This study examined sexual dimorphism in 19 species of nectar feeding bats of the family Phyllostomidae, representing four subfamiles Glossophaginae, Brachyphyllinae, Phyllonycterinae and Lonchophyllinae subfamilies. Most nectar feeding bats have elongated muzzles and tongues, and reduced post-canine teeth, which are specializations for this diet. However, many have large canine teeth, despite the lack of hard food items in their diet. Across mammals, sexually dimorphic traits are often related to various mating strategies. This study used measurements of forearm length, skull size, and canine tooth size, taken from 892 individuals in museums to determine patterns of sexual dimorphism in these bats. Of the 19 species, only three were monomorphic in all three variables (skull size, canine size, and forearm length). The remaining 15 species exhibited some degree of sexual dimorphism in one or two of the measurements, but no species showed dimorphism across all three traits. There were three instances where the forearms of females were significantly longer than those of the males, and two different instances, where females had significantly larger skulls than males. In all 13 species with significant size differences in canines, the larger canines were found on the males. Only 3 species had males that were significantly larger in both skull size and canine tooth size. The smaller of the species generally did not show dimorphism in the size of their canines, while canine dimorphism was common in larger bats. These results will be analyzed in a phylogenetic and behavioral context.
Diptera species like midges and mosquitoes produce lots of offspring and, as they provide no parental care, the initial choice of where they eggs is crucial to their offspring’s survival. Although mosquito and midge adults are terrestrial, their larvae are aquatic and adult females may use multiple factors to help select an ideal pool of water for eggs, including the pool’s likelihood of evaporating, its available nutrients, and the presence of predators. One midge, Metriocnemus knabi, and one mosquito, Wyeomyia smithii, are so particular that it only lays eggs in the leaves of the purple pitcher plant, Sarracenia purpurea, a pitcher plant species that hosts a community of invertebrate species in its pitchers instead of digesting these insects as prey. In the mountains of North Carolina, S. purpurea var. montana hybridizes with the mountain sweet pitcher, S. jonesii, a species that does not harbor aquatic invertebrates. Over two years, I examined whether ovipositing female flies avoided hybrids, as expected if hybrids provide a less hospitable environment for larvae. In 2013, I found both species of fly were more common in S. purpurea var. montana pitchers. In 2014, I examined whether adult females avoid hybrids due to characteristics of their pitcher water, or of the leaf itself. I tested this hypothesis by swapping water from S. purpurea var. montana pitchers with water from hybrids. I found that hybrids had fewer larvae regardless of which type of water they held, suggesting that females select sites based on characteristics of the leaves, themselves.
Many amphibians lay their eggs in seasonal ponds. To avoid the threat of suffocation and intense resource competition that is typical of these ephemeral ponds, the ability to quickly grow and metamorphose is of paramount importance. Typically, low dissolved oxygen presents an unfavorable condition to larvae, prolonging time until general metamorphic size is reached. Therefore, they may respond by either taking longer to metamorphose, yet reaching a favorable size, or by metamorphosing smaller, but leaving the highly competitive pond environment sooner. We studied Ambystoma maculatum, who like most Ambystomatids, lays its eggs aquatically. We also studied Ambystoma opacum, who lays its eggs terrestrially under moist soil. We examined embryonic development of these two species under laboratory conditions, comparing growth, development, and hatching success under a range of dissolved oxygen concentrations as well as in a moist, terrestrial environment. Because A. maculatum is used to developing in aquatic environments with lower dissolved oxygen than the terrestrial habitats of A. opacum, we hypothesized that their embryos and larvae will develop faster and/or be larger at metamorphosis, particularly in lower oxygen environments. Our study concluded that while A. maculatum did develop faster in all treatments, the difference between species was greatest in higher oxygen treatments, instead of the predicted lower treatments. Treatment differences showed no significant effect on mass except in A. maculatum (2013) where larvae in low oxygen treatment were significantly larger than those in higher oxygen treatments.
Cell attachment, migration, and proliferation are essential processes for both normal tissue growth and malignant development. Guanine nucleotide-binding (G) proteins and integrins play key roles in the regulation of these processes. The G proteins Gα12 and Gα13 have 66% amino acid homology, yet differ in their downstream signaling function. While these proteins have been extensively studied in their own roles, the mechanisms by which they interact have only been partially examined. Other investigators have shown C-terminal binding of β3 integrin to Gα13, but not Gα12. Utilizing epitope-tagged forms of Gα12 and Gα13, we examined their affinity for β3 integrin. We found that Gα12 showed greater binding affinity for β3 integrin than did Gα13, in both myc- and GFP-tagged forms. Furthermore, we utilized constitutively active and inactive forms of Ga12 and Gα13, as well as wild-type Gα12/13, to determine if this differential was due to the activation state. For Gα13, β3 integrin did not differ in affinity for the QL or wild type, and still showed no binding overall. Yet for Gα12, β3 integrin exhibited stronger binding for the GTP-bound form than the wild-type or GDP-bound forms. These findings suggest that Gα12 must be in the GTP-bound form to adequately bind β3 integrin in cells. Lastly, we tested NAAIRS sextet mutations to attain greater knowledge of specific structural features of Gα12 required for interaction with β3 integrin (Richie et al. 2013). Surprisingly, we found that two amino acid substitution mutants of Gα12 exhibited impaired binding to β3 integrin. This provides new structural details and serves as a basis for further research in examining these regions to specifically identify the properties that disallow binding action. As both Gα12 and Gα13 have been implicated in cancer pathways, understanding these interactions is critical for complete appreciation of the metastatic potential of epithelial cancer cells.
