Fatty acid composition and N2 solubility in triacylglycerol-rich adipose tissue: the likely importance of intact molecular structure.

Diving tetrapods (sea turtles, seabirds and marine mammals) are a biologically diverse group, yet all are under similar constraints: oxygen limitation and increased hydrostatic pressure at depth. Adipose tissue is important in the context of diving because nitrogen gas (N2) is five times more soluble in fat than in blood, creating a potential N2 sink in diving animals. Previous research demonstrates that unusual lipid composition [waxes and short-chained fatty acids (FA)] in adipose tissue of some whales leads to increased N2 solubility. We evaluated the N2 solubility of adipose tissue from 12 species of diving tetrapods lacking these unusual lipids to explore whether solubility in this tissue can be linked to lipid structure. Across all taxonomic groups, the same eight FA accounted for 70-80% of the entire lipid profile; almost all adipose tissues were dominated by monounsaturated FA (40.2-67.4 mol%). However, even with consistent FA profiles, there was considerable variability in N2 solubility, ranging from 0.051±0.003 to 0.073±0.004 ml N2 ml-1 oil. Interestingly, differences in N2 solubility could not be attributed to taxonomic group (P=0.06) or FA composition (P>0.10). These results lead to two main conclusions: (1) in triacylglycerol-only adipose tissues, the FA pool itself may not have a strong influence on N2 solubility; and (2) samples with similar FA profiles can have different N2 solubility values, suggesting that 3D arrangement of individual FA within a triacylglycerol molecule may have important roles in determining N2 solubility.

Microvessel density, lipid chemistry and N2 solubility in human and pig adipose tissue.

Decompression sickness (DCS) occurs when nitrogen gas (N2) comes out of solution too quickly, forming bubbles in the blood and tissues. These bubbles can be a serious condition; thus it is of extreme interest in the dive community to model DCS risk. Diving models use tissue compartments to calculate tissue partial pressures, often using data obtained from other mammalian species (i.e., pigs). Adipose tissue is an important compartment in these models because N2 is five times more soluble in fat than in blood; at any blood/tissue interface N2 will diffuse into the fat and can lead to bubble formation on ascent. Little is known about many characteristics of adipose tissue relevant to diving physiology. Therefore, we measured microvessel density and morphology, lipid composition, and N2 solubility in adipose tissue from humans and pigs. Human adipose tissue has significantly higher microvascular density (1.79 ± 0.04 vs. 1.21 ± 0.30%), vessel diameter (10.25 ± 0.28 vs. 6.72 ± 0.60 µm), total monounsaturated fatty acids (50.1 vs. 41.2 mol%) and N2 solubility (0.061 ± 0.003 vs. 0.054 ± 0.004 mL N2 mL⁻ ¹ oil) compared to pig tissue. Pig adipose tissue has significantly higher lipid content (76.1 ± 4.9 vs. 64.6 ± 5.1%) and total saturated fatty acids (38.8 vs. 29.5 mol%). Though two important components in gas kinetics within adipose tissue during diving (blood flow rates and degree of perfusion) are not well understood, our results indicate differences between the adipose tissue of humans and pigs. This suggests data from swine may not exactly predict gas dynamics for estimating DCS in humans.

Nitrogen solubility in odontocete blubber and mandibular fats in relation to lipid composition.

Understanding toothed whale (odontocete) diving gas dynamics is important given the recent atypical mass strandings of odontocetes (particularly beaked whales) associated with mid-frequency naval sonar. Some stranded whales have exhibited gas emboli (pathologies resembling decompression sickness) in their specialized intramandibular and extramandibular fat bodies used for echolocation and hearing. These tissues have phylogenetically unique, endogenous lipid profiles with poorly understood biochemical properties. Current diving gas dynamics models assume an Ostwald nitrogen (N2) solubility of 0.07 ml N2 ml(-1) oil in odontocete fats, although solubility in blubber from many odontocetes exceeds this value. The present study examined N2 solubility in the blubber and mandibular fats of seven species across five families, relating it to lipid composition. Across all species, N2 solubility increased with wax ester content and was generally higher in mandibular fats (0.083 ± 0.002 ml N2 ml(-1) oil) than in blubber (0.069 ± 0.007 ml N2 ml(-1) oil). This effect was more pronounced in mandibular fats with higher concentrations of shorter, branched fatty acids/alcohols. Mandibular fats of short-finned pilot whales, Atlantic spotted dolphins and Mesoplodon beaked whales had the highest N2 solubility values (0.097 ± 0.005, 0.081 ± 0.007 and 0.080 ± 0.003 ml N2 ml(-1) oil, respectively). Pilot and beaked whales may experience high N2 loads during their relatively deeper dives, although more information is needed about in vivo blood circulation to mandibular fats. Future diving models should incorporate empirically measured N2 solubility of odontocete mandibular fats to better understand N2 dynamics and potential pathologies from gas/fat embolism.

