Although it was once thought that neurons solely rely on glucose as a substrate for cellular energy production, it is now known that small monocarboxylate molecules, like pyruvate, lactate, and ketone bodies, are also utilized. Monocarboxylates are transported across plasma membranes via facilitated diffusion using a family of transport proteins known as monocarboxylate transporters (MCTs). Four MCTs (MCT1, MCT2, MCT3, and MCT4) are expressed within neural tissues. Expression of the MCTs has been tied to co-expression of a cell adhesion molecule belonging to the Basigin subset of the immunoglobulin superfamily (IgSF). Basigin gene products are known to interact with MCT1 and MCT4 in the mammalian neural retina and this association is essential to support the cellular energy needs of photoreceptors. A previous study indicated that Basigin gene products use hydrophobic amino acids within specific regions of the transmembrane domain to interact with MCT1. In the present study, it is hypothesized that the same amino acids within the transmembrane domain are used to interact with MCT4, but that no association exists with MCT2, which typically interacts with a different member of the IgSF subset. Therefore, the purpose of the present study was to assess the association between Basigin gene products and MCT4, and with MCT2. Recombinant proteins corresponding to the transmembrane domain of Basigin gene products were used in in vitro binding assays with endogenous MCT2 and MCT4 from mouse brain protein lysates. Contrary to the hypothesis, it was determined that the transmembrane domain of Basigin gene products binds to both MCT2 and MCT4 in vitro. Different amino acids within the transmembrane domain of Basigin gene products are used for each association and the pattern is different from that used in the association with MCT1. The data suggest that Basigin plays multiple roles in the nervous system.

Although tiger sharks are an important apex predator in many ecosystems, little is known about their reproduction. The goal of this study was to determine the size-at-maturity and the reproductive seasonality of tiger sharks in the western Atlantic Ocean. This was achieved using a combination of ultrasonography and measurements of plasma hormone concentrations; in particular, testosterone for males and estradiol and progesterone for females. Steroid hormone concentrations were measured using chemiluminescent assays (CLIA). Maturity was also examined through histology of reproductive organs in females and clasper calcification in males. Females were found to mature between 270 and 310 cm total length and males were found to mature between 260 and 300 cm total length. Mating was determined to occur in October/November, based on the presence of mating wounds on females and increased concentrations of testosterone in males. Some females were shown to exhibit increased plasma estradiol concentrations also during October/November; however, we do not believe that ovulation takes place until May or June based on ultrasonography data. This suggests a period of sperm storage although histological examination of the oviducal gland was not able to confirm this. Ultrasonography data, showing increasing embryo size over the course of a year, and data on minimum size of tiger sharks caught in longline surveys suggested that parturition occurs between June and September with pups being born as small as 56 cm fork length. The findings from this study show that some tiger sharks reach reproductive maturity at sizes smaller than what has been previously suggested. Additionally, the possibility of tiger sharks storing sperm suggests that their reproductive cycle is a minimum of two years long and could be up to three years in duration. This information is important for management of the species in the future. Additionally, this study adds to the limited knowledge about reproduction of elasmobranchs and how patterns of reproductive steroids can correlate with different reproductive events.