Heterotrimeric guanine nucleotide binding proteins (G proteins), upon activation by cell surface G protein coupled receptors (GPCRs) via external stimuli, transmit signals to downstream pathways regulating a variety of cellular responses, such as cell proliferation and cytoskeletal rearrangements. The G12/13 subfamily of G proteins, consisting of Gα12 and Gα13, has been demonstrated to promote uncontrolled growth of cells and play a role in metastatic cancer progression. Evolutionary comparison of proteins in the G12/13 subfamily of G proteins showed a highly conserved Gln/Gln amino acid pair at a precise structural position in G12/13 proteins in species ranging from sea sponge to humans. Surprisingly, mammalian Gα13 has lost these amino acids and reverted to the Glu/Lys pair common to other G protein subfamilies. To study the effects of this reversion on the ability of Gα13 to bind its effector proteins, we engineered a myc-tagged Gα13 containing a Glu/Lys to Gln/Gln mutation and are testing this in binding assays with a number of proteins that have been shown to interact exclusively with Gα13 or with both Gα12 and Gα13. For this purpose, we constructed several new GST-fusions of Gα13 binding proteins, including Hax-1, JLP, and RGS16. The genes encoding Gα13 binding proteins were isolated from a brain cDNA library, amplified by PCR, and inserted into a vector. The plasmids were transformed into bacterial cells for protein synthesis, and the proteins were purified for ongoing interaction experiments.
Purple pitcher plant (Sarracenia purpurea L.) is a carnivorous species that is widespread throughout the coastal plain of eastern North America. In western North Carolina, S. purpurea var. montana (mountain purple pitcher plant) exhibits a much more limited distribution and is found only in isolated montane bogs and fens, which serve as ecosystem islands. Population sizes in mountain bogs are fairly small, ranging from as few as 2 up to approximately 300 individuals. In addition, many bogs are spatially isolated, with S. purpurea var. montana located up to 14 km from the next known population. We worked to characterize the genetic structure of eight S. purpurea var. montana demes in Western North Carolina using microsatellite loci. DNA was extracted from leaf tissue using Qiagen kits, and 5 microsatellite primer sets were used to amplify polymorphic regions of the genome. PCR products were run through agarose gel electrophoresis to visualize and quantify band sizes. Data from this project will help the United States Fish and Wildlife Service determine S. purpurea var. montana’s suitability as a candidate species for conservation and preservation programs.
The river otter species, Lontra canadensis, was considered endangered in North Carolina until 2008. These otters are now part of a captive breeding program aimed at supplementing rivers with low populations. Because of the limited number of individuals in wild populations, the maintenance of genetic diversity must be an important consideration for those undertaking conservation efforts. The Western North Carolina Nature Center in Asheville, NC has two otters that they believe are unrelated enough to breed according the Species Conservation Act. The objective of this project was to determine the relatedness of these two individuals and establish their breeding suitability. DNA was extracted from fecal samples, and PCR was used to amplify five specific genetic loci. The PCR products were run on 3% agarose gel, and the coefficient of relatedness (R) was calculated. The coefficient of relatedness was used to determine whether or not the otters are unrelated enough to breed according to the WNC Nature Center’s guidelines. If sufficient measures are not taken to avoid inbreeding of closely related individuals, the breeding of captive river otters in order to introduce offspring into wild populations could have a detrimental impact on the genetic diversity of future populations. Therefore, the results of this project have important evolutionary implications for river otter conservation.
