Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Tracking the Development of Muscular Myoglobin Stores in Mysticete Calves

  • Rachel Cartwright ,

    rachel.cartwright@csuci.edu

    Affiliations California State University Channel Islands, Camarillo, California, United States of America, The Keiki Kohola Project, Lahaina, Hawaii, United States of America

  • Cori Newton,

    Affiliation California State University Channel Islands, Camarillo, California, United States of America

  • Kristi M. West,

    Affiliation Hawaii Pacific University Stranding Program, College of Natural and Computational Sciences, Hawaii Pacific University, Kaneohe, Hawaii, United States of America

  • Jim Rice,

    Affiliation Oregon Marine Mammal Stranding Network, Marine Mammal Institute, Oregon State University, Newport, Oregon, United States of America

  • Misty Niemeyer,

    Affiliation International Fund for Animal Welfare, Yarmouth Port, Massachusetts, United States of America

  • Kathryn Burek,

    Affiliation Alaska Veterinary Pathology Services, Eagle River, Alaska, United States of America

  • Andrew Wilson,

    Affiliation California State University Channel Islands, Camarillo, California, United States of America

  • Alison N. Wall,

    Affiliation California State University Channel Islands, Camarillo, California, United States of America

  • Jean Remonida-Bennett,

    Affiliation California State University Channel Islands, Camarillo, California, United States of America

  • Areli Tejeda,

    Affiliation California State University Channel Islands, Camarillo, California, United States of America

  • Sarah Messi,

    Affiliation California State University Channel Islands, Camarillo, California, United States of America

  • Lila Marcial-Hernandez

    Affiliation California State University Channel Islands, Camarillo, California, United States of America

Tracking the Development of Muscular Myoglobin Stores in Mysticete Calves

  • Rachel Cartwright, 
  • Cori Newton, 
  • Kristi M. West, 
  • Jim Rice, 
  • Misty Niemeyer, 
  • Kathryn Burek, 
  • Andrew Wilson, 
  • Alison N. Wall, 
  • Jean Remonida-Bennett, 
  • Areli Tejeda
PLOS
x

Abstract

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.

Introduction

In marine mammals, the ability to tolerate apnea for extended periods is a highly adaptive trait, primarily facilitating foraging within the marine realm[1]. Elevated levels of muscular myoglobin characterize this trait [2]: Myoglobin (Mb), a hemo-protein found within muscle cells, essentially allows for the storage of oxygen that can be used to maintain aerobic respiration and extend aerobic diving capacity. Typically, muscular Mb levels in marine mammals are between 10 and 20 times greater than their terrestrial counterparts [2,3]. Within marine mammals, increasing muscular Mb levels closely track the extension of aerobic dive limits [4] and increase as individuals mature [59]. Elevated muscular Mb levels generally comprise one element within a diverse suite of physiological, morphological or behavioral adaptations that facilitate the extension of the dive capacity in marine mammals. Notwithstanding, high Mb levels are a consistent hallmark of the ability to maintain extended aerobic dives [10] and consequently, increases in muscular Mb comprise a key component in the respiratory ontogeny of marine mammals.

To date, the majority of work documenting respiratory ontogeny and the development of muscular Mb in marine mammals has focused on pinnipeds, perhaps reflecting the relative ease of access to younger animals during land-based periods of early development. These studies document a high degree of temporal variability: Periods of rapid increase in muscular Mb levels may occur during the nursing period (e.g. Steller sea lions (Eumetopias jubatus); [8]) or during the post weaning fast (e.g. Northern elephant seals (Mirounga angustirostris); [11]). Alternatively, Mb increases may be delayed until the onset of foraging (e.g. harbor seals (Phoca vitulina); [12]) or until later periods of post-weaning activity (e.g. subantarctic fur seals (Arctocephalus tropicalis); [13]). Notably, there are also some exceptions to the typical trajectory of continually increasing Mb levels; for example, in Weddell seals (Leptonychotes weddellii), Mb levels peak during the juvenile stage, then subsequently drop to slightly lower levels in mature adults [9].

Within cetacea, documentation of the physiological aspects of respiratory ontogeny is currently limited to a selection of small odontocetes, specifically Fraser’s dolphin (Lagenodelphis hosei) [5], spinner dolphin (Stenella longirostris) [5], bottlenose dolphin (Tursiops truncatus) [7,14] and harbor porpoise (Phocoena phocoena) [15]. In these examples, while increasing respiratory capacity and concurrent increases in muscular Mb track increasing age, interspecific variability is equally as pronounced as seen in pinnipeds. For example in harbor porpoise, neonate (estimated age < 2 weeks) muscular Mb levels comprise 50% of adult levels and calves attain adult Mb levels by age 9 to 10 months. By comparison, in bottlenose dolphin, neonate Mb levels comprise around 10% of adult levels and do not reach adult levels until 1.5 years of age [15]. For mysticetes, reports on the physiological ontogeny of the respiratory capacity are sparse. Details of muscular Mb levels for immature age classes of mysticetes are limited to 2 examples, both of which provide details of muscular Mb levels for immature gray whales (Eschrichtius robustus) [3,6]. Given the high degree of variation reported for both pinnipeds and odontocetes, differences in development trajectories between different mysticete species likely exist, but remain undocumented to date.

At the molecular level, the underlying biochemical mechanisms that control production of myoglobin within muscle tissue have been studied extensively. Numerous studies have detected interplay between exercise, hypoxia and increased myoglobin production (e.g. [1623]). Recent tissue-based investigations have provided a detailed description of the biochemical pathway involved. Essentially, during muscular activity calcium is released from within the sarcoplasmic reticulum into the cytosol of the muscle cell. This activates the enzyme calcineurin, causing dephosphorylation of NFAT (nuclear factor of activated T-cells), which in turn translocates into the nucleus of the cell where it promotes expression of myoglobin [24,25]. Hypoxia alone actually impedes production of Mb in muscle cells [24], however in combination with exercise, these two stimuli are associated with increased rates of production of Mb [24,26]. The availability of lipids, acquired during nursing [27] or directly from the diet [28], may also augment the expression of Mb. Comparatively, hypoxia combined with lipid supplementation leads to modest increases in Mb [26]. Exercise then acts as a secondary stimulus, leading to the elevated levels of muscular Mb that characterize marine mammals [26,29].

