How Marine Mammals Fight off Infection and Disease

Project Title: Investigation of the Marine Mammal Immune System

The immune system protects the body from harmful substances, bacteria, viruses, and other things that might cause illness. For many years, little was known about the immune systems in marine mammals and how they function. Although researchers are making much progress in this area in recent year, there is still much information to be learned about the marine mammal immune system.

One of the main issues with studying marine mammal immune systems is that reagents that are available to study the immune system of terrestrial mammals cannot be used to investigate the marine mammal immune system. The Mystic Aquarium research team has helped solve that problem. They have focused on studying the bottlenose dolphin and beluga whale immune systems and have developed dolphin and whale-specific tools and reagents to characterize the cetacean immune system. These include molecular probes, functional assays, and cell markers.

Mystic Aquarium scientists are also cloning genes of interest from marine mammals. Cloning genes enable the comparison of immune sequences between marine mammals and humans and/or other terrestrial mammals to gain insight into the evolution of these important proteins and the immune system. By studying the immune system of marine mammals we may also gain important information on the human immune system.

Important Chemical Messengers

Project Title: Measurement of Hormones in the Blood, Saliva, and Feces of Beluga Whales

Hormones are chemical messengers that send signals to the body’s bloodstream and tissues. Hormones regulate body processes, including growth, development, metabolism, reproduction, and other functions. For most hormones, having too much or too little of them causes health issues. Mystic Aquarium scientists are studying hormones that play major roles in metabolism, reproduction, and adrenal (stress) response in beluga whales. The traditional way to measure hormones is to collect blood samples and analyze them. However, this is impractical in free-ranging beluga whales. To collect the sample, the animal needs to be handled, which can be challenging for researchers and stressful on the animal. To overcome this challenge, researchers are developing non-invasive methods to monitor hormone activity. These include measuring the levels of these chemical signals in feces, saliva, and exhaled breath (in cetaceans, this is referred to as “blow”). For this project, Mystic Aquarium scientists are investigating the feasibility of measuring hormones in saliva, blow, and feces of belugas as a non-invasive alternative to blood collection.

Currently, the team is working with resident beluga whales to refine and test these methods. Once fully tested and validated, we will transition the techniques to sample free-ranging whales. For example, hormone levels in samples obtained from breath (blow) samples from endangered whales will provide information on their reproductive and health status.

Genes and Health

Project Title: Molecular Signatures of Health in Marine Mammals

The health of an animal is written, in part, in its genes. Genetics play a role in an organism’s susceptibility to illness and disease, as well as its overall health. Although genes alone do not define physical condition – environment and lifestyle are also crucially important – genes do provide insight into health, both at an individual level and population level.

Mystic Aquarium scientists are developing molecular tests to identify genes that play a critical role in marine mammal health. Genetic markers in both blood and skin can reveal important information such as health consequences of exposure to environmental stressors, such as chemical or noise pollution.

Genes and Health

Project Title: Skin Transcriptome Analysis as a Potential Tool for Health Assessment in Belugas

Mystic Aquarium scientists are investigating the feasibility of skin as a health assessment tool through this study, since skin can be obtained through remote biopsy. The endangered Cook Inlet (CI) beluga whale stock is currently still declining despite efforts to promote recovery. Identification of alternative indicators is critical to help understand their health challenges to help with conservation efforts. This pilot study utilized skin transcriptomics using RNA sequencing technology to identify the biological and cellular processes that are activated or inhibited. The overarching goal is to expand this pilot study with sample numbers to provide extensive genomic resources to explore the links between genes and health including stress levels, infection and inflammation, and other immune and physiologic imbalances.

