Received: 16 April 2019 Revised: 31 July 2019 Accepted: 9 September 2019 DOI: 10.1002/jmor.21089 RESEARCH ARTICLE Locomotor muscle morphology of three species of pelagic delphinids Jacqueline P. Kroeger Brandy P. Velten | | William A. McLellan | Logan H. Arthur | Emily M. Singleton | Stephen T. Kinsey | D. Ann Pabst Department of Biology and Marine Biology, University of North Carolina Wilmington, Wilmington, North Carolina Correspondence Jacqueline P. Kroeger, Department of Biology and Marine Biology, University of North Carolina Wilmington, 601 S. College Road, Wilmington, NC 28403. Email: kroegerj92@gmail.com Abstract The locomotor muscle morphology of diving mammals yields insights into how they utilize their environment and partition resources. This study examined a primary locomotor muscle, the longissimus, in three closely related, similarly sized pelagic delphinids (n = 7–9 adults of each species) that exhibit different habitat and depth preferences. The Atlantic spotted dolphin (Stenella frontalis) is a relatively shallow diver, inhabiting continental shelf waters; the striped (Stenella coeruleoalba) and shortbeaked common (Delphinus delphis) dolphins are sympatric, deep-water species that dive to different depths. Based upon comparative data from other divers, it was hypothesized that the locomotor muscle of the deepest-diving S. coeruleoalba would exhibit a higher percentage of slow oxidative fibers, larger fiber diameters, a higher myoglobin concentration [Mb], and a lower mitochondrial density than that of the shallow-diving S. frontalis, and that the muscle of D. delphis would display intermediate values for these features. As expected, the locomotor muscle of S. coeruleoalba exhibited a significantly higher proportion of slow (57.3 ± 3.9%), oxidative (51.7 ± 2.5%) fibers and higher [Mb] (8.2 ± 0.7 g/100 g muscle) than that of S. frontalis (41.3 ± 3.9%, 31.0 ± 3.2%, 4.7 ± 0.05 g/100 g muscle, respectively). There were no differences in fiber size or mitochondrial density among these species. Like other deep divers, S. coeruleoalba displayed locomotor muscle features that enhance oxygen storage capacity and metabolic efficiency but did not display features that limit aerobic capacity. These results suggest a previously undescribed muscle design for an active, small-bodied, deep-diving cetacean. Highlights • The locomotor muscle features displayed by the striped dolphin, which are unique among deep divers, enhance oxygen stores but do not limit aerobic capacity. This novel muscle design may facilitate the active lifestyle of this small-bodied deep diver. KEYWORDS delphinid, diving, fiber type, muscle, myoglobin 170 © 2020 Wiley Periodicals, Inc. wileyonlinelibrary.com/journal/jmor Journal of Morphology. 2020;281:170–182. 171 KROEGER ET AL. 1 | I N T RO DU CT I O N divers, such as the common bottlenose dolphin (Tursiops truncatus) and harbor seal (Phoca vitulina), are composed predominantly of fast Animal movement is a complex phenomenon reliant, in part, upon the fibers, including fast glycolytic (FG) fibers and fast oxidative glycolytic morphology and biochemistry of locomotor muscle. This reliance is (FOG) fibers (Kanatous, DiMichele, Cowan, & Davis, 1999; Kielhorn particularly important in marine mammals, which mechanically uncou- et al., 2013). In contrast, the primary locomotor muscles of most deep ple respiration and locomotion while swimming and diving on a divers, including the pygmy sperm whale (Kogia breviceps), narwhal breath-hold. While diving, these marine mammals display a suite of (Monodon monoceros), Weddell seal (Leptonychotes weddellii), and ele- physiological responses, known as the “dive response,” including bra- phant seal (Mirounga angustirostris) are composed predominantly of dycardia and peripheral vasoconstriction, which reduces convective slow oxidative (SO) fibers (Kanatous et al., 