RESTRICTABLE DNA FROM SLOUGHED CETACEAN SKIN; ITS POTENTIAL FOR USE IN POPULATION ANALYSIS

Abstract: Several species of cetaceans naturally slough visible quantities of skin. We have investigated the prospect of using this material as a viable alternative to the use of biopsy darts for the collection of samples for molecular analysis. Pieces of skin were collected from free-ranging individuals of three different species—the humpback ( Megaptera novaeangliae) , sperm ( Physeter macrocephalus ) and right whales ( Eubalaena glacialis ). DNA was extracted from 11 pieces of sloughed skin and DNA "fingerprint" profiles prepared. All samples contained DNA of both sufficient quality and quantity for genetic analysis. The applicability of this approach is discussed generally in relation to answering problems about the population structure and breeding systems of cetaceans.     

Molecular analysis of the efficiency of sloughed skin sampling in whale population genetics

Sloughed whale skin contains enough DNA for genetic analysis, and offers a nonintrusive method for collecting tissue. Here, we examine the efficiency of sloughed skin sampling using 1460 samples collected from free-ranging humpback whales. Samples were sexed and screened for up to 10 microsatellite markers. The vast majority of samples appear genetically compatible with field observations. About 1% of groups revealed more genotypes than whales, but we argue that this is more likely to be due to unobserved whales than to contamination. Sloughed skin sampling is particularly effective when applied to active groups and offers a viable alternative to biopsy darting in regions where darting is either not permitted or otherwise undesirable.     

Skin in the game: Epidermal molt as a driver of long-distance migration in whales

Long-distance migration in whales has historically been described as an annual, round-trip movement between high-latitude, summer feeding grounds, and low-latitude, winter breeding areas, but there is no consensus about why whales travel to the tropics to breed. Between January 2009 and February 2016, we satellite-tagged 62 antarctic killer whales (Orcinus orca) of four different ecotypes, of which at least three made short-term (6–8 weeks), long-distance (maximum 11,000 km, round trip), essentially nonstop, migrations to warm waters (SST 20°C–24°C), and back. We previously suggested that antarctic killer whales could conserve body heat in subfreezing (to −1.9°C) waters by reducing blood flow to their skin, but that this might preclude normal (i.e., continuous) epidermal molt, and necessitate periodic trips to warm waters for routine skin maintenance (“skin molt migration,” SMM). In contrast to the century-old “feeding/breeding” migration paradigm, but consistent with a “feeding/molting” hypothesis, the current study provides additional evidence that deferred skin molt could be the main driver of long-distance migration for antarctic killer whales. Furthermore, we argue that for all whales that forage in polar latitudes and migrate to tropical waters, SMM might also allow them to exploit rich prey resources in a physiologically challenging environment and maintain healthy skin.     

Non-geographically based population structure of South Pacific sperm whales: dialects, fluke-markings and genetics

1. This study addresses the issue of structure in sperm whale ( Physeter macrocephalus Linnaeus) populations and whether it is geographically based.
2. During a survey around the South Pacific Ocean, we collected sloughed skin for genetic analyses, recorded coda vocalizations, and photographed fluke markings.
3. Groups of female and immature sperm whales had characteristic mitochondrial haplotypes, coda repertoires, and fluke-mark patterns, but there was no clear geographical structure in any of these attributes.
4. However, similarities of coda repertoire and mitochondrial haplotype distribution were significantly correlated among pairs of groups in a manner that was not geographically based. There was also a significant canonical correlation coefficient between coda repertoire and fluke-mark patterns.
5. These results suggest that attributes (such as vocal repertoire and techniques of predator defence) which are acquired matrilineally, and probably culturally, are conserved during the fission and dispersal of groups.     