Challenges in small cetacean telemetry: an attempt at developing a remotely deployed attachment device for single-pin dorsal fin satellite transmitters.

Satellite telemetry is critical for collecting fine-scale temporal and spatial data on individual animals that has broad-scale applicability at population and species levels. There have been significant advances in the remote deployment of satellite telemetry devices on large cetacean species. However, the development of comparable remote attachment methodologies for small cetaceans is still limited. Currently, satellite tag attachment for small cetaceans requires manual capture that increases the risk to the target animal, can be logistically challenging, and cost prohibitive. The goal of this project was to develop a novel tool to remotely attach single-pin satellite telemetry devices to the dorsal fin of individual small cetaceans. Three different spring-loaded designs and one pneumatic version of the remote attachment device were built in an iterative process to identify a successful deployment methodology. Ultimately, as a result of logistical challenges associated with a Category 5 hurricane, the COVID-19 pandemic, and engineering complexities related to dorsal fin morphology and small cetacean behavior, the objective of this project was not met. However, lessons learned from these attempts to develop this new sampling tool have applicability for future researchers in the successful completion of a safe and effective methodology for remote attachment of satellite tags to small cetacean dorsal fins.

Solubility of nitrogen in marine mammal blubber depends on its lipid composition.

Understanding the solubility of nitrogen gas in tissues is a crucial aspect of diving physiology, especially for air-breathing tetrapods. Adipose tissue is of particular interest because of the high solubility of nitrogen in lipids. Surprisingly, nothing is known about nitrogen solubility in the blubber of any marine mammal. We tested the hypothesis that N(2) solubility is dependent on the lipid composition of blubber; most blubber is composed of triacylglycerols, but some toothed whales deposit large amounts of waxes in blubber instead. The solubility of N(2) in the blubber of 13 toothed whale species ranged from 0.062 to 0.107 ml N(2) ml(-1) oil. Blubber with high wax ester content had higher N(2) solubility, observed in the beaked (Ziphiidae) and small sperm (Kogiidae) whales, animals that routinely make long, deep dives. We also measured nitrogen solubility in the specialized cranial acoustic fat bodies associated with echolocation in a Risso's dolphin; values (0.087 ml N(2) ml(-1) oil) were 16% higher here than in its blubber (0.074 ml N(2) ml(-1) oil). As the acoustic fats of all Odontocetes contain waxes, even if the blubber does not, these tissues may experience greater interaction with N(2). These data have implications for our understanding and future modeling of diving physiology in Odontocetes, as our empirically derived values for nitrogen solubility in toothed whale adipose were up to 40% higher than the numbers traditionally assumed in marine mammal diving models.

Microvascular anatomy suggests varying aerobic activity levels in the adipose tissues of diving tetrapods.

Adipose tissue has many important functions including metabolic energy storage, endocrine functions, thermoregulation and structural support. Given these varied functions, the microvascular characteristics within the tissue will have important roles in determining rates/limits of exchange of nutrients, waste, gases and molecular signaling molecules between adipose tissue and blood. Studies on skeletal muscle have suggested that tissues with higher aerobic capacity contain higher microvascular density (MVD) with lower diffusion distances (DD) than less aerobically active tissues. However, little is known about MVD in adipose tissue of most vertebrates; therefore, we measured microvascular characteristics (MVD, DD, diameter and branching) and cell size to explore the comparative aerobic activity in the adipose tissue across diving tetrapods, a group of animals facing additional physiological and metabolic stresses associated with diving. Adipose tissues of 33 animals were examined, including seabirds, sea turtles, pinnipeds, baleen whales and toothed whales. MVD and DD varied significantly (P < 0.001) among the groups, with seabirds generally having high MVD, low DD and small adipocytes. These characteristics suggest that microvessel arrangement in short duration divers (seabirds) reflects rapid lipid turnover, compared to longer duration divers (beaked whales) which have relatively lower MVD and greater DD, perhaps reflecting the requirement for tissue with lower metabolic activity, minimizing energetic costs during diving. Across all groups, predictable scaling patterns in MVD and DD such as those observed in skeletal muscle did not emerge, likely reflecting the fact that unlike skeletal muscle, adipose tissue performs many different functions in marine organisms, often within the same tissue compartment.

