Pathology of short-term dorsal fin tag-attachments in tagged and re-stranded short-beaked common dolphins Delphinus delphis on Cape Cod, MA.

Odontocetes are difficult to study in the wild, making tagging and remote tracking a valuable practice. However, evaluations of host responses at tagging sites have been primarily limited to visual observations in the field. Here we explore the macro- and microscopic pathology of dorsal fin tag attachments in 13 stranded and released short-beaked common dolphins Delphinus delphis from Cape Cod, MA that later re-stranded and died or were euthanized 1-28 d post-tagging. Tags were attached to stranded dolphins' dorsal fins using 2 methods: core biopsy or piercing. Grossly, the piercing method resulted in epidermal compression into the dermis. One tag site had a necrotic border 28 d after application. Grossly, the biopsy method resulted in minimal to no tissue reaction. Two tag sites had granulation tissue accumulation 4 and 12 d after tagging. Histopathologic findings for all tag types and animals consisted of focal epithelial loss, dermal edema, perivascular edema, inflammation and hyperplasia, and inter- and extracellular edema in the adjacent epidermis. Minor expected pathological changes given the procedure were also observed: superficial epidermal necrosis in 3 cases, and superficial bacterial colonization in 2 cases. There was no evidence of sepsis and tagging was not related to cause of re-stranding or death in any case. These gross and histopathologic findings support previous observational conclusions in small delphinids that with appropriate sterile technique, the impacts of single pin dorsal fin tagging on the animal can be minimal and localized. Of the 2 methods, core biopsy may be a better tagging method.

Brucella ceti sequence type 23, 26, and 27 infections in North American cetaceans.

Brucella ceti infection is associated with a variety of disease outcomes in cetaceans globally. Multiple genotypes of B. ceti have been identified. This retrospective aimed to determine if specific lesions were associated with different B. ceti DNA sequence types (STs). Characterization of ST was performed on 163 samples from 88 free-ranging cetaceans, including common bottlenose dolphin Tursiops truncatus (T.t.; n = 73), common short-beaked dolphin Delphinus delphis (D.d.; n = 7), striped dolphin Stenella coeruleoalba (n = 3), Pacific white-sided dolphin Lagenorhynchus obliquidens (n = 2), sperm whale Physeter macrocephalus (n = 2), and harbour porpoise Phocoena phocoena (n = 1), that stranded along the coast of the US mainland and Hawaii. ST was determined using a previously described insertion sequence 711 quantitative PCR. Concordance with 9-locus multi-locus sequence typing was assessed in a subset of samples (n = 18). ST 26 was most commonly identified in adult dolphins along the US east coast with non-suppurative meningoencephalitis (p = 0.009). Animals infected with ST 27 were predominately perinates that were aborted or died shortly after birth with evidence of in utero pneumonia (p = 0.035). Reproductive tract inflammation and meningoencephalitis were also observed in adult T.t. and D.d. with ST 27, though low sample size limited interpretation. ST 23 infections can cause disease in cetacean families other than porpoises (Phocoenidae), including neurobrucellosis in D.d. In total, 11 animals were potentially infected with multiple STs. These data indicate differences in pathogenesis among B. ceti STs in free-ranging cetaceans, and infection with multiple STs is possible.

Longitudinal analysis of pinnipeds in the northwest Atlantic provides insights on endemic circulation of phocine distemper virus.

Phocine distemper virus (PDV) is a morbillivirus that circulates within pinnipeds in the North Atlantic. PDV has caused two known unusual mortality events (UMEs) in western Europe (1988, 2002), and two UMEs in the northwest Atlantic (2006, 2018). Infrequent cross-species transmission and waning immunity are believed to contribute to periodic outbreaks with high mortality in western Europe. The viral ecology of PDV in the northwest Atlantic is less well defined and outbreaks have exhibited lower mortality than those in western Europe. This study sought to understand the molecular and ecological processes underlying PDV infection in eastern North America. We provide phylogenetic evidence that PDV was introduced into northwest Atlantic pinnipeds by a single lineage and is now endemic in local populations. Serological and viral screening of pinniped surveillance samples from 2006 onward suggest there is continued circulation of PDV outside of UMEs among multiple species with and without clinical signs. We report six full genome sequences and nine partial sequences derived from harbour and grey seals in the northwest Atlantic from 2011 through 2018, including a possible regional variant. Work presented here provides a framework towards greater understanding of how recovering populations and shifting species may impact disease transmission.