SPERM WHALE PHYLOGENY REVISITED: ANALYSIS OF THE MORPHOLOGICAL EVIDENCE

Some recent analyses of three mitochondrial DNA regions suggest that sperm whales are the sister group to baleen whales and, therefore, the suborder Odontoceti (toothed whales) constitutes a paraphyletic group. I cladistically analyzed the available morphological data, including that from relevant fossil taxa, for all families of extant cetaceans to test this hypothesis. The results of this analysis unambiguously support a monophyletic Odontoceti including the sperm whales. All synapomorphies that support the Odontoceti node are decisive, not related to the evolution of highly correlated characters, and provide the same result regardless of what order of mammals is used as an outgroup. These numerous, anatomically diverse, and unambiguous characters make this clade one of the best-supported higher-level groupings among mammals. In addition, the fossil evidence refutes a sperm whale/baleen whale clade. Both the molecular and morphological data produce the same unrooted tree. The improper rooting of the molecular tree appears to be producing these seemingly incongruent phylogenies.     

OPEN-BOAT WHALING ON THE STRAITS OF GIBRALTAR GROUND AND ADJACENT WATERS

Logbooks ( n = 317) from whaling expeditions made in the North Atlantic during the 19th century were examined to investigate activity in the Gibraltar Straits grounds. At least forty expeditions of whaling vessels from European and American ports visited the area. In all cases the main target was the sperm whale, but pilot whales, dolphins, sea turtles, and even a blue whale were also taken. Whaling effort concentrated on the Atlantic side of the Straits; only two expeditions ventured into the Mediterranean Sea, obtaining negligible catches. The whaling season extended during spring and summer and peaked in June–July. This seasonality appeared not to be governed by changes in whale density but by the trade winds necessary to sail southward or westward to cross the Atlantic. Searching effort continued while trying out, but the rate of sighting cetaceans was about half that of searching periods. However, the rate of sighting or capturing a sperm whale remained unchanged during processing, probably because the gregarious habits of the species produced clumping of catches. For every whale secured, 1.31 whales were struck. After correcting for struck but lost whales and for "gammed" vessels, the minimum number of removals of sperm whales during 1862–1889 is estimated at 237.     

Temperature regulation of marine mammals

A mathematical model of heat loss from an aquatic animal to the surrounding water is presented. Heat is generated in metabolically active tissues and distributed by circulating blood and by conduction. The time dependent radial temperature profile of the animal is numerically solved from heat transfer equations by a computer. The model is applied to large whales, porpoises, and seals. For the whales, blood circulation to the dermal layer below appendage and body skin surfaces proved to be essential for sufficient heat dissipation. When decreasing the blood flow below a certain value (dependent on sea temperature and whale activity) the large whales would overheat. Blubber thickness was found to be of minor importance in whale thermoregulation, because the blubber coat can be bypassed by blood circulation. On the other hand, it is in general not possible for small porpoises and seals to stay warm in the coldest waters using normal mammalian resting metabolic rates, even if the peripheral circulation is shut off (or artery-vein heat exchangers used). Heat loss can be reduced if the outermost tissue layers are allowed to cool. This is achieved by minimizing convective radial heat flow via the circulation. (For large whales even minute radial blood flow raises the muscle temperatures to the core temperature level.) Seasonal acclimatization of harbour seals is explained by changes in their effective insulation thickness. Differences in whale activity induce changes in the temperature profile mainly within the first few centimeters from the skin surface. These superficial temperatures, if known, could be used to estimate whale metabolic rates. Since they drop close to the sea water temperature within minutes after whale death, the measurements should be done of live whales.     

KILLER WHALE PREDATION ON SPERM WHALES: OBSERVATIONS AND IMPLICATIONS

In October 1997 we observed a herd of approximately 35 killer whales ( Orcinus orca ) attack a pod of nine sperm whales ( Physeter macrocephalus ) 130 km off the coast of central California. During the four hours we watched, adult female killer whales, including some with calves, attacked in waves of four to five animals in what was apparently a "wound and withdraw" strategy. Adult male killer whales stood by until the very end when one charged in and quickly killed a seriously wounded sperm whale that had been separated from the group. The sperm whales appeared largely helpless: their main defensive behavior was the formation of a rosette ("marguerite"-heads together, tails out). When the killer whales were successful in pulling an individual out of the rosette, one or two sperm whales exposed themselves to increased attack by leaving the rosette, flanking the isolated individual, and leading it back into the formation. Despite these efforts, one sperm whale was killed and eaten and the rest were seriously, perhaps mortally, wounded. We also present details of two other encounters between sperm whales and killer whales that we observed. Although sperm whales, because of various behavioral and morphological adaptations, were previously thought to be immune to predation, our observations clearly establish their vulnerability to killer whales. We suggest that killer whale predation has potentially been an important, and underrated, selective factor in the evolution of sperm whale ecology, influencing perhaps the development of their complex social behavior and at-sea distribution patterns.     