2002; Williams, Noren, & delivery of oxygen to locomotor muscle, decreases the diving meta- Glenn, 2011; Kielhorn et al., 2013; Moore et al., 2014; see Table 1). bolic rate, and extends the aerobic dive limit (ADL; reviewed by Thus, the locomotor muscle of most deep divers rely upon the more Kooyman, 1989; Davis, 2014). The ADL is the maximum amount of metabolically efficient oxidative, as opposed to glycolytic, pathway to time an air-breathing vertebrate can dive while utilizing only aerobic fuel contraction. metabolic pathways (Kooyman, Wahrenbrock, Castellini, Davis, & Sin- Muscle fiber size influences its metabolic rate. Johnston et al. nett, 1980). The ADL can also be calculated (cADL) as the ratio of (2003, 2004) proposed that muscle fibers reach an “optimal” size that onboard oxygen stores and diving metabolic rate (Kooyman et al., permits a balance between the decreased metabolic energy demand 1980). Features of the locomotor muscle, including fiber type, fiber associated with large fibers and the short diffusion distances associ- size, mitochondrial density, and myoglobin concentration, influence ated with small fibers. Larger fibers are energetically “cheaper” these two measures and differ across shallow, short-duration versus because their lower surface area to volume ratio results in fewer ATP- deep, long-duration mammalian divers (reviewed by Kanatous et al., consuming ion pumps required to maintain the membrane potential 2002; Kielhorn et al., 2013). per unit of cell volume (Jimenez, Dasika, Locke, & Kinsey, 2011; Fiber type composition influences the speed of contraction Jimenez, Dillaman, & Kinsey, 2013). Deep-diving mammals do possess and the efficiency of oxygen utilization of muscle (Barnard, Edgerton, larger fiber diameters than do shallow divers. For example, meso- Furukawa, & Peter, 1971; Brooke & Kaiser, 1970; Gauthier & Lowey, plodont beaked whales, M. monoceros, and K. breviceps all possess 1977; Gauthier & Padykula, 1966). The locomotor muscles of shallow larger fiber diameters than does the shallow-diving T. truncatus T A B L E 1 Percent of slow oxidative (SO), slow oxidative glycolytic (SOG), fast glycolytic (FG), and fast oxidative glycolytic (FOG) fibers by area; myoglobin concentration ([Mb]); mitochondrial volume density (Vmt), and diameter of type II (fast-twitch) fibers of primary locomotor muscles of various species of cetaceans and pinnipeds, roughly in order of relative diving ability (depth and duration) Species % SO % SOG % FG % FOG [Mb] (g/100 g muscle) Vmt TII fiber diameter (μm) 44.8 ± 2.2 – 48.8 ± 2.0 6.4 ± 1.8 3.21 ± 0.12 – 59.7 ± 1.9 55.7 ± 1.8 – 44.3 ± 1.8 0 5.92 ± 0.41 – 81.7 ± 2.1 33 33 33 0 6.82 ± 0.43 7.3 ± 4.6 66.8 ± 13.3 Cetaceans Tursiops truncatusa Kogia breviceps a Globicephala macrorhynchusb Monodon monoceros c Mesoplodon spp.b d 87.8 ± 6.3 – 12.2 ± 6.3 7.87 ± 1.72 – 60.3 ± 16.2 21.5 ± 9.6 0 78.5 ± 9.6 0 7.34 ± 0.87 2.4 ± 2.2 79.6 ± 11.5 40.3 ± 5.0 – 54.7 ± 4.3 5.0 ± 1.7 3.74 ± 0.17 9.7 ± 0.5 – 67 ± 4.7 – 0 33.6 ± 5.6 4.59 ± 0.33 3.13 ± 0.28 94.4 ± 26.2h 100j – 0 – 5.91 ± 0.41 – 118.7 ± 21.1h Pinnipeds Phoca vitulinae,f Leptonychotes weddellii g Mirounga angustirostrisi Values are mean ± SE (M. monoceros diameter value is ±1 SD). Dashes (−) denote features not explicitly stained for (e.g., SOGs) or measured (Vmt and fiber size). a Kielhorn et al. (2013). b Velten, Dillaman, Kinsey, McLellan, and Pabst (2013). c Williams et al. (2011). d Percentage is all fast fibers combined, not differentiated between FG and FOG. e Reed, Butler, and Fedak (1994). f Kanatous et al. (1999). g Kanatous et al. (2002). h Fiber diameter is an average of all (slow and fast) longissimus fibers. i Moore et al. (2014). j Fiber type is presumed, as fibers were only stained using SDH protocol. 