Understanding worldwide declines in reptiles due to factors such as habitat loss and emerging infectious disease has become an increasingly important focus in conservation biology. Here, I use novel approaches from the field of landscape genetics to combine spatial genetic data with landscape data at both regional and local spatial scales to explore natural and anthropogenic landscape features that shape population structure and gene flow in a federally threatened reptile, Gopherus polyphemus. I also utilize approaches from the field of spatial epidemiology to examine the extent to which environmental variables can be used to predict the seroprevalence of an associated pathogen Mycoplasma agassizzi in gopher tortoise populations. Using mitochondrial data, I find evidence of a historical barrier to gene flow that appears to coincide with the Apalachicola River. I also discover low genetic diversity and evidence of population bottlenecks in the western portion of the range. My evaluation at the regional scale shows that dispersal is limited by geographic distance, areas of low elevation and major roads ways. A finescale study reveals no evidence of spatial genetic structure within a 14 x 35 km area. However, soil type is significantly correlated with pairwise genetic distances between individuals, suggesting that this variable influences fine-scale population structure in the gopher tortoise. In addition to soil, high density canopy cover is an important factor impeding gene flow at the local level for females, while land cover type explains some of the genetic variance between males. Finally, temperature and precipitation appear to be important predictors of the seroprevalence of the pathogen Mycoplasma agassizii in gopher tortoises. The probability of an individual testing seropositive for exposure to this disease increased with high temperature and low precipitation values. The methods presented in this dissertation evaluate novel approaches for assessing the influence of environmental variables on population structure, dispersal and disease occurrence and could be applied in future studies of other threatened and endangered taxa.

ABSTRACT This thesis was divided into two parts. The purpose of part I was to clone and sequence the full-length ependymin gene from the invertebrate Limulus polyphemus, or portions of the gene, and to use RT-PCR to determine whether expression of this gene increases during leg regeneration. PCR was chosen as the method for obtaining the gene due to the success our lab had previously characterizing several ependymin genes using this approach. Three sets of primers were designed based on the conserved domains between teleost fish and three invertebrate ependymin sequences. “Sea primers" were designed based on the nucleotide sequence of the sea cucumber H. glaberrima for each conserved domain, and these primers produced all four of the expected size amplicons with Limulus DNA, but surprisingly only one such band with the sea cucumber Sclerodactyla briareus. The consensus primers (con-primers) were designed based on the most conserved nucleotide among all known ependymin species at each particular position in the conserved domains. Primers designated“5-11 primers" were designed based on the absolutely conserved domains among the three known invertebrate ependymins. Neither con-primers nor 5-11 primers produced any bands of the expected size; this was true for both species of DNA. One very strong band was produced using“5-11" primer pair 6/10 with both species. One of the bands from this reaction from Limulus was cloned and sequenced, and showed a very strong homology (88% over 292 bp) with mouse FGF-14, a neurotrophic factor involved in mouse neurogenesis. The expression of this gene during leg regeneration will be tested in future experiments. Limulus GAPDH was also cloned and sequenced, and a genomic intron was identified for the first time in this study. This Limulus housekeeping gene will be used in future studies for gene expression comparisons. The purpose of part two of this thesis was to study the up-regulation of growth-related genes induced by treatment of a human neuroblastoma SH-SY5Y cell line with a human ependymin peptide mimetic (hEPN-1), in an attempt to help provide a basis for using human EPN mimetics as therapeutics in stroke and neurodegenerative diseases. The sequence of this mimetic is derived from an area of human MERP-1 analogous to goldfish mimetic CMX-8933. The human mimetic was previously found to up-regulate growth related genes L-19, EF-2 and ATP Synthase in the mouse neuroblastoma cell line Nb2a (Saif, 2004). The expression levels of genes encoding ribosomal proteins and ribosomal RNA were studied using RT-PCR as hallmarks of proliferating cells. hEPN-1 was found to increase the expression of the nuclear-encoded ribosomal proteins S-19 and S-12, an average of 2.76 fold and 1.74 fold, with statistically significant p-values of 0.031 and 0.015 (<0.05), respectively. The expression levels of nuclear-encoded 5.8S ribosomal RNA (p = 0.018) and the mitochondrial-encoded 16S RNA (p = 0.046) were found to be increased an average of 14.04 fold and 3.91 fold, respectively. Thus, human ependymin mimetic hEPN-1 appears to stimulate growth-related genes, a property which can be useful to regenerate neuronal tissue after injury.