Live animal studies provide support for these conclusions, although notable interspecific variability within this general framework persists. For example Geiseler et al. [30], comparing rates of increase in muscular Mb levels in active vs. sedentary hooded seals, (Cystophora cristata) during early ontogeny, demonstrated that active, freely diving young seals exhibited faster rates of increase of muscular Mb compared to pups initially raised in a sedentary setting. The active pups also exhibited ultimately higher adult Mb levels. Similarly, Ponganis et al. [31] documented differences in levels of muscular Mb in captive vs. wild emperor penguins (Aptenodytes forster). In this study, animals raised in captivity, where free-diving was restrained and exposure to hypoxia was limited, exhibited lower adult levels of muscular Mb in comparison to their free-diving, unrestrained wild counterparts. In contrast, Noren et al. [32] recorded moderate increases in muscular Mb levels in young gray seals during the post weaning fast, when seal pup activity is low and pups are not exposed to hypoxia. However pups do have access to a ready supply of lipids during this time, sourced from their lipid-rich blubber, which is laid down during the nursing period. Finally, Noren et al. [15] documented a rapid rise in Mb in very young harbor porpoise, wherein neonate calves achieved 50% of adult levels of Mb within two weeks of parturition. During this period young harbor porpoise would be exposed to hypoxia and they would receive lipid supplementation of the diet through nursing. Additionally, they would be exposed to consistent and sustained exercise, as neonates swim continually alongside their fast-moving mothers [33].

A comparative review of the dynamics of the development of muscular myoglobin stores in mysticetes has the potential to contribute to these investigations and extend our understanding of the ontogeny of the respiratory capacity across the range of marine mammals. In this study, we focus on three species of mysticetes; specifically minke (Balaenoptera acutorostrata), humpback (Megaptera novaeangliae) and gray (Eschrichtius robustus) whales. All three are migratory capital breeders, making extensive forays between high latitude feeding regions and low latitude breeding regions [3436]. For all three species, the ontogeny of the respiratory capacity is a vital component of their life history, influencing predation avoidance [37], costs of travel [38] and foraging success of the mother and offspring pair [3942]. Here, we provide updated and refined estimates of adult Mb levels for the three focal species and describe the trajectories for the development of muscular Mb in these species. We compare levels of Mb within and between muscles, as these details may provide insight into the potential role of exercise in the development of muscular Mb in these species. Finally, we review changes in muscular Mb in the youngest mysticetes, and compare these changes to the differing life histories and early behavior in these three species of mysticetes.

Method

Ethics statement

Animal tissues were provided by member organizations within the National Marine Mammal Stranding Network. The work of these groups is overseen and approved by the Office of Protected Resources, National Oceanic and Atmospheric Administration; marine mammal parts are collected under the authority of a NMFS Stranding Agreement issued to each of the co-operating organizations. The laboratory analysis of the tissues was conducted under a Marine Mammal Parts Handling Authorization, provided by regional offices of National Marine Fisheries Service. After reviewing the planned study, each regional office provided a letter of authorization that allowed the shipment of tissues to the lab. As this prior review had been conducted by experts in this field further ethical review by the co-operating institution, California State University Channel Islands, was not required.

Tissue samples

The Hawaii Pacific University Stranding Program (HPUSP), the Oregon Marine Mammal Stranding Network (OMMSN) the International Fund for Animal Welfare (IFAW) and the Alaska Stranding Network (ASN) provided tissue samples from stranded, deceased animals for this study. Muscle samples were taken from the longissimus dorsi, from directly below the dorsal fin (see Fig 1; site b—adapted from [43]). Although details were not always available, the depth of the sample and the precise location of the sampling site were noted whenever possible. For some immature humpback whales (n = 5), samples included the entire longitudinal muscle core at site b, allowing identification of inner and outer portions of the muscle. Additionally, for 3 minke whales and 1 humpback whale calf, samples from the same animal were obtained for a range of positions along the epaxial and hypaxial portions of the major swimming muscles (see Fig 1; sites a—d). All animals were reported as Smithsonian code state 2 or better at the time of the necropsy. Details of dates for each stranding, including causes of death, are provided in S1 Table.

thumbnail
Fig 1. Sampling sites used for the provision of muscle tissue samples from mysticetes.

(based on Polasek and Davis [43]). Picture credit: Yvette Hansen.

http://dx.doi.org/10.1371/journal.pone.0145893.g001

Samples were frozen on collection and stored at -80°C until analysis. Tissue samples were obtained from 18 different whales. Age classes were estimated based on 1) body length details in comparison to published growth curves and / or age-length records (for minke whales; [44], for humpback whales;[45] and for gray whales;[46]), and 2) through the interpretation of field records such as time and location of stranding (See S1 Table).

Myoglobin Concentration Analysis

Myoglobin levels were assessed following established protocols [3,47]. One gram samples were minced, homogenized in 19.25 ml of 0.04M phosphate buffer solution (pH6.6), sonicated for 30 seconds (using a Power Gen 125 sonicator; Fisher Scientific, Pittsburgh, PA, USA) and centrifuged at 13,000g for 1 hour 15 minutes. The supernatant was collected and bubbled with carbon monoxide for 8 minutes, with 0.02g of sodium thiosulphate added at the mid-point. Absorbance was measured at two key wavelengths, 536 nm and 568nm on a light spectrophotometer (Genesys 10V light spectrophotometer; Thermo Fisher Scientific, Waltham, MA, USA). Reynafarje’s equation was applied to estimate Mb concentration based on the differences in absorbance at the two key wavelengths. All assays were run in triplicate, samples of harbor porpoise muscle tissue were included in each run to verify consistency and estimate the precision of the assay protocol and lyophilized horse myoglobin (Sigma, MD) was used as an additional control to confirm the reliability of the protocol.

Data Analysis

Mean estimates of myoglobin levels were compiled for each specific age and / or species of whale, for tissues from different muscle sites and from different portions of the muscle. Where tissues were obtained from multiple muscle sites, values obtained for site b (see Fig 1) were used in comparative analyses. As site b is the standard sampling site typically used in field necropsies, this could potentially reduce the impact of differing muscle sites where the sample site was unknown. Where samples were obtained from different depths of the muscle at site b, mean values for all samples from different depths at site b were used. Values for myoglobin levels are reported throughout in milligrams per gram of thawed tissue (mg Mb g-1) ± 1 standard error of the mean (S.E.) or ± 1 standard deviation (S.D.) as indicated.

Standard parametric and non-parametric tests were applied using SPSS software to detect significant differences between species, age groups and between different muscle sites, with the level of significance set at 0.05. General linear models (GLM) models were used to detect interactions between factors, and log transformations were used where data was non-normal or variances were unequal. To compensate as much as possible for the small sample sizes in this study, available data from the literature was incorporated into the data analysis wherever possible, and inferential analysis as well as hypothesis based testing was incorporated into data analysis and interpretation.