172 KROEGER ET AL. (Kielhorn et al., 2013; Williams et al., 2011), and a similar pattern is using an appropriate combination of histochemical stains, although seen within pinnipeds (see Table 1). Thus, large muscle fibers, and they did not identify any such fibers in this classic work. SOGs their concomitant lower resting metabolic costs, appear to be a shared appear to be exceedingly rare in mammals, having been previously morphology across a diversity of deep divers (reviewed by Pabst, described in the esophageal (Whitmore, 1982) and rarely in some McLellan, & Rommel, 2016). Mitochondrial volume density (Vmt) also influences locomotor hindlimb (Suzuki & Hayama, 1991) muscles of macaques. For G. macrorhynchus, a relatively large percentage of SOG fibers may allow muscle metabolic rate and dictates its aerobic capacity—its maxi- them to conserve oxygen to sustain deep, long-duration dives while mum rate of oxygen usage (Burpee, Bardsley, Dillaman, Watanabe, & providing the ability to perform burst activity when foraging. Kinsey, 2010; Hoppeler et al., 1985). While few studies have directly In addition to these differences in locomotor muscle morphology, (Kanatous et al., 1999, 2002, 2008; Velten et al., 2013) or indirectly it is also important to note that species that achieve deeper dive (Kielhorn et al., 2013) measured mitochondrial density in mammalian depths tend to exhibit larger body sizes than do shallow-diving spe- divers, the locomotor muscles of deep divers typically exhibit a sig- cies (Noren & Williams, 2000). This increased body size allows for nificantly lower Vmt than do those of shallow divers (see Table 1). greater absolute oxygen storage capacity and a lower mass-specific Thus, oxygen conservation via low muscle mitochondrial density metabolic rate (Kleiber, 1932), features that work in concert to extend appears to be a common feature that extends the ADL of deep dive duration. To date, the locomotor muscle morphology of a phylogenetically divers. Breath-hold divers must also store onboard the oxygen they broad sample of species, exhibiting a wide range of body sizes, has require during a dive, and their locomotor muscles have high concen- been investigated. The current study sought to control for the effects trations of myoglobin relative to those of terrestrial animals, a feature of body size and phylogeny by investigating three similarly sized, considered a hallmark of diving mammals and birds (Kooyman & closely related pelagic delphinids that display dramatic differences in Ponganis, 1998). Deep divers, in particular, must possess high onboard their diving behavior—the Atlantic spotted dolphin (Stenella frontalis), oxygen stores within their locomotor muscles to remain aerobic for short-beaked common dolphin (Delphinus delphis), and striped dolphin their long-duration dives. For example, the locomotor muscles of the (S. coeruleoalba; Table 2). short-finned pilot whale (Globicephala macrorhynchus), M. monoceros, Stenella frontalis is a relatively shallow-diving odontocete that and mesoplodont beaked whales exhibit more than twice the concen- inhabits the waters over the continental shelf and upper continental tration of myoglobin than that of T. truncatus (see Table 1). slope (Jefferson et al., 2008; Read et al., 2014), and that feeds pre- Increasing metabolic efficiency via a predominantly SO muscle dominantly on epi- and meso-pelagic fishes and squids (Davis et al., fiber profile, decreasing muscle resting metabolic rate via large 1996). Delphinus delphis is a highly gregarious, pelagic odontocete typ- fibers, decreasing aerobic capacity via low mitochondrial densities, ically found hundreds to thousands of kilometers offshore (Jefferson and increasing onboard oxygen stores via high myoglobin concentra- et al., 2008) that primarily feeds on epipelagic schooling fishes and tions (Table 1) allows deep divers to extend their ADL and facilitates squids, and on those species associated with the deep scattering layer longer, deeper