Project Information File Number #22629 *Project Title Health-related Research on beluga whales conducted at Mystic Aquarium to contribute knowledge and inform management and recovery of wild beluga populations, including the ESA-endangered Cook Inlet and MMPA-depleted Sakhalin Bay-Nikolaya Bay-Amur River beluga populations. *Project Status New Previous Federal or State Permit # NMFS Parts Permit #21966 (Issued 3/30/2018) Scientific Research/Enhancement Permit #42-1642 (Expired 10/15/2007) Scientific Research/Enhancement Permit #42-1908 (Expired 1/31/2014) *Permits Requested MMPA Research permit *Where Will the Activities Occur? Transport of animals will occur from Marineland, Niagara Falls, Ontario, Canada to Mystic Aquarium, Mystic, CT USA. Research activities will occur at Mystic Aquarium, 55 Coogan Boulevard, Mystic, CT 06355 USA. Mystic Aquarium has a formal partnership agreement with Georgia Aquarium that includes research, animal exchanges, education and PR initiatives (See Appendix 1 Georgia_Mystic_Partnership). If deemed in the best interest of an individual beluga or the US beluga population for social, health, or welfare reasons, animals may be moved to Georgia Aquarium (Georgia Aquarium, 225 Baker Street NW, Atlanta, GA USA) which has agreed to participate in the described research studies and sample collections that can be conducted as part of the routine health monitoring of the belugas housed at the aquarium. Georgia Aquarium is an accredited facility with experience in the care of belugas. *Research Timeframe The requested duration of the permit is for a 5-year period: January 1, 2020 through December 31, 2025. *Sampling Season/Project Duration Import is proposed to occur in January 2020. This permit is for continuous marine mammal health related research conducted on a daily basis at Mystic Aquarium. Research will occur continuously; there is no specific season or duration of the research. To gain the maximum knowledge that can be applied for beluga conservation, we are requesting the 5-year maximum for the permit. Training 1 for research activities is already part of the Mystic Aquarium husbandry protocol and will occur on a daily basis during regular scheduled training sessions. *Abstract We request to import 5 aquarium-born belugas (Delphinapterus leucas) from Marineland Canada to Mystic Aquarium for the purpose of conducting scientific health-related research over a 5 year period that will contribute knowledge and inform management and recovery of beluga populations in the wild, including the ESA-endangered Cook Inlet and MMPA-depleted Sakhalin Bay- Nikolaya Bay-Amur River beluga populations. Given the escalating pace of environmental and anthropogenic changes in the Arctic, it is critical to act as soon as possible. We are prepared to identify causes, measure impacts, and gather important data to help with the recovery of Cook Inlet, Sakhalin Bay-Nikolaya Bay-Amur River and other beluga populations. Certain information can only be gained from studying belugas under controlled conditions. It is essential for Aquarium scientists to collaborate with wildlife biologists and managers in order to utilize our collective expertise and resources. Through this type of collaborative research we can further our understanding of beluga biology and make strides in population recovery and conservation. The research includes investigations on 1) the neuroimmunological response to environmental and anthropogenic stressors, 2) the development of novel non-invasive techniques to assess health in free-ranging, stranded and endangered belugas, 3) the hearing and physiological response to anthropogenic sound, 4) morphometrics to inform photogrammetry studies, 5) diving physiology, 6) microbiome, 7) behavior and reproduction, and 8) testing of prototype telemetry and imaging devices before deployment on wild whales. Project Description Page *Project Purpose: Hypothesis/Objectives and Justification Study 1 (Neuroimmunological Response to Environmental and Anthropogenic Stressors) Specific Purpose: To continue to develop and ground truth biomarkers, reagents and diagnostics in belugas under controlled conditions in an aquarium setting in order to assess and monitor health and interpret findings in wild whales Hypothesis: Anthropogenic and environmental stressors elicit physiological responses, which may affect the immune system or health in marine mammals. Objectives: • To characterize and investigate the beluga nervous and immune systems • To continue to develop and evaluate hormone, immune system assays and reagents, and diagnostics for health assessments in belugas • To investigate the effects of anthropogenic and environmental stressors on the nervous and immune systems (i.e. health) of belugas under controlled