Skin tissue, which is easily accessible in wild cetaceans, is a rich source of gene expression with diverse biological functions that are also represented in other tissue types. For this purpose, skin samples collected from endangered CI and stable Bristol Bay (BB), Eastern Chukchi Sea (ECS) beluga stocks through remote biopsy, health assessment studies and subsistence hunts were utilized for RNA sequencing. Comparisons of the transcriptomes obtained from these stocks displayed significant gene expression differences as reflected in principle component and heatmap analyses. This preliminary analysis resulted in identification of over 1,000 significantly regulated genes (p-adjusted<0.05, log2 fold-change>1) in CI stock when compared to BB or ECS stocks. The topmost differentially expressed genes were related with immune response and skin structural components showing downregulation in Cook Inlet belugas. A pathway analysis was then carried out showing the footprint of the dataset on established signaling and metabolic (canonical) pathways. Significantly enriched (p-adjusted<0.05, z-score≥|2|) canonical pathways were identified in CI belugas in relation to the other two stocks, mostly including inhibited immune activity and cell signaling.

This pilot study provides promising results towards understanding of global gene expression differences among populations. Through the North Pacific Research Board funding, additional samples will be analyzed in conjunction with other health-related data to further investigate these pathways. Overall, this study proposes skin transcriptomics as a powerful tool to pinpoint key gene expression changes between populations, with potential to inform management decisions for conservation success of CI belugas. 

Diving and the Immune System

Project Title: Impact of Diving on the Immune System of Marine Mammals

Reports of marine mammal strandings and disease have been increasing in recent decades, leading to questions over whether human activities are negatively impacting marine mammal health. There is concern that human activities may serve as stressors, interrupting adaptations to diving in marine mammals and leaving them more susceptible to injury and disease. This has highlighted the need to further understand the relationship between marine mammal health, environmental challenges, and human activities.

Mystic Aquarium scientists are studying how marine mammal immune cells function during diving and how additional stressors may alter this function, thus impacting marine mammal health. Blood samples are exposed to simulated dives using a small pressure chamber, and the response of immune cells to changes in pressure are measured. In addition, we collect blood samples from belugas after diving Blood samples are also being obtained from beluga whales at the Aquarium after diving with activity and without to determine the impact on the immune system and health.

Non-Invasive Measurement of Stressors in Walruses

Project Title: Development of a Non-Invasive Method to Measure the Response to Stressors in the Pacific Walrus

Pacific walruses live in the Bering and Chuckchi Seas.  Like many Arctic species, their lives depend on sea ice, which is where the walruses rest, give birth, and nurse their calves.  However, sea ice is disappearing as a direct result of global climate change – threatening the survival of this unique species.  Less sea ice means that the walruses have to perform many of these activities on land where human disturbance can trigger deadly stampedes, trampling over each other as they flee into the Arctic waters.  The impact of these changes is unknown. At the Mystic Aquarium research laboratory we are validating tests to measure the response to chronic stressors by using fecal samples collected noninvasively from walruses housed at other U.S. zoos and aquariums.  Studying walruses under human care will allow us to collect baseline hormone data that will contribute to our knowledge of walrus physiology with the ultimate goal to monitor stress levels in wild walrus and contribute to their conservation and management.

Health Assessment of Wild Belugas in Alaska

Project Title: Health Assessment of Beluga Whales in the Chukchi Sea and Bristol Bay, Alaska

Climate change, pollution, offshore oil and gas exploration, and other human factors are challenges for whales in the wild and pose potential threats to Alaska’s beluga populations. In the fall of 2008, beluga whales in Cook Inlet were listed as critically endangered. In collaboration with the Department of Wildlife Management, North Slope Borough, Utqiagvik, Alaska this project aims to establish baseline health measurements of wild belugas with studies of live capture-released belugas in Point Lay, Alaska via satellite tracking, blood sampling, and other biological sample collection. A similar study is being conducted with belugas in Bristol Bay. Moreover, samples and natural history data are collected from whales taken during native subsistence hunts. Establishing baseline health measures of belugas in the wild before further oil and gas exploration and development, and the impact of climate change is critical. After an adverse event, such as an oil spill, it is too late to understand impacts if baseline levels of contaminants, pathogens are not available. This study enables us to gather information on belugas over time and reveal health status of belugas under different environmental pressures. Data can eventually be compared with information on the endangered Cook Inlet beluga population. Human health impacts are also studied given that native peoples consume the beluga for food. For the first time ever, in collaboration with National Geographic, a critter cam (camera placed on a beluga) was placed on a wild beluga to get footage of beluga behavior.

