Evaluating beluga (Delphinapterus leucas) blow samples as a potential diagnostic for immune function gene expression within the respiratory system.

Evaluating respiratory health is important in the management of cetaceans, which are vulnerable to respiratory diseases. Quantifying the expression of genes related to immune function within the respiratory tract could be a valuable tool for directly assessing respiratory health. Blow (exhale) samples allow DNA analysis, and we hypothesized that RNA could also be isolated from blow samples for gene expression studies of immune function. We evaluated the potential to extract RNA from beluga blow samples and tested whether transcripts associated with immune function could be detected with endpoint polymerase chain reaction. A total of 54 blow samples were collected from clinically healthy aquarium belugas (n = 3), and 15 were collected from wild belugas temporarily restrained for health assessment in Bristol Bay, Alaska (n = 9). Although RNA yield varied widely (range, 0-265.2 ng; mean = 85.8; SD = 71.3), measurable RNA was extracted from 97% of the samples. Extracted RNA was assessed in 1-6 PCR reactions targeting housekeeping genes (Rpl8, Gapdh or ActB) or genes associated with immune function (TNFα, IL-12p40 or Cox-2). Fifty of the aquarium samples (93%) amplified at least one transcript; overall PCR success for housekeeping genes (96/110, 87%) and genes associated with immune function (90/104, 87%) were similarly high. Both RNA yield and overall PCR success (27%) were lower for wild beluga samples, which is most likely due to the reduced forcefulness of the exhale when compared with trained or free-swimming belugas. Overall, the high detection rate with PCR suggests measuring gene expression in blow samples could provide diagnostic information about immune responses within the respiratory tract. While further study is required to determine if quantitative gene expression data from blow samples is associated with disease states, the non-invasive nature of this approach may prove valuable for belugas, which face increasing anthropogenic disturbances.

Skin microbiome of beluga whales: spatial, temporal, and health-related dynamics.

BACKGROUND: Host-specific microbiomes play an important role in individual health and ecology; in marine mammals, epidermal microbiomes may be a protective barrier between the host and its aqueous environment. Understanding these epidermal-associated microbial communities, and their ecological- or health-driven variability, is the first step toward developing health indices for rapid assessment of individual or population health. In Cook Inlet, Alaska, an endangered population of beluga whales (Delphinapterus leucas) numbers fewer than 300 animals and continues to decline, despite more than a decade of conservation effort. Characterizing the epidermal microbiome of this species could provide insight into the ecology and health of this endangered population and allow the development of minimally invasive health indicators based on tissue samples. RESULTS: We sequenced the hypervariable IV region of bacterial and archaeal SSU rRNA genes from epidermal tissue samples collected from endangered Cook Inlet beluga whales (n = 33) and the nearest neighboring population in Bristol Bay (n = 39) between 2012 and 2018. We examined the sequences using amplicon sequence variant (ASV)-based analyses, and no ASVs were associated with all individuals, indicating a greater degree of epidermal microbiome variability among beluga whales than in previously studied cetacean species and suggesting the absence of a species-specific core microbiome. Epidermal microbiome composition differed significantly between populations and across sampling years. Comparing the microbiomes of Bristol Bay individuals of known health status revealed 11 ASVs associated with potential pathogens that differed in abundance between healthy individuals and those with skin lesions or dermatitis. Molting and non-molting individuals also differed significantly in microbial diversity and the abundance of potential pathogen-associated ASVs, indicating the importance of molting in maintaining skin health. CONCLUSIONS: We provide novel insights into the dynamics of Alaskan beluga whale epidermal microbial communities. A core epidermal microbiome was not identified across all animals. We characterize microbial dynamics related to population, sampling year and health state including level of skin molting. The results of this study provide a basis for future work to understand the role of the skin microbiome in beluga whale health and to develop health indices for management of the endangered Cook Inlet beluga whales, and cetaceans more broadly.

Comparing progesterone in blubber and serum to assess pregnancy in wild beluga whales (Delphinapterus leucas).

