generated additional molecular sequence data for the mitochondrial genes CYTB and ND2, as well as for the nuclear genes RAG, ACM, and CMOS- a total of several thousand base pairs of data across most species of Puerto Rican Sphaerodactylus. Our combined analyses using Bayesian and Maximum Likelihood analyses support recognition of a new species of dwarf gecko from the Virgin Islands distinct from S. macrolepis, which we are in the process of naming. Subsequent studies will determine morphological, behavioral, and phenotypic uniqueness of this species.


Tuesday April 24, 2018 12:00pm - 1:30pm PDT
Sherrill Center Concourse

12:00pm PDT

Constructing A New Phylogenetic Tree To Trace The Evolution Of Ontogenetic Color Change In The Boas (Booidae)
Ontogenetic color change (OCC) in animals is the irreversible change in coloration occurring as an individual matures and develops from a juvenile to an adult. Because coloration is such an important aspect of the ecology, behavior, and natural history of an animal, OCC is often associated with either a shift in habitat use, diet, or sexual maturity. This trait has been observed across many vertebrate taxa from birds to fishes but has not been studied at all in the boas (Reptilia; superfamily Booidae) despite some dramatic known examples of OCC in this group. We compiled a database of all currently-recognized boa species and assessed whether they exhibit OCC or not using evidence from the primary and secondary literature, photographs of different life stages, and information from breeders. We then aligned genetic sequence data from an 1100 basepair mitochondrial gene sequenced across 90% of boa species (including newly-sequenced species never included in a molecular phylogeny before) and constructed a Bayesian phylogeny for the boas. We then mapped the presence of OCC onto the phylogeny as a binary trait using stochastic character mapping and analyzed whether OCC showed phylogenetic signal by performing a Pagel’s λ test. We also reconstructed ancestral character states (whether or not ancestors were likely to have exhibited OCC) and tested whether OCC evolved according to standard evolutionary models. Our results will provide insight into the evolution of OCC in the boas and allow for further research into the selective pressures that precipitated the evolution of this trait, whether this trait has evolved once or a multitude of times, and how it might affect fitness in some species.

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Tuesday April 24, 2018 12:00pm - 1:30pm PDT
Sherrill Center Concourse

12:00pm PDT

Fire-Regime Management In Western North Carolina
Records of forest fire disturbance are relatively short (~50-100 years) in many regions across the southeastern United States. For much of the southeast, the historical fire records only cover approximately the past 50 years. Therefore, there is a need to develop proxy records of fire history to better understand the natural variability of fire regimes. This research will attempt to develop proxy fire histories using bog sediment records collected in Western North Carolina in the Southern Appalachian Mountains. Understanding the fire history will help to identify the underlying controls of the local fire regime, and to determine how ecosystems have responded to past changes in climate so that this information can be used to improve land-use and forest management plans in the future. Records of fire activity were determined by analyzing sedimentary macroscopic (>125 µm) charcoal preserved in the sediments of the Panthertown Valley Wetland Complex in Sapphire, NC. The sediment records were dated using 14C dating at Woods Hole - National Ocean Sciences Accelerator Mass Spectrometry (NOSAMS) facility, MA. Analysis of the regions fire histories will inform decision makers about the management of forest resources and guide the use of prescribed fire as a management tool in the region.


Tuesday April 24, 2018 12:00pm - 1:30pm PDT
Sherrill Center Concourse

12:00pm PDT

Method For Isolation Of Vomeronasal-2 Receptors From Vomeronasal Organ Cdna Library For Eventual Expression In Mammalian Cells
The vomeronasal organ (VNO) is a chemosensory organ present in amphibians, reptiles, and non-primate mammals. In mice, vomeronasal neurons express vomeronasal-1 receptors (V1R) or vomeronasal-2 receptors (V2R), both of which are G protein-coupled receptors involved in pheromone detection. V2Rs are expressed by the basal neurons of the VNO. They are of special interest because they are used to detect protein pheromones, the Major Urinary Proteins (MUPs), which induce intermale aggression, female responsiveness to mating, and territory marking behaviors. Because V2Rs use combinatorial coding instead of a labelled line coding strategy, linking pheromone responses to the correct V2R has so far been difficult. We designed primers for each V2R to amplify separate individual sequences from a cDNA library, which could then be transfected into cells using vectors. This cell culture method would allow for deorphanization of V2Rs by creating entire cell populations which only express a single V2R. With V2Rs deorphanized, mapping of pathways can begin from a bottom-up method, instead of the more difficult top-down approach. Here we show how to isolate and clone V2Rs for individual eventual expression in mammalian cells using DNA purification, Zero Blunt TOPO cloning PCR kit and ligation into mammalian vector. V2R 83 was successfully cloned into a bacterial vector with a complete sequence. Phosphatase treatments aided correct insert directionality when the V2R was transferred into mammalian vectors using blunt end cloning. Our results demonstrate progress towards setting up a cell culture based V2Rs expression system. This system will allow for further research to deorphanize individual V2Rs by matching them to their corresponding ligands. Stimulation of V2Rs reproducibly activate specific neural circuits in mouse brains, allowing for reliable study of how external environmental cues can direct behaviors. Cloning V2Rs is the first step to identify receptor-ligand interactions, which can be utilized for future research.