Gopherus polyphemus populations are diminishing throughout their range due to urbanization, fragmentation, and poor management of habitats. Increased population densities, poor habitat quality, and lack of fire may influence disease transmission. Parasite roles within wild tortoise populations are largely unknown, despite evidence these pathogens may pose health risks. This study provides a baseline of gopher tortoise endoparasites across South Florida and reports on how varying environmental and tortoise characteristics may affect endoparasite species prevalence, approximate loads, and overall distributions. Tortoise fecal samples were taken from five differing SF habitats. Seven species of intestinal parasites were discovered from 123 tortoises. Identified parasites include endo-helminths such as cyathostomes, pinworms, ascarids, flukes, and protozoans including Eimeria, Cryptosporidium, and Amoeba species. Significant differences in parasite prevalence and loads were seen between sampling years, seasons, size classes, and sites, however, overall parasite distributions suggest parasitism remains relatively ubiquitous throughout most host and site characteristics. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

Information regarding spawning site fidelity and movement patterns of the American horseshoe crab (Limulus polyphemus) is crucial for developing effective conservation and management strategies on the correct spatial scale. To investigate the spatial ecology of American horseshoe crabs, 75 adult animals were tracked off the coast of Chatham, Cape Cod, MA from June 2010 to November 2011 using acoustic telemetry. Two groups of horseshoe crabs were tagged in spawning habitats (separated by ~2.0 km) with differing commercial harvesting pressure: one group inside Stage Harbor, where harvesting is permitted and the other within a Marine Protected Area (MPA) where harvesting is prohibited. Network analysis revealed that horseshoe crabs exhibited fidelity to spawning habitat, but not necessarily to the habitat where they were initially tagged. Fifty-nine percent of horseshoe crabs tagged inside Stage Harbor were detected in the MPA and 13% of horseshoe crabs tagged in the MPA were detected inside Stage Harbor. Although horseshoe crabs were utilizing both spawning habitats, predictive modeling revealed little temporal overlap, suggesting that horseshoe crabs from the two spawning habitats represent local populations. Isolated and local populations are more susceptible to overexploitation than are larger populations with many migrants. To protect against overharvest and extinction of isolated and local populations, the correct identification of management units (MUs) must be a priority of fisheries managers. Horseshoe crab populations around Cape Cod, Massachusetts and New England behave differently, requiring the collection of more information so that conservation tools such as MUs or MPAs can be used most effectively.

Gopher tortoise (Gopherus polyphemus; hereinafter, tortoise) is listed as threatened under the Endangered Species Act in the western portion of its range. Across the species’ range, approximately 70 % of potential habitat is privately owned, and these properties are often managed primarily for timber production. However, tortoise ecology on private, working forest landscapes remains poorly understood. To provide a better understanding of tortoise response to active forest management, I evaluated population demographics, movement ecology, and habitat selection of two tortoise populations: former Ben’s Creek Wildlife Management Area (BC) in Washington Parish, Louisiana and Perry County Gopher Tortoise Management Unit (PCGTMU) in Perry County, Mississippi. At BC, tortoises were generally clustered along utility rights-of- way and roadways. At PCGTMU, tortoises were clustered within forest stands with high quality soils. Low recruitment has been documented at BC for the last 25 years. However, PCGTMU appears to have a stable population with active recruitment.

