In the present investigation two container breeding mosquitoes, Aedes aegypti and Aedes albopictus were exposed to Sindbis virus (SINV). Mosquitoes were proffered a viremic bloodmeal using a vertical membrane feeder in an attempt to infect females while preserving virus-gut interactions that are speculated to be responsible for amplification and selective roles encountered in nature. Cohorts of 50 mosquitoes of each species were exposed to SINV strain TR339 or EMEM in a bloodmeal and incubated in insectary conditions for 30 days. Once infected, the mosquito remains persistently infected for life, accumulating mutations in the virus RNA genome. Plaque size variants may indicate changes to the virus genome, therefore in this investigation TR339 persistent disseminated isolates (TR339-PDIs) were isolated from mosquito legs 30 days post infection (p.i.) and assayed for plaque size differences. These TR339-PDIs represent virus selected for by the whole mosquito. TR339-PDIs following a long term infection were used to study virus adaptations selected for during persistent infection of the mosquito host. The two mosquito species exhibited different host responses to SINV; survivorship, dissemination rates, and immunohistochemistry of mosquito midguts and hindguts were assayed at 30 days p.i. Aedes aegypti and Ae. albopictus along with SINV strain TR339 provide a good model system to study the selective pressures that occur within the whole mosquito. Greater knowledge of the mechanisms of virus persistence in the mosquito host is vital for controlling arthropod-borne disease at the vector level.

Bald Cypress (Taxodium distichum (L.) Rich.) is native to freshwater wetlands of Florida. The vitality of cypress within coastal freshwater wetlands is threatened by saltwater intrusion. Biomarkers to detect sub-lethal salinity stress were developed using a controlled greenhouse study. Cypress saplings maintained at elevated salinities of 4 and 8‰ exhibited a decrease in maximum quantum yield (MQY) and an increase in non-photochemical quenching (NPQ). Cypress leaves exhibited an increase in Na+, H2O2, and free proline content compared to plants maintained in freshwater. These biomarkers were used to detect salinity stress within a population of cypress associated with the lower St. Johns River where saltwater intrusion is occurring. Cypress in a basin swamp exhibited signs of salinity stress with low MQY and elevated NPQ values compared to Cypress at other sites. Cypress leaves at the basin swamp also had the highest Na+, lipid peroxidation, and proline content compared to plants at other sites. Detached Cypress leaf experiments were conducted to explore the mechanisms of salt tolerance. Detached cypress leaves were first exposed to elevated NaCl concentrations for 24, 48, or 72 hours. Elevated salinity caused a decrease in leaf transpiration for all times tested. Total peroxidase activity exhibited an increase in response to salt stress after 48 hours. Enhanced peroxidase activity was found to be associated with the induction of a ~37 kDa peroxidase isoform. Treatment of leaves with clofibrate caused an increase in activity of the ~37 kDa peroxidase. Pre-treatment of leaves with brefeldin A (BFA) blocked the induction of the ~37 kDa peroxidase associated with salt stress. Pre-treatment of Cypress leaves with diphenyliodonium (DPI) blocked the decrease in transpiration associated with salt stress, suggesting that H2O2 is enzymatically produced within the stomata in response to salt stress

Reproductive biology is a necessary element for the management of elasmobranch fisheries. Traditionally, characterization of elasmobranch reproduction has involved lethal sampling to examine gross reproductive structures and development of embryos. However, this method is counterproductive to the conservation of shark populations. One non-lethal alternative is the measurement of serum hormones, which often vary according to reproductive events. Radioimmunoassay (RIA) has been used to measure hormone concentrations in reproductive endocrinology, but can be problematic for researchers. Alternatively, chemiluminescence immunoassays (CLIA) are routinely used for measuring circulating hormone concentrations in low-volume, non-extracted human serum samples. However these assays have not been previously examined for use with elasmobranch blood. In the first component of this study, I examined whether CLIA was a suitable alternative for detecting seasonal profiles of these hormones in the bonnethead, Sphyrna tiburo. This was accomplished by collecting serum from sexually mature male (n = 35) and female (n = 32) bonnetheads , measuring reproductive organs for maturity and reproductive stage, and measuring concentrations of testosterone (T) in males, and 17β-estradiol (E2) and progesterone (P4) in females using RIA and CLIA. CLIA was successfully validated for use with shark serum by assessing parallelism and spike recovery. CLIA-derived measurements were significantly correlated with those obtained with RIA (r = 0.809, 0.773, and 0.908 for T, E2, and P4, respectively; p

Skeletal muscle atrophy can occur at any age and as a result of numerous physiological conditions and thus, it was necessary to better identify the molecular underpinnings of the atrophy cascade so that new therapeutic targets to treat muscle wasting might be identified. MuRF1 was first identified as a marker of skeletal muscle atrophy over a decade ago; however, recent work suggests that this E3 ubiquitin ligase may participate in muscle wasting by regulating the transcriptional activity of genes differentially expressed in response to muscle atrophy. Dusp4, a dual-specificity phosphatase and member of the MAPK cascade, is induced in response to neurogenic atrophy; however, this induction is significantly blunted in the MuRF1-null mice which are resistant to muscle atrophy. The research presented in this thesis aims to characterize the mechanism by which MuRF1 may transcriptionally regulate Dusp4 and characterizes the function of Dusp4 in skeletal muscle.