Results

1. Developmental changes in muscular myoglobin levels

Looking collectively at the indications from this study, muscular Mb levels increased with age in each of the three species of mysticetes included here (Table 1, Fig 2). Using data from this study alone (n = 18 whales), in minke whales, calf Mb levels represented 17.0% of the mean adult levels, in humpback whales, mean calf levels represented 31.3% of adult levels and in gray whales, mean calf levels represented 21.2% of adult levels. When data available in the literature are included (n = 3; see Table 1), values for minke whales remain unchanged, humpback whale calf levels represented 22.8% of adult levels and gray whale calf levels represented 17.3% of adult levels. Using all 21 samples (i.e. those obtained from this study and from the literature), a GLM was constructed, incorporating muscular Mb as the dependent, and age class and species as fixed factors. All calf data for each species were compiled into a single age cohort (calves). Mb levels were log transformed to adjust for unequal variances in the raw data. The model indicated that differences according to age and species were significant (GLM; for age class ANOVA F2 = 25.532, p = 0.000, for species ANOVA F2 = 5.671, p = 0.018). Post-hoc tests indicated that significant differences according to age were between calf vs. adult age groups and calf vs. juveniles, while differences between juveniles and adults were marginally non-significant (Tukey test; p < 0.001, p = 0.005 and p = 0.068 respectively). Significant differences between species lay principally between minke vs. humpback whales and minke vs. gray whales (Tukey; p = 0.001 in both cases), while differences between humpback and gray whales were not significant (Tukey; p = 0.930). The GLM indicated that the interaction between age and species was not significant (ANOVA F4 = 0.446, p = 0.774). Based on values obtained for the control tissue used (harbor porpoise samples taken from site b), estimated precision was 97.4%.

thumbnail
Table 1. Muscular Mb levels according to age class for three species of mysticetes.

http://dx.doi.org/10.1371/journal.pone.0145893.t001

thumbnail
Fig 2. The development trajectory of muscular Mb levels in three species of mysticetes.

Differences between age classes and between species were significant (GLM; for age class ANOVA F2 = 25.532, p = 0.000, for species ANOVA F 2 = 5.671, p = 0.018). Error bars indicate +/- 1 S.E.

http://dx.doi.org/10.1371/journal.pone.0145893.g002

2. Differences between muscle sites

Comparing Mb levels pairwise between epaxial and hypaxial muscles for samples taken from the same animal, levels were consistently higher in epaxial muscles (Friedman’s test; X2 = 7.800, d.f. = 3, p = 0.05; see Table 2). Differences were more pronounced in the younger animals; for the humpback calf and the juvenile minke whale, hypaxial muscle Mb levels represented 59% and 46% of the mean epaxial muscle Mb levels respectively, while in the two adult minke whales, hypaxial muscle Mb levels represented 92% of the mean for epaxial muscle Mb levels.

thumbnail
Table 2. Differences in myoglobin levels between epaxial and hypaxial muscles in mysticete whales at different age stages.

http://dx.doi.org/10.1371/journal.pone.0145893.t002

3. Differences according to depth

Comparing muscle samples taken from different depths within the same muscle (epaxial; site b), muscular Mb levels were consistently higher in the inner portions of the muscle (paired t test; t = 4.020, d.f. = 4, p = 0.016; Table 3). Percentage increases between outer and inner portions were highly variable, ranging from 13% to a more than 100% increase. Differences were more pronounced as levels of Mb increased. It should be noted that all five samples included in the statistical analysis came from humpback whale calves.

thumbnail
Table 3. Differences in myoglobin levels between inner and outer portions of the major swimming muscles.

http://dx.doi.org/10.1371/journal.pone.0145893.t003

Anecdotally, the humpback whale adult tissue sample provided for this study (AK2014085) came from a region within the vicinity of the dorsal fin (Site b—Fig 1) however in this sample, the muscle tissue occurred in small bundles and had a bubbled and loose appearance. This is characteristic of the fascia boundary to the blubber (J. Rice; personal observation), suggesting that the tissue was peripheral muscle tissue. Comparing this value to the current published estimate of adult humpback whale Mb levels [48], this value was considerably lower than the current estimate (See Table 3). The single muscle sample used for the previously published estimate did not come from a known sampling site; however, the authors could confirm that the appearance was typical of interior portions of the muscle (S. Helbo; personal communication).

4. Early ontogeny of myoglobin levels in mysticete calves

In total, using data from this study along with data available in the literature (See S2 Table; n = 2 gray whale calves [3,6,49]), details of Mb levels in 12 mysticete calves were compiled. Ages ranged from neonates (estimated age < 2 weeks) to migrating calves (estimated age 3–5 months). To examine rates of change of Mb levels within this subset, a general linear model (GLM) was constructed, using muscular Mb as the dependent; relative calf age (classified as neonate, young or migrating calf) and species were included as fixed factors. Results indicated that Mb levels varied significantly between relative calf age cohorts and between species (for relative calf age; ANOVA F2 = 21.919, p = 0.002, for species; F2 = 10.281, p = 0.006). Using all three species, the interaction between species and age was not significant (ANOVA F4 = 3.938, p = 0.094). Unfortunately, only a single minke whale calf was included in the study, so to further investigate differences in rate of accumulation of Mb, the analysis was limited to a comparison of humpback and gray whale calves. Assigning approximate ages to the relative calf age classes (for neonates age = 0 .5 months, for young calves, approximate age = 1.5 months and for migrating calves, approximate age = 4 months) and comparing rates of change of muscular Mb levels in humpback and gray whale calves, the rate of increase in Mb levels was faster in humpback vs. gray whale calves (Fig 3; for humpback whales β = 0.98 and for gray whales β = 0.41). A GLM including mean Mb as the dependent, relative age in months as a covariate and species as a fixed factor suggested a significant interaction between species and relative calf age (ANOVA F1 = 14.903, p = 0.002) for humpback vs. gray whale calves over this period.

thumbnail
Fig 3. Early ontogeny of Mb levels in three species of mysticetes.

Calf age classes based on growth curves described by a [44], b [45] and c [46], along with additional information from the field site of the necropsy. Estimated ages: Neonates < 2 weeks, young calves between 2 weeks to 3 months and stranded in breeding areas, migrating calves between 3 to 5 months and stranded in migratory corridors. Levels of Mb in the neonate minke whale lay beyond the upper limits of the 95% confidence interval for mean Mb levels in neonate humpback and gray whale calves (4.2 mg Mb g-1 vs. 0.0<1.5<3.0 mg Mb g-1). Levels of Mb rose faster in humpback vs. gray whale calves between the neonate, young and migratory age classes (for humpback whales β = 0.98 and for gray whales β = 0.41). Error bars indicate S.D.

http://dx.doi.org/10.1371/journal.pone.0145893.g003

Examining differences in calf Mb levels between relative age classes using confidence intervals provides similar inferences. Comparing Mb levels in neonates, while differences between the humpback and the gray whale neonates were marginal (see Table 1), the single value for the neonate minke whale calf Mb level (Mb = 4.2 mg Mb g1) fell above the upper limits of the 95% C.I. for the mean muscular Mb for humpback and gray neonates combined (95% CI for the mean muscular Mb for neonate humpback and gray whale neonate calves; 0.0<1.5<3.0 mg Mb g-1), inferring a significant difference in Mb levels between minke vs. humpback and gray whale neonates. Looking at changes across this time period, in humpback whale calves, the mean Mb level for the migrating humpback whale calf fell above the 95% CI for the mean Mb for breeding ground (neonate and young) humpback calves (4.6 mg Mb g1 vs. 1.8<2.3<3.6 mg Mb g1), inferring that there was significant increase in Mb levels between the breeding grounds and early migration in humpback whale calves. By comparison, in the gray whale calves, the estimate for Mb levels in the migrating calf fell within the 95% CI for the mean Mb for breeding ground gray whale calves (3.2 mg Mb g1 vs. 0.5<1.9<3.2 mg Mb g1).