Canis lupus, the gray wolf, is a highly adaptable species found in zoos and nature centers across the United States. Though Canis lupus once thrived throughout Asia, Europe, and North America, it was rapidly over-hunted and by the late 1970s, was reclassified as endangered. This mandated its increased regulation and protection. The Western North Carolina Nature Center in Asheville, NC has two Canis lupus cubs whose grandparents are of unknown geographic origin. Their animal curator hopes to determine if their grandparents came from North America, as this will require a higher protection status and increased regulation (such as experimental populations and controlled geographic regions, among many others) of the cubs. To test this, fur samples were collected from both cubs, and DNA was extracted. Primers (ATP8-1F, ATP8-2R, ATP-8-lupus-F) which can differentiate between Old World and New World Canis lupus subspecies were used to amplify a region of the wolves’ mitochondrial DNA by polymerase chain reaction (PCR). The MT-ATP8 gene will be sequenced and compared to previously identified sequences of this gene in other Canis lupus subspecies via a GenBank® Blast. This comparative analysis will determine the origin of the grandparents, which in turn will allow the WNC-NC to take the appropriate conservation steps for the Canis lupus cubs.
Owls generally exhibit little sexual dimorphism: therefore, sex determination often requires analysis beyond morphological differences. The staff of the Western North Carolina Nature Center in Asheville, NC is currently debating the sexes of two barn owls, Tyto alba, and one screech owl, Megascops asio, after discovering eggs in an enclosure thought to have only contained males. In this study, total genomic DNA was extracted from feathers sampled from each of these birds and amplified with polymerase chain reaction (PCR) using P2 and P8 primers to amplify the CHD1 gene, which is used to determine sex. The DNA product was concentrated and run through 2.5% agarose gel electrophoresis, sequenced, and then examined to determine chromosomal sex. Individuals corresponding to single (ZZ) bands were confirmed to be male, while those exhibiting double (ZW) bands were determined to be female. Sex determination is an important component in the conservation and development of breeding programs for many bird species. Efficient procedures for examining chromosomal DNA in monomorphic species, such as these owls, could therefore be vital to further studies.
Molecular genetic techniques have been implemented to differentiate between two morphologically similar species of snake, the pine snake (Pituophis melanolecus) and a congeneric gopher snake (Pituophis catenifer). The genetic identity of a display snake specimen is currently being debated by staff at the Western North Carolina Nature Center in Asheville, NC. Total genomic DNA was extracted from snakeskin, and the ND4 mitochondrial DNA region was amplified using polymerase chain reaction (PCR). The PCR product was confirmed using gel electrophoresis, sequenced, and compared to known mtDNA sequences of the congeners. Because both potential candidates contain multiple subspecies with convergent morphologies, the ability to distinguish between closely related species of Pituophis could provide a substantial molecular basis to support taxonomic claims.
Sexual dimorphism occurs in many mammals and has been shown to occur in common Vespertilionid bats, with females larger in size than males. Female mammals are sometimes larger than males in order to be able to carry their offspring while pregnant. Males exhibit larger size in order to compete for a mate or territory, as seen in lions. Measuring sexual dimorphism can help explain mating and roosting behavior of species that are not well known, and to see if patterns of dimorphism are constrained from a phylogenetic context. Vampire bats’ teeth are highly specialized to extract blood from their prey, meaning that they would not be expected to be dimorphic between males and females. Any dimorphisms found in this study will be useful to better understand the extent of competition and self-protection present in vampire bats. This study investigated patterns of sexual dimorphisms in three different vampire bat species: Desmodus rotundus, Diphylla eucaudata, and Diaemus youngi. Skull size and canine size of males and females of each species were measured from skulls of museum collections and digital photographs. In all three species, male canines were found to be significantly larger than female canines. Diphylla eucaudata, Diaemus youngi, and Desmodus rotundus male canines were an average of .19, .27, and .23 mm longer than females, respectively. In Desmodus rotundus, the male skulls were found to be 3% smaller than the females. These results show that males use their canines for more than just feeding, which motivates further research on this subject.
There has been a rapid decline in populations of the Eastern hellbender salamander and the endangered subspecies, the Ozark salamander, due to a highly infectious fungal disease caused by Batrachochytrium dendrobatidis. The U.S. Fish and Wildlife service currently considers the Eastern hellbender a species of concern, while the Ozark subspecies was federally listed as endangered in 2011. Declining populations of both species have prompted conservation efforts throughout the hellbender’s range in the Eastern United States, from Arkansas to New York. The Western North Carolina Nature Center (WNCNC) believe that they have an Eastern hellbender salamander in their care. However, WNCNC requested genetic testing to confirm that it is an Eastern hellbender and eliminate the possibility that it is the endangered Ozark hellbender subspecies. If the salamander is found to be an Ozark hellbender instead of an Eastern hellbender, then a special permit to house an endangered species will be required. To determine the subspecies of the WNCNC hellbender, a thick mucosal excretion sample was collected from the organism and DNA was extracted using the DNeasy Tissue and Blood Kit. The extracted DNA was then amplified at the cytochrome B region of mitochondrial DNA, and the resulting PCR product was sequenced. Results were compared with known DNA sequences from both hellbender subspecies to rectify uncertainty over the salamander’s identity. This species classification is essential for the proper care of the specimen and to ensure that the correct animal curation paperwork is on file.