Through this project an educational cultural exchange program was established in which high school students from Point Lay and Mashantucket tribal students from southeastern Connecticut assist the scientists in the field with collection of data and then travel to Mystic Aquarium to participate hands-on with the analysis. Students also participate in the Aquarium’s other educational programs and spend time on the Mashantucket .

This project seeks to further investigate current health status in Chukchi Sea and Bristol Bay belugas and initiates steps to study how stressors impact beluga health; furthermore, the project develops baseline data for future studies, and broadens public awareness of current threats for belugas in the wild.

Beluga Microbiome

Project Title: Understanding the Beluga Microbiome

The term microbiome refers to the collection of microorganisms that live in or on a host species. A microbiome occupies a specific niche, such as the skin or gut, each harboring different bacterial communities. These bacteria make up the normal flora of the host and help protect them from invading pathogens. Microbiomes play critical roles in host nutrition, immune function, and can even affect brain development. A microbiome naturally shifts (in number and type of bacteria) over the course of the lifetime of the host. However, disruptions in a microbiome can lead to a dysbiosis that can alter host health. Although scientists have extensively studied microbiomes in humans and terrestrial animals, little is known about the microbiomes of marine mammals.

Mystic Aquarium researchers are investigating the microbial communities of belugas under controlled conditions at the Aquarium. We are characterizing the bacteria and other organisms that make up the beluga microbiome and see if there are differences associated with health status, season, sex, and age. We expect that marine mammals, like the beluga, harbor specific microbial core communities that are necessary for physiological health, and changes or disruptions in these communities can be an indicator of illness, disease, or chronic stress.

For this study, the team is documenting the bacterial composition of the oral, respiratory, skin, vaginal, and gastrointestinal microbiomes of belugas; defining differences in microbial communities based on age, sex, and environmental parameters; and comparing the microbiome profiles with available clinical data to identify any changes that can be correlated to illness or disease.

Photogrammetry of Belugas

Project Title: Photogrammetry Body Condition Studies

Photogrammetry is a technique that uses quantitative (countable or measurable) information from aerial photographs to measure features of wild cetaceans, such as length, growth and body condition. This information can be used to infer health information on individual animals and determine group abundance and structure. One way to obtain photogrammetry data is with Unmanned Autonomous Vehicles (UAVs).  Photogrammetry with UAVs has recently been used or is in development for monitoring beluga populations such as Cook Inlet and St. Lawrence Estuary belugas. Mystic Aquarium researchers are participating in a study to provide longitudinal photogrammetry and morphometric data from two known individual belugas (an older female and a younger male) to inform photogrammetry studies of wild beluga populations. The data will aid in developing a minimally invasive method to assess body condition in beluga whales in the wild using photographs taken with UAVs. We will also obtain photographs and morphometric measurements on additional belugas at different life stages for body condition, to contribute to development, validation, and interpretation of photogrammetry on wild belugas.

The team is working with the belugas under controlled conditions at Mystic Aquarium to help inform, validate, and interpret photogrammetry of wild belugas. This helps ground truth photogrammetry data obtained from belugas in the wild particularly regarding body condition and pregnancy status.

For this study, we are obtaining photographs of belugas as they swim underneath a camera at a minimum of 6 m in height, collecting a series of morphometric measurements and body weights that coincide with the photographs. We are sharing this information with biologists conducting photogrammetry studies on wild belugas, especially endangered beluga populations.