Relevant life history details for the three focal species over this period were compiled to review the degree of variability in early activity levels and other key developmental features, such as growth rates and length of lactation periods (see Table 4). Looking comparatively, minke whale calves are likely the most mobile of the three species in terms of swimming speeds [37], while humpback calves engage in frequent surface activity, such as extended sequences of breaching [50] and gray whale calves are typically quiescent during early development [51, 52]. Within the three focal species, relative growth rates are highest in gray whale calves [44], and lactation periods vary from 4–6 months in minke whale calves [44] to 10–12 months in humpback whale calves [34].

thumbnail
Table 4. Early life history traits for three species of mysticete calves.

http://dx.doi.org/10.1371/journal.pone.0145893.t004

Discussion

This study provides the first outlines of the developmental trajectories of muscular Mb stores for three species of mysticetes. As muscular Mb levels potentially dictate the aerobic dive limit in mysticetes, these trajectories may influence a wide range of related aspects of developmental and life history traits.

In the three species of mysticetes included in this study, the development of muscular Mb stores was protracted. Calves exhibited notably low levels of Mb and weaned juveniles carried only a portion of their eventual adult complement. Levels of Mb varied between and within muscles, with differences most pronounced in younger animals. Comparing the three species of mysticete calves included in this study, early levels and rates of accumulation of muscular Mb varied considerably between species. Comparing these differences to differences in life histories suggests that high levels of activity during early development may enhance the development of Mb stores during this period.

One of the main challenges inherent in this study is the small sample size, reflecting the challenges of obtaining usable tissue samples from stranded mysticetes. Notwithstanding, the sample includes three different species and three different age cohorts. Currently, available data on Mb levels in mysticetes are extremely limited and as a consequence, very early assessments of Mb levels from single animals (e.g. [53,54]) or theoretical, calculated estimates of myoglobin levels based on respiratory patterns (e.g. [55]) have been used in a wide range of comparative studies on the evolution, energetics and physiology of diving in mysticetes (e.g. [49,5658]). This study therefore adds new and useful information to the limited dataset documenting mysticete Mb levels.

Levels of Mb in adult mysticetes

Comparing the results of this study to previous reports, the minke whale estimate provided here is substantially higher than previously reported (24.2 (22.1–26.4) mg Mb g-1 vs. 7 mg Mb g-1 [48]. In the previous study, researchers reported the presence of an unexplained precipitate during the assay and suggested that this may have influenced the results. In this study, consistent results were obtained from a range of different muscles and from two different adult minke whales. Consequently, the results from this study would seem to be the more robust estimate.

The humpback whale estimate obtained in this study is considerably lower than the previously published estimate (9.4 mg Mb g-1 vs. 15.9 mg Mb g-1 [48]). Here, a feasible explanation may be based on field necropsy notes and the appearance of the tissue. The tissue sample used in this study had a bubbled, loose appearance, characteristic of peripheral tissue close to the fascia boundary to the blubber. The tissue sample used in the original study did not exhibit these characteristics (S Helbo; pers. comm.) Presuming that this tissue was taken from the interior regions of the muscle, this suggests that a pronounced gradient in Mb may exist within the muscle. The relatively high variability in gray whale adult Mb levels (9.2–16.3 mg Mb g-1) recorded in this study also infers that there may be a high degree of intra-muscular variability in Mb levels in mature mysticetes. To adequately assess intra-muscular variability in Mb levels, specific samples would need to be collected from a full range of known muscle sites during necropsies. In large mysticetes, this is certainly a challenge. However, as the results from such a study would provide valuable details for use in accurate, updated assessments of mysticete aerobic dive capacity, the investment of time and effort involved would certainly be well-justified.

Taken comparatively, differences in adult Mb between the three species included in this study fit well within the confines of previously reported trends in Mb levels across diving mammals with respect to increasing body mass. Essentially, as large body size confers increased metabolic efficiency this offsets the aerobic demands of extended dives [2, 49, 58]. Minke whales, the smallest species of the mysticetes, are considerably smaller than both humpback and gray whales (see table 4). Consequently, in order to sustain equivalent diving capabilities, minke whales would require higher Mb levels than seen in other, larger mysticetes, while the lower Mb levels reported here for humpback and gray whales may reflect the offset in the demands of aerobic dive capacity attributed to their larger body size.

The developmental trajectory of Mb

Within the limited sample in the study, Mb levels increased with age and there were no indications of non-linear trends between specific age cohorts, as seen in other species of marine mammals such as Weddell seals [9]. Mysticete calf Mb levels ranged from 17–22.8% of adult levels and juveniles levels ranged between 28–60% of adult levels. As aerobic dive capacity tracks Mb levels [4], these low Mb levels likely contribute to the reduced dive times reported in younger mysticetes [46,50].

Ecologically, the consequences of reduced dive capacity are far-reaching: Predation risks increase [37,59] and increases in surfacing frequency and persistence raise the energetic costs of travel [6062]. Additionally, foraging success for maternal females with dependent calves is impacted, due to the competing needs of offspring vigilance vs. diving [42]. These constraints persist through the juvenile phase, when constrained aerobic dive capacity would similarly increase travel costs and reduce foraging success [63].

Interestingly, the behavioral ecology of mysticetes includes a range of compensatory adaptive traits that offset these challenges. Predation risks are postponed as these three species of mysticetes are seasonal migrants and relocate to regions beyond the range of their key predator, the transient Orca, during the earliest periods of calf development. During travel, calves draft alongside maternal females [50,64] which provides hydrodynamic lift and reduces the energetic costs of travel [65]. Additionally, as capital breeders, maternal females use stored energy reserves during early calf development and maternal foraging is delayed until the latter periods of calf development in humpback and gray whales or until after weaning in minke whales. Furthermore, in juveniles, high levels of juvenile site fidelity to maternal feeding grounds [66,67] mean that newly weaned and juvenile whales benefit from maternal experience in prey choice and selection of foraging habitat during periods of limited dive capacity. Even key aspects of the juvenile mysticete morphology, specifically the shortened head and modified, smaller feeding apparatus, reduce the energetic costs of foraging during this life stage [63].