Natural products are bioactive secondary metabolites produced by living organisms and are prevalently utilized as pharmaceutical drugs. Marine adapted organisms are a promising source of new natural products possessing unique chemical structures and biological activities. By studying the biosynthetic pathways employed by living organisms to produce natural products, insights into new strategies to generate molecules to combat disease and overcome drug resistance may be gained. This thesis study aimed to uncover the biosynthetic pathway employed by a marine actinomycete, Nocardiopsis sp. CMB-M0232, to catalyze the assembly of the nocardioazines. These molecules are a group of 2,5-diketopiperazine natural products that feature structurally unique functional groups. Nocardioazine A, the hypothesized end product of the nocardioazine biosynthetic pathway, exhibits anticancer activity. Bioinformatics analyses revealed three biosynthetic gene clusters from Nocardiopsis encoding proteins with hypothesized roles in nocardioazine A biosynthesis. Two cyclodipeptide synthases (CDPSs), NozA and NcdA, were biochemically characterized in vivo and in vitro to reveal that both are substrate specific enzymes that utilize tryptophan-charged tRNA substrates to catalyze assembly of cyclo(L-Trp-L-Trp), a proposed precursor of nocardioazines. Fidelity is uncommon amongst characterized CDPSs, making NozA and NcdA important CDPS family additions. This study also aimed to characterize NozD and NozE, two cytochrome P450 homologs with predicted roles as diketopiperazine-tailoring enzymes. Heterologous expression of these enzymes in Streptomyces strains was not able to confirm the functions of NozD and NozE but set the stage for future studies to optimize conditions for probing their roles in nocardioazine A biosynthesis. The results gathered from this study, along with future work to better understand the engineering of unique functional groups from Nocardiopsis may provide opportunities to produce new bioactive molecules.

Invasive species are a top threat to global biodiversity. Lionfish (Pterois volitans/miles) are a marine invasive predator that are now established in the Western Atlantic, Caribbean Sea and Gulf of Mexico. Studies that have documented impacts of lionfish suggest they can reduce fish recruitment and native species biomass by up to 80%. Research on lionfish impacts, biology and ecology is heavily focused in tropical and subtropical systems, with considerably less research effort occurring in the temperate regions of their invaded range, such as the South Atlantic Bight. Lionfish life history estimates are important to modeling lionfish population growth, modeling future dispersal and evaluating the efficacy of different removal strategies. Since lionfish life history will vary with a suite of environmental, biological and ecological factors, estimates of lionfish life history should be collected in all regions of their invaded range. The purpose of this study was to collect baseline information of lionfish biology and ecology in the southern portion of the South Atlantic Bight, an unstudied region for this species. As such, chapter one uses a quantitative approach to determine lionfish life history estimates important to management. Some important results of chapter one are: lionfish are recruiting at one main time throughout the year, growth changes seasonally and the population is relatively young (< 3 years of age). Chapter two takes the first step in determining lionfish impacts in this region by quantifying their diet. The main conclusion of this chapter is that round scad (Decapterus punctatus), sand perch (Diplectrum formosum) and black sea bass (Centropristis striata) are the most important prey items in the lionfish diet. Black sea bass are an important fishery in the South Atlantic Bight, and lionfish could be negatively affecting recruitment. Together, these chapters provide important insight into lionfish ecology in this region and in general.

Purified brush border membrane vesicles (BBMV) were isolated to characterize primary cellular transport mechanisms for white shrimp. The ultimate goal is to determine the effective components of a shrimp’s diet, thereby enhancing growth, as well as nutrient content. Juvenile shrimp are dependent on plant material as a food source. Potassium is a key component of plants, thus it may play a role in nutrient transport. In addition, divalent metals have been shown to act as co-transporters in several other organisms, thus they may serve as a transport mechanism for shrimp. Fresh, live, white or brown shrimp were obtained, and from them 15-30 hepatopancreases were dissected to prepare the BBMV. Methods for preparing BBMV were based on the Mg2+ precipitation technique developed by Kessler et al., (1978) and Biber et al. (1981) for mammalian eipithelia and applied to crustaceans. The results suggest that there is a sodium/potassium-dependent glucose transport system that resembles the SGLT1 system of vertebrates, except the shrimp transporter can accept both sodium and potassium as cofactors, while the vertebrate system is restricted to sodium stimulation. Potassium showed strong stimulation of L-histidine uptake by shrimp BBMV, suggesting that a crustacean isoform of the insect potassium-dependent carrier protein (KAAT1) might be present in shrimp, and contribute to amino acid uptake. Amino acids also appear to form bis-complexes with divalent metals, that are transported by an analog of the dipeptide transporter (PEPT1). The metals appear to be accommodated, with varying affinities. PEPT1 has been described as a very non-specific carrier process because it transports such a wide range of di- and tripeptide combinations.