Telemetry and Cameras on Beluga Whales

Project Title: Telemetry and Cameras on Beluga Whales

Marine telemetry is the use of sensors on aquatic animals to record information about where they go and what their environment is like. Telemetry devices collect information on the environment (temperature, salinity, location, etc.) as well as activity (dive frequency, depth, movements, etc.) of marine mammals. These technologies are constantly being improved, and it is helpful to test some of these devices in a controlled environment before deploying them in wild marine mammals.

Mystic Aquarium researchers are working with technology developers to test new telemetry devices and cameras on the resident beluga whales before these devices are deployed on wild whales and dolphins. Some of the factors the team is determining are optimal body placement, size, duration (how long the tag will stay on), as well as noting any changes in animal behavior with the tag in place. 

Previously, in collaboration with one of our scientists-in-residence, Mystic Aquarium tested Critter Cam on one resident beluga. The whale was trained to station to receive the camera which was adhered via suction cups. Tests were run to determine the optimal placement on the body. Two different sized prototypes were tested. Moreover, adherence was tested utilizing different sizes and configurations of suction cups to determine which worked the best. Observations were made while the whale was swimming and then after the whale was asked to carry out more rigorous activity such as breaching and increasing swim speed. Given results from the testing on the beluga, the engineers are currently modifying the camera, and it will undergo additional refinements before deployment to track and monitor cetaceans in the wild.

Influenza in the Ocean

Project Title: Ecology and Evolution of Influenza Virus in the Marine Environment

Influenza is a globally important pathogen leading to significant morbidity and mortality which has implications for both wildlife and human health. The coastal environment provides an interface between marine and terrestrial habitats where avian reservoirs of influenza collide (sea ducks, gulls, and shorebirds) and overlap spatio-temporally with marine mammals, providing an opportunity for interspecies transmission of influenza virus. The repeated infections in marine mammals during the last 30-40 years in New England and the associated sampling and laboratory studies have been the foundation of our understanding of influenza in marine mammals. However, the infection and/or exposure to influenza viruses in marine mammal populations outside of these mass mortalities and in other parts of the world remain limited. In collaboration with Dr. Jonathan Runstadler and Wendy Puryear from Tufts University, Mystic Aquarium is participating in a continuous, multiyear, and long-term (5-10 year plus) project to assess the role of influenza exposure to marine mammal health.

Mystic Aquarium is collecting samples from seals for viral analyses that come into the animal rescue clinic as well as participating in live capture release studies of gray seals off the Massachusetts coast.  From the wild seals, the Mystic Aquarium scientists are investigating immune function in the seals paired with the viral information. This project seeks to develop collaborations with researchers and rehabilitation networks so that a larger team can work together to collect samples and run laboratory analyses on a continuous, long-term basis to better understand the persistence of marine influenza in the environment and its impact on marine mammal health.

Pathogen Detection

Project Title: The Impact of Marine Origin Brucella on Marine Mammal and Human Health

Brucella, a bacterial pathogen known for centuries for its devastating reproductive and general health effects in humans and livestock, is now recognized in marine mammals. Although it is now found in marine environments, little is known about its full physiologic effects in marine mammals. Though similar to terrestrial origin brucellosis, marine origin Brucella infection will likely act as a significant factor in low conception rates and increased spontaneous abortion and stillbirth rates in marine mammal populations. Marine origin Brucella is also zoonotic, meaning it can be transmitted to humans who come into close contact with marine mammal body fluids and tissues, such as researchers, veterinarians, and animal care specialists.

To appreciate the potential for health problems associated with marine origin Brucella, we must understand the magnitude of the problem in marine mammal populations. Past studies involving marine mammal brucellosis have used traditional livestock diagnosis methods, and the results are frequently inconsistent. Thus this disease has likely been under-diagnosed in marine mammals, and the true impact of infection has not been properly accounted for in studies that seek to document significant causes of population decline in marine mammal populations. The diagnosis of marine Brucella exposure in humans uses techniques similar to those in veterinary medicine, likely repeating the problem of under-diagnosis.