Speakers
Sponsors

Tuesday April 24, 2018 12:00pm - 1:30pm PDT
Sherrill Center Concourse

12:00pm PDT

Molecular Phylogenetic Identification Of An Enigmatic Tree Frog From Conception Bank, Bahamas
During a 2017 survey of the remote Conception Island Bank, a small toepad sample of an unidentifiable tree frog (Hylide: Osteopilus) was collected by Dr. R. Graham Reynolds. Specimens of Osteopilus from this region appear much larger in size and much more colorful than populations throughout the rest of the Bahamas Archipelago. Due to the morphological ambiguity of the organism, we sought to investigate its phylogenetic affinities in comparison to other geographically proximal Osteopilus species. To do so, we extracted DNA from the toepad tissue of the unknown sample and one known sample of O. septentrionalis from Crooked Island, Bahamas using the Qiagen Wizard SV Kit, tested primers for the 16S mitochondrial gene obtained from a literature search, and then sequenced the sample using PCR. We then aligned the sequence with other Osteopilus species mined from the online resource GenBank and constructed a maximum-likelihood phylogenetic tree of West Indian hylid frogs using the RaxML algorithm in Geneious 10.0®. A BLAST search of the resulting sequence data yielded 98% sequence similarity to a confirmed O. septentrionalis specimen. Our phylogenetic tree revealed that the unknown population on Conception Bank is indeed O. septentrionalis. This raises some interesting questions with regard to the observed unique phenotype of frogs from this island. The large body size and unusual color might be owing to founder effects or local selective pressures. To deepen the mystery, other herpetofauna from the same island also exhibit gigantism- the Brown Anole has the largest body size for the species.

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Tuesday April 24, 2018 12:00pm - 1:30pm PDT
Sherrill Center Concourse

12:00pm PDT

Phylogenetic Comparative Analysis Of DNA Methylation Rates In Reptiles
In addition to playing a role in genomic function, DNA methylation influences evolution by regulating transcription. Technological advances, such as High-Performance Liquid Chromatography (RP-HPLC), have allowed scientists to explore genomic regulatory changes that contribute to species diversity and phenotypic variability. Epigenetic modifications of notable interest include 5-methylcytosine (5mC) and CpG (GC), as they are related to neutral selection on the cellular level. To understand how these regulatory changes evolve in a phylogenetic context, we analyzed these quantitative traits phylogenetically by mapping them to a mitochondrial phylogeny inferred de novo across 28 reptile species. Previous studies in reptiles concluded that there was no significant correlation between DNA methylation and environmental stimuli, but these studies did not correct for the non-independence of evolutionarily related species and thus violated a fundamental statistical assumption. To model these traits, we corrected these traits and ran phylogenetic comparative analyses in RStudio®. First, we examined the extent of the phylogenetic non-independence problem by estimating measures of phylogenetic signal for each quantitative trait. We then repeated regressions from a previous study, following phylogenetic correction, and inferred correlation between our two epigenetic modifications. Finally, we fit a series of evolutionary models to examine the evolution of these traits across the reptile phylogeny and selected the best model using an AICc model selection procedure. We found phylogenetic signal in GC but not 5mC, and that phylogenetic correction did not affect results, likely owing to the relatively small number of tips and the lack of phylogenetic signal in one of the traits. The evolution of these traits is best approximated by an Ornstein-Uhlenbeck model, suggesting that local optima exist for these quantitative characters and predicting a loss of phylogenetic signal (convergence or homoplasy). This study is important because the results can be used to understand the modifications to the genome influencing phenotypic diversity.

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Tuesday April 24, 2018 12:00pm - 1:30pm PDT
Sherrill Center Concourse