Milton, Sarah L.

The dinoflagellate Karenia brevis is one organism responsible for harmful algal blooms (HABs) that severely impact marine life. K. brevis produces a suite of neurotoxins referred to as brevetoxins (PbTx) which bind to voltage-gated sodium channels (VGSCs) in excitable tissues, affecting cellular permeability leading to a suite of symptoms and potentially cell death. Brevetoxicosis is difficult to treat in sea turtles as the physiological impacts have not been investigated and the magnitude and duration of brevetoxin exposure are generally unknown. Due to their threatened and endangered status, experimental exposures cannot be performed to determine the fate of brevetoxin in sea turtle tissues, making it difficult to design appropriate treatments. The freshwater turtle, Trachemys scripta, was utilized as a model for brevetoxin exposure in turtles. Turtles were exposed to intratracheal instillation (10.53μg/kg) or oral dosing (33.48μg/kg) of PbTx-3 3x weekly over a period of 2-4 weeks. Tissues and fluids were collected for ELISA to determine PbTx-3 uptake and distribution, routes of excretion and rates of clearance (1h-1wk post-exposure). Tissues were also preserved for histopathology. Primary turtle neuronal cell cultures were exposed to PbTx-3 in the presence and absence of various agonists and antagonists to determine brevetoxin’s mode of action. PbTx-3 was widely distributed in all tissues and fluids following both intratracheal and oral exposures, but was largely cleared from the system within 24 hours; PbTx-3 moved into the bile and feces over 48h post exposure indicating that this is the main route of excretion. While exposed animals showed clear behavioral symptoms of toxicity including muscle twitching, swimming in circles, and ataxia, there was no evident tissue pathology. Despite the evident behavioral effects, turtle neurons are surprisingly resistant to PbTx-3, with an EC50 significantly higher than is seen in mammalian neurons. While PbTx-3 exposure resulted in significant Ca2+ influx, various antagonists prevented Ca2+ influx when added with PbTx-3 confirming the mechanism of action through VGSCs. Upregulation of Hsp72 in the turtle brain could be enhancing cell survival. Based on results, intralipid treatment post PbTx-3 exposure rapidly decreases symptoms and proves to be a suitable treatment for toxin exposure.

The channelized Kissimmee River experiences seasonally low dissolved oxygen levels
which have changed the ecosystem to favor air-breathing species, while gamefish
populations have declined greatly. In 2001 , the central portion of the river (Pool C) was
physically restored.
To evaluate the usefulness of various stress markers and assess the success of the partial
restoration, the stress responses were analyzed (brain monoamines, heat shock proteins,
and plasma cortisol) of air-breathing fish (bowfin, Florida gar), versus gamefish
(largemouth bass and bluegill sunfish). Fish were collected from hypoxic and normoxic
water at three sites in the Kissimmee River. Statistical analysis (MANOV A) showed
overall significantly higher stress response in hypoxic gamefish than those in normoxia,
with higher hsp72 levels, DOPAC, and Dopamine turnover (DOPAC:DA). Hsp60 and
cortisol was significantly higher only in hypoxic bass while serotonin and 5-HIAA were
significantly higher in hypoxic bluegill sunfish. Air-breathers did not show higher stress
levels during hypoxia.

Fibropapillomatosis (FP) is a devastating pandemic characterized by benign
cutaneous neoplasias that is afflicting marine turtles worldwide. This study evaluated the
expression of HSP72, GRP96, and Bcl-2 in tumor and healthy biopsies. These proteins
are found in high levels in some tumors and prevent apoptosis, allowing tumor cells to
survive. HSP72 and GRP96 are also known to initiate an immune response and may
contribute to the regression often observed in FP tumors. Results were found that
showed all three protective proteins had significantly higher levels in actively growing
tumor tissue compared to healthy tissue. Specifically, cauliflower-like tumors, thought to
be actively growing, were found to have higher levels of HSP72 and GRP96 compared to
healthy skin, whereas smooth tumors, thought to be regressing, did not. These results
offer insight into the molecular mechanisms behind the development of FP tumors and
open a number of avenues for future research.

Following nest emergence, sea turtle hatchling dispersal can be disrupted by
artificial light or skyglow from urban areas. Mis- or disorientation increases exposure to
predation, thermal stress, and dehydration, could consume valuable energy, and thus
influence the likelihood of survival. This study utilized laboratory simulations of
extended crawl distances and field observations to investigate the energy cost of
disorientation crawling on loggerhead (Caretta caretta) and green (Chelonia mydas) sea
turtle hatchlings and the impact on the subsequent swim frenzy. Extended crawls
increased oxygen consumption, decreased plasma glucose, and reduced green hatchling
swim performance following a 200m crawl. Surprisingly, plasma lactate concentrations
did not increase with crawl distance, likely due to frequent rest periods that would,
however, increase time on the beach and thus exposure to predators. This research may
provide managers with physiological data to determine best practices for sea turtle
conservation and adds to the biological knowledge of these animals.