Taken cumulatively, these examples suggest that the limitations of the protracted ontogeny of the respiratory capacity through the calf and juvenile stages are integrated into the larger life history strategies and adaptive traits that characterize mysticetes. Notwithstanding though, in many ways this further underscores the costs and challenges associated with limited aerobic capacity during early development. Understanding the physiological mechanisms that may drive development of Mb in mysticetes provides the opportunity for further insight into this key aspect of ontogeny in young mysticetes.

At the molecular level, three factors are currently recognized as drivers in the development of Mb stores during early ontogeny, namely hypoxia, lipid supplementation and exercise, [2426,29]. As all mysticetes are born into water and maintain an entirely aquatic lifestyle, exposure to hypoxia immediately following parturition is ubiquitous among mysticetes. Based on analysis of cetacean milk samples, while differences in milk composition are seen towards the later stages of lactation, proportions of lipid in the early diet are also consistent in the three species included here [27]. However, levels of exercise vary interspecifically between the three focal species and cumulatively, a range of results from this study suggest that exercise assumes a driving role in the development of muscular Mb stores in young mysticetes.

Firstly, comparing Mb levels in different muscles of the same animal, in young animals, levels of Mb were greater in the epaxial vs. hypaxial portions of the muscle. Both of these muscles play a role in providing propulsion during swimming; the epaxial muscles provide the upstroke of the fluke and the hypaxial muscles are responsible for the downstroke [68]. Although equivalent levels of propulsion are provided during the up- and down stroke [69], drag is greater during the upstroke vs. the downstroke [70], so levels of exertion are likely higher during the upstroke [71]. Higher levels of Mb in the epaxial muscles therefore correspond to higher levels of exertion during swimming. In marine mammals, the costs of swimming scales inversely with body size [38]. Despite the benefits of drafting alongside the mother during swimming [65], high fluke beat rates compared to conspecific adults are a distinct characteristic of young cetaceans [65,72] and would potentially amplify differences in exertion between the epaxial and hypaxial muscles. Looking at these results collectively, as differences were less pronounced in the adults included in this study, a tentative inference could be that heterogeneity in Mb levels between muscle sites in mysticetes is an ontogenetic phenomenon reflecting the exertion of swimming during early development. As the costs of swimming diminish with increasing body size [38], the difference may then become less pronounced as whales mature.

A second connection between levels of exercise and levels of Mb emerges when comparing samples from different depths within the same muscle site. In this study, confirmed site-specific samples were only available from humpback whale calves (n = 5). Within this group, Mb levels were significantly higher in the deepest regions of the epaxial muscle compared to outer regions of the same muscle. The inner portions of the muscle lying closest to the skeleton are under the greatest stress and exertion during exercise [43], so muscle activity would be elevated relative to adjacent areas. Higher Mb levels in this area therefore correspond to the levels of exertion and exercise in the inner vs. the outer portions of the same muscle.

Finally, reviewing interspecific differences in temporal changes in Mb levels during early development, differing rates of development of Mb stores correspond to interspecific differences in levels of activity and exercise between the three mysticete species included in this study. In minke whales, although reports of female-calf behavior are exceptionally sparse, adult minke whales are remarkably fast swimmers [37]. Assuming that maternal females maintain relatively fast swimming speeds and calves maintain the close proximity and consistent activity that is typical across cetacea [33], young minke whales would be exposed to high levels of exercise from birth, which could contribute to elevated Mb levels. Notwithstanding, given the elevated Mb levels in the minke neonate in comparison to the other two species, additional mechanisms that may somehow prime the muscles prior to parturition may be at play, as suggested by other researchers [15]. Both the diminutive body size of neonate minke whales in comparison to other mysticete calves, and the comparatively short lactation period relative to other mysticetes, correspond to life-history trends seen in species of odontocetes that exhibit very early maturation of muscular Mb stores.

Comparing humpback and gray whale calves, in both cases, Mb levels in the youngest calves were extremely low in comparison to the minke calf. As calves matured, Mb levels increased, however rates of increase were significantly faster for humpback whale calves in comparison to gray whale calves (see Fig 3). By the beginning of the natal migration, the migrating humpback whale calf had significantly higher levels of muscular Mb than seen in breeding regions while gains in gray whale calves over this period were marginal.

Levels of exercise in young humpback and gray whales differ markedly during this period. During their time in breeding regions, gray whale calves are described as quiescent and aerial behaviors such as breaching, though seen, are very rare [51,52]. In contrast, humpback whale calves are highly active, especially during the earliest periods of development [50,73]. Extended sequences of breaching are common and frequently include 30 or more consecutive breaches [50,73]. While single breaches may not be energetically costly, sequences of breaching behavior carry high energetic costs [74] and the social contexts that may justify the costs of this behavior in adults are not relevant to young calves [74,75]. Previous researchers focusing on early development in mysticete calves have speculated on a possible connection between exercise during early development and the ontogeny of the respiratory capacity [52,76]. With the mechanism that links exercise to the production of Mb via the calcium-calcineurin pathway now established, this potentially justifies the diversion of finite energy reserves to the support high levels of activity that optimize the rate of development of the respiratory capacity in young humpback whale calves. Notably, growth rates in gray whale calves exceed growth rates in humpback calves during this period, despite the overall smaller size of gray whales [44]. Implicitly, this suggests a trade-off between optimal growth and exercise during this crucial developmental period. Potentially this trade-off plays out differently in these two mysticetes, perhaps reflecting the differing threats and pressures of the impending natal migration.

In summary, this study provides new updates on muscular Mb levels in a range of mysticete adults and provides the first outlines of key factors that may drive the ontogeny of the respiratory capacity of young mysticetes. Still, this study leaves many questions unanswered. For example, the ability of mysticetes to utilize alternate respiratory pathways and support anaerobic respiration during extended dives has yet to be established. Additionally, proportions and changes in types of muscle tissue (i.e. type 1 vs type 2, slow vs fast twitch tissue) are also un-documented at this point. Based on recent simulations of mysticete dive capacities [63] these factors may prove to be very pertinent in discussions of breath-hold capacity and the associated foraging tactics used by both young and mature mysticetes. Further studies in this field will clearly be required for a full understanding of mysticete respiratory capacity. For now, as marine resources enter a period of unprecedented change [77,78], these results bring focus to the physiological constraints on diving and foraging in young and maturing mysticetes, allowing informed insight into their resilience to the impending challenges facing marine fauna.