Prevalence of alopecia in gray seals Halichoerus grypus atlantica in Massachusetts, USA, 2004-2013.

There has been an increase in the presence of alopecia among gray seals Halichoerus grypus atlantica in Massachusetts, USA. To understand the prevalence and describe the presentation of this condition, data records and photographs of 10070 gray seals from 2004-2013 were reviewed; there were sufficient data to confidently assess the presence or absence of alopecia in 2134 seals. Mild hair loss presented in multifocal patches with minimal to no skin lesions or erythema. In severe cases, alopecia was concentrated over the dorsal head, neck, and shoulders and extended down the ventrum, affecting >50% of the body. Associated skin lesions and erythema were often present. Alopecia was documented in 7.1% of the surveyed seals, and was centered in Nantucket. Alopecia was more prevalent in stranded/sighted animals in spring and summer, with 81% of cases documented from April to July. There was no sex bias, and weanlings were the most affected age class (38%). The etiology for alopecia in Massachusetts gray seals is unknown. Possible causes of alopecia in wildlife are infectious disease, nutritional deficiencies, endocrinopathies, or chronic physiologic stress. High population density around Nantucket may escalate intraspecific competition for resources, which may indirectly lead to stress-induced immunosuppression or nutritional deficiencies. Crowded haul-out sites increase the opportunity for disease transmission. The weanling age class may be prone to alopecia due to naïve immune systems and inexperienced foraging capabilities. Diagnostic sample collection from gray seals will be required to characterize the etiology, pathogenesis, and significance of alopecia in this population.

Discrimination between bycatch and other causes of cetacean and pinniped stranding.

The challenge of identifying cause of death in discarded bycaught marine mammals stems from a combination of the non-specific nature of the lesions of drowning, the complex physiologic adaptations unique to breath-holding marine mammals, lack of case histories, and the diverse nature of fishing gear. While no pathognomonic lesions are recognized, signs of acute external entanglement, bulging or reddened eyes, recently ingested gastric contents, pulmonary changes, and decompression-associated gas bubbles have been identified in the condition of peracute underwater entrapment (PUE) syndrome in previous studies of marine mammals. We reviewed the gross necropsy and histopathology reports of 36 cetaceans and pinnipeds including 20 directly observed bycaught and 16 live stranded animals that were euthanized between 2005 and 2011 for lesions consistent with PUE. We identified 5 criteria which present at significantly higher rates in bycaught marine mammals: external signs of acute entanglement, red or bulging eyes, recently ingested gastric contents, multi-organ congestion, and disseminated gas bubbles detected grossly during the necropsy and histologically. In contrast, froth in the trachea or primary bronchi, and lung changes (i.e. wet, heavy, froth, edema, congestion, and hemorrhage) were poor indicators of PUE. This is the first study that provides insight into the different published parameters for PUE in bycatch. For regions frequently confronted by stranded marine mammals with non-specific lesions, this could potentially aid in the investigation and quantification of marine fisheries interactions.

A comparative analysis of marine mammal tracheas.

In 1940, Scholander suggested that stiffened upper airways remained open and received air from highly compressible alveoli during marine mammal diving. There are few data available on the structural and functional adaptations of the marine mammal respiratory system. The aim of this research was to investigate the anatomical (gross) and structural (compliance) characteristics of excised marine mammal tracheas. Here, we defined different types of tracheal structures, categorizing pinniped tracheas by varying degrees of continuity of cartilage (categories 1-4) and cetacean tracheas by varying compliance values (categories 5A and 5B). Some tracheas fell into more than one category along their length; for example, the harbor seal (Phoca vitulina) demonstrated complete rings cranially, and as the trachea progressed caudally, tracheal rings changed morphology. Dolphins and porpoises had less stiff, more compliant spiraling rings while beaked whales had very stiff, less compliant spiraling rings. The pressure-volume (P-V) relationships of isolated tracheas from different species were measured to assess structural differences between species. These findings lend evidence for pressure-induced collapse and re-inflation of lungs, perhaps influencing variability in dive depth or ventilation rates of the species investigated.