Lipid signature of neural tissues of marine and terrestrial mammals: consistency across species and habitats.

Marine mammals are exposed to O2-limitation and increased N2 gas concentrations as they dive to exploit habitat and food resources. The lipid-rich tissues (blubber, acoustic, neural) are of particular concern as N2 is five times more soluble in lipid than in blood or muscle, creating body compartments that can become N2 saturated, possibly leading to gas emboli upon surfacing. We characterized lipids in the neural tissues of marine mammals to determine whether they have similar lipid profiles compared to terrestrial mammals. Lipid profiles (lipid content, lipid class composition, and fatty acid signatures) were determined in the neural tissues of 12 cetacean species with varying diving regimes, and compared to two species of terrestrial mammals. Neural tissue lipid profile was not significantly different in marine versus terrestrial mammals across tissue types. Within the marine species, average dive depth was not significantly associated with the lipid profile of cervical spinal cord. Across species, tissue type (brain, spinal cord, and spinal nerve) was a significant factor in lipid profile, largely due to the presence of storage lipids (triacylglycerol and wax ester/sterol ester) in spinal nerve tissue only. The stability of lipid signatures within the neural tissue types of terrestrial and marine species, which display markedly different dive behaviors, points to the consistent role of lipids in these tissues. These findings indicate that despite large differences in the level of N2 gas exposure by dive type in the species examined, the lipids of neural tissues likely do not have a neuroprotective role in marine mammals.

Reference genome and demographic history of the most endangered marine mammal, the vaquita.

The vaquita is the most critically endangered marine mammal, with fewer than 19 remaining in the wild. First described in 1958, the vaquita has been in rapid decline for more than 20 years resulting from inadvertent deaths due to the increasing use of large-mesh gillnets. To understand the evolutionary and demographic history of the vaquita, we used combined long-read sequencing and long-range scaffolding methods with long- and short-read RNA sequencing to generate a near error-free annotated reference genome assembly from cell lines derived from a female individual. The genome assembly consists of 99.92% of the assembled sequence contained in 21 nearly gapless chromosome-length autosome scaffolds and the X-chromosome scaffold, with a scaffold N50 of 115 Mb. Genome-wide heterozygosity is the lowest (0.01%) of any mammalian species analysed to date, but heterozygosity is evenly distributed across the chromosomes, consistent with long-term small population size at genetic equilibrium, rather than low diversity resulting from a recent population bottleneck or inbreeding. Historical demography of the vaquita indicates long-term population stability at less than 5,000 (Ne) for over 200,000 years. Together, these analyses indicate that the vaquita genome has had ample opportunity to purge highly deleterious alleles and potentially maintain diversity necessary for population health.

Propagation of narrow-band-high-frequency clicks: measured and modeled transmission loss of porpoise-like clicks in porpoise habitats.

Estimating the range at which harbor porpoises can detect prey items and environmental objects is integral to understanding their biosonar. Understanding the ranges at which they can use echolocation to detect and avoid obstacles is particularly important for strategies to reduce bycatch. Transmission loss (TL) during acoustic propagation is an important determinant of those detection ranges, and it also influences animal detection functions used in passive acoustic monitoring. However, common assumptions regarding TL have rarely been tested. Here, TL of synthetic porpoise clicks was measured in porpoise habitats in Canada and Denmark, and field data were compared with spherical spreading law and ray-trace (Bellhop) model predictions. Both models matched mean observations quite well in most cases, indicating that a spherical spreading law can usually provide an accurate first-order estimate of TL for porpoise sounds in porpoise habitat. However, TL varied significantly (+/-10 dB) between sites and over time in response to variability in seafloor characteristics, sound-speed profiles, and other short-timescale environmental fluctuations. Such variability should be taken into account in estimates of the ranges at which porpoises can communicate acoustically, detect echolocation targets, and be detected via passive acoustic monitoring.

