Cytochemical studies using 1-(4-chloromercuriphenylazo)-naphthol-2 (Mercury Orange), a specific reagent for the detection of sulfhydryl groups, were carried out on the giant multi-nucleated amoeba, Pelomyxa carolinensis. Distribution of -SH groups was compared in normal well-fed organisms, starved amoebas and in sodium arsenite-treated individuals. The hypothesis that spindle fibre formation during mitosis involves, in part, oxidation of -SH groups to -SS- bonds and that peripheral nuclear granules play a role in spindle fibre formation was investigated. Preliminary investigations on mouse and toad muscle fibres were carried out for the purpose of confirming Bennett's results as well as to test the specificity of Bennett's Mercury Orange technique for -SH. Considerable time was spent in observing the living amoeba. Included in these observations was feeding behaviour, reaction to various stimuli and reproduction. Prior to the -SH studies, the general morphology of P. carolinensis was investigated in considerable detail using various fixation and staining techniques. Likewise, mitosis in this amoeba was thoroughly studied. Synchronous division of the many nuclei (as observed by Kudo and Schaeffer) was confirmed. Relationship of the amoeba's contour to the stage of nuclear division was studied. This relationship proved helpful in the study of -SH groups in the dividing nucleus since it provided a means of choosing a specifically desired mitotic stage. / Science, Faculty of / Zoology, Department of / Graduate

This thesis examines how Eastern grey squirrels, Sciurus carolinensis, modify their foraging and hoarding behaviour in relation to different risks, particularly those which involve a trade-off between securing food resources and avoiding a negative outcome with a competitor. While foraging for food to eat and hoard, squirrels must compete with conspecifics and heterospecifics for access to resources, and they must ensure the safety of their food hoards from onlookers or opportunistic pilferers. While engaging in these behaviours in the most efficient way, they must also avoid being predated upon. Five studies were conducted to further understanding of grey squirrel foraging, hoarding and pilferage behaviours, and how they are affected by different risk factors. The data in this thesis provide experimental evidence that grey squirrels respond directly to conspecific presence as a cue of pilferage risk and adjust their behaviour in ways that may help to reduce cache theft. The data also support the view that conspecific and heterospecific competitors pose risks to foraging and caching, with squirrels modifying their behaviour in ways that serve to avoid negative competitive interactions. Predation risk was found to be particularly disruptive to foraging behaviour, and it also had a seasonal effect upon pilferage rates of experimenter-made caches. A variety of strategies that squirrels might use to pilfer caches were investigated, however, the data did not provide a clear indication of pilferage strategy used by squirrels; they did not seem to use observational spatial memory, and they did not simply pilfer in profitable foraging locations. This thesis raises questions about the mechanisms grey squirrels use to assess pilferage risk and how they engage in pilferage in comparison to other caching species; the studies conducted illustrate different methods that future research could use to investigate food hoarding and pilfering behaviour in wild and captive squirrels.

Successful management of an invasive species requires in depth knowledge of the invader, the invaded ecosystem, and their interactions. The complexity of the species-system interactions can be reduced and represented in ecological models for better comprehension. In this study, a spatially explicit population model was created using the RAMAS software package to simulate the past and future invasion dynamics of the eastern grey squirrel (Sciurus carolinensis) in the fragmented habitat in case study areas in Ireland. This invasive squirrel species causes economic damage by bark stripping forest crops and is associated with the decline of its native congener (S. vulgaris). Three combinations of demographic and dispersal parameters, which best matched the distribution of the species shortly after introduction, were used to simulate invasion dynamics. Future population expansion was modeled under scenarios of no control and two different management strategies: fatal culls and immunocontraceptive vaccination programmes. In the absence of control, the grey squirrel range is predicted to expand to the south and southwest of Ireland endangering internationally important habitats, vulnerable forest crops, and the native red squirrel. The model revealed that region-wide intensive and coordinated culls would have the greatest impact on grey squirrel populations. Control strategies consisting solely of immunocontraceptive vaccines, often preferred by public interest groups, are predicted to be less effective. Complete eradication of the grey squirrel from Ireland is not economically feasible and strategic evidence-based management is required to limit further range expansion. Ecological models can be used to choose between informed management strategies based on predicted outcomes.

