Application of endocrine biomarkers to update information on reproductive physiology in gray whale (Eschrichtius robustus).

Most of our knowledge on reproductive biology of gray whales dates back to scientific research conducted during commercial whaling in the late 1950s and 1960s. The goal of the present study was to provide updated insights on reproductive physiology of gray whales, using progesterone and testosterone as biomarkers. We measured hormone concentrations using enzyme immunoassay (EIA) techniques in blubber biopsies collected from 106 individual whales from March to November over a span of 12 years (2004-2016) between California and Alaska. We found testosterone concentrations in males to increase significantly with age (P = 0.03). Adult males showed significantly elevated testosterone concentrations when sampled in the fall compared to the summer (P = 0.01), likely indicating physiological preparation for mating. We measured testosterone concentrations in females of different age classes, but no statistical differences were found. We found significantly higher progesterone concentrations in pregnant females compared to non-pregnant females and adult males (P< 0.001), indicating progesterone is a valid biomarker for pregnancy in gray whales. Both female and male calves had elevated progesterone concentrations, suggesting maternal transfer via lactation. We fit a mixture of two normal distributions to progesterone data from all non-calf females to identify clusters of high and low progesterone and estimated the probability of being pregnant for whales of unknown reproductive status. With this approach we identified likely pregnant and non-pregnant animals. This study represents an important milestone on reproductive profiles in this population, that can be used to estimate more accurate and precise reproductive parameters to be used for better understanding population dynamics of gray whales.

Gray whales strand more often on days with increased levels of atmospheric radio-frequency noise.

Evidence from live gray whale strandings suggests that their navigation may be disrupted by increased radio frequency noise generated by solar storms, suggesting the potential for magnetoreception in this species.

Quantifying hormones in exhaled breath for physiological assessment of large whales at sea.

Exhaled breath analysis is a non-invasive assessment tool that has shown promise in human diagnostics, and could greatly benefit research, management, and conservation of large whales. However, hormone assessment of whale respiratory vapor (blow) has been challenged by variable water content and unknown total volume of collected samples. To advance this technique, we investigated urea (a compound present in narrow range in circulation) as a normalizing factor to correct for blow sample concentration. Normalized progesterone, testosterone, and cortisol concentrations of 100 blow samples from 46 photo-identified North Atlantic right whales (Eubalaena glacialis) were more biologically relevant compared to absolute estimates, varying by sex, age class, or individual. Progesterone was elevated in adult females compared with other cohorts and highest in one independently confirmed pregnant female. For both sexes, testosterone was two-fold higher in reproductively mature whales but studied adult females showed the widest variation. Cortisol was present in relatively low concentrations in blow and demonstrated variation between individual whales, suggesting potential for studies of individual differences in adrenal activity. Incorporation of methodologies that normalize sample concentration are essential for blow hormone analysis of free-swimming whales, and measurement of urea could be used to optimize non-invasive physiological assessment of whales.

Aural exostoses (surfer's ear) provide vital fossil evidence of an aquatic phase in Man's early evolution.

For over a century, otolaryngologists have recognised the condition of aural exostoses, but their significance and aetiology remains obscure, although they tend to be associated with frequent swimming and cold water immersion of the auditory canal. The fact that this condition is usually bilateral is predictable since both ears are immersed in water. However, why do exostoses only grow in swimmers and why do they grow in the deep bony meatus at two or three constant sites? Furthermore, from an evolutionary point of view, what is or was the purpose and function of these rather incongruous protrusions? In recent decades, paleoanthropological evidence has challenged ideas about early hominid evolution. In 1992 the senior author suggested that aural exostoses were evolved in early hominid Man for protection of the delicate tympanic membrane during swimming and diving by narrowing the ear canal in a similar fashion to other semiaquatic species. We now provide evidence for this theory and propose an aetiological explanation for the formation of exostoses.

Noninvasive Respiratory Metabolite Analysis Associated with Clinical Disease in Cetaceans: A Deepwater Horizon Oil Spill Study.