Supporting Information

S1 Table. Stranding details for sources of tissue samples.

doi:10.1371/journal.pone.0145893.s001

(DOCX)

S2 Table. Mysticete muscular Mb levels sourced from the literature.

doi:10.1371/journal.pone.0145893.s002

(DOCX)

Acknowledgments

We are very grateful for support provided by the Biology Department, the Provost’s Faculty Resource Fund and Project Acceso, California State University Channel Islands. Additional thanks to Michael Mahoney and Catherine Hutchinson for their assistance during laboratory work, to Blake Gillespie, for assistance at the out-set of this study and to Phil Clapham, for support in the final stages of this study. We would also like to thank the many volunteers of the Hawaii Pacific University Stranding Program, the Oregon Marine Mammal Stranding Network, the International Fund for Animal Welfare and the Alaska Stranding Network for the provision of tissue samples used in the study. Additionally, thanks go to Michelle Berman, Santa Barbara Natural History Museum, for the provision of additional tissue samples for use as controls in the study.

Author Contributions

Conceived and designed the experiments: RC. Performed the experiments: AW ANW JRB AT SM LMH. Analyzed the data: RC. Contributed reagents/materials/analysis tools: KMW JR MN KB. Wrote the paper: RC CN. Supervised laboratory work: RC CN.

