Craig Harms

Sea turtles inhabit temperate and tropical coastal and offshore environments across the globe. These habitats are increasingly permeated with anthropogenic noise from activities such as shipping, military activity, and energy development, which may affect sea turtles. Recent measurements of underwater hearing sensitivity indicate that sea turtles are able to detect frequencies and levels of sound overlapping with those produced by various low-frequency underwater anthropogenic activities. Sea turtle hearing, however, remains poorly understood compared to other taxa, and the potential for these sources to cause temporary or permanent hearing loss or physiological damage to sea turtles remains unclear. The objective of this proposed research is to address priority data gaps in the knowledge of sea turtles hearing. Using auditory evoked potential techniques, we will measure the hearing sensitivities (frequencies and sound pressure levels of Kemp??????????????????s ridley sea turtles (Lepidochelys kempii) and juvenile loggerhead (Caretta caretta) and green sea turtles (Chelonia mydas). Results will provide researchers, managers, and stakeholders critical data to improve estimates of noise impacts to sea turtles and guide the development of appropriate mitigation measures to reduce potential harm to sea turtles from low-frequency anthropogenic sound.

Probiotics and prebiotics are live microbes and microbial substrates, respectively, which may be supplemented in the diet to alter the gastrointenstinal tract microbiota to favorable species such as lactic acid bacteria. These supplements have been shown in some terrestrial and aquatic species to confer numerous favorable effects on production efficiency, immuno-stimulation, disease resistance and gut morphology. Very little effort has been directed to evaluation of the efficacy of these products during hatchery rearing from the early larval stages through metamorphosis to the fingerling stage. Two commercially available probiotics (Aqua Blend from bio-CAt and Bactocell from Lallemand0; and two commercially available prebiotics (GroBiotic-A from IIC and SiLO Health from BASF) will be administered. In vitro clinical trials and in vivo hatchery trials will be conducted. Data will include length, weight gain, feed palatability and survival. Results of the project will be distributed to aquaculturists through refereed journal publication, articles in trade journals, conferences and a Southern Regional Aquaculture Center fact sheet.

Sea turtles inhabit temperate and tropical coastal and offshore environments across the globe. These habitats are increasing permeated with anthropogenic noise, potentially impacting these animals. Sea turtle species face many threats, and in U.S. waters are listed as endangered or threatened under the Endangered Species Act. Recent measurements of underwater hearing sensitivity indicate that sea turtles are able to detect frequencies and levels of sound overlapping with those produced by various low-frequency underwater anthropogenic activities, including shipping, underwater explosions, pile driving and other construction activities, low-frequency active sonar, and oil exploration and extraction. However, the potential for these sources to cause temporary or permanent hearing loss or physiological damage to sea turtle hearing remains unknown. The objective of this proposed research is to determine how to effectively measure temporary auditory threshold shifts in turtles and potentially provide the initial sound exposure levels which induce these temporary threshold shifts in sea turtles. Loggerhead sea turtles (Caretta caretta) will be the primary model taxa; other sea turtle species available in the research location (green sea turtles, Chelonia mydas, and Kemp??????????????????s ridley sea turtles, Lepidochelys kempii) will be tested depending upon time and availability. Initial tests and methods development will be conducted with the eastern painted turtle and red-eared slider, both of which can be collected from the wild prior or rehabilitation facilities prior to conducting research. Results will provide researchers, managers, and stakeholders critical data to improve estimates of noise impacts to sea turtles and guide the development of appropriate mitigation measures to reduce potential harm to sea turtles from low-frequency anthropogenic sound. Statement of Work Dr. Craig Harms of NC State Center for Marine Sciences and Technology, and his Research Assistant Dr. Maria Serrano, will play an advisory technical role in the restraint, sedation and anesthesia components of the study, in order to facilitate successful AEP measurements in water. This includes helping or advising on planning, logistics and supplies acquisitions for in-water restraint, sedation and anesthesia of turtles, advising an in-state veterinarian on the protocols, traveling for on-site conduct of initial trials, and refining protocols as necessary. He will also participate in writing reports and publications, particularly for materials and methods.