Microvascular characteristics of the acoustic fats: Novel data suggesting taxonomic differences between deep and shallow-diving odontocetes.

Odontocetes have specialized mandibular fats, the extramandibular (EMFB) and intramandibular fat bodies (IMFB), which function as acoustic organs, receiving and channeling sound to the ear during hearing and echolocation. Recent strandings of beaked whales suggest that these fat bodies are susceptible to nitrogen (N2 ) gas embolism and empirical evidence has shown that the N2 solubility of these fat bodies is higher than that of blubber. Since N2 gas will diffuse from blood into tissue at any blood/tissue interface and potentially form gas bubbles upon decompression, it is imperative to understand the extent of microvascularity in these specialized acoustic fats so that risk of embolism formation when diving can be estimated. Microvascular density was determined in the EMFB, IMFB, and blubber from 11 species representing three odontocete families. In all cases, the acoustic tissues had less (typically 1/3 to 1/2) microvasculature than did blubber, suggesting that capillary density in the acoustic tissues may be more constrained than in the blubber. However, even within these constraints there were clear phylogenetic differences. Ziphiid (Mesoplodon and Ziphius, 0.9 ± 0.4% and 0.7 ± 0.3% for EMFB and IMFB, respectively) and Kogiid families (1.2 ± 0.2% and 1.0 ± 0.01% for EMFB and IMFB, respectively) had significantly lower mean microvascular densities in the acoustic fats compared to the Delphinid species (Tursiops, Grampus, Stenella, and Globicephala, 1.3 ± 0.3% and 1.3 ± 0.3% for EMFB and IMFB, respectively). Overall, deep-diving beaked whales had less microvascularity in both mandibular fats and blubber compared to the shallow-diving Delphinids, which might suggest that there are differences in the N2 dynamics associated with diving regime, phylogeny, and tissue type. These novel data should be incorporated into diving physiology models to further understand potential functional disruption of the acoustic tissues due to changes in normal diving behavior.

Seasonal variation in the spatial distribution of basking sharks (Cetorhinus maximus) in the lower Bay of Fundy, Canada.

The local distribution of basking sharks in the Bay of Fundy (BoF) is unknown despite frequent occurrences in the area from May to November. Defining this species' spatial habitat use is critical for accurately assessing its Special Concern conservation status in Atlantic Canada. We developed maximum entropy distribution models for the lower BoF and the northeast Gulf of Maine (GoM) to describe spatiotemporal variation in habitat use of basking sharks. Under the Maxent framework, we assessed model responses and distribution shifts in relation to known migratory behavior and local prey dynamics. We used 10 years (2002-2011) of basking shark surface sightings from July-October acquired during boat-based surveys in relation to chlorophyll-a concentration, sea surface temperature, bathymetric features, and distance to seafloor contours to assess habitat suitability. Maximum entropy estimations were selected based on AICc criterion and used to predict habitat utilizing three model-fitting routines as well as converted to binary suitable/non-suitable habitat using the maximum sensitivity and specificity threshold. All models predicted habitat better than random (AUC values >0.796). From July-September, a majority of habitat was in the BoF, in waters >100 m deep, and in the Grand Manan Basin. In October, a majority of the habitat shifted southward into the GoM and to areas >200 m deep. Model responses suggest that suitable habitat from July - October is dependent on a mix of distance to the 0, 100, 150, and 200 m contours but in some models on sea surface temperature (July) and chlorophyll-a (August and September). Our results reveal temporally dynamic habitat use of basking sharks within the BoF and GoM. The relative importance of predictor variables suggests that prey dynamics constrained the species distribution in the BoF. Also, suitable habitat shifted minimally from July-September providing opportunities to conserve the species during peak abundance in the region.

Microvascular patterns in the blubber of shallow and deep diving odontocetes.