Vitellogenin (Vtg) is a precursor to yolk-proteins produced in the liver of many invertebrates and non-mammalian vertebrates; its synthesis is stimulated by the hormone estradiol (E2). This study is the first to characterize vitellogenin synthesis in a placental viviparous elasmobranch, the yolk-sac placental bonnethead shark, Sphyrna tiburo. This study focused on determining where and when Vtg is produced, as well as what hormonal factors regulate Vtg production. The liver was confirmed as the site of Vtg production via immunohistochemistry. Immunoreactivity was also observed within granulosa cells of ovarian follicles; further experimentation is needed to determine if this is indicative of Vtg production by these cells. Using immunoblotting, the highest proportions of Vtg positive females were found in March, with Vtg production continuing into April and May. Putative Vtg production was found to begin in August for some individuals, with production continuing throughout the fall and winter months. In regards to hormonal regulation, immunohistochemical analysis identified receptors for E2 and progesterone (P4) within the liver. Comparison of the monthly E2 and Vtg cycles provides evidence that E2 stimulates Vtg production in S. tiburo, as high or increasing concentrations of E2 correlated with Vtg presence in the plasma. Preliminary results also suggest in vitro production of Vtg by liver tissue when exposed to E2. Comparison of the monthly P4 and Vtg cycles suggests P4 may inhibit Vtg synthesis, with higher levels of P4 found in the months when Vtg production declines. Additionally, the methods developed for this study were able to identify Vtg in the plasma of other elasmobranch species. Vtg detection in plasma may thus be an ideal new, nonlethal method for characterizing elasmobranch reproductive periodicity, which will aid in assessing population growth and allow for managers to possess more accurate information to make appropriate decisions for the populations.

If we want to understand how meroplankton utilize the water column and how their vertical distribution may influence horizontal advection, it is important to study their behavior in the various environments where they exist. In a well-mixed system with physical cues dampened, and no vertical layering, these organisms will have to depend on environmental cues such as light, tidal current, and tide cycle, as well as their own swimming ability to migrate vertically. Plankton and water samples were collected at three depths (near surface, midwater, near bottom) during the summers of 2013 and 2014 from sites within the main channel of the Intracoastal Waterway. Six taxonomic groups were collected including polychaetes, bivalves, gastropods, barnacles, tunicates, and crabs, and fell into one of three categories of vertical distribution. Certain preferences for vertical distribution, and habitat, of sessile invertebrates can increase, or provide refuge from, competition. To assess the potential competition for spatial resources between native and nonnative bivalves in the Guana Tolomato Matanzas estuary, settlement collectors with settlement plates at different depths were deployed for one month periods during the summers of 2013 and 2014 at two main channel sites and two feeder creek sites. Competition would likely be highest subtidally and within the main channel due to all species occurring in that habitat in higher numbers than the feeder creek.

Dusp4 and Ppme1 have been identified as novel genes in skeletal muscle that are upregulated in response to neurogenic atrophy in a mouse model. Overexpression (OE) of Dusp4 wild-type (Dusp4-WT), as well as a Dusp4 Dominant Negative (Dusp4-DN) in C2C12 cells inhibits proper muscle differentiation potentially through its attenuation of ERK-MAPK signaling. Co-immunoprecipitation analysis shows Dusp4-DN associating with ERK1/2 but not p38, suggesting Dusp4 is specific for ERK1/2 but not p38. Quantitative PCR as well as Western blot analysis confirm that Ppme1 is expressed uniformly during muscle cell proliferation and differentiation. Interestingly, Ppme1 mRNA levels appear to decrease as differentiation proceeds whereas the protein levels remain constant throughout proliferation and differentiation. Transcriptional regulation of Ppme1 was observed by cloning proximal promoter fragments of the gene. The Ppme1 promoter is highly active on its own but when myogenic regulatory factors are ectopically expressed they repress promoter activity. Furthermore, mutation of a conserved E-box sequence inhibits full induction of the Ppme1 reporter gene , suggesting this E-box element is necessary for full Ppme1 expression. Additionally, inhibition of Ppme1 using a pharmacologic inhibitor results in delayed muscle cell differentiation and significant attenuation of AP-1 reporter activity. However, Ppme1 inhibition does not result in significant effects on phosphorylation of ERK1/2, c-Jun, or AKT. Additionally, C2C12 cells were treated with ERK1/2 and MEK1/2 inhibitors to compare results to that of Ppme1 inhibition and Dusp4 OE. While all of these conditions exhibit attenuation of AP-1 reporter activity, western blotting showed Dusp4 OE and Ppme1 inhibition do not mimic the results of ERK1/2 and MEK1/2 inhibition. Thus, Dusp4 and Ppme1 likely do not just block phosphorylation but act through more complex protein-protein interactions.