Abundance and Distribution of Sperm Whales in the Canary Islands: Can Sperm Whales in the Archipelago Sustain the Current Level of Ship-Strike Mortalities?

Sperm whales are present in the Canary Islands year-round, suggesting that the archipelago is an important area for this species in the North Atlantic. However, the area experiences one of the highest reported rates of sperm whale ship-strike in the world. Here we investigate if the number of sperm whales found in the archipelago can sustain the current rate of ship-strike mortality. The results of this study may also have implications for offshore areas where concentrations of sperm whales may coincide with high densities of ship traffic, but where ship-strikes may be undocumented. The absolute abundance of sperm whales in an area of 52933 km², covering the territorial waters of the Canary Islands, was estimated from 2668 km of acoustic line-transect survey using Distance sampling analysis. Data on sperm whale diving and acoustic behaviour, obtained from bio-logging, were used to calculate g(0) = 0.92, this is less than one because of occasional extended periods when whales do not echolocate. This resulted in an absolute abundance estimate of 224 sperm whales (95% log-normal CI 120–418) within the survey area. The recruitment capability of this number of whales, some 2.5 whales per year, is likely to be exceeded by the current ship-strike mortality rate. Furthermore, we found areas of higher whale density within the archipelago, many coincident with those previously described, suggesting that these are important habitats for females and immature animals inhabiting the archipelago. Some of these areas are crossed by active shipping lanes increasing the risk of ship-strikes. Given the philopatry in female sperm whales, replacement of impacted whales might be limited. Therefore, the application of mitigation measures to reduce the ship-strike mortality rate seems essential for the conservation of sperm whales in the Canary Islands.     

Sedation at Sea of Entangled North Atlantic Right Whales (Eubalaena glacialis) to Enhance Disentanglement

Background

The objective of this study was to enhance removal of fishing gear from right whales (Eubalaena glacialis) at sea that evade disentanglement boat approaches. Titrated intra muscular injections to achieve sedation were undertaken on two free swimming right whales.

Methodology/Principal Findings

Following initial trials with beached whales, a sedation protocol was developed for right whales. Mass was estimated from sighting and necropsy data from comparable right whales. Midazolam (0.01 to 0.025 mg/kg) was first given alone or with meperidine (0.17 to 0.25 mg/kg) either once or four times over two hours to whale #1102 by cantilevered pole syringe. In the last attempt on whale #1102 there appeared to be a mild effect in 20–30 minutes, with duration of less than 2 hours that included exhalation before the blowhole fully cleared the water. Boat avoidance, used as a measure of sedation depth, was not reduced. A second severely entangled animal in 2009, whale #3311, received midazolam (0.03 mg/kg) followed by butorphanol (0.03 mg/kg) an hour later, delivered ballistically. Two months later it was then given midazolam (0.07 mg/kg) and butorphanol (0.07 mg/kg) simultaneously. The next day both drugs at 0.1 mg/kg were given as a mixture in two darts 10 minutes apart. The first attempt on whale #3311 showed increased swimming speed and boat avoidance was observed after a further 20 minutes. The second attempt on whale #3311 showed respiration increasing mildly in frequency and decreasing in strength. The third attempt on whale #3311 gave a statistically significant increase in respiratory frequency an hour after injection, with increased swimming speed and marked reduction of boat evasion that enabled decisive cuts to entangling gear.

Conclusions/Significance

We conclude that butorphanol and midazolam delivered ballistically in appropriate dosages and combinations may have merit in future refractory free swimming entangled right whale cases until other entanglement solutions are developed.     