dives. While the patterns described above have been (Jefferson et al., 2008; Pusineri et al., 2007). One study has investi- observed across a wide range of mammalian divers, novel fiber type gated the locomotor muscle of this species in Japanese waters and compositions have been recently described in two extreme-diving found that it was composed of approximately 50% FG fibers and 50% odontocete cetaceans. Beaked whales are the deepest diving of all SO fibers (Suzuki, Tsuchiya, Takahashi, & Tamate, 1983). Suzuki et al. mammals recorded to date (Falcone et al., 2017; Schorr, Falcone, (1983) also report that the SO fibers had both enhanced oxidative and Moretti, & Andrews, 2014; Tyack, Johnson, Soto, Sturlese, & glycolytic capacity, suggesting they may be SOG fibers, although Madsen, 2006). In contrast to other deep divers, however, the long- these authors did not identify them as such. Stenella coeruleoalba is a issimus muscle of mesoplodont beaked whales exhibit a sprinter's deep-diving, fast-swimming, pelagic odontocete that, unlike D. delphis, fiber type profile, composed of approximately 80% FG fibers, which feeds on mesopelagic and bathypelagic fishes and squids (Archer II & are extremely large in size and possess exceedingly low mitochon- Perrin, 1999; Jefferson et al., 2008). drial volume densities (Velten et al., 2013; see Table 1). These features of the FG fibers likely decrease dramatically the metabolic rate and aerobic capacity, respectively, of those fibers (Velten et al., 2013). Globicephala macrorhynchus is another extreme deep diver T A B L E 2 Maximum adult total length, mass, and dive depth for the species investigated herein that displays a unique swimming pattern at depth. Unlike other deep Species Length (m)a Mass (kg)a Depth (m) divers that routinely exhibit slow swim speeds (e.g., Tyack et al., Stenella frontalis 2.3 140 60b 2006), this species is known to sprint at the deepest part of their Delphinus delphis 2.7 200 280c dive (Soto et al., 2008). While its longissimus muscle does exhibit a Stenella coeruleoalba 2.6 160 700d predominantly slow fiber type profile, approximately 33% of the slow fibers are slow oxidative glycolytic (SOG) fibers (Velten et al., 2013). Peter, Barnard, Edgerton, Gillespie, and Stempel (1972) suggested that this fiber type was possible and could be identified a Jefferson, Webber, and Pitman (2008). Davis, Worthy, Würsig, and Lynn (1996) (rehabilitated dolphin). c Evans (1971). d Iwasaki (2003). b 173 KROEGER ET AL. These three delphinids exhibit similar body sizes but different diving behaviors. Stenella frontalis and S. coeruleoalba are congeners but McLellan, Dillaman, Frierson Jr., & Pabst, 2000; Etnier, Dearolf, McLellan, & Pabst, 2004;Kielhorn et al., 2013 ; Velten et al., 2013). inhabit different environments and achieve different dive depths. Delphinus delphis and S. coeruleoalba are sympatric species that exhibit different dive depths. This study investigated whether the deeper- 2.2.1 | Myosin adenosine triphosphatase staining diving species could display features of their locomotor muscle that extend the ADL. Based upon the morphologies exhibited across most Myosin adenosine triphosphatase (ATPase) staining was used to differ- shallow and deep divers, this study tested the hypotheses that the entiate slow versus fast muscle fibers (Guth & Samaha, 1970). For six locomotor muscle of (a) S. coeruleoalba will exhibit a higher percentage individuals of each species (see Table S1), two to three slides, with three of SO fibers, larger fibers, a higher myoglobin concentration, and to four cross-sections each, were preincubated in an alkaline solution lower mitochondrial densities than that of S. frontalis and (b) D. delphis (pH 10.1, 10.2, 10.3; 53 mmol l−1 NaCl, 45 mmol l−1 NaOH, 32 mmol l−1 will exhibit values for these features that are intermediate to those of CaCl, and 