As the human population in Florida continues to expand, development follows, and tree farms give way to homes and businesses. As parks are established, restoration of these semi-natural plantations may provide critical habitat for species conservation. This study evaluates vegetation response to restoration treatments at two study sites, formerly tree farms, now preserves in NE Florida. Treatments included thinning, clearing, or control (no treatment) within 10m^2 plots. Thinning reduced tree canopy to 20% (2-3 pines/plot) and removed all other vegetation; clearing treatments removed all biomass to bare soil; no herbicides were used. Within these plots richness and abundance was assessed by establishing two parallel transects and counting ramets on a bi-annual basis. Tree diameter at breast height (dbh) was also measured (cm). It was hypothesized that release of resources (thinning and clearing) would increase overall diversity (more so in graminoids and forbs), and encourage more robust tree growth versus control groups. Diversity ANOVA (Simpons & Shannon indices) showed significant differences due to survey date (p< 0.05) at the McGirts Creek site and a significant (p= 0.056) effect for the interaction term at the Tigers Point site. Tree dbh also increased at a significantly greater rate in thinned, versus control groups at the Tigers Point site (p= 0.03) perhaps due to higher initial tree density, but not at the McGirts Creek site (p= 0.85). Placing species into guilds revealed both sites reflected high levels of graminoids in cleared plots, which is consistent with early successional species (pioneer plants). McGirts followed hypothesis as forbs and graminoids were dominant in both thinned and cleared plots and the Tigers Point site had higher levels of vines and shrubs than expected. Restoration goals of increasing vegetative diversity, especially in r-selected species, and robust growth can be met by techniques used in this study.

Epiphytes are organisms attached to plants and are responsible for the majority of primary productivity in many aquatic systems. While epiphytes serve as a valuable food resource to herbivores, they may prove deleterious to the host plant by competing for light and nutrients, as well as increasing sheer stress. This study evaluated the impacts of nutrient additions, nitrogen and phosphorus, on the epiphytic algal community on Spartina alterniflora over the course of two growing seasons. Three nutrient treatments (N, P, and N+P) and one control treatment were placed in a salt marsh in the Tolomato River during the growing seasons of 2011 and 2012. To assess community development, we examined biomass, ash-free dry mass (AFDM), chlorophyll-a levels, cell counts, and community diversity by algal division. The nutrient additions did not significantly alter any of the measured parameters in either sampling year. However, the sampling month did have a significant (pa, and community composition. A total of 155 infrageneric taxa were identified. Biomass tended to be dominated by diatoms and red algae, while cyanobacteria were most abundant. In both years, biomass was highest in the spring with a second smaller pulse in the fall. Conversely, chlorophyll-a levels varied between the years and did not show the same monthly patterns as AFDM. A laboratory study subjecting S. alterniflora to the same nutrient additions also found no significant effects of increased nutrients, but did observe temporal changes in biomass and chlorophyll-a levels. Overall, epiphytic growth was not influenced by nutrient additions in this study suggesting that this and other similar salt marsh systems may be resilient to anthropogenic eutrophication. Instead, other factors, such as light and herbivory, likely played a key role in determining epiphytic algal growth and community composition.

Arthropod-borne-viruses (arboviruses) pose a global threat due to their ability to be transmitted by hematophagous insects to vertebrate hosts resulting in a range of serious infectious diseases. Sindbis virus (SINV) is the prototype arbovirus of the genus Alphavirus in the family Togaviridae. The purpose of this study was to investigate the use of a fluorescent tagged reporter virus in both in vitro and in vivo environments. The fluorescent protein GFP was inserted between the Capsid and PE2 in the genome of TR339; SINV TaV-GFP (Wm. Klimstra Lab). This virus construct should have the same infectivity and virulence as wild type TR339, leaving a fluorescent ‘path’ in infected cells that may reveal virus transit. Virus stocks were grown in BHK-21 vertebrate cells and C7-10 mosquito cells. Two Aedes albopictus mosquito cell lines, C7-10 and C6/36, were then challenged with vertebrate and mosquito grown reporter virus. Evidence of GFP were seen as early as 6 hours post infection (p.i.) in all samples. Infected C7-10 cells with the vertebrate grown reporter virus were fixed for 1 hour in chilled 4% buffered paraformaldehyde; GFP was shown to be resilient to both fixation and light quenching. Ultimately, Ae. aegypti mosquitoes were challenged with a viremic bloodmeal at a titer of 107 PFU/ml and midguts were dissected over several days. The presence of GFP was observed in midgut columnar epithelial cells as early as day 3 p.i. and remained localized even at day 30 p.i. This is in agreement with published work on the interaction of TR339 in Ae. aegypti gut, signaling this viral construct as a means to visualize wild-type infection.