Health assessments of wild cetaceans can be challenging due to the difficulty of gaining access to conventional diagnostic matrices of blood, serum and others. While the noninvasive detection of metabolites in exhaled breath could potentially help to address this problem, there exists a knowledge gap regarding associations between known disease states and breath metabolite profiles in cetaceans. This technology was applied to the largest marine oil spill in U.S. history (The 2010 Deepwater Horizon oil spill in the Gulf of Mexico). An accurate analysis was performed to test for associations between the exhaled breath metabolome and sonographic lung abnormalities as well as hematological, serum biochemical, and endocrine hormone parameters. Importantly, metabolites consistent with chronic inflammation, such as products of lung epithelial cellular breakdown and arachidonic acid cascade metabolites were associated with sonographic evidence of lung consolidation. Exhaled breath condensate (EBC) metabolite profiles also correlated with serum hormone concentrations (cortisol and aldosterone), hepatobiliary enzyme levels, white blood cell counts, and iron homeostasis. The correlations among breath metabolites and conventional health measures suggest potential application of breath sampling for remotely assessing health of wild cetaceans. This methodology may hold promise for large cetaceans in the wild for which routine collection of blood and respiratory anomalies are not currently feasible.

Nanospray liquid chromatography/tandem mass spectrometry analysis of steroids from gray whale blubber.

RATIONALE: Analysis of steroids from precious blubber biopsies obtained from marine mammals, especially endangered species, can provide valuable information on their endocrine status. Challenges with currently used ELISA methodology include lack of absolute quantitation and incompatibility with multiple steroids analysis due to limited biopsy mass. Development of a sensitive, accurate analytical method for this purpose is critical. METHODS: A nanospray liquid chromatography/tandem mass spectrometry (nanoLC/MS/MS) method was validated for sensitive, specific and quantitative analysis of three steroid hormones, without derivatization, extracted from 50 mg blubber samples. Data was acquired with an LTQ XL ion trap mass spectrometer in positive ion mode, using single reaction monitoring. All three steroids were analyzed in a single run. Cholic acid was used as a surrogate internal standard for quantitation due to its steroidal structure and lack of measurable endogenous levels in blubber. RESULTS: The lowest limits of quantitation for progesterone, testosterone, and hydrocortisone were significantly improved compared to previous studies using conventional LC/MS/MS. The lowest limit of detection was 7 fg/µL using a 1 µL injection volume. Calibration curves for steroid quantification showed good linearity (r2 >0.99) between 14 and 3620 fg/µL, and accuracy was <20% for interday and <10% for intraday. After validation, the method was successfully applied to quantification of steroids in gray whale blubber samples. CONCLUSIONS: The nanoLC/MS/MS method is more sensitive than traditional LC/MS/MS for steroid analysis. It is also compatible with other important biopsy analyses due to its small blubber mass requirement. This will benefit the reproductive and stress assessments for all marine mammals, particularly endangered populations. Copyright © 2017 John Wiley & Sons, Ltd.

Body density and diving gas volume of the northern bottlenose whale (Hyperoodon ampullatus).

Diving lung volume and tissue density, reflecting lipid store volume, are important physiological parameters that have only been estimated for a few breath-hold diving species. We fitted 12 northern bottlenose whales with data loggers that recorded depth, 3-axis acceleration and speed either with a fly-wheel or from change of depth corrected by pitch angle. We fitted measured values of the change in speed during 5 s descent and ascent glides to a hydrodynamic model of drag and buoyancy forces using a Bayesian estimation framework. The resulting estimate of diving gas volume was 27.4±4.2 (95% credible interval, CI) ml kg(-1), closely matching the measured lung capacity of the species. Dive-by-dive variation in gas volume did not correlate with dive depth or duration. Estimated body densities of individuals ranged from 1028.4 to 1033.9 kg m(-3) at the sea surface, indicating overall negative tissue buoyancy of this species in seawater. Body density estimates were highly precise with ±95% CI ranging from 0.1 to 0.4 kg m(-3), which would equate to a precision of <0.5% of lipid content based upon extrapolation from the elephant seal. Six whales tagged near Jan Mayen (Norway, 71°N) had lower body density and were closer to neutral buoyancy than six whales tagged in the Gully (Nova Scotia, Canada, 44°N), a difference that was consistent with the amount of gliding observed during ascent versus descent phases in these animals. Implementation of this approach using longer-duration tags could be used to track longitudinal changes in body density and lipid store body condition of free-ranging cetaceans.

First Direct Evidence for Natal Wintering Ground Fidelity and Estimate of Juvenile Survival in the New Zealand Southern Right Whale Eubalaena australis.