A CETACEAN BIOPSY SYSTEM USING LIGHTWEIGHT PNEUMATIC DARTS, AND ITS EFFECT ON THE BEHAVIOR OF KILLER WHALES

Lightweight untethered pneumatic darts were used to biopsy killer whales, Orcinus orca , for genetic and toxicological analysis. Samples of epidermal, dermal, and hypodermal tissue weighing approximately 0.5 g were obtained by 65% of the 91 darts fired during the study. Sufficient DNA for multiple analyses was extracted from the biopsies, which were also used for fatty acid and toxic contaminant analyses. Reactions such as momentary shakes or accelerations were observed after 81% of the dart hits and 53% of the misses. Aversion to the research vessel was assessed by reapproaching target whales after the sampling attempts. In 6% of the hits and 8% of the misses aversion to the research boat increased immediately following the attempt. No similar increases in aversion were seen when killer whales were reapproached one day to one year after being hit. The darts were also tested successfully on humpback whales, Megaptera novaeangliae . In view of the simplicity of the system, its effectiveness in acquiring multipurpose samples, and the apparently short-term disturbance it caused, it is recommended for future cetacean biopsy studies.     

Global distribution of fin whales Balaenoptera physalus in the post‐whaling era (1980–2012)

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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).     

The simultaneous detection of mitochondrial DNA damage from sun-exposed skin of three whale species and its association with UV-induced microscopic lesions and apoptosis

Due to life history and physiological constraints, cetaceans (whales) are unable to avoid prolonged exposure to external environmental insults, such as solar ultraviolet radiation (UV). The majority of studies on the effects of UV on skin are restricted to humans and laboratory animals, but it is important to develop tools to understand the effects of UV damage on large mammals such as whales, as these animals are long-lived and widely distributed, and can reflect the effects of UV across a large geographical range. We and others have used mitochondrial DNA (mtDNA) as a reliable marker of UV-induced damage particularly in human skin. UV-induced mtDNA strand breaks or lesions accumulate throughout the lifespan of an individual, thus constituting an excellent biomarker for cumulative exposure. Based on our previous studies in human skin, we have developed for the first time in the literature a quantitative real-time PCR methodology to detect and quantify mtDNA lesions in skin from sun-blistered whales. Furthermore the methodology allows for simultaneous detection of mtDNA damage in different species. Therefore using 44 epidermal mtDNA samples collected from 15 blue whales, 10 fin whales, and 19 sperm whales from the Gulf of California, Mexico, we quantified damage across 4.3 kilobases, a large region of the ~ 16,400 base pair whale mitochondrial genome. The results show a range of mtDNA damage in the skin of the three different whale species. This previously unreported observation was correlated with apoptotic damage and microscopic lesions, both of which are markers of UV-induced damage. As is the case in human studies, this suggests the potential use of mtDNA as a biomarker for measuring the effect of cumulative UV exposure in whales and may provide a platform to help understand the effects of changing global environmental conditions.     

Molecular genetic identification of whale and dolphin products from commercial markets in Korea and Japan