The Cook Inlet population of beluga whales (Delphinapterus leucas) is listed as endangered and continues to decline for largely unknown reasons; however, there is some evidence that poor reproductive success is a contributing factor. Pregnancy is difficult to detect through observation, and, there is reluctance to capture endangered beluga whales for reproductive tract imaging via ultrasound or to obtain suitable samples for pregnancy assessments. An endocrine analysis of blubber biopsies collected by remote darting could represent a minimally invasive way to identify pregnant females and compare pregnancy rates among years or populations. Studies have validated the use of blubber biopsies to identify pregnant females in other cetacean species, but not beluga whales; therefore, validation of blubber progesterone levels to proven tests that reliably detect pregnancy was needed for this species. As part of a larger study, we sampled blood and blubber from live-captured beluga whales (21 females, 26 males) in Bristol Bay, Alaska. Progesterone levels were determined in serum samples obtained from all animals and in blubber samples from a subset (14 females, 13 males) to determine pregnancy status, estimate the stage of pregnancy, and evaluate the suitability of using blubber alone for these assessments. In general, there was distinct separation of high levels of progesterone in serum and blubber for presumed pregnant females and low levels for males and presumed non-pregnant females. Blubber progesterone levels in two females (14% of females tested) were intermediate (i.e. ambiguous); their corresponding serum levels were consistent with being pregnant in one case and not being pregnant in the other. Except for these two intermediate values, pregnancy status of beluga whales could be determined from blubber alone, thereby providing a valuable tool to better understand reproduction dynamics from populations that cannot be captured for examination.

Variation in hearing within a wild population of beluga whales (Delphinapterus leucas).

Documenting hearing abilities is vital to understanding a species' acoustic ecology and for predicting the impacts of increasing anthropogenic noise. Cetaceans use sound for essential biological functions such as foraging, navigation and communication; hearing is considered to be their primary sensory modality. Yet, we know little regarding the hearing of most, if not all, cetacean populations, which limits our understanding of their sensory ecology, population level variability and the potential impacts of increasing anthropogenic noise. We obtained audiograms (5.6-150 kHz) of 26 wild beluga whales to measure hearing thresholds during capture-release events in Bristol Bay, AK, USA, using auditory evoked potential methods. The goal was to establish the baseline population audiogram, incidences of hearing loss and general variability in wild beluga whales. In general, belugas showed sensitive hearing with low thresholds (<80 dB) from 16 to 100 kHz, and most individuals (76%) responded to at least 120 kHz. Despite belugas often showing sensitive hearing, thresholds were usually above or approached the low ambient noise levels measured in the area, suggesting that a quiet environment may be associated with hearing sensitivity and that hearing thresholds in the most sensitive animals may have been masked. Although this is just one wild population, the success of the method suggests that it should be applied to other populations and species to better assess potential differences. Bristol Bay beluga audiograms showed substantial (30-70 dB) variation among individuals; this variation increased at higher frequencies. Differences among individual belugas reflect that testing multiple individuals of a population is necessary to best describe maximum sensitivity and population variance. The results of this study quadruple the number of individual beluga whales for which audiograms have been conducted and provide the first auditory data for a population of healthy wild odontocetes.

De novo transcriptome assembly and RNA-Seq expression analysis in blood from beluga whales of Bristol Bay, AK.

Assessing the health of marine mammal sentinel species is crucial to understanding the impacts of environmental perturbations on marine ecosystems and human health. In Arctic regions, beluga whales, Delphinapterus leucas, are upper level predators that may serve as a sentinel species, potentially forecasting impacts on human health. While gene expression profiling from blood transcriptomes has widely been used to assess health status and environmental exposures in human and veterinary medicine, its use in wildlife has been limited due to the lack of available genomes and baseline data. To this end we constructed the first beluga whale blood transcriptome de novo from samples collected during annual health assessments of the healthy Bristol Bay, AK stock during 2012-2014 to establish baseline information on the content and variation of the beluga whale blood transcriptome. The Trinity transcriptome assembly from beluga was comprised of 91,325 transcripts that represented a wide array of cellular functions and processes and was extremely similar in content to the blood transcriptome of another cetacean, the bottlenose dolphin. Expression of hemoglobin transcripts was much lower in beluga (25.6% of TPM, transcripts per million) than has been observed in many other mammals. A T12A amino acid substitution in the HBB sequence of beluga whales, but not bottlenose dolphins, was identified and may play a role in low temperature adaptation. The beluga blood transcriptome was extremely stable between sex and year, with no apparent clustering of samples by principle components analysis and <4% of genes differentially expressed (EBseq, FDR<0.05). While the impacts of season, sexual maturity, disease, and geography on the beluga blood transcriptome must be established, the presence of transcripts involved in stress, detoxification, and immune functions indicate that blood gene expression analyses may provide information on health status and exposure. This study provides a wealth of transcriptomic data on beluga whales and provides a sizeable pool of preliminary data for comparison with other studies in beluga whale.

Seasonal and developmental differences in blubber stores of beluga whales in Bristol Bay, Alaska using high-resolution ultrasound.