Juvenile survival and recruitment can be more sensitive to environmental, ecological and anthropogenic factors than adult survival, influencing population-level processes like recruitment and growth rate in long-lived, iteroparous species such as southern right whales. Conventionally, Southern right whales are individually identified using callosity patterns, which do not stabilise until 6-12 months, by which time the whale has left its natal wintering grounds. Here we use DNA profiling of skin biopsy samples to identify individual Southern right whales from year of birth and document their return to the species' primary wintering ground in New Zealand waters, the Subantarctic Auckland Islands. We find evidence of natal fidelity to the New Zealand wintering ground by the recapture of 15 of 57 whales, first sampled in year of birth and available for subsequent recapture, during winter surveys to the Auckland Islands in 1995-1998 and 2006-2009. Four individuals were recaptured at the ages of 9 to 11, including two females first sampled as calves in 1998 and subsequently resampled as cows with calves in 2007. Using these capture-recapture records of known-age individuals, we estimate changes in survival with age using Cormack-Jolly-Seber models. Survival is modelled using discrete age classes and as a continuous function of age. Using a bootstrap method to account for uncertainty in model selection and fitting, we provide the first direct estimate of juvenile survival for this population. Our analyses indicate a high annual apparent survival for juveniles at between 0.87 (standard error (SE) 0.17, to age 1) and 0.95 (SE 0.05: ages 2-8). Individual identification by DNA profiling is an effective method for long-term demographic and genetic monitoring, particularly in animals that change identifiable features as they develop or experience tag loss over time.

Passive Acoustic Monitoring of the Environmental Impact of Oil Exploration on Marine Mammals in the Gulf of Mexico.

The Gulf of Mexico is a region densely populated by marine mammals that must adapt to living in a highly active industrial environment. This paper presents a new approach to quantifying the anthropogenic impact on the marine mammal population. The results for sperm and beaked whales of a case study of regional population dynamics trends after the Deepwater Horizon oil spill, derived from passive acoustic-monitoring data gathered before and after the spill in the vicinity of the accident, are presented.

Bioaccumulation of persistent organochlorine pesticides (OCPs) by gray whale and Pacific walrus from the western part of the Bering Sea.

The feeding habits of a gray whale (Eschrichtius robustus) and a Pacific walrus (Odobenus rosmarus divergens), caught from the western Bering Sea in the summers of 2010 and 2011, have been studied, and concentration of persistent organochlorine pesticides (OCPs) in their organs determined. The total OCP concentration (∑HCH+∑DDT) in muscles and liver of the gray whales varies from 297 to 3581 and from 769 to 13,808 ng/g lipids, respectively. The total OCP concentration (∑HCH+∑DDT) in muscles and liver of the Pacific walruses varies from 197 to 5659 and from 4856 to 90,263 ng/g lipids, respectively. The specifics of diet as a source of pesticide accumulation in these two marine mammal species are discussed.

Comparative cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human and sperm whale (Physeter macrocephalus) skin cells.

Chromium (Cr) is a global marine pollutant, present in marine mammal tissues. Hexavalent chromium [Cr(VI)] is a known human carcinogen. In this study, we compare the cytotoxic and clastogenic effects of Cr(VI) in human (Homo sapiens) and sperm whale (Physeter macrocephalus) skin fibroblasts. Our data show that increasing concentrations of both particulate and soluble Cr(VI) induce increasing amounts of cytotoxicity and clastogenicity in human and sperm whale skin cells. Furthermore, the data show that sperm whale cells are resistant to these effects exhibiting less cytotoxicity and genotoxicity than the human cells. Differences in Cr uptake accounted for some but not all of the differences in particulate and soluble Cr(VI) genotoxicity, although it did explain the differences in particulate Cr(VI) cytotoxicity. Altogether, the data indicate that Cr(VI) is a genotoxic threat to whales, but also suggest that whales have evolved cellular mechanisms to protect them against the genotoxicity of environmental agents such as Cr(VI).

Differential antioxidant protection in tissues from marine mammals with distinct diving capacities. Shallow/short vs. deep/long divers.