Modeling the complex relationships between habitat characteristics and a species' habitat preferences pose many difficult problems for ecological researchers. These problems are complicated further when information is collected over a range of time or space. Additionally, the variety of factors affecting these choices is difficult to understand and even more difficult to accurately collect information about. In light of these concerns, we evaluate the performance of current standard habitat preference models that are based on Bayesian methods and then present some extensions and supplements to those methods that prove to be very useful. More specifically, we demonstrate the value of extending the standard Bayesian hierarchical model using finite mixture model methods. Additionally, we demonstrate that an extension of the Bayesian hierarchical changepoint model to allow for estimating multiple changepoints simultaneously can be very informative when applied to data about multiple habitat locations or species. These models allow the researcher to compare the sites or species with respect to a very specific ecological question and consequently provide definitive answers that are often not available with more commonly used models containing many explanatory factors. Throughout our work we use a complex data set containing information about horseshoe crab spawning habitat preferences in the Delaware Bay over a five-year period. These data epitomize some of the difficult issues inherent to studying habitat preferences. The data are collected over time at many sites, have missing observations, and include explanatory variables that, at best, only provide surrogate information for what researchers feel is important in explaining spawning preferences throughout the bay. We also looked at a smaller data set of freshwater mussel habitat selection preferences in relation to bridge construction on the Kennerdell River in Western Pennsylvania. Together, these two data sets provided us with insight in developing and refining the methods we present. They also help illustrate the strengths and weaknesses of the methods we discuss by assessing their performance in real situations where data are inevitably complex and relationships are difficult to discern. / Ph. D.

In conjunction with Cambrex, a biomedical company that utilizes horseshoe crabs for the production of Limulus Amoebocyte Lysate (LAL), a study was conducted to investigate movement patterns and population distributions of horseshoe crabs to increase understanding of mid-Atlantic horseshoe crab populations. In addition, areas of the shoreline of Tom's Cove, Assateague Island, Virginia were investigated as possible locations for annual spawning surveys. Twelve thousand five hundred horseshoe crabs were tagged and released in Chincoteague, Virginia and Ocean City, Maryland as part of a movement study; 431 (3.45%) were reported as resights. The mean distance between site of release and site of recapture for all resighted crabs was 68.3 km; maximum distance moved was 493.7 km. During 1999-2004, demographic data were collected from horseshoe crabs harvested in Chincoteague, VA and Ocean City, MD. The proportion of females (p < 0.0001) and juveniles (p < 0.0001) sampled varied from year-to-year, but no trends were observed. This study also showed that a greater proportion of females were observed in the juvenile cohort sampled compared to the adults sampled (p < 0.0001). The spawning survey revealed that spawning activity in Tom's Cove varied between years. On May 30th, 2003, 1,192 horseshoe crabs were observed spawning on the northern shoreline of Tom's Cove. The maximum number of horseshoe crabs observed spawning in the same area in 2004 was 94. This study provides no evidence for isolated subpopulations in Chincoteague, VA or Ocean City, MD. It also shows that horseshoe crab sex and age ratios fluctuate annually, therefore requiring a long time series of data to detect trends. / Master of Science

C-reactive protein (CRP) is an acute phase protein of the innate immune system that has been evolutionarily conserved. Human CRP is known to exist in two different pentameric conformations; native CRP and non-native CRP that possess differential ligand recognition functions. The structure of CRP evolved from arthropods to humans, in terms of subunit composition, disulfide bonds, and glycosylation pattern. Along with change in structure, the gene expression pattern of CRP also evolved from a constitutive protein in lower invertebrates to an acute phase protein in humans. The objective of this study was to determine the function of a non-native pentameric CRP, that binds to atherogenic LDL, in atherosclerosis and compare the ligand recognition functions of human pentameric CRP with an evolutionary distant CRP for understanding the evolution of the structure of CRP. Additionally, in vitro reporter gene assays were used to gain further insight into the regulation of human CRP gene expression by an IL-6 inducible transcription factor, STAT3. We observed that CRP, in its non-native pentameric conformation, binds to atherogenic LDL and slows the progression of atherosclerosis in a site-specific manner in high fat diet fed LDLR-/- mice. Further, we observed that the ligand recognition function of CRP from an evolutionary conserved species, Limulus polyphemus, is different than that of native pentameric human CRP, but overlaps that of non-native pentameric human CRP. Lastly, we screened the proximal 300 bp region of the CRP promoter and identified a novel STAT3 binding site at position -134 located upstream of the previously identified, transcriptionally active STAT3 site at -108. In conclusion, non-native pentameric human CRP is an atheroprotective molecule whose ligand recognition functions exhibit similarity with CRP from an evolutionarily distant species. IL-6 mediated transcriptional regulation of human CRP is modulated, in part, by STAT3 binding to two distinct positions on the CRP promoter.