References

  1. 1. Kooyman GL. Diverse Divers: Physiology and Behaviour.Berlin: Springer-Verlag. 1989.
  2. 2. Kooyman GL, Ponganis PJ. The physiological basis of diving to depth: birds and mammals. Annu Rev Physiol. 1998;60: 19–32. doi: 10.1146/annurev.physiol.60.1.19. pmid:9558452
  3. 3. Castellini MA, Somero GN. Buffering capacity of vertebrate muscle: Correlations with potentials for anaerobic function. J Comp Physiol Part B. 1981;143: 191–198
  4. 4. Snyder GK. Respiratory adaptations in diving mammals. Respir Physiol. 1983;54: 269–294. doi: 10.1016/0034-5687(83)90072-5. pmid:6369460
  5. 5. Dolar ML, Suarez P, Ponganis PJ, Kooyman GL. Myoglobin in pelagic small cetaceans. J Exp Biol. 1999;202: 227–36. pmid:9882635
  6. 6. Noren SR, Williams TM, Pabst DA, McLellan WA, Dearolf JL. The development of diving in marine endotherms: preparing the skeletal muscles of dolphins, penguins, and seals for activity during submergence. J Comp Physiol B Biochem Syst Environ Physiol. 2001;171: 127–134. doi: 10.1007/s003600000161.
  7. 7. Noren SR, Lacave G, Wells RS, Williams TM. The development of blood oxygen stores in bottlenose dolphins (Tursiops truncatus): implications for diving capacity. J Zool. 2002;258: 105–113. doi: 10.1017/S0952836902001243.
  8. 8. Richmond JP, Burns JM, Rea LD. Ontogeny of total body oxygen stores and aerobic dive potential in Steller sea lions (Eumetopias jubatus). J Comp Physiol B. 2006;176: 535–45. doi: 10.1007/s00360-006-0076-9. pmid:16514541
  9. 9. Kanatous SB, Hawke TJ, Trumble SJ, Pearson LE, Watson RR, Garry DJ, et al. The ontogeny of aerobic and diving capacity in the skeletal muscles of Weddell seals. J Exp Biol. 2008;211: 2559–65. doi: 10.1242/jeb.018119. pmid:18689409
  10. 10. Ponganis PJ. Diving mammals. Compr Physiol. 2011;1: 447–65. doi: 10.1002/cphy.c091003. pmid:23737181
  11. 11. Thorson P., Le Boeuf BJ. Elephant Seals: Population Ecology, Behavior, and Physiology. University of California Press; 1994.
  12. 12. Prewitt JS, Freistroffer D V., Schreer JF, Hammill MO, Burns JM. Postnatal development of muscle biochemistry in nursing harbor seal (Phoca vitulina) pups: limitations to diving behavior? J Comp Physiol B. 2010;180: 757–766. doi: 10.1007/s00360-010-0448-z. pmid:20140678
  13. 13. Verrier D, Guinet C, Authier M, Tremblay Y, Shaffer S, Costa DP, et al. The ontogeny of diving abilities in subantarctic fur seal pups: developmental trade-off in response to extreme fasting? Funct Ecol. 2011;25: 818–828. doi: 10.1111/j.1365-2435.2011.01846.x.
  14. 14. Etnier SA, Dearolf JL, McLellan WA, Pabst DA. Postural role of lateral axial muscles in developing bottlenose dolphins (Tursiops truncatus). Proc Biol Sci. 2004;271: 909–18. doi: 10.1098/rspb.2004.2683. pmid:15255045
  15. 15. Noren SR, Noren DP, Gaydos JK. Living in the fast lane : rapid development of the locomotor muscle in immature harbor porpoises (Phocoena phocoena). J Comp Physiol B. 2014;184: 1065–1076. doi: 10.1007/s00360-014-0854-8. pmid:25150059
  16. 16. Millikan GA. Muscle hemoglobin. Physiol Rev. 1939;19: 503–523.
  17. 17. Reynafarje B. Myoglobin content and enzymatic activity of muscle and altitude adaptation. J Appl Physiol. 1962;17: 301–305. pmid:14491693
  18. 18. Stephenson BYR, Turnert DL, Butler PJ. The relationship between diving activity and oxygen storage capacity in the tufted duck (Aythya fuligula). J Exp Biol. 1989;141: 265–275.
  19. 19. McIntyre IW, Campbell KL, MacArthur RA. Body oxygen stores, aerobic dive limits and diving behaviour of the star-nosed mole (Condylura cristata) and comparisons with non-aquatic talpids. J Exp Biol. 2002;205: 45–54. pmid:11818411
  20. 20. Reed JZ, Butler PJ, Fedak MA. The metabolic characteristics of the locomotory muscles of grey seals (Halichoerus grypus), harbour seals (Phoca vitulina) and Antarctic fur seals (Arctocephalus gazella). J Exp Biol. 1994;194: 33–46. pmid:7964404
  21. 21. Kanatous SB, Davis RW, Watson R, Polasek L, Williams TM, Mathieu-Costello O. Aerobic capacities in the skeletal muscles of Weddell seals: key to longer dive durations? J Exp Biol. 2002;205: 3601–8. pmid:12409486
  22. 22. MacArthur R. Seasonal changes in the oxygen storage capacity and aerobic dive limits of the muskrat (Ondatra zibethicus). J Comp Physiol B. 1990;160. doi: 10.1007/BF00258987.
  23. 23. Lestyk KC, Folkow LP, Blix AS, Hammill MO, Burns JM. Development of myoglobin concentration and acid buffering capacity in harp (Pagophilus groenlandicus) and hooded (Cystophora cristata) seals from birth to maturity. J Comp Physiol B. 2009;179: 985–96. doi: 10.1007/s00360-009-0378-9. pmid:19565249
  24. 24. Kanatous SB, Mammen PPA, Rosenberg PB, Martin CM, White MD, Dimaio JM, et al. Hypoxia reprograms calcium signaling and regulates myoglobin expression. Am J Physiol Cell Physiol. 2009;296: 393–402. doi: 10.1152/ajpcell.00428.2008.
  25. 25. Wittenberg BA. Both hypoxia and work are required to enhance expression of myoglobin in skeletal muscle. Focus on “Hypoxia reprograms calcium signaling and regulates myoglobin expression”. Am J Physiol Cell Physiol. 2009;296: C390–C392. doi: 10.1152/ajpcell.00002.2009. pmid:19144863
  26. 26. De Miranda MA, Schlater AE, Green TL, Kanatous SB. In the face of hypoxia: myoglobin increases in response to hypoxic conditions and lipid supplementation in cultured Weddell seal skeletal muscle cells. J Exp Biol. 2012;215: 806–813. doi: 10.1242/jeb.060681. pmid:22323203
  27. 27. Oftedal OT. Lactation in whales and dolphins: evidence of divergence between baleen- and toothed-species. J Mammary Gland Biol Neoplasia. 1997;2: 205–230. doi: 10.1023/a:1026328203526. pmid:10882306
  28. 28. Thiemann GW, Iverson SJ, Stirling I. Variation in blubber fatty acid composition among marine mammals in the Canadian Arctic. Mar Mammal Sci. 2008;24: 91–111. doi: 10.1111/j.1748-7692.2007.00165.x.
  29. 29. Schlater AE, De Miranda MA, Frye MA, Trumble SJ, Kanatous SB. Changing the paradigm for myoglobin: a novel link between lipids and myoglobin. J Appl Physiol. 2014;117: 307–315. doi: 10.1152/japplphysiol.00973.2013. pmid:24925978
  30. 30. Geiseler SJ, Blix AS, Burns JM, Folkow LP. Rapid postnatal development of myoglobin from large liver iron stores in hooded seals. J Exp Biol. 2013;216: 1793–8. doi: 10.1242/jeb.082099. pmid:23348948
  31. 31. Ponganis PJ, Welch TJ, Welch LS, Stockard TK. Myoglobin production in emperor penguins. J Exp Biol. 2010;213: 1901–6. doi: 10.1242/jeb.042093. pmid:20472777
  32. 32. Noren SR, Iverson SJ, Boness DJ, Noren SR, Iverson SJ, Boness DJ. Development of the blood and muscle oxygen stores in gray seals (Halichoerus grypus): implications for juvenile diving capacity and the necessity of a terrestrial postweaning fast. Physiol Biochem Zool. 2013;78: 482–490. doi: 10.1086/430228.
  33. 33. Lyamin O, Pryaslova J, Lance V, Siegel J. Animal behaviour: continuous activity in cetaceans after birth. Nature. 2005;435: 1177. doi: 10.1038/4351177a. pmid:15988513
  34. 34. Chittleborough R. The Breeding Cycle of the Female Humpback Whale, Megaptera nodosa (Bonnaterre). Mar Freshw Res.1958;9: 1. doi: 10.1071/MF9580001.
  35. 35. Rice DW, Wolman AA. The life history and ecology of the gray whale (Eschrichtius robustus). American Society of Mammalogists; 1971.
  36. 36. Horwood JW. Biology and Exploitation of the Minke Whale. CRC Press; 1989.
  37. 37. Ford JK, Reeves RR. Fight or Flight: antipredator strategies of baleen whales. Mamm Rev. 2008;38: 50–86.
  38. 38. Williams TM. The evolution of cost efficient swimming in marine mammals: limits to energetic optimization. Philos Trans R Soc B Biol Sci. 1999;354: 193–201. doi: 10.1098/rstb.1999.0371.