Melon and rostral muscle morphology of Gervais' beaked whale (Mesoplodon europaeus): Alternating patterns of bilateral asymmetry.

Toothed whales utilize specialized nasal structures such as the lipid-rich melon to produce sound and propagate it into the aquatic environment. Very little nasal morphology of mesoplodont beaked whales has been described in the literature, and the anatomy of the melon and associated musculature of Gervais' beaked whale (Mesoplodon europaeus) remains undescribed. Heads of three (n = 3) Gervais' beaked whales were examined in detail via dissection as well as computed tomography (CT) and magnetic resonance imaging (MRI). Two additional Gervais' beaked whale individuals (n = 2) were studied via archived CT and MRI scans. Representative transverse dissection sections of the melon were processed for polarized light imaging to verify the presence of tendons inserting into the melon tissue. Three-dimensional (3D) CT reconstructions of the melon, rostral muscles, and associated structures were performed to assess morphology and spatial relationships. In all individuals, the melon's main body demonstrated a bilaterally asymmetrical, curvilinear geometry. This curvilinear shape was defined by a pattern of alternating asymmetry in the medial rostral muscles that projected into the melon's tissue. In transverse polarized light imaging, a network of tendons originating from these asymmetrical rostral muscle projections was observed permeating the melon's lipid tissue. This curvilinear melon morphology and associated asymmetrical musculature suggest a means of lengthening the lipid pathway within a relatively short dimensional footprint. In addition, the species-specific arrangement of muscular projections suggests complex fine-tuning of the melon's geometry during echolocation. Further studies may lend additional insight into the function of this unusual melon morphology.

Static inflation and deflation pressure-volume curves from excised lungs of marine mammals.

Excised lungs from eight marine mammal species [harp seal (Pagophilus groenlandicus), harbor seal (Phoca vitulina), gray seal (Halichoerus grypush), Atlantic white-sided dolphin (Lagenorhynchus acutus), common dolphin (Delphinus delphis), Risso's dolphin (Grampus griseus), long-finned pilot whale (Globicephala melas) and harbor porpoise (Phocoena phocoena)] were used to determine the minimum air volume of the relaxed lung (MAV, N=15), the elastic properties (pressure-volume curves, N=24) of the respiratory system and the total lung capacity (TLC). Our data indicate that mass-specific TLC (sTLC, l kg(-1)) does not differ between species or groups (odontocete vs phocid) and agree with that estimated (TLC(est)) from body mass (M(b)) by applying the equation: TLC(est)=0.135 M(b)(0.92). Measured MAV was on average 7% of TLC, with a range from 0 to 16%. The pressure-volume curves were similar among species on inflation but diverged during deflation in phocids in comparison with odontocetes. These differences provide a structural basis for observed species differences in the depth at which lungs collapse and gas exchange ceases.

An assessment of temporal, spatial and taxonomic trends in harmful algal toxin exposure in stranded marine mammals from the U.S. New England coast.

Despite a long-documented history of severe harmful algal blooms (HABs) in New England coastal waters, corresponding HAB-associated marine mammal mortality events in this region are far less frequent or severe relative to other regions where HABs are common. This long-term survey of the HAB toxins saxitoxin (STX) and domoic acid (DA) demonstrates significant and widespread exposure of these toxins in New England marine mammals, across multiple geographic, temporal and taxonomic groups. Overall, 19% of the 458 animals tested positive for one or more toxins, with 15% and 7% testing positive for STX and DA, respectively. 74% of the 23 different species analyzed demonstrated evidence of toxin exposure. STX was most prevalent in Maine coastal waters, most frequently detected in common dolphins (Delphinus delphis), and most often detected during July and October. DA was most prevalent in animals sampled in offshore locations and in bycaught animals, and most frequently detected in mysticetes, with humpback whales (Megaptera novaeangliae) testing positive at the highest rates. Feces and urine appeared to be the sample matrices most useful for determining the presence of toxins in an exposed animal, with feces samples having the highest concentrations of STX or DA. No relationship was found between the bloom season of toxin-producing phytoplankton and toxin detection rates, however STX was more likely to be present in July and October. No relationship between marine mammal dietary preference and frequency of toxin detection was observed. These findings are an important part of a framework for assessing future marine mammal morbidity and mortality events, as well as monitoring ecosystem health using marine mammals as sentinel organisms for predicting coastal ocean changes.