Anolis carolinensis has been a model organism for ecology and evolutionary biology since the seventies, yet there are still understudied aspects of their ecology. A five-year study has provided microsatellite genotypes to be used in building a pedigree and assess relatedness, enabling us to evaluate the spatial distribution of an urban population of A. carolinensis. Results indicate no correlation between a male’s size and the distance others keep from it; however, males belonging in the heavyweight morph are dictating the spatial distribution in this population. In addition, juvenile dispersal of male offspring and partial philopatry of female offspring are key in this dynamic, where a single heavyweight male will actively defend a small area that contains multiple females, some of which are be daughters, and multiple unrelated males, most likely sneaker males.

Displays are ubiquitous throughout the animal kingdom. While many have been thoroughly documented, the factors affecting the expression of such displays are still not fully understood. We tested the hypotheses that display production would be affected by ecological context (i.e. the identity of the receiver) and intrinsic qualities of the signaler (i.e. heavyweight and lightweight size class) in the green anole lizard, Anolis carolinensis. Our results supported these predictions and show that a) ecological context, specifically displaying to conspecifics, has the greatest impact on display production; b) size class influenced display rate with heavyweight males displaying more to green females and lightweight males displaying more to green males in similar frequency between the two size classes to their respective target stimuli. Furthermore, our results provide empirical support for differential use of the three major display types (A, B and C displays), and uncover unexpected complexity in green anole display production.

abstract: Postnatal skeletal muscle repair is dependent on the tight regulation of an adult stem cell population known as satellite cells. In response to injury, these quiescent cells are activated, proliferate and express skeletal muscle-specific genes. The majority of satellite cells will fuse to damaged fibers or form new muscle fibers, while a subset will return to a quiescent state, where they are available for future rounds of repair. Robust muscle repair is dependent on the signals that regulate the mutually exclusive decisions of differentiation and self-renewal. A likely candidate for regulating this process is NUMB, an inhibitor of Notch signaling pathway that has been shown to asymmetrically localize in daughter cells undergoing cell fate decisions. In order to study the role of this protein in muscle repair, an inducible knockout of Numb was made in mice. Numb deficient muscle had a defective repair response to acute induced damage as characterized by smaller myofibers, increased collagen deposition and infiltration of fibrotic cells. Satellite cells isolated from Numb-deficient mice show decreased proliferation rates. Subsequent analyses of gene expression demonstrated that these cells had an aberrantly up-regulated Myostatin (Mstn), an inhibitor of myoblast proliferation. Further, this defect could be rescued with Mstn specific siRNAs. These data indicate that NUMB is necessary for postnatal muscle repair and early proliferative expansion of satellite cells. We used an evolutionary compatible to examine processes controlling satellite cell fate decisions, primary satellite cell lines were generated from Anolis carolinensis. This green anole lizard is evolutionarily the closet animal to mammals that forms de novo muscle tissue while undergoing tail regeneration. The mechanism of regeneration in anoles and the sources of stem cells for skeletal muscle, cartilage and nerves are poorly understood. Thus, satellite cells were isolated from A. carolinensis and analyzed for their plasticity. Anole satellite cells show increased plasticity as compared to mouse as determined by expression of key markers specific for bone and cartilage without administration of exogenous morphogens. These novel data suggest that satellite cells might contribute to more than muscle in tail regeneration of A. carolinensis. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2012

The grey squirrel (Sciurus carolinensis) has wildtype and melanic (dark) colour morphs. Melanism is associated with variations in the melanocortin-1 receptor (MC1R) gene in a number of species. The MC1R protein is a G-protein coupled receptor, predominantly expressed in melanocytes, where it is a key regulator of pigment production. To investigate the genetic and molecular basis of melanism, the MC1R genes of the wildtype and melanic grey squirrel were sequenced. The wildtype (MC1R-wt) and melanic (MC1RΔ24) variants of the MC1R were then functionally characterised in a cell-based assay. The MC1R gene of the grey squirrel was found to have a 24 base pair (bp) deletion associated with melanism. The MC1R is typically activated by its agonist, the alpha-melanocyte stimulating hormone (α-MSH), which stimulates dark pigment production by raising intracellular cAMP levels. Conversely, the MC1R is inactivated by its inverse agonist, the agouti signalling protein (ASIP), which stops dark pigment production by lowering intracellular cAMP levels. To investigate the effects that the 24 bp deletion have on receptor function, MC1R-wt and MC1RΔ24 genes were transfected into HEK293 cells. Cells expressing either MC1R-wt or MC1RΔ24 were stimulated with α-MSH or ASIP and intracellular cAMP levels were measured. Unstimulated MC1RΔ24 cells showed higher basal activity than the MC1R-wt cells. Both MC1R-wt and MC1RΔ24 cells responded to α-MSH with a concentration-dependent increase in intracellular cAMP. However, while the MC1Rwt cells responded to ASIP with a concentration-dependent decrease in intracellular cAMP, MC1RΔ24 cells responded with an increase in cAMP. Melanism in the grey squirrel is associated with a 24 bp deletion in the MC1R. Cells expressing MC1RΔ24 have higher basal levels of cAMP than MC1R-wt cells. ASIP acts as an inverse agonist to the MC1R-wt but as an agonist to the MC1RΔ24. As MC1RΔ24 cells have higher levels of cAMP, and higher levels of cAMP lead to dark pigment production, the 24 bp deletion is the likely molecular cause of melanism in the grey squirrel.