Diving mammals use blubber for a variety of structural and physiological functions, including buoyancy, streamlining, thermoregulation, and energy storage. Estimating blubber stores provides proxies for body condition, nutritional status, and health. Blubber stores may vary topographically within individuals, across seasons, and with age, sex, and reproductive status; therefore, a single full-depth blubber biopsy does not provide an accurate measure of blubber depth, and additional biopsies are limited because they result in open wounds. We examined high-resolution ultrasound as a noninvasive method for assessing blubber stores by sampling blubber depth at 11 locations on beluga whales in Alaska. Blubber mass was estimated as a proportion of body mass (40% from the literature) and compared to a function of volume calculated using ultrasound blubber depth measurements in a truncated cone. Blubber volume was converted to total and mass-specific blubber mass estimates based on the density of beluga blubber. There was no significant difference in mean total blubber mass between the 2 estimates (R2 = 0.88); however, body mass alone predicted only 68% of the variation in mass-specific blubber stores in juveniles, 7% for adults in the fall, and 33% for adults in the spring. Mass-specific blubber stores calculated from ultrasound measurements were highly variable. Adults had significantly greater blubber stores in the fall (0.48±0.02kg/kgMB) than in the spring (0.33±0.02kg/kgMB). There was no seasonal effect in juveniles. High-resolution ultrasound is a more powerful, noninvasive method for assessing blubber stores in wild belugas, allowing for precise measurements at multiple locations.

Measuring Hearing in Wild Beluga Whales.

We measured the hearing abilities of seven wild beluga whales (Delphinapterus leucas) during a collection-and-release experiment in Bristol Bay, AK. Here we summarize the methods and initial data from one animal and discuss the implications of this experiment. Audiograms were collected from 4 to 150 kHz. The animal with the lowest threshold heard best at 80 kHz and demonstrated overall good hearing from 22 to 110 kHz. The robustness of the methodology and data suggest that the auditory evoked potential audiograms can be incorporated into future collection-and-release health assessments. Such methods may provide high-quality results for multiple animals, facilitating population-level audiograms and hearing measures in new species.

CIRCULATING CONCENTRATIONS OF THYROID HORMONE IN BELUGA WHALES (DELPHINAPTERUS LEUCAS): INFLUENCE OF AGE, SEX, AND SEASON.

Thyroid hormones play a critical physiologic role in regulating protein synthesis, growth, and metabolism. To date, because no published compilation of baseline values for thyroid hormones in beluga whales (Delphinapterus leucas) exists, assessment of thyroid hormone concentrations in this species has been underused in clinical settings. The purpose of this study was to document the concentrations of total thyroxine (tT4) and total triiodothyronine (tT3) in healthy aquarium-maintained and free-ranging beluga whales and to determine the influence of age, sex, and season on the thyroid hormone concentrations. Archived serum samples were collected from healthy aquarium-maintained (n=43) and free-ranging (n=39) belugas, and serum tT4 and tT3 were measured using chemiluminescence immunoassay. The mean tT4 concentration in aquarium-maintained belugas was 5.67±1.43 µg/dl and the mean tT3 concentration was 70.72±2.37 ng/dl. Sex comparisons showed that aquarium-maintained males had significantly greater tT4 and tT3 (9.70±4.48 µg/dl and 92.65±30.55 ng/dl, respectively) than females (7.18±2.82 µg/dl and 77.95±20.37 ng/dl) (P=0.004 and P=0.013). Age comparisons showed that aquarium-maintained whales aged 1-5 yr had the highest concentrations of tT4 and tT3 (8.17±0.17 µg/dl and 105.46±1.98 ng/dl, respectively) (P=0.002 and P<0.001). tT4 concentrations differed significantly between seasons, with concentrations in winter (4.59±1.09 µg/dl) being significantly decreased compared with spring (P=0.009), summer (P<0.0001), and fall (P<0.0001) concentrations. There was a significant difference in tT4 and tT3 concentrations between aquarium-maintained whales (5.67±1.43 µg/dl and 70.72±15.57 ng/dl, respectively) and free-ranging whales (11.71±3.36 µg/dl and 103.38±26.45 ng/dl) (P<0.0001 and P<0.001). Clinicians should consider biologic and environmental influences (age, sex, and season) for a more accurate interpretation of thyroid hormone concentrations in belugas. The findings of this study provide a baseline for thyroid health monitoring and comprehensive health assessments in both aquarium-maintained and free-ranging beluga whales.

Blow collection as a non-invasive method for measuring cortisol in the beluga (Delphinapterus leucas).