Algae are a group of highly diverse photosynthetic organisms found in variety of habitats. As the primary energy base in ecosystems, knowledge of the diversity and presence of certain algal lineages is paramount to our understanding of the trophic state of aquatic habitats. In recent years, the state of Florida has seen an increase of both marine and freshwater algal blooms. Similarly, filamentous algae have begun outcompeting vascular macrophytes throughout many of Florida’s springs as nutrient enrichment from anthropogenic sources increases. Traditionally, the Florida algal spring communities have been assessed using classic morphological methods, which may underrepresent the true biodiversity present. Therefore, the goal of this study was to conduct a more complete diversity assessment implementing next-generation sequencing techniques (NGS) with morphological analyses and culturing methods. While morphological methods identified a wide variety of algal taxa, belonging to 4 phyla (Bacillariophyta, Charophyta, Chlorophyta, and Cyanobacteria), next-generation sequencing techniques provided greater detail of the diatom community. This is particularly important as many diatom taxa are used as indicators of water quality. We noted discrepancies between these two methods, highlighting how NGS techniques may complement the use of morphological analyses when analyzing algal diversity in this system. Culturing methods also revealed the presence of two taxa new to science (Nodosilinea fontisand Brasilonema variegatus), indicating these springs may represent a potential source of novel cyanobacteria. Taken together, this study showcases Florida springs are rich in algal diversity and a combination of methods is required for more complete biodiversity assessments. Future studies implementing such methods will aid in the preservation and conservation of these ecosystems.

Basigin and Embigin are members of the immunoglobulin superfamily that function as cell adhesion molecules. Studies of Basigin null mice revealed reproductive sterility, increased pain sensitivity, and blindness. It is thought that the mechanism causing blindness involves misexpression of monocarboxylate transporter 1 (MCT1) in the absence of Basigin. It is known that the transmembrane domain of Basigin interacts with MCT1. In the absence of Basigin, MCT1 does not localize to the plasma membrane of expressing cells and photoreceptor function is disrupted. Studies of the Basigin null mouse brain suggest that MCT1 is properly expressed, which suggests a separate mechanism causes the increased pain sensitivity in these animals, and also that a different protein directs MCT1 to the plasma membrane of expressing cells in mouse brain. Embigin is known to interact with MCT2 in neurons and with MCT1 in erythrocytes. It is not known, however, if Embigin normally interacts with MCT1 in the mouse brain or if Embigin acts to compensate for the lack of Basigin in the Basigin null animals. Therefore, the purpose of this study was to determine if Embigin normally interacts with MCT1, 2, or 4 in the mouse brain and if so, whether the interaction is similar to that between Basigin and MCT1. Expression of Basigin, Embigin, MCT1, MCT2, and MCT4 in mouse brain was assessed via immunoblotting and immunohistochemical analyses. In addition, recombinant protein probes corresponding to the Embigin transmembrane domain were generated for ELISA binding assays using endogenous mouse brain MCTs. It was determined that the proteins in question are rather ubiquitously expressed throughout the mouse brain, and that the cell adhesion molecules Basigin and Embigin may be co-expressed in the same cells as the MCT2 and MCT4 transporter proteins. In addition, it was determined that the Embigin transmembrane domain does not interact with the MCTs. The data therefore suggest that MCTs do not require Basigin or Embigin for plasma membrane expression in mouse brain.