We report the methods and results of molecular genetic identification of the species and, in some cases, geographical origins of whale and dolphin products purchased from retail markets and restaurants in Japan and South Korea. As reported previously (Baker & Palumbi 1994), we used the polymerase chain reaction (PCR) and a portable laboratory to amplify, purify and later sequence a portion of the mitochondrial DNA control region from 16 commercial products purchased in Japan. This ‘spot check’ revealed a surprising variety of species for sale, including minke, fin and humpback whales and one or two species of dolphins sold as ‘kujira’ or whale. In the Korean survey, DNA amplifications were conducted by two of us (C.S.B. and F.C.) working with independent equipment and reagents. The two sets of DNA amplifications were returned to our respective laboratories and sequenced independently for cross-validation. Among the total of 17 species-specific sequences we found a dolphin, a beaked whale, 13 Northern Hemisphere minke whales (representing at least seven distinct individuals) and two whales which are closely related to the recognized sei and Bryde's whales but could not be identified as either using available type sequences. We suggest that these two specimens represent a currently unrecognized species or subspecies of Bryde's whale, possibly the so-called ‘small-form’ reported from the tropical waters of the Indo-Pacific. We conclude that molecular systematic analyses of DNA sequences have tremendous utility for the identification of whale and dolphin products. However, there are certain constraints on the application of these techniques for monitoring whaling or trade in whale products. First, PCR and DNA sequencing can generate misleading artefacts. These can generally be recognized or eliminated through experimental controls. Second, phylogenetic reconstructions of DNA sequences can be misinterpreted if the database of type sequences is inadequate or the taxonomy of the group is incomplete. This constraint is, at present, a more serious obstacle to molecular monitoring of whaling. Our results highlight uncertainties about the taxonomic status of oceanic populations and morphological forms of two species (or species complexes) targeted by legal and illegal hunting, the minke and Bryde's whales. Despite these uncertainties, it is difficult to reconcile some of the species available in Japanese and Korean commercial markets with recent catch records made available to the International Whaling Commission. It is particularly disturbing that two specimens of an unrecognized species or subspecies of baleen whale were for sale in a restaurant in South Korea in October, 1994, 8 years after the acceptance of an international moratorium on commercial whaling.     

BEHAVIORAL RESPONSE OF FOUR SPECIES OF BALAENOPTERID WHALES TO BIOPSY SAMPLING

Behavioral responses to biopsy sampling of four species of northwestern Atlantic balaenopterid whales summering in the estuary and Gulf of St. Lawrence, Quebec, from 1990 to 1995 were studied to determine if this technique was an important disturbance to the whales. A total of 447 biopsy samples were taken using a small punch-type biopsy tip fired from a crossbow. Biopsies were successfully taken from 91.2% of the whales approached. Whales displayed no reaction to 45.2% of the successful biopsy attempts. Whales that responded to biopsy sampling typically resumed their normal behavior immediately or within a few minutes. Most humpback whales displayed a hard tail flick, and the majority of fin and blue whales submerged following biopsy sampling. Significantly different frequencies and intensities of responses were found between whale species. Minke and humpback whales were found to be more sensitive to biopsy sampling than fin and blue whales. Response frequencies were similar between females and males for all species, with the exception of fin whales where females had a higher response frequency than males. Biopsy sample length, i. e., penetration depth, did not explain variations in response intensity but may influence response frequency to biopsy sampling. Group size, geographical region, and number of biopsies taken per whale were not factors that explained variation in behavioral responses. The biopsy technique was found to be an efficient method for obtaining high-quality whale skin and blubber samples with limited behavioral disturbance to balaenopterid whales.     

Molecular studies on two variant repeat types of the common cetacean DNA satellite of the sperm whale, and the relationship between Physeteridae (sperm whales) and Ziphiidae (beaked whales)

In the sperm whale (Physeter macrocephalus) two different repeat types (A and B) of the common cetacean DNA satellite were identified. The evolution of each group of repeats appears to be independent from that of the other. The sequence similarity between the two groups is less than the similarity between group A and repeats of the satellite in related whale species. The systematic relationship within and between the families Physeteridae (sperm whales) and Ziphiidae (beaked whales) was addressed by both sequence analysis of the satellite and comparisons with the families Delphinidae and Phocoenidae. The mysticete blue whale (Balaenoptera musculus) was used as an outgroup in the comparisons. The molecular phylogeny, when maximum-parsimony analysis and the neighbor-joining method were used, grouped together species of each family. At the family level the ziphiids grouped closet to the families Phocoenidae and Delphinidae. The similarities between the common cetacean satellite of the blue whale and the sperm whale were greater than those between the blue whale and the other odontocetes included, suggesting that the evolution of the satellite is slower in the sperm whale than in the other odontocetes.      