Non-invasive sampling techniques are increasingly being used to monitor glucocorticoids, such as cortisol, as indicators of stressor load and fitness in zoo and wildlife conservation, research and medicine. For cetaceans, exhaled breath condensate (blow) provides a unique sampling matrix for such purposes. The purpose of this work was to develop an appropriate collection methodology and validate the use of a commercially available EIA for measuring cortisol in blow samples collected from belugas (Delphinapterus leucas). Nitex membrane stretched over a petri dish provided the optimal method for collecting blow. A commercially available cortisol EIA for measuring human cortisol (detection limit 35 pg ml-1) was adapted and validated for beluga cortisol using tests of parallelism, accuracy and recovery. Blow samples were collected from aquarium belugas during monthly health checks and during out of water examination, as well as from wild belugas. Two aquarium belugas showed increased blow cortisol between baseline samples and 30 minutes out of water (Baseline, 0.21 and 0.04 µg dl-1; 30 minutes, 0.95 and 0.14 µg dl-1). Six wild belugas also showed increases in blow cortisol between pre and post 1.5 hour examination (Pre 0.03, 0.23, 0.13, 0.19, 0.13, 0.04 µg dl-1, Post 0.60, 0.31, 0.36, 0.24, 0.14, 0.16 µg dl-1). Though this methodology needs further investigation, this study suggests that blow sampling is a good candidate for non-invasive monitoring of cortisol in belugas. It can be collected from both wild and aquarium animals efficiently for the purposes of health monitoring and research, and may ultimately be useful in obtaining data on wild populations, including endangered species, which are difficult to handle directly.

Baseline hearing abilities and variability in wild beluga whales (Delphinapterus leucas).

While hearing is the primary sensory modality for odontocetes, there are few data addressing variation within a natural population. This work describes the hearing ranges (4-150 kHz) and sensitivities of seven apparently healthy, wild beluga whales (Delphinapterus leucas) during a population health assessment project that captured and released belugas in Bristol Bay, Alaska. The baseline hearing abilities and subsequent variations were addressed. Hearing was measured using auditory evoked potentials (AEPs). All audiograms showed a typical cetacean U-shape; substantial variation (>30 dB) was found between most and least sensitive thresholds. All animals heard well, up to at least 128 kHz. Two heard up to 150 kHz. Lowest auditory thresholds (35-45 dB) were identified in the range 45-80 kHz. Greatest differences in hearing abilities occurred at both the high end of the auditory range and at frequencies of maximum sensitivity. In general, wild beluga hearing was quite sensitive. Hearing abilities were similar to those of belugas measured in zoological settings, reinforcing the comparative importance of both settings. The relative degree of variability across the wild belugas suggests that audiograms from multiple individuals are needed to properly describe the maximum sensitivity and population variance for odontocetes. Hearing measures were easily incorporated into field-based settings. This detailed examination of hearing abilities in wild Bristol Bay belugas provides a basis for a better understanding of the potential impact of anthropogenic noise on a noise-sensitive species. Such information may help design noise-limiting mitigation measures that could be applied to areas heavily influenced and inhabited by endangered belugas.

Variation in hematologic and serum biochemical values of belugas (Delphinapterus leucas) under managed care.

Blood analytes are critical for evaluating the general health of cetacean populations, so it is important to understand the intrinsic variability of hematology and serum chemistry values. Previous studies have reported data for follow-up periods of several years in managed and wild populations, but studies over long periods of time (> 20 yr) have not been reported. The study objective was to identify the influences of partitioning characteristics on hematology and serum chemistry analytes of apparently healthy managed beluga (Delphinapterus leucas). Blood values from 31 managed belugas, at three facilities, collected over 22 yr, were assessed for seasonal variation and aging trends, and evaluated for biologic variation among and within individuals. Linear mixed effects models assessed the relationship between the analytes and sex, age, season, facility location, ambient air temperature, and photoperiod. Sex differences in analytes and associations with increasing age were observed. Seasonal variation was observed for hemoglobin, hematocrit, mean corpuscular volume, monocytes, alkaline phosphatase, total bilirubin, cholesterol, and triglycerides. Facilities were associated with larger effects on analyte values compared to other covariates, whereas age, sex, and ambient temperature had smaller effects compared to facility and season. Present findings provide important baseline information for future health monitoring efforts. Interpretation of blood analytes and animal health in managed and wild populations over time is aided by having available typical levels for the species and reference intervals for the degree to which individual animals vary from the species average and from their own baseline levels during long-term monitoring.