Both captive and free??????????????????ranging sea turtles are frequently anesthetized for diagnostic, therapeutic, and research purposes. While general anesthesia with inhalant anesthetics is a common means of restraint in these species,reports of prolonged recovery times following inhalant anesthesia abound (Chittick, 2002; Moon, 1996; Sladky,2012; Vigani, 2014). Delayed time to full anesthetic recovery can result in increased morbidity and mortality during the peri??????????????????anesthetic period as well as increased personnel time and labor costs. Identifying methods to shorten the duration of inhalant anesthetic recovery would be advantageous for both sea turtles and their human caretakers. While presumably multifactorial, delayed anesthetic recovery time in sea turtles is likely influenced by the unique chelonian cardiopulmonary anatomy and physiology and response to changing oxygen levels. To this end, previous reports have hypothesized that exposure to high concentrations of oxygen (eg, 100% oxygen)will prolong reptilian anesthetic recovery, however, results of prospective studies are mixed and none have used a chelonian model (Bertelsen, 2005; Diethelm, 1999; O, 2015). Inhalant anesthetic characteristics may also affect recovery time and, in mammals, the use of a more poorly soluble inhalant anesthetic (eg, desflurane) will hasten emergence from inhalant anesthesia (Steffey, 2015); however, the effects of desflurane use in sea turtles is currently unknown. As the use of a poorly soluble inhalant anesthetic and lower concentrations of oxygen, either alone or in combination, may be advantageous in the anesthesia of sea turtles, the objective of this study is to investigate physiologic effects and anesthetic recovery time following use of desflurane or isoflurane in either 21% oxygen or 100% oxygen in green sea turtles. We hypothesize that sea turtles receiving desflurane in 21% oxygen will have significantly faster times to anesthetic recovery without significant negative physiologic effects.

To provide medical, surgical, and preventive care support to the collection animals of the North Carolina Museum of Natural Sciences. Said care will include physical examination, diagnostic techniques, the administration of medications, surgery, and necropsy examination when appropriate.

The proposal is to apply advanced NMR-based metabolomics techniques developed in our laboratories to samples already collected during a traditional prospective crude oil and dispersant (Corexit) exposure trial to accomplish the objective of identifying useful biomarkers of low level oil exposure with potential to improve oil contamination diagnosis, evaluation of treatment protocols, and understanding of the mechanisms of oil toxicity.

Sea turtles are frequently presented to rehabilitation centers (including NCAq STAR and the other NC Aquariums) with severe traumatic injuries, including limb amputations, long bone and shell fractures, head injuries, and entanglements resulting in limb necrosis and amputation. While sea turtles have an amazing capacity to heal from these injuries with minimal intervention, strong evidence exists for the capacity of reptiles to perceive pain and discomfort that may be expected with these injuries. The responsible and ethical provision of veterinary care to these animals dictates that this potential for pain should be addressed to the best extent available. Particularly in cases of head trauma, where jaw movement may exacerbate pain and depress feeding response, provision of pain-relieving medications to injured sea turtles has anecdotally shortened time to return to feeding, and food intake is essential for wound healing and recovery. Ketoprofen is a non-steroidal anti-inflammatory drug (NSAID) that is widely used in clinical veterinary medicine for musculoskeletal pain relief, including empirical use in loggerhead turtles. Doses currently in use for these animals were previously extrapolated from available pharmacokinetic (PK) studies in other species, mostly mammalian, that may or may not be applicable to sea turtles. In a recent study funded by a North Carolina Aquarium Society Conservation Grant, we determined PK parameters for single and multiple doses of ketoprofen in loggerheads (Thompson et al. 2018). These findings have improved the safety for this important therapeutic intervention by providing solid data on plasma concentrations achieved, and we have employed it frequently. However, all nonsteroidal antiinflammatory drugs (NSAIDs) like ketoprofen are known to have some potential side effects such as gastrointestinal ulceration, kidney damage, and bleeding (Plumb 2018), and pre-existing conditions or concurrent treatments can increase the risk of side effects causing clinical complications. Therefore, we currently limit the duration of treatment to 5 days to ensure a margin of safety, and less in the presence of pre-existing conditions such as dehydration, stingray spines, and cold stunning. For the latter two conditions, hemostatic (clotting) disorders can occur, including persistent bleeding from the (painful) spine wound, or disseminated intravascular coagulation with internal bleeding occasionally associated with cold stunning (Innis and Staggs 2017). In those situations, any marginal reduction in clotting function could lead to serious additional and potentially lethal hemorrhage. Until recently, there were no diagnostic tests to assess clotting function in sea turtles, or any reptiles. Our research group has successfully adapted a hemostasis function test widely used in mammals, thromboelastography, or TEG, for use in sea turtles, through development of sea turtle specific reagents (Barratclough et al. 2016). Using TEG, we have demonstrated dramatically reduced clotting function in cold-stunned sea turtles (Barratclough et al. 2018). The objective of the proposed study is to determine the effects of a 5-day course of ketoprofen on blood clotting function in loggerheads, as determined by TEG. Results will help to guide treatments for serious injury and painful conditions in sea turtles, to keep such treatments within parameters of safe as well as humane use.