Myoglobin Concentration and Oxygen Stores in Different Functional Muscle Groups from Three Small Cetacean Species.

Compared with terrestrial mammals, marine mammals possess increased muscle myoglobin concentrations (Mb concentration, g Mb · 100g-1 muscle), enhancing their onboard oxygen (O2) stores and their aerobic dive limit. Although myoglobin is not homogeneously distributed, cetacean muscle O2 stores have been often determined by measuring Mb concentration from a single muscle sample (longissimus dorsi) and multiplying that value by the animal's locomotor muscle or total muscle mass. This study serves to determine the accuracy of previous cetacean muscle O2 stores calculations. For that, body muscles from three delphinid species: Delphinus delphis, Stenella coeruleoalba, and Stenella frontalis, were dissected and weighed. Mb concentration was calculated from six muscles/muscle groups (epaxial, hypaxial and rectus abdominis; mastohumeralis; sternohyoideus; and dorsal scalenus), each representative of different functional groups (locomotion powering swimming, pectoral fin movement, feeding and respiration, respectively). Results demonstrated that the Mb concentration was heterogeneously distributed, being significantly higher in locomotor muscles. Locomotor muscles were the major contributors to total muscle O2 stores (mean 92.8%) due to their high Mb concentration and large muscle masses. Compared to this method, previous studies assuming homogenous Mb concentration distribution likely underestimated total muscle O2 stores by 10% when only considering locomotor muscles and overestimated them by 13% when total muscle mass was considered.

Cyanobacterial neurotoxin BMAA and brain pathology in stranded dolphins.

Dolphin stranding events occur frequently in Florida and Massachusetts. Dolphins are an excellent sentinel species for toxin exposures in the marine environment. In this report we examine whether cyanobacterial neurotoxin, ß-methylamino-L-alanine (BMAA), is present in stranded dolphins. BMAA has been shown to bioaccumulate in the marine food web, including in the muscles and fins of sharks. Dietary exposure to BMAA is associated with the occurrence of neurofibrillary tangles and ß-amyloid plaques in nonhuman primates. The findings of protein-bound BMAA in brain tissues from patients with Alzheimer's disease has advanced the hypothesis that BMAA may be linked to dementia. Since dolphins are apex predators and consume prey containing high amounts of BMAA, we examined necropsy specimens to determine if dietary and environmental exposures may result in the accumulation of BMAA in the brains of dolphins. To test this hypothesis, we measured BMAA in a series of brains collected from dolphins stranded in Florida and Massachusetts using two orthogonal analytical methods: 1) high performance liquid chromatography, and 2) ultra-performance liquid chromatography with tandem mass spectrometry. We detected high levels of BMAA (20-748 µg/g) in the brains of 13 of 14 dolphins. To correlate neuropathological changes with toxin exposure, gross and microscopic examinations were performed on cortical brain regions responsible for acoustico-motor navigation. We observed increased numbers of ß-amyloid+ plaques and dystrophic neurites in the auditory cortex compared to the visual cortex and brainstem. The presence of BMAA and neuropathological changes in the stranded dolphin brain may help to further our understanding of cyanotoxin exposure and its potential impact on human health.

Tracking the Development of Muscular Myoglobin Stores in Mysticete Calves.