Fecal pathogen pollution: sources and patterns in water and sediment samples from the upper Cook Inlet, Alaska ecosystem.

Fecal pathogens are transported from a variety of sources in multi-use ecosystems such as upper Cook Inlet (CI), Alaska, which includes the state's urban center and is highly utilized by humans and animals. This study used a novel water quality testing approach to evaluate the presence and host sources of potential fecal pathogens in surface waters and sediments from aquatic ecosystems in upper CI. Matched water and sediment samples, along with effluent from a municipal wastewater treatment facility, were screened for Salmonella spp., Vibrio spp., Cryptosporidium spp., Giardia spp., and noroviruses. Additionally, Bacteroidales spp. for microbial source tracking, and the fecal indicator bacteria Enterococcus spp. as well as fecal coliforms were evaluated. Overall, Giardia and Vibrio were the most frequently detected potential pathogens, followed by Cryptosporidium and norovirus, while Salmonella was not detected. Sample month, matrix type, and recent precipitation were found to be significant environmental factors for protozoa or host-associated Bacteroidales marker detection, whereas location and water temperature were not. The relative contribution of host-associated markers to total fecal marker concentration was estimated using a Monte Carlo method, with the greatest relative contribution to the Bacteroidales marker concentration coming from human sources, while the remainder of the universal fecal host source signal was uncharacterized by available host-associated assays, consistent with wildlife fecal sources. These findings show how fecal indicator and pathogen monitoring, along with identifying contributing host sources, can provide evidence of coastal pathogen pollution and guidance as to whether to target human and/or animal sources for management.

Seasonal hematology and serum chemistry of wild beluga whales (Delphinapterus leucas) in Bristol Bay, Alaska, USA.

We collected blood from 18 beluga whales (Delphinapterus leucas), live-captured in Bristol Bay, Alaska, USA, in May and September 2008, to establish baseline hematologic and serum chemistry values and to determine whether there were significant differences in hematologic values by sex, season, size/age, or time during the capture period. Whole blood was collected within an average of 19 min (range=11-30 min) after the net was set for capture, and for eight animals, blood collection was repeated in a later season after between 80-100 min; all blood was processed within 12 hr. Mean hematocrit, chloride, creatinine, total protein, albumin, and alkaline phosphatase were significantly lower in May than they were in September, whereas mean corpuscular hemoglobin concentration, monocytes, phosphorous, magnesium, blood urea nitrogen, alanine aminotransferase, aspartate aminotransferase, γ-glutamyltranspeptidase, and creatinine kinase were significantly higher. Mean total protein, white blood cell count, neutrophils, and lymphocytes were significantly higher early in the capture period than they were later. No significant differences in blood analyte values were noted between males and females. Using overall body length as a proxy for age, larger (older) belugas had lower white blood cell, lymphocyte, and eosinophil counts as well as lower sodium, potassium, and calcium levels but higher creatinine levels than smaller belugas. These data provide values for hematology and serum chemistry for comparisons with other wild belugas.

Fractal analysis of narwhal space use patterns.

Quantifying animal movement in response to a spatially and temporally heterogeneous environment is critical to understanding the structural and functional landscape influences on population viability. Generalities of landscape structure can easily be extended to the marine environment, as marine predators inhabit a patchy, dynamic system, which influences animal choice and behavior. An innovative use of the fractal measure of complexity, indexing the linearity of movement paths over replicate temporal scales, was applied to satellite tracking data collected from narwhals (Monodon monoceros) (n = 20) in West Greenland and the eastern Canadian high Arctic. Daily movements of individuals were obtained using polar orbiting satellites via the ARGOS data location and collection system. Geographic positions were filtered to obtain a daily good quality position for each whale. The length of total pathway was measured over seven different temporal length scales (step lengths), ranging from one day to one week, and a seasonal mean was calculated. Fractal dimension (D) was significantly different between seasons, highest during summer (D = 1.61, SE 0.04) and winter (D = 1.69, SE 0.06) when whales made convoluted movements in focal areas. Fractal dimension was lowest during fall (D = 1.34, SE 0.03) when whales were migrating south ahead of the forming sea ice. There were no significant effects of size category or sex on fractal dimension by season. The greater linearity of movement during the migration period suggests individuals do not intensively forage on patchy resources until they arrive at summer or winter sites. The highly convoluted movements observed during summer and winter suggest foraging or searching efforts in localized areas. Significant differences between the fractal dimensions on two separate wintering grounds in Baffin Bay suggest differential movement patterns in response to the dynamics of sea ice.