The development of effective management plans for animal populations relies on an understanding of how the population is utilizing the habitat as well as the identification of any critical habitat areas. The St. Johns River (SJR), an urban estuary with a high level of anthropogenic disturbance, is home to a resident population of bottlenose dolphins (Tursiops truncatus). In chapter one, SJR dolphin habitat use patterns, the factors that influenced these patterns, and the critical habitat areas were identified. Significant associations were found in most pair-wise comparisons between season, behavioral state, group size, water depth, and location, indicating that the overall habitat use patterns of SJR dolphins were influenced by complex interactions among these variables. Additionally, two critical habitat areas were identified. Both critical habitats had high levels of anthropogenic activity and the SJR will undergo further development during the Jacksonville Port expansion project. In conjunction with increasing levels of activity, anthropogenic sound can have numerous effects on cetaceans including the masking of signals, alterations in behavior, abandonment of critical habitats, and physiological stress. In chapter two, the soundscape of the SJR was characterized to evaluate the potential impacts of anthropogenic sound on SJR dolphins. Sound levels in the SJR were consistently high and anthropogenic sound was pervasive throughout the river. Therefore, the dolphins in the SJR are at risk of experiencing long-term behavioral and physiological stress due to anthropogenic sound. Together, this work provides valuable knowledge about dolphin habitat use and the soundscape ecology of an urbanized estuary that will enable more informed management decisions and hopefully lead to more effective conservation practices.

Sucralose is a zero calorie sweetener developed and manufactured by Tate and Lyle Sweetener Company in the 1980’s. They sell the sweetener compounded with maltodextrin and dextrose under the brand name Splenda®. Sucralose was developed as a low cost artificial sweetener that is non-metabolizable in humans and can withstand changes in pH and temperature. It is not degraded by the waste water treatment process. Since the molecule can withstand heat, acidification and microbial degradation it is accumulating in the environment, and has been found in waste water, estuaries, rivers and the Gulf Stream. The highest concentration of environmental sucralose detected to date is 300 ng/L (Torres et al., 2009). Our lab has isolated six bacterial species from areas that may have been exposed to sucralose, given that sucralose has been detected throughout the aquatic environment (Mead et al., 2009). These isolates were cultured in the presence of sucralose looking for potential sucralose metabolism or growth acceleration. Sucralose was found to be nonnutritive, and we found bacteriostatic effects on all six isolates. This inhibition was directly proportional to the concentration of sucralose exposure. The amount of the growth inhibition appears to be species specific. The bacteriostatic effect may be due to a decrease in sucrose uptake by bacteria exposed to sucralose. We have determined that sucralose inhibits invertase and sucrose permease. These enzymes cannot catalyze hydrolysis or be effective in transmembrane transport of the sugar substitute. As sucralose builds up in the environment we must consider it a contaminant due to its bacteriostatic effect. Sucralose may also destabilize or shift the compositions of the bacterial communities in microenvironments such as the mammalian gut.

Estuaries are widely recognized as important habitats for the early life history stages of commercially and recreationally important marine fish species. In general the estuaries of northeast Florida are understudied, and there is a need to characterize the ichthyoplankton community at this important faunal boundary between temperate and tropical marine zones. To determine community structure and temporal patterns in the distribution and abundance of larval fish ingressing in to the Guana-Tolomato-Matanzas (GTM) estuary through its two inlets (St. Augustine and Matanzas), ichthyoplankton were sampled bi-weekly for one year at both inlets during nighttime spring flood tides beginning in March 2012. Samples were collected with a plankton net (1 m diameter with 1mm mesh) suspended 1 m below the surface in the water column. Over 30,000 individuals were collected, representing 74 taxa. Four families made up 90 % of the collection: Gobiidae (34.8 %), Sciaenidae (26.1 %), Engraulidae (19.3 %), and Gerreidae (9.3 %). Examination of the ichthyoplankton community revealed seasonal trends in species richness and in larval fish density; species richness and larval fish density were generally greatest during the protracted summer season. Spring and summer pulses in recruitment were evident in nearshore summer spawners (e.g., gobiids and engraulids), and winter peaks in recruitment were evident in marine spawned species (e.g., sciaenids, sparids, haemulids). The variety of taxa collected, and the patterns in the seasonal species assemblage of the ichthyoplankton community of the GTM estuary align with its geographical position near a faunal boundary.