For marine mammals, the ability to tolerate apnea and make extended dives is a defining adaptive trait, facilitating the exploitation of marine food resources. Elevated levels of myoglobin within the muscles are a consistent hallmark of this trait, allowing oxygen collected at the surface to be stored in the muscles and subsequently used to support extended dives. In mysticetes, the largest of marine predators, details on muscular myoglobin levels are limited. The developmental trajectory of muscular myoglobin stores has yet to be documented and any physiological links between early behavior and the development of muscular myoglobin stores remain unknown. In this study, we used muscle tissue samples from stranded mysticetes to investigate these issues. Samples from three different age cohorts and three species of mysticetes were included (total sample size = 18). Results indicate that in mysticete calves, muscle myoglobin stores comprise only a small percentage (17-23%) of conspecific adult myoglobin complements. Development of elevated myoglobin levels is protracted over the course of extended maturation in mysticetes. Additionally, comparisons of myoglobin levels between and within muscles, along with details of interspecific differences in rates of accumulation of myoglobin in very young mysticetes, suggest that levels of exercise may influence the rate of development of myoglobin stores in young mysticetes. This new information infers a close interplay between the physiology, ontogeny and early life history of young mysticetes and provides new insight into the pressures that may shape adaptive strategies in migratory mysticetes. Furthermore, the study highlights the vulnerability of specific age cohorts to impending changes in the availability of foraging habitat and marine resources.

Humpback whale populations share a core skin bacterial community: towards a health index for marine mammals?

Microbes are now well regarded for their important role in mammalian health. The microbiology of skin--a unique interface between the host and environment--is a major research focus in human health and skin disorders, but is less explored in other mammals. Here, we report on a cross-population study of the skin-associated bacterial community of humpback whales (Megaptera novaeangliae), and examine the potential for a core bacterial community and its variability with host (endogenous) or geographic/environmental (exogenous) specific factors. Skin biopsies or freshly sloughed skin from 56 individuals were sampled from populations in the North Atlantic, North Pacific and South Pacific oceans and bacteria were characterized using 454 pyrosequencing of SSU rRNA genes. Phylogenetic and statistical analyses revealed the ubiquity and abundance of bacteria belonging to the Flavobacteria genus Tenacibaculum and the Gammaproteobacteria genus Psychrobacter across the whale populations. Scanning electron microscopy of skin indicated that microbial cells colonize the skin surface. Despite the ubiquity of Tenacibaculum and Psychrobater spp., the relative composition of the skin-bacterial community differed significantly by geographic area as well as metabolic state of the animals (feeding versus starving during migration and breeding), suggesting that both exogenous and endogenous factors may play a role in influencing the skin-bacteria. Further, characteristics of the skin bacterial community from these free-swimming individuals were assembled and compared to two entangled and three dead individuals, revealing a decrease in the central or core bacterial community members (Tenacibaculum and Psychrobater spp.), as well as the emergence of potential pathogens in the latter cases. This is the first discovery of a cross-population, shared skin bacterial community. This research suggests that the skin bacteria may be connected to humpback health and immunity and could possibly serve as a useful index for health and skin disorder monitoring of threatened and endangered marine mammals.

Compositional discrimination of decompression and decomposition gas bubbles in bycaught seals and dolphins.

Gas bubbles in marine mammals entangled and drowned in gillnets have been previously described by computed tomography, gross examination and histopathology. The absence of bacteria or autolytic changes in the tissues of those animals suggested that the gas was produced peri- or post-mortem by a fast decompression, probably by quickly hauling animals entangled in the net at depth to the surface. Gas composition analysis and gas scoring are two new diagnostic tools available to distinguish gas embolisms from putrefaction gases. With this goal, these methods have been successfully applied to pathological studies of marine mammals. In this study, we characterized the flux and composition of the gas bubbles from bycaught marine mammals in anchored sink gillnets and bottom otter trawls. We compared these data with marine mammals stranded on Cape Cod, MA, USA. Fresh animals or with moderate decomposition (decomposition scores of 2 and 3) were prioritized. Results showed that bycaught animals presented with significantly higher gas scores than stranded animals. Gas composition analyses indicate that gas was formed by decompression, confirming the decompression hypothesis.