Hemocyanins (Hcs) and phenoloxidases (POs) are both members of the type-3 copper protein family, possessing di-cupric active sites which facilitate the binding of dioxygen. While Hcs and POs share a high degree of sequence homology, Hcs have been associated traditionally with oxygen transport whereas POs are catalytic proteins with a role in innate immunity. Evidence gathered in recent years details numerous immune functions for Hc, including an inducible PO activity. Unlike the pro-phenoloxidase activation cascade in arthropods, the endogenous mechanism(s) involved in the conversion of Hc into an immune enzyme is lacking in detail. The overall aim of this research was to characterise the physiological circumstances in which Hc is converted into a PO-like enzyme during immune challenge. A series of biochemical, biophysical and cellular techniques were used to assess the ability of phospholipid liposomes to mimic the well-characterised induction of PO activity in Hc by SDS micelles. Incubation of Hc purified from Limulus polyphemus, in the presence of phosphatidylserine (PS) liposomes, yielded ~ 90% of the PO activity observed upon incubation of Hc with the non-physiological activator, SDS. Phospholipid–induced PO activity in Hc was accompanied by secondary and tertiary structural changes similar to those observed in the presence of SDS. Subsequent analysis revealed that electrostatic interactions appear to be important in the PS-Hc activation complex. In vivo, PS-Hc interactions are assumed to be limited in quiescent cells. However, amebocytes undergoing apoptosis redistribute PS onto the outer leaflet of the plasma membrane, resulting in the potential for increased Hc-PS interactions. In the absence of a reliable culturing technique for L. polyphemus amebocytes, in vitro conditions were optimised for the short term maintenance of this labile cell type. Amebocytes retained viability and functionality in a medium that mimicked most-closely, the biochemical properties of L. polyphemus hemolymph. When presented with a fungal, bacterial or synthetic challenge, ~9% of amebocytes in vitro were found to be phagocytically active. Target internalisation was confirmed via the use of fluorescent quenchers and membrane probes. Within 4 hours of target internalisation, amebocytes underwent apoptosis, characterised by the loss of plasma and mitochondrial membrane potential, increased caspase-3 activity and extracellularisation of PS. Phagocytosis-induced cell death led to a proportional increase in the level of Hc-derived PO activity, suggesting that Hc may be interacting with PS present on terminal amebocyte membranes. The PO activity of Hc was investigated further in order to address an economically important issue; hyperpigmentation in commercial shellfish. While PO enzymes are thought to be the cause of hyperpigmentation in Nephrops norvegicus, evidence presented here suggests that cellular PO is inactivated after freeze-thawing, while extracellular Hc retains stability and displays a heightened level of inducible PO activity under similar treatments. Known PO inhibitors were used successfully to reduce Hc-derived PO activity, with inhibitors assumed to bind Hc in a manner similar to PO-inhibitor complexes. Structural and functional studies of hemocyanins and immune cells presented here provide new insights into the interactions of hemocyanin-activator complexes in invertebrates.