Florida’s sea turtle populations are increasing due to conservation efforts;
however, sea turtle species are vulnerable to climate change. Turtles exhibit temperaturedependent
sex determination, in which nest environment influences sex. Environmental
changes may produce altered sex ratios that limit reproduction potential; therefore
hatchling sex ratios should be monitored. Hatchlings are not externally sexually
dimorphic, making sex identification difficult. This study established baseline plasma
hormone concentrations in hatchling and post-hatchling green (Chelonia mydas) and
loggerhead (Caretta caretta) sea turtles using High Performance Liquid Chromatography.
Five hormones were assayed and were present in the majority of samples (testosterone:
N.D.-10.12, progesterone: N.D.-0.43, estradiol: N.D.-4.78, estriol: N.D.-5.55 and estrone:
N.D.-1.67 μg/mL). Plasma hormones did not distinguish hatchling sex because male and
female ranges overlapped. Hormone concentrations varied with sex but also with incubation temperature, indicating that climate change could impact hatchling and posthatchling
hormone profiles and thus could impact future fitness.

Oxidant stress and injury is inherent in many human diseases such as ischemic vascular and respiratory diseases, heart failure, myocardial infarction, stroke, perinatal and placental insufficiencies, diabetes, cancer, and numerous psychiatric and neurodegenerative disorders. Finding novel therapeutics to combat the deleterious effects of oxidative stress is critical to create better therapeutic strategies for many conditions that have few treatment options. This study used the anoxia-tolerant fruit fly, Drosophila melanogaster, to investigate endogenous cellular protection mechanisms and potential interactions to determine their ability to regulate synaptic functional tolerance and cell survival during acute oxidative stress. The Drosophila larval neuromuscular junction (NMJ) was used to analyze synaptic transmission and specific motor axon contributions. Drosophila Schneider 2 (S2) cells were used to assess viability. Acute oxidative stress was induced using p harmacological paradigms that generate physiologically relevant oxidant species: mitochondrial superoxide production induced by sodium azide (NaN3) and hydroxyl radical formation via hydrogen peroxide (H2O2). A combination of genetic and pharmacological approaches were used to explore the hypothesis that endogenous protection mechanisms control cellular responses to stress by manipulating ion channel conductance and neurotransmission. Furthermore, this study analyzed a group of marine natural products, pseudopterosins, to identify compounds capable of modulating synaptic transmission during acute oxidative stress and potential novel neuromodulatory agents.

This dissertation examined the natal origins, home-range, and in-situ foraging behavior of an aggregation of sub-adult hawksbill turtles (Eretmochelys imbricata) found off the coast of Palm Beach County, Florida. Surveys were conducted on approximately 30 linear km of reef between 15 and 30 m in depth. Tissue samples were retrieved from 112 turtles for mtDNA haplotype determination. GPS-linked satellite transmitters were deployed on six resident sub-adults, resulting in both minimum convex polygon (MCP) and 95%, 50%, and 25% kernel density estimates (KDE) of home-range size. A foraging ethogram was developed, and sequential analysis performed on thirty videos (141 total minutes) of in-situ foraging behavior. Seventeen total haplotypes were identified in this aggregation, the majority (75%) of which represented rookeries on Mexico’s Yucatan Peninsula. Other sources, from most to least important, include Barbados, Costa Rica, Puerto Rico, Antigua, and the U.S. Virgin Islands.

Loggerhead (Caretta caretta) and green (Chelonia mydas) turtles recruit to nearshore environments as juveniles. These often degraded habitats are associated with emerging diseases such as green turtle fibropapillomatosis (GTFP), however there are few studies on immune function in sea turtles. The objective of this research was to
quantify phagocytosis of the innate immune system by flow cytometry and compare
levels between animals from a degraded habitat (the Indian River Lagoon, FL) to a more pristine environment (the Trident Basin, Port Canaveral, FL), and across a range of temperatures. While in vitro temperatures did not alter rates of phagocytosis, it was
higher in samples obtained in the summer than winter. Rates of phagocytosis in sea
turtles with GTFP and from degraded environments with increased prevalence of GTFP
were low compared to animals from the more pristine environment, suggesting that the
environment can alter innate immunological function and thus contribute to the
development of disease.

Sea turtles have temperature-dependent sex determination, with males being produced at cooler temperatures and females at warmer ones. Thus, sex ratios are often estimated on average nest temperatures, but this is unreliable. Therefore, many studies have begun to look to alternative methods to identify sex ratios. Other methods used to determine sex require hatchling sacrifice or are labor intensive. This study utilized high performance liquid chromatography (HPLC) to investigate correlations between steroid hormone levels in hatchling plasma and allantoic fluid, nest temperature and sex. Hatchling sex was determined laparoscopically to ground-truth hormone profiles. No correlation was found between hormones, nest temperature and sex. However, as hormones were readily detectable by HPLC, the technique may be applicable to juvenile or adult turtles with mature profiles.

Specific dynamic action (SDA) describes a post-feeding increase in oxygen consumption where most energy derived from food is allocated to post-absorptive processes, including growth and maintenance. SDA and growth in hatchling sea turtles is of special interest because they experience drastic changes in diet, oxygen availability and habitat during the first several months of life. This study investigated SDA variation between species and among age groups. SDA was quantified in two species of sea turtle (Chelonia mydas, Caretta caretta) at 7, 30, 60, & 90 days post-hatching. Turtles were fed a known amount of food, and oxygen consumption was monitored using a metabolic chamber. SDA percentage of total energy did not increase with age (species pooled), although magnitude of SDA was higher in C. mydas when compared to C. caretta (ages pooled). These results imply that SDA does not increase proportionally with age, and that hatchling C. mydas use more absolute energy for SDA than C. caretta .