The diving response in marine mammals results in bradycardia and peripheral vasoconstriction, with blood flow redistributing preferentially to nervous and cardiac tissues. Therefore, some tissues are rendered ischemic during a dive; with the first breath after a dive, blood flow to all tissues is reestablished. In terrestrial mammals, reactive oxygen species (ROS) production increases in response to ischemia/reperfusion and oxidative damage can occur. The capacity of marine mammals to tolerate repeated ischemia/reperfusion cycles associated with diving appears to be due to an enhanced antioxidant system. However, it is not known if diving depth and/or duration elicit differences in tissue capacity to produce ROS and antioxidant defenses in marine mammals. The objective of this study was to analyze ROS production, antioxidant defenses and oxidative damage in marine mammal species that perform shallow/short vs. deep/long dives. We measured production of superoxide radical (O(2)(•-)), oxidative damage to lipids and proteins, activity of antioxidant enzymes, and glutathione levels in tissues from shallow/short divers (Tursiops truncatus) and deep/long divers (Kogia spp.). We found that differences between the diving capacity of dolphins and Kogia spp. are reflected in O(2)(•-) production and antioxidant levels. These differences suggest that shallow/short and deep/long divers have distinct mechanisms to successfully maintain redox balance.

Methodology for in situ gas sampling, transport and laboratory analysis of gases from stranded cetaceans.

Gas-bubble lesions were described in cetaceans stranded in spatio-temporal concordance with naval exercises using high-powered sonars. A behaviourally induced decompression sickness-like disease was proposed as a plausible causal mechanism, although these findings remain scientifically controversial. Investigations into the constituents of the gas bubbles in suspected gas embolism cases are highly desirable. We have found that vacuum tubes, insulin syringes and an aspirometer are reliable tools for in situ gas sampling, storage and transportation without appreciable loss of gas and without compromising the accuracy of the analysis. Gas analysis is conducted by gas chromatography in the laboratory. This methodology was successfully applied to a mass stranding of sperm whales, to a beaked whale stranded in spatial and temporal association with military exercises and to a cetacean chronic gas embolism case. Results from the freshest animals confirmed that bubbles were relatively free of gases associated with putrefaction and consisted predominantly of nitrogen.

Acute sun damage and photoprotective responses in whales.

Rising levels of ultraviolet radiation (UVR) secondary to ozone depletion are an issue of concern for public health. Skin cancers and intraepidermal dysplasia are increasingly observed in individuals that undergo chronic or excessive sun exposure. Such alterations of skin integrity and function are well established for humans and laboratory animals, but remain unexplored for mammalian wildlife. However, effects are unlikely to be negligible, particularly for species such as whales, whose anatomical or life-history traits force them to experience continuous sun exposure. We conducted photographic and histological surveys of three seasonally sympatric whale species to investigate sunburn and photoprotection. We find that lesions commonly associated with acute severe sun damage in humans are widespread and that individuals with fewer melanocytes have more lesions and less apoptotic cells. This suggests that the pathways used to limit and resolve UVR-induced damage in humans are shared by whales and that darker pigmentation is advantageous to them. Furthermore, lesions increased significantly in time, as would be expected under increasing UV irradiance. Apoptosis and melanocyte proliferation mirror this trend, suggesting that whales are capable of quick photoprotective responses. We conclude that the thinning ozone layer may pose a risk to the health of whales and other vulnerable wildlife.

Could beaked whales get the bends? Effect of diving behaviour and physiology on modelled gas exchange for three species: Ziphius cavirostris, Mesoplodon densirostris and Hyperoodon ampullatus.

A mathematical model, based on current knowledge of gas exchange and physiology of marine mammals, was used to predict blood and tissue tension N2 (P(N2)) using field data from three beaked whale species: northern bottlenose whales, Cuvier's beaked whales, and Blainville's beaked whales. The objective was to determine if physiology (body mass, diving lung volume, dive response) or dive behaviour (dive depth and duration, changes in ascent rate, diel behaviour) would lead to differences in P(N2) levels and thereby decompression sickness (DCS) risk between species. Diving lung volume and extent of the dive response had a large effect on end-dive P(N2). The dive profile had a larger influence on end-dive P(N2) than body mass differences between species. Despite diel changes in dive behaviour, P(N2) levels showed no consistent trend. Model output suggested that all three species live with tissue P(N2) levels that would cause a significant proportion of DCS cases in terrestrial mammals. Cuvier's beaked whale diving behaviour appears to put them at higher risk than the other species, which may explain their prevalence in strandings after the use of mid-frequency sonar.

Theoretical paper: exploring overlooked natural mitochondria-rejuvenative intervention: the puzzle of bowhead whales and naked mole rats.