Blubber, a specialized form of subdermal adipose tissue, surrounds marine mammal bodies. Typically, adipose tissue is perfused by capillaries but information on blubber vascularization is lacking. This study's goals were to: 1) describe and compare the microvasculature (capillaries, microarterioles, and microvenules) of blubber across odontocete species; 2) compare microvasculature of blubber to adipose tissue; and 3) examine relationships between blubber's lipid composition and its microvasculature. Percent microvascularity, distribution, branching pattern, and diameter of microvessels were determined from images of histochemically stained blubber sections from shallow-diving bottlenose dolphins (Tursiops truncatus), deeper-diving pygmy sperm whales (Kogia breviceps), deep-diving beaked whales (Mesoplodon densirostris; Ziphius cavirostris), and the subdermal adipose tissue of domestic pigs (Sus scrofa). Tursiops blubber showed significant stratification in percent microvascularity among the superficial, middle, and deep layers and had a significantly higher percent microvascularity than all other animals analyzed, in which the microvasculature was more uniformly distributed. The percent microvasculature of Kogia blubber was lower than that of Tursiops but higher than that of beaked whales and the subdermal adipose tissue of domestic pigs. Tursiops had the most microvascular branching. Microvessel diameter was relatively uniform in all species. There were no clear patterns associating microvascular and lipid characteristics. The microvascular characteristics of the superficial layer of blubber resembled the adipose tissue of terrestrial mammals, suggesting some conservation of microvascular patterns in mammalian adipose tissue. The middle and deep layers of blubber, particularly in Tursiops, showed the greatest departure from typical mammalian microvascular arrangement. Factors such as metabolics or thermoregulation may be influencing the microvasculature in these layers.

Seasonal patterns of heat loss in wild bottlenose dolphins (Tursiops truncatus).

This study investigated patterns of heat loss in bottlenose dolphins (Tursiops truncatus) resident to Sarasota Bay, FL, USA, where water temperatures vary seasonally from 11 to 33 degrees C. Simultaneous measurements of heat flux (HF) and skin surface temperature were collected at the body wall and appendages of dolphins during health-monitoring events in summer (June 2002-2004) and winter (February 2003-2005). Integument thickness was measured and whole body conductance (W/m(2) degrees C) was estimated using HF and colonic temperature measurements. Across seasons, HF values were similar at the appendages, but their distribution differed significantly at the flipper and fluke. In summer, these appendages displayed uniformly high values, while in winter they most frequently displayed very low HF values with a few high HF values. In winter, blubber thickness was significantly greater and estimated conductance significantly lower, than in summer. These results suggest that dolphins attempt to conserve heat in winter. In winter, though, HF values across the body wall were similar to (flank) or greater than (caudal keel) summer values. It is likely that higher winter HF values are due to the steep temperature gradient between the body core and colder winter water, which may limit the dolphin's ability to decrease heat loss across the body wall.

The relationship between heat flow and vasculature in the dorsal fin of wild bottlenose dolphins Tursiops truncatus.

The dorsal fin of the bottlenose dolphin Tursiops truncatus contains blood vessels that function either to conserve or to dissipate body heat. Prior studies have demonstrated that heat flux, measured from a single position on the dorsal fin, decreases during body cooling and diving bradycardia and increases after exercise and at the termination of the dive response. While prior studies attributed changes in heat flux to changes in the pattern of blood flow, none directly investigated the influence of vascular structures on heat flux across the dorsal fin. In this study we examined whether heat flux is higher directly over a superficial vein, compared to a position away from a vein, and investigated the temporal relationship between heart rate, respiration and heat flux. Simultaneous records of heat flux and skin temperature at three positions on the dorsal fins of 19 wild bottlenose dolphins (with the fin in air and submerged) were collected, together with heart rate and respiration. When the fin was submerged, heat flux values were highest over superficial veins, usually at the distal tip, suggesting convective delivery of heat, via blood, to the skin's surface. Conversely, in air there was no relationship between heat flux and superficial vasculature. The mean difference in heat flux (48 W m(-2)) measured between the three fin positions was often equal to or greater than the heat flux that had been recorded from a single position after exercising and diving in prior studies. Tachycardia at a respiratory event was not temporally related to an increase in heat flux across the dorsal fin. This study suggests that the dorsal fin is a spatially heterogeneous thermal surface and that patterns of heat flux are strongly influenced by underlying vasculature.