The distribution of short-tailed shrews, Blarina carolinensis and B. brevicauda, demonstrates contiguous allopatry in peninsular situations in the Coastal Plain of Virginia, and in other areas. The distribution of these two species is unusual in that they do not exhibit syntopy except within a narrow corridor along the borders of their ranges. Interestingly, along the Coastal Plain of Virginia, B. carolinensis is generally found only at the distal ends of the peninsular areas, existing as local populations that appear to be isolated from each other and from the primary population that exists in south-central Virginia. It is thought that the disjunct distribution of B. carolinensis populations results from the unique physiography of Chesapeake Bay, namely peninsular water barriers, and that the intervening distribution of its congener, B. brevicuada, should promote morphometric and population differentiation due to isolation. Whether populations of B. carolinensis along the Coastal Plain of Virginia are truly isolated (and possibly morphologically and/or genetically dissimilar to the continuous southeastern Unites States distribution) is unknown. I examined cranial morphometrics and molecular genetic characters (mitochondrial DNA control region and exon-primed intron-crossing nuclear DNA, EPIC) from peninsular populations of B. carolinensis and compared them to shrews of the mainland population to address this question. The sources of specimens were shrews ensnared in discarded bottles along roadsides, museum skins, and specimens frozen in alcohol. Principle coordinate analyses did not indicate a correlation between cranial morphology and genetic structuring of the populations. MANOVA analyses correlate distinct geographic regions with variation of four cranial measurements related to length. A 492 base pair region of the mitochondrial control region was obtained for many specimens and found to be polymorphic resulting in 27 unique haplotypes evenly partitioned across populations reflecting low levels of differentiation. Nuclear DNA markers were not found to exhibit repeatable amplification and therefore were not useful. Genetic variance detected among populations ranged from ΦPT = 0.001 to 0.045. The effective number of migrants (Nem), as estimated from the standard relationship with ФPT values, indicated panmixia. Both sets of observations indicated that physiography and B. brevicauda are not as severe factors limiting migration among the Coastal Plain populations of B. carolinensis as was thought.

Due to its significance in many cellular functions, fatty acid binding protein 7 (FABP7) has become a rising topic of interest for many scientists. Immunocytochemistry was used to map the distribution of FABP7 and test whether the amount of FABP7 immunoreactivity (FABP7-IR) differed in animals that were defeated in a fight, as compared to control animals that did not engage in any social interaction. The male green anole was used as the subject because its natural tendency to establish social classes within its species provides an ideal model to observe for variation in FABP7-IR. The results showed FABP7-IR in cells and fibers of the cortex, hypothalamus, thalamus, medial preoptic area, dorsoventricular ridge, amygdala, suprachiasmatic nucleus, nucleus accumbens, nucleus rotundus, habenular area, tectum, dorsal noradrenergic and lateral forebrain bundles, and lining the third and lateral ventricles. Qualitative observation suggested higher FABP7 levels in socially defeated males than controls in all areas.

The aim of the study was to analyze the infectious potential of the newly described species, B. americana and B. carolinensis, studied on the laboratory model mammals mice. Our goal was to analyze and compare the vectorial capacity of two different tick vectors, Amblyomma americanum and Ixodes ricinus, in acquiring and transmition of both spirochete species to the host. The results of this study confirmed that ticks A. americanum and I. ricinus are capable to maintain and transmit B. americana and B.carolinensis.We confirmed that both analysed spirochete species, B. carolinensis and B. americana, showed the potential to develop the disease in laboratory model mammal, which indirectly support the fact that both spirochete species might be concidered as the risk factors in the area where they are distributed. Our results shows that A. americanum is able to transmit both spirochete species, which increases that risk of acquiring the Lyme disease to human population in the area of distribution of A. americanum