Fibropapillomatosis (FP) is a debilitating neoplastic disease that has reached panzootic proportions in green sea turtles (Chelonia mydas), and also affects other sea turtle species including loggerheads (Caretta caretta) and Kemp??????????????????s ridleys (Lepidochelys kempii). Chelonid herpesvirus 5 (ChHV5) is the presumed etiologic agent of sea turtle FP, as it is consistently identified in FP tumors using various molecular techniques. The proposed research investigates under-studied aspects of ChHV5 pathobiology in these sea turtles, including subclinical infections and infection with different viral variants. The objective of this study is to assess foraging and rehabilitating sea turtles in North Carolina for evidence of subclinical ChHV5 infection. We will use quantitative polymerase chain reaction (qPCR) to identify and quantitate four ChHV5 gene segments in whole blood samples taken from free-ranging and rehabilitating loggerhead, green, and Kemp??????????????????s ridley sea turtles encountered in North Carolina. All qPCR-positive samples will be verified using Sanger sequencing. The proposed work is a continuation of a North Carolina Aquarium Society-funded project that was initiated last year. Based on our previous findings, we predict that a small subset of the turtles sampled will test positive for ChHV5 DNA, and that the distribution of viral variants will differ from that observed in more southern areas of the southeastern United States. Better understanding of the pathobiology and epidemiology of ChHV5 will benefit rehabilitators, researchers, and population managers by informing handling, husbandry and treatment protocols that aim to avoid viral transmission. In turn, these measures will help us to prevent FP outbreaks, predict case outcomes, and inform prognoses in both free-ranging and captive sea turtles. On a broader scale, the principles, approach and techniques detailed herein are widely applicable within the fields of wildlife disease biology and ecology, and can be replicated in future studies to investigate viral diseases of diverse marine and terrestrial species across the world.

This proposal seeks funding to use proton nuclear magnetic resonance techniques (1H-NMR) to analyze diagnostically and physiologically relevant samples that were collected as an adjunct to a well-designed, traditional exposure study of petroleum and dispersant kinetics using a hatchling loggerhead sea turtle (Caretta caretta) model developed in our laboratories. We have in hand tissue samples properly collected and stored for metabolomic analysis from 20 animals exposed at 3-days posthatching, the time expected for nest emergence, to a 96-hour cutaneous exposure to either crude oil (Gulf Coast ?????????????????? Mixed Crude Oil Sweet, CAS #8002-05-9, 0.833 mL/L), Corexit (Corexit 9500A, 0.083 mL/L), crude oil/Corexit 9500A combination, or unexposed aged seawater control in a traditional exposure study. Our objective is to better understand the mechanisms underlying the complex effects of crude oil and the dispersant, Corexit-9500A on the metabolism and physiology of sea turtles. Through these studies, we expect to identify biomarkers of crude oil exposure with the potential to improve our ability to diagnose oil contamination, assess outcomes of treatment protocols, and further our understanding of the mechanisms of oil toxicity in sea turtles through future prospective research. The hypothesis for our proposed first year of research was: the metabolome of hatchling loggerhead sea turtles exposed to crude oil will be altered compared to that of unexposed hatchlings in tissues suitable for future field assessment. During the first year of studies we characterized whole blood and skeletal muscle of sea turtle hatchlings in the oil treatment and control groups after refining techniques to achieve complete hemolysis of blood and optimized extraction for the muscle samples. Good spectra were achieved and 8 and 12 compounds identified in control spectra. No additional or missing metabolites were identified blood or muscle from oil exposed hatchlings. Routine PCA using uniform and smart binning did not identify markers of separation between treatment groups. In the 3 months remaining on the original first year schedule we are using a combination of key metabolite targeting techniques to focus on metabolic pathways in muscle published research suggests are affected by petroleum exposure (energy and osmoregulation). The hypothesis we propose to pursue in year 2: the metabolome of tissues and fluids known to have roles in detoxifying polycyclic aromatic hydrocarbons will be altered compared to that of unexposed hatchlings will be addressed by 1H-NMR study of archived hepatic and bile samples from the same hatchlings using the refinements in approaches to PCA we developed in our first year work. In the third year we propose to analyze the heart muscle to evaluate the hypothesis: the constant contractions of cardiac muscle will alter the metabolomic changes seen in energetics and oxidative pathways from exposure to crude oil from those detected in skeletal muscle. We will also be able to compare Corexit-9500A only and Corexit-9500A plus oil exposures to oil only and controls for all 5 tissues. The use of 1H-NMR techniques represents a rapidly emerging and new approach to the evaluation of petroleum exposure, which promises to generate new methods to detect, quantify, and document oil exposure in wildlife, even for chronic and very low-dose oil exposures, and to compile many useful biomedical health parameters of a species increasingly susceptible to oil exposure. The studies proposed will both generate and help validate novel methods to detect and quantify oil exposure in wildlife and document their effects including those that persist after release from treatment. The design of the proposed study also examines the impact of the application of Corexit and will allow us to directly assess the impacts of one chemical countermeasure on wildlife.

To provide medical, surgical, and preventive care to a collection of confiscated reptiles housed at the North Carolina Museum of Natural Sciences. Said care will include physical examination, diagnostic techniques, the administration of medications, surgery, and necropsy examination when appropriate.