conditions to better interpret findings in wild belugas 2 Justification and Summary of published findings related to the objectives: Anthropogenic and environmental stressors continue to increase and pose significant threats to marine mammals. These stressors include but aren’t limited to emerging pollutants, changes in climate resulting in shifts in prey availability, changes in predator/prey relationships, emerging pathogens, and increases in noise due to dredging and construction, shipping, oil and gas exploration and drilling, sonars, geophysical surveys and oceanographic research (Bossart, 2011; Letcher et al., 2010; Moore and Huntington, 2008; Van Bressem et al., 2009; Kyhn et al., 2014). Our laboratory was the first to characterize an anatomical link between the nervous and immune system in cetaceans and specifically the beluga (Romano et al., 1994; 2002) whereby hormones and neurotransmitters released during stress could have an effect on the immune system and an animal’s ability to fight off infection and disease. In order to characterize the nervous and immune system in cetaceans, specific reagents and assays were developed, adapted and standardized in our laboratory utilizing the beluga and bottlenose dolphin as representative cetaceans (Romano et al., 1992, 1999; Mancia et al., 2007; Keogh et al., 2011; Peden-Adams et al., 2012; Spoon and Romano, 2012; Levin et al., 2014). We now have a ‘tool box” to help assess health in belugas but continued development of reagents and assays are needed (e.g. additional markers for lymphocyte subsets, natural killer cell functional assays, etc.). Moreover, our laboratory developed marine specific diagnostic assays to detect disease and pathogens such as Brucella (bacteria that cause reproductive failure) in marine mammals (Meegan et al., 2010, 2012). These tests have been useful in the management of marine mammals under professional care as well as investigating the prevalence in wild counterparts. In order to study the physiological response to stressors and impact on the immune system in marine mammals, we have studied and are continuing to conduct controlled studies on belugas under professional care as well as live capture released cetaceans and subsistence hunted belugas (Spoon and Romano, 2012; Romano et al., 2004; Bossart et al., 2008; Reif et al., 2009; St. Aubin et al., 2013; Fair et al., 2014; Thompson et al., 2014). Additional studies similar to the above are needed to increase the number of whales being investigated as well as further provide information on the health impacts of anthropogenic and environmental stressors on belugas. The continued development of reagents and assays, including diagnostic assays, to detect disease and pathogens will enable us to monitor the health of wild beluga populations. Importantly, through application of developed reagents and assays, the health of different beluga populations can be compared with the ESA-endangered Cook Inlet (CI) and the MMPA-depleted Skahalin Bay-Nikolaya Bay-Amur River beluga populations i.e. in the event of live-capture release studies, strandings (live and dead), and/or the application of non-invasive techniques (see Study 2) to collect samples from free-ranging whales. Moreover, continued research and development and validation of these “tools” will help with interpretation of health measurements obtained from other tissues such as skin and breath. Overall, these studies will contribute to the Cook Inlet Beluga Whale Recovery Plan including Action #s 4 (“Increase efforts to identify and monitor individual CI belugas, coordinating photo-identification, stranding data, genetic studies, and body condition assessments via biopsy samples of skin and blubber”); #6 (“Conduct regular biopsy surveys of CI belugas to monitor changes in condition and reproductive success in relation to environmental changes”); #25 (“Using currently available information, compare data on diseases from CI belugas 3 with other beluga populations to determine if there are abnormal levels or atypical types of disease agents present in Cook Inlet affecting CI belugas”) and #26 (“Determine types and sources of disease agents identified to be of concern specifically to CI belugas and assess management actions targeted at mitigating the disease agents”) identified in the Cook Inlet Beluga Whale Recovery Plan. The health information gained through the development and validation of health-related reagents and assays and monitoring the physiological response to challenges in a controlled setting will help enable interpretation of health measurements/biomarkers in wild whales. Moreover, these reagents and assays will contribute to information and interpretation of data from individual Cook Inlet belugas through skin biopsy studies (Action