Scientists at Mystic Aquarium developed and utilized  multiple marine origin brucella diagnostic tools, including culture, PCR, and newly developed serological studies to improve our knowledge of the past and present prevalence of marine origin Brucella exposure/infection and its effects in both wild and marine mammals in professional care.

Measuring Stress in African Penguins

Project Title: Monitoring of Health and Welfare in the Endangered African Penguin through Fecal Glucocorticoid and Microbiome Analysis

African penguins live in South Africa, Namibia, and a handful of small islands off the southern coast of the continent. African penguins are critically endangered with less than 22,000 breeding pairs left in the wild.  Initial declines were caused by harvesting their nests, which are made of guano and can be used for fertilizer.  Today, African penguins are threatened by multiple stressors, such as food shortages, oil spills, and climate change.  When an animal (or penguin) encounters a stressor, they respond by releasing hormones known as glucocorticoids into the blood stream.  This stress response results in various behavioral and physiological changes that allow the animal to focus its energy on escaping the “stressor.” This is both beneficial and necessary for survival. However, chronic release of these hormones can have negative effects, including reduced immune function, reproductive activity, and survival. These circulating hormones are eventually broken down and their metabolites are excreted in the feces as fecal glucocorticoid metabolites (FGM). FGM can therefore be used as a biomarker for measuring the response to stressors.  Chronic stress has also been shown to disrupt the bacteria that live in the gastrointestinal (GI) tract and make up the GI microbiome.  In some animals, a decrease in the types of different bacteria that inhabit the GI tract have been linked to poor health.

Fecal sampling is a noninvasive technique that can be applied to a variety of situations where traditional blood sampling is not possible, and FGM analysis has been used to measure chronic stress in many wildlife populations.  By using fecal samples collected from the African penguins at Mystic Aquarium, we are learning how these hormones change throughout the year as the penguins transition through their natural lifecycle (breeding and molt).  We are also investigating the relationship between FGM levels and what types of bacteria live in the gut of African penguins, as well as determining  certain types of bacteria that are necessary for penguin health.

The goal of this research is to apply this combined approach of monitoring elevated GC levels and disruptions in the GI microbiome in wild penguin colonies under different stressors, thus targeting and playing a key role in conservation strategies.

Deadly Fungal Infections

Project Title: Amphibian Conservation – Chytrid Monitoring and Microbiome Analysis

Chytridiomycosis is an infectious disease of amphibians caused by the fungal zoospore Batrachochytrium dendrobatidis (Bd). This disease occurs worldwide and has been responsible for dramatic declines in amphibian populations, with some populations suffering complete loss.  Connecticut Department of Energy & Environmental Protection has confirmed the presence of Bd in frog and toad populations throughout the state; however, there has been limited sampling of the newly emerging Batrachochytrium salamandrivorans (Bsal) in local species.

This project employs citizen scientist volunteers, through our Environmental Stewardship program, to conduct amphibian surveys in a variety of wetland habitats throughout Northeast Connecticut.  During these surveys, project participants will assist staff in the collection of samples to be tested for the presence of Bd and Bsal to further conservation of amphibian species in the region.  Samples will be collected from the diverse amphibian species found in the region, including the eastern red-backed salamander, spotted salamander, American bullfrog, and wood frogs.  Assessing the prevalence of chytrid in the area will help in early identification of threats to amphibian biodiversity and to minimize potentially disastrous population loss.

While chytridiomycosis can be devastating to many amphibian populations, some species seem to be more resistant to infection.  What makes these amphibians more protected than others remains unknown.  One possibility may be the bacteria that live on their skin, which collectively make up the skin microbiome.  By utilizing the same swabbing technique for chytrid monitoring, we can analyze these bacterial populations to determine if certain groups of bacteria may be responsible for resistance to this disease.

With this combined approach, we can further conservation of amphibian species in the region and beyond.