There is an imperative need for exploring and implementing mitochondria-rejuvenative interventions that can bridge the current gap toward the step-by step realization of strategies for engineered negligible senescence (SENS) agenda. Recently discovered in mammals, natural mechanism mitoptosis-a selective "suicide" of mutated mitochondria-can facilitate continuous purification of mitochondrial pool in an organism from the most reactive oxygen species (ROS)-producing mitochondria. Mitoptosis, which is considered to be the first stage of ROS-induced apoptosis, underlies follicular atresia (a "quality control" mechanism in female germline cells that eliminates most germinal follicles in female embryos). Mitoptosis can be also activated in adult postmitotic somatic cells by evolutionary conserved phenotypic adaptations to intermittent oxygen restriction (IOR) and synergistically acting intermittent caloric restriction (ICR). IOR and ICR are common in mammals and seem to underlie extraordinary longevity and augmented cancer resistance in bowhead whales (Balena mysticetus) and naked mole rats (Heterocephalus glaber). Furthermore, in mammals IOR can facilitate continuous stromal stem cells-de-pendent tissue repair. A comparative analysis of IOR and ICR mechanisms in both mammals, in conjunction with the experience of decades of biomedical and clinical research on emerging preventative, therapeutic, and rehabilitative modality-the intermittent hypoxic training/therapy (IHT)-indicates that the notable clinical efficiency of IHT is based on the universal adaptational mechanisms that are common in mammals. Further exploration of natural mitochondria-preserving and -rejuvenating strategies can help refinement of IOR- and ICR-based synergistic protocols, having value in clinical human rejuvenation.

Structure of the integument of southern right whales, Eubalaena australis.

Skin (integument) anatomy reflects adaptations to particular environments. It is hypothesized that cetacean (whale) integument will show unique anatomical adaptations to an aquatic environment, particularly regarding differences in temperature, density, and pressure. In this study, the gross and histological structure of the southern right whale integument is described and compared with terrestrial mammals and previous descriptions of mysticete (baleen whale) and odontocete (toothed whale) species. Samples were taken of the integument of 98 free-swimming southern right whales, Eubalaena australis, and examined by both light and electron microscopy. Results show that three epidermal layers are present, with the stratum corneum being parakeratotic in nature. As in bowhead whales, southern right whales possess an acanthotic epidermis and a notably thick hypodermis, with epidermal rods and extensive papillomatosis. However, unlike bowhead whales, southern right whales possess an uninterrupted hypodermal layer. Surprisingly, the integument of balaenids (right and bowhead mysticetes) in general is more like that of odontocetes than that of the more closely related balaenopterids (rorqual mysticetes). Similarities to odontocetes were found specifically in the collagen fibers in a fat-free zone of the reticular dermal layer and the elastic fibers in the dermal and hypodermal layers. Callosities, a distinctive feature of this genus, have a slightly thicker stratum corneum and are usually associated with hairs that have innervated and vascularized follicles. These hairs may function as vibrissae, thus aiding in aquatic foraging by allowing rapid detection of changes in prey density. Although the thick insulatory integument makes right whales bulky and slow-moving, it is an adaptation for living in cold water. Epidermal thickness, presence of epidermal rods, and callosities may act as barriers against mechanical injury from bodily contact with conspecifics or hard surfaces in the environment (e.g., rocks, ice).

Use of chemical tracers in assessing the diet and foraging regions of eastern North Pacific killer whales.

Top predators in the marine environment integrate chemical signals acquired from their prey that reflect both the species consumed and the regions from which the prey were taken. These chemical tracers-stable isotope ratios of carbon and nitrogen; persistent organic pollutant (POP) concentrations, patterns and ratios; and fatty acid profiles-were measured in blubber biopsy samples from North Pacific killer whales (Orcinus orca) (n=84) and were used to provide further insight into their diet, particularly for the offshore group, about which little dietary information is available. The offshore killer whales were shown to consume prey species that were distinctly different from those of sympatric resident and transient killer whales. In addition, it was confirmed that the offshores forage as far south as California. Thus, these results provide evidence that the offshores belong to a third killer whale ecotype. Resident killer whale populations showed a gradient in stable isotope profiles from west (central Aleutians) to east (Gulf of Alaska) that, in part, can be attributed to a shift from off-shelf to continental shelf-based prey. Finally, stable isotope ratio results, supported by field observations, showed that the diet in spring and summer of eastern Aleutian Island transient killer whales is apparently not composed exclusively of Steller sea lions.