  39. 39. Stelle LL, Megill WM, Kinzel MR, Science MM, Stelle LL, Megill WM, et al. Activity budget and diving behavior of gray whales (Eschrichtius robustus) in feeding grounds off coastal British Columbia. Mar Mammal Sci. 2008;24: 462–478. doi: 10.1111/j.1748-7692.2008.00205.x.
  40. 40. Tyson RB, Friedlaender AS, Ware C, Stimpert AK, Nowacek DP. Synchronous mother and calf foraging behaviour in humpback whales Megaptera novaeangliae: Insights from multi-sensor suction cup tags. Mar Ecol Prog Ser. 2012;457: 209–220. doi: 10.3354/meps09708.
  41. 41. Goldbogen JA, Calambokidis J, Shadwick RE, Oleson EM, McDonald MA, Hildebrand JA. Kinematics of foraging dives and lunge-feeding in fin whales. J Exp Biol. 2006;209: 1231–44. doi: 10.1242/jeb.02135. pmid:16547295
  42. 42. Szabo A, Duffus D. Mother-offspring association in the humpback whale, Megaptera novaeangliae: following behaviour in an aquatic mammal. Anim Behav. 2008;75: 1085–1092
  43. 43. Polasek LK, Davis RW. Heterogeneity of myoglobin distribution in the locomotory muscles of five cetacean species. J Exp Biol. 2001;204: 209–15. pmid:11136607
  44. 44. Best PB. Seasonal abundance, feeding, reproduction, age and growth in minke whales off Durban (with incidental observations from the Antarctic). Rep Int Whal Commn. 1982;32: 759–786.
  45. 45. Stevick PT. Age-length relationships in humpback whales: A comparison of strandings in the western North Atlantic with commercial catches. Mar Mammal Sci. 1999;15: 13. doi: 10.1111/j.1748-7692.1999.tb00839.x.
  46. 46. Sumich JL. Growth in Young Gray Whales (Eschrichtius Robustus). Mar Mammal Sci. 1986;2: 145–152. doi: 10.1111/j.1748-7692.1986.tb00035.x.
  47. 47. Reynafarje B. Simplified method for the determination of myoglobin. J Lab Clin Med. 1963;61: 138–45. pmid:13981912
  48. 48. Helbo S, Fago A. Functional properties of myoglobins from five whale species with different diving capacities. J Exp Biol. 2012;215: 3403–3410. doi: 10.1242/jeb.073726. pmid:22693033
  49. 49. Noren SR, Williams TM. Body size and skeletal muscle myoglobin of cetaceans: adaptations for maximizing dive duration. Comp Biochem Physiol A Mol Integr Physiol. 2000;126: 181–91. pmid:10936758
  50. 50. Cartwright R, Sullivan M. Behavioral ontogeny in humpback whale (Megaptera novaeangliae) calves during their residence in Hawaiian waters. Mar Mammal Sci. 2009;25: 659–680. doi: 10.1111/j.1748-7692.2009.00286.x.
  51. 51. Swartz SL, Jones M Lou. Gray Whale (Eschrichtius robustus) Calf Production and Mortality in the Winter Range. Report of the International Whaling Commission. 1983;1981: 503–507.
  52. 52. Norris KS, Goodman RM, VillaRamírez B, Hobbs L. Behavior of california gray whale, Eschrichtius-robustus, in Southern Baja California, Mexico. Fish Bull. 1977;75: 159–172.
  53. 53. Tawara T. Respiratory pigments of whales. Yakugaku Zasshi-Journal Pharm Soc Japan. 1949;69: 539–542.
  54. 54. Lawrie RA. Biochemical Differences between Red and White Muscle. Nature. 1952;170: 122–123. doi: 10.1038/170122a0. pmid:14957045
  55. 55. Scholander PF. Experimental investigations on the respiratory function in diving mammals and birds. Hvalrad Skr. 1940;22: 1–131.
  56. 56. Croll DA, Acevedo-Gutiérrez A, Tershy BR, Urbán-Ramírez J. The diving behavior of blue and fin whales: is dive duration shorter than expected based on oxygen stores? Comp Biochem Physiol A Mol Integr Physiol. 2001;129: 797–809. pmid:11440866
  57. 57. Goldbogen JA, Calambokidis J, Croll DA, McKenna MF, Oleson E, Potvin J, et al. Scaling of lunge-feeding performance in rorqual whales: mass-specific energy expenditure increases with body size and progressively limits diving capacity. Funct Ecol. 2012;26: 216–226. doi: 10.1111/j.1365-2435.2011.01905.x.
  58. 58. Mirceta S, Signore A V., Burns JM, Cossins AR, Campbell KL, Berenbrink M. Evolution of mammalian diving capacity traced by myoglobin net surface charge. Science. 2013;340. doi: 10.1126/science.1234192.
  59. 59. Ellis M, Morton B, Ford JKB, Ellis GM, Matkin DR, Balcomb KC, et al. Killer whale attacks on minke whales: prey capture and anitpredator tactics. Mar Mammal Sci. 2005;21: 603–618. doi: 10.1111/j.1748-7692.2005.tb01254.x.
  60. 60. Williams TM, Davis RW, Fuiman LA, Francis J, Le Boeuf BJ, Calambokidis J, et al. Sink or Swim : Strategies for Diving by Mammals. 2000;288: 133–136. pmid:10753116
  61. 61. Hertel H. Structure, form, movement. Reinhold; 1966;
  62. 62. Blake RW. Energetics of leaping in dolphins and other aquatic animals. J Mar Biol Assoc United Kingdom. Cambridge University Press; 2009;63: 61. doi: 10.1017/S0025315400049808.
  63. 63. Potvin J, Goldbogen JA, Shadwick RE. Metabolic Expenditures of Lunge Feeding Rorquals Across Scale: Implications for the Evolution of Filter Feeding and the Limits to Maximum Body Size. PLoS One. 2012;7. doi: 10.1371/journal.pone.0044854.
  64. 64. Rodríguez de la Gala-Hernández S, Heckel G, Sumich JL. Comparative swimming effort of migrating gray whales (Eschrichtius robustus) and calf cost of transport along Costa Azul, Baja California, Mexico. Can J Zool. 2008;86: 307–313. doi: 10.1139/Z07-141.
  65. 65. Noren SR, Biedenbach G, Redfern J V., Edwards EF. Hitching a ride: the formation locomotion strategy of dolphin calves. Funct Ecol. 2008;22: 278–283. doi: 10.1111/j.1365-2435.2007.01353.x.
  66. 66. Calambokidis J, Laake JL, Klimek A. Updated analysis of abundance and population structure of seasonal gray whales in the Pacific Northwest, 1998–2010. 2010. Paper SC/62/BRG32 presented to the International Whaling Commission Scientific Committee. Available at www.iwcoffice.org.
  67. 67. Barendse J, Best PB, Carvalho I, Pomilla C. Mother knows best: occurrence and associations of resighted humpback whales suggest maternally derived fidelity to a southern hemisphere coastal feeding ground. PLoS One. 2013;8: e81238. doi: 10.1371/journal.pone.0081238. pmid:24349047
  68. 68. Pabst DA. Intramuscular morphology and tendon geometry of the epaxial swimming muscles of dolphins. Journal of Zoology. 1993; 230: 159–176.
  69. 69. Arkowitz R, Rommel S. Force and bending moment of the caudal muscles in the shortfin pilot whale. Mar Mammal Sci. 1985;1: 203–209. doi: 10.1111/j.1748-7692.1985.tb00009.x.
  70. 70. Videler J, Kamermans P. Differences between upstroke and downstroke in swimming dolphins. J Exp Biol. 1985;119: 265–274. pmid:4093758
  71. 71. Fish FEF, Hui CCA. Dolphin swimming–a review. Mamm Rev. 1991;21: 181–195. doi: 10.1111/j.1365-2907.1991.tb00292.x.
  72. 72. Edel RK, Winn HE. Observations on underwater locomotion and flipper movement of the humpback whale Megaptera novaeangliae. Mar Biol. 1978;48: 279–287. doi: 10.1007/BF00397155.
  73. 73. Cartwright R. A comparative study of the behaviour and dynamics of humpback whale (Megaptera novaeangliae) mother and calf pairs during their residence in nursery waters. Ph. D. Thesis. Manchester Metropolitan University. 2005.
  74. 74. Whitehead H. Humpback whale breaching. Investig cetacea. 1985;17: 117–155.
  75. 75. Pacheco AS, Silva S, Alcorta B, Balducci N, Guidino C, Llapapasca MA, et al. Aerial behavior of humpback whales Megaptera novaeangliae at the southern limit of the southeast Pacific breeding area. Rev Biol Mar Oceanogr. 2013;48: 185–191.
  76. 76. Taber SM, Thomas PO. Mother-Infant Interaction and Behavioral Development in Southern Right Whales, Eubalaena Australis. Behaviour. 1984;88: 42–60. doi: 10.1163/156853984X00470.
  77. 77. Hoegh-Guldberg O, Bruno JF. The impact of climate change on the world’s marine ecosystems. Science. 2010;328: 1523–1528. doi: 10.1126/science.1189930. pmid:20558709
  78. 78. Boyd PW, Lennartz ST, Glover DM, Doney SC. Biological ramifications of climate-change-mediated oceanic multi-stressors. Nat Clim Chang. 2015;5: 71–79.