The Mitochondrial Genome of the Sperm Whale and a New Molecular Reference for Estimating Eutherian Divergence Dates

Extant cetaceans are systematically divided into two suborders: Mysticeti (baleen whales) and Odontoceti (toothed whales). In this study, we have sequenced the complete mitochondrial (mt) genome of an odontocete, the sperm whale (Physeter macrocephalus), and included it in phylogenetic analyses together with the previously sequenced complete mtDNAs of two mysticetes (the fin and blue whales) and a number of other mammals, including five artiodactyls (the hippopotamus, cow, sheep, alpaca, and pig). The most strongly supported cetartiodactyl relationship was: outgroup,((pig, alpaca),((cow, sheep),(hippopotamus,(sperm whale,(baleen whales))))). As in previous analyses of complete mtDNAs, the sister-group relationship between the hippopotamus and the whales received strong support, making both Artiodactyla and Suiformes (pigs, peccaries, and hippopotamuses) paraphyletic. In addition, the analyses identified a sister-group relationship between Suina (the pig) and Tylopoda (the alpaca), although this relationship was not strongly supported. The paleontological records of both mysticetes and odontocetes extend into the Oligocene, suggesting that the mysticete and odontocete lineages diverged 32–34 million years before present (MYBP). Use of this divergence date and the complete mtDNAs of the sperm whale and the two baleen whales allowed the establishment of a new molecular reference, O/M-33, for dating other eutherian divergences. There was a general consistency between O/M-33 and the two previously established eutherian references, A/C-60 and E/R-50. Cetacean (whale) origin, i.e., the divergence between the hippopotamus and the cetaceans, was dated to ≈55 MYBP, while basal artiodactyl divergences were dated to ≥65 MYBP. Molecular estimates of Tertiary eutherian divergences were consistent with the fossil record. Received: 12 July 1999 / Accepted: 28 February 2000     

WHY DO BALEEN WHALES MIGRATE?1

The annual migrations of baleen whales are a conspicuous but unexplained feature of their behavioral repertoire. Some hypotheses offered to explain whale migration focus on direct benefits to the calf (thermoregulation, calm water) and some do not (resource tracking, and the “evolutionary holdover” hypothesis). Here, we suggest that a major selective advantage to migrating pregnant female baleen whales is a reduced risk of killer whale (Orcinus orca) predation on their newborn calves in low-latitude waters. Killer whale abundance in high latitudes is substantially greater than that in lower latitudes, and most killer whales do not appear to migrate with baleen whales. We suggest that the distribution of killer whales is determined more by their primary marine mammal prey, pinnipeds, and that following the baleen whale migrations would remove them from their pinniped prey. There are problems with all current hypotheses, most of which stem from a lack of directed research. We explore variation in migratory habits between species, populations, and individuals that may provide a “natural laboratory” for discriminating among the competing hypotheses.     

What influences the worldwide genetic structure of sperm whales (Physeter macrocephalus)?

The interplay of natural selection and genetic drift, influenced by geographic isolation, mating systems and population size, determines patterns of genetic diversity within species. The sperm whale provides an interesting example of a long‐lived species with few geographic barriers to dispersal. Worldwide mtDNA diversity is relatively low, but highly structured among geographic regions and social groups, attributed to female philopatry. However, it is unclear whether this female philopatry is due to geographic regions or social groups, or how this might vary on a worldwide scale. To answer these questions, we combined mtDNA information for 1091 previously published samples with 542 newly obtained DNA profiles (394‐bp mtDNA, sex, 13 microsatellites) including the previously unsampled Indian Ocean, and social group information for 541 individuals. We found low mtDNA diversity (π = 0.430%) reflecting an expansion event <80 000 years bp, but strong differentiation by ocean, among regions within some oceans, and among social groups. In comparison, microsatellite differentiation was low at all levels, presumably due to male‐mediated gene flow. A hierarchical amova showed that regions were important for explaining mtDNA variance in the Indian Ocean, but not Pacific, with social group sampling in the Atlantic too limited to include in analyses. Social groups were important in partitioning mtDNA and microsatellite variance within both oceans. Therefore, both geographic philopatry and social philopatry influence genetic structure in the sperm whale, but their relative importance differs by sex and ocean, reflecting breeding behaviour, geographic features and perhaps a more recent origin of sperm whales in the Pacific. By investigating the interplay of evolutionary forces operating at different temporal and geographic scales, we show that sperm whales are perhaps a unique example of a worldwide population expansion followed by rapid assortment due to female social organization.