The American horseshoe crab (HSC), Limulus polyphemus, is one of four species of horseshoe crabs found throughout the world, and the only one found in North America. It is an economically and ecologically important species throughout its native range from Maine to the Yucatan Peninsula. Harvested for fertilizer and livestock feed in the 19th century, the species is now harvested as bait for whelk and eel fisheries, and for their blood by the biomedical industry. The Atlantic States Marine Fisheries Commission (ASMFC) started to formally manage HSC in 1998 with its Interstate Fisheries Management Plan (IFMP). Unique emphasis and harvest limits have been placed on the Delaware Bay stock, as it is commercially exploited and a critical food source for the threatened red knot, Calidris canutus rufa. Previously, estimates of relative and total abundance of HSC in the Delaware Bay area were based on a design-based approach using a stratified random sampling design. In Chapter 1 of this work, I developed hurdle models for each of the six HSC demographic groups to standardize catch-per-unit-effort (CPUE) and estimate relative abundance using a model-based approach. It was determined that while the two approaches resulted in mostly convergent estimates of relative abundance, external factors such as month, time-of-day, and average depth have major effects on the observed CPUE of all demographic groups. Chapter 2 involved the development of hurdle models for the three species of bycatch frequently caught in our trawls, channeled whelk (Busysotypus canaliculatus), knobbed whelk (Busycon carica), and summer flounder (Paralichthys dentatus). It was found that channeled whelk relative abundance has been at a historical low since 2016, while summer flounder has been at a consistent high. Recent estimates of knobbed whelk relative abundance have been less variable than previously seen, with estimates since 2016 being similar to those seen before 2012. These results provide the first estimates for whelk population trends in the mid-Atlantic region and add to the growing knowledge of summer flounder relative abundance in the area. In Chapter 3, I applied the hurdle models developed in Chapter 1 to estimate the total abundance of HSC in the Delaware Bay area. For this work, I developed two spatio-temporal variograms to estimate bottom temperature and bottom salinity at unmeasured cells per month in the time series. The results showed that night estimates of total abundance were consistently higher than daytime estimates, and estimates from September or November resulted in the highest estimated catch for all demographic groups. The results suggest that when comparing September model-based estimates at night to those of the design-based approach, nearly a third of all previous design-based estimates significantly underestimated the total abundance of HSC in the Delaware Bay area. This result suggests that the ASMFC can recommend increased harvest limits for mature individuals if that action aligns with the goals of their adaptive resource management (ARM) framework. / Master of Science / The American horseshoe crab (HSC), Limulus polyphemus, is one of four species of horseshoe crabs found throughout the world, and the only one found in North America. It is an economically and ecologically important species throughout its native range from Maine to the Yucatan Peninsula. Harvested by the millions in the 19th century, the species is still harvested as bait for whelk and eel fisheries, and for their blood by the biomedical industry, on the order of hundreds of thousands. Formal management of HSC by the Atlantic States Marine Fisheries Commission (ASMFC) began in 1998, and allowable catch and landings have decreased since 1999. A strong focus has been placed on the Delaware Bay population, as it is the center of HSC abundance and provides a critical food source for the threatened red knot, Calidris canutus rufa, while also being commercially exploited. To effectively manage the species, it is important to accurately estimate relative and total abundance so that proper harvest limits can be set. Previously, estimates of relative and total abundance of HSC in the Delaware Bay area were based on a design-based approach using a stratified random sampling design. In Chapter 1 of this work, I developed hurdle models (a generalized linear model that models the probability of observations and the observed positive counts using two separate regression models that are then combined) for each of the six HSC demographic groups to remove the effect of external factors (year, latitude, longitude, depth [inshore/offshore], topography, average trawl depth, time-of-day, month, bottom temperature, bottom salinity, and distance from shore) on our observed catch-per-unit-effort (CPUE) and estimate relative abundance using a model-based approach. It was determined that while the two approaches resulted in mostly convergent estimates of relative abundance, factors like month, time-of-day, and average depth had major effects on the observed CPUE of all demographic groups. Chapter 2 involved developing similar hurdle models for three species of bycatch frequently caught in our trawls, i.e., channeled whelk (Busysotypus canaliculatus), knobbed whelk (Busycon carica), and summer flounder (Paralichthys dentatus). It was found that channeled whelk relative abundance has been at a historical low since 2016, while summer flounder has been at a consistent high. Recent estimates of knobbed whelk relative abundance have been less variable than previously seen, with estimates since 2016 being similar to those seen before 2012. These results provide the first estimates for whelk population trends in the mid-Atlantic region and add to the growing knowledge of summer flounder relative abundance in the area. In Chapter 3, I applied the hurdle models developed in Chapter 1 to estimate the total abundance of HSC in the Delaware Bay area. To do this, the bottom temperature and salinity had to be estimated for each geographic cell. This was accomplished by developing two spatio-temporal variograms which allowed me to estimate either variable at an unmeasured point and time based on its spatial and temporal distance from a measured value in a process known as spatio-temporal kriging. The results showed that night estimates of total abundance were consistently higher than daytime estimates and that estimates from September or November resulted in the highest estimated total abundance for all demographic groups. The results suggest that when using September model-based estimates at night to compare against the design-based approach, nearly a third of all previous design-based estimates significantly underestimated the total abundance of HSC in the Delaware Bay area. This outcome could justify ASMFC increasing recommended harvest limits for mature individuals if that action aligns with the goals of their adaptive resource management (ARM) framework.