53 mmol l−1 glycine) at 37 C (MyTemp Mini Incubator, Bench- these two stenellids. Based upon the results of Velten et al. (2013) mark Scientific) for 10 min while two to three other slides were pre- and Suzuki et al. (1983), this study also tested the hypothesis that the incubated in an acidic solution (pH 4.2–4.3, 43.5 mmol l−1 barbital locomotor muscle of the deeper-diving D. delphis and S. coeruleoalba acetate, and 43.5 mmol l−1 HCl) at 37 C for 5 min following methods will exhibit SOG fibers. outlined in Velten et al. (2013) (Guth & Samaha, 1970). After alkaline preincubation, slides were rinsed in superwater (distilled water at pH 8.5–9.0), and after acidic preincubation, slides were 2 METHODS | rinsed in 0.02 mmol l−1 sodium barbital buffer. All slides were then incubated in an ATP solution at 37 C for 30 min and thereafter under- 2.1 | Specimens and sample collection went a series of rinses including superwater, 1% CaCl, and 2% CoCl for 3 min each. Slides were then stained in (NH4)2S for 3 min and This study relied upon an archive of high-quality frozen muscle sam- rinsed in cold running water for 5 min. After dehydration using ples collected from dolphins that stranded between 2003 and 2017 in increasingly concentrated ethanol solutions, the slides were rinsed in North Carolina and Virginia (UNCW stranding response was carried toluene for 3 min and coverslips mounted using Permount (Kielhorn out under NOAA stranding agreements and UNCW IACUC protocols et al., 2013; Velten et al., 2013). #2003-13, #2006-15, #A0809-19, #A1112-013, and #A1415-015). Specifically, the longissimus muscle was investigated, which is the largest of the epaxial muscles (Pabst, 1990), and the muscle utilized in 2.2.2 | Succinate dehydrogenase staining multiple previous studies (e.g., Dolar, Suarez, Ponganis, & Kooyman, 1999; Kielhorn et al., 2013; Noren & Williams, 2000; Reed et al., Succinate dehydrogenase (SDH) staining was used to identify oxida- 1994; Velten et al., 2013; Williams et al., 2011). Freshly stranded tive muscle fibers (Nachlas, Tshou, De Souza, Cheng, & Seligman, (Smithsonian Institution Code 1 or 2; Geraci & Lounsbury, 2005), adult 1957). Slides were incubated in 0.2 mol L−1 phosphate buffer with Stenella coeruleoalba (Meyen, 1833), S. frontalis (Cuvier, 1829), and nitro blue tetrazolium (NBT) and 0.32 mol L−1 sodium succinate for Delphinus delphis (Linnaeus, 1758) exhibiting good body condition 30 or 60 min at 37 C. The slides were then rinsed in a saline solution were used in this study (n = 7–9 adults of each species; see Table S1). for 3 min and fixed in a formalin-saline solution for 3 min. After sec- From each specimen, a cross-section of epaxial muscle was collected tions were dehydrated using 15% ethanol, coverslips were mounted at the level of the dorsal fin. This cross-section was wrapped in using Kaiser's glycerin jelly (Kielhorn et al., 2013; Nachlas et al., 1957; ® Saran™ Premium Wrap, placed in two Ziplock Freezer bags, and Velten et al., 2013). stored at −20 C until subsampled for this study. 2.2 | 2.2.3 | staining Muscle Histochemistry and fiber diameter Alpha-glycerophosphate dehydrogenase A 0.5 × 0.5 × 1 cm sample of the longissimus muscle was taken from Alpha-glycerophosphate dehydrogenase (α-GPD) staining was used to deep within the frozen cross-section at a position just ventral to the identify glycolytic muscle fibers (Wattenberg & Leong, 1960). Slides superficial tendon, immediately mounted on a microtome chuck using were incubated in NBT in 0.2 mol L−1 phosphate buffer with man- Optimal Cutting Temperature compound (Sakura Finetek, Torrance, adione and α-GPD for 45 min at 37 C and then rinsed with CA), and rapidly frozen in liquid nitrogen-cooled isopentane. The sam- dH2O. After slides were rinsed in 50% acetone, coverslips were ple was sectioned at 10 μm using a Leica CM 1860 freezing micro- mounted ® tome (Leica Microsystems, Germany) at −19 C, placed on Superfrost  using Kaiser's glycerin jelly (Velten et al., 2013; Wattenberg & Leong, 1960). Plus glass slides (Fisher Scientific, Pittsburgh, PA), and stored in an air- This combination of histochemical staining was used to identify tight container at 4 C until processing later that day (Dearolf, fibers both on their presumed contractile capabilities and on 174 KROEGER ET AL. metabolic pathways. For example, a fiber that stained intensely for 2.3 | Myoglobin concentration ATPase after alkaline preincubation, stained weakly for SDH, and stained intensely for α-GPD was identified as a FG fiber. For four to six individuals of each species (see Table S1), the concentration of myoglobin [Mb], in grams of Mb per 100 g wet muscle, was measured using methods of Reynafarje (1963) as adapted by 2.2.4 | Fiber type composition and fiber diameter Noren and Williams (2000) and Etnier et al. (2004). Samples (0.5 g, three replicates for each individual) of the longissimus muscle at a Sections were viewed with the 10× objective using a light microscope similar position as those used for fiber type analyses were thawed (BX60, Olympus Corporation), and digital micrographs were captured and minced, and connective tissue was removed. The samples were using a SPOT RT camera (Diagnostic Instruments, Inc.) and stored as then placed in cold 0.04 mol L−1 phosphate buffer (39.25 ml buffer uncompressed TIFF files. Fiber type composition was determined using a per gram of muscle), homogenized (PowerGen 125, Fisher Scientific), stereological method to measure the cross-sectional area of the muscle and centrifuged at 4 C for 50 min at 15,000 rpm (J2-21M/E, made up by each fiber type (Cotten et al., 2008; Dearolf et al., 2000; Beckman; Avanti JXN-26, Beckman-Coulter). Supernatant (5 ml) was Russ, 1986). The micrographs were exported to Adobe Photoshop bubbled with carbon monoxide (CO) for 8 min. Sodium dithionite (Adobe Systems Inc.) and overlaid with a Mertz curvilinear grid. Points was quickly added to the supernatant, which was then vortexed for found in specific fibers and white space were counted until at least 5 s. The supernatant was bubbled with CO for an additional 2 min 500 points within fibers were counted. Achieving this count required to ensure complete reduction. The samples stayed on ice when not examining between 3 and 5 muscle sections. Fiber type-specific counts being homogenized, vortexed, and centrifuged. One milliliter of the were divided by the total fiber count (i.e., excluding white space) to bubbled supernatant was then transferred to a cuvette, and the determine fiber type composition (Cotten et al., 2008; Dearolf et al., absorbance was read at 538 and 568 nm using a spectrophotometer 2000; Etnier et al., 2004; Kielhorn et al., 2013; Velten et al., 2013). The (Ultrospec 8000, General Electric Healthcare Bio-Sciences Corp.). grids were scaled relative to the microscopic image so that no two grid Three spectrophotometric measurements were obtained for each of points fell within the same fiber and so that fibers were not missed. the replicates from an individual. The difference between these This stereological method was used for all three histochemical absorbance values was multiplied by a constant, which depended protocols and, thus, yielded three separate quantifications of fiber upon the exact initial buffer volume (Reynafarje, 1963) to determine type profile. The myosin ATPase method identified slow versus fast the myoglobin content (Etnier et al., 2004; Kielhorn et al., 2013; fibers, while the SDH and α-GPD methods identified oxidative and Velten et al., 2013). glycolytic fibers, respectively. Together, these metrics provide insights into the percentage of locomotor muscle formed by fibers of different contractile and metabolic states. If all slow fibers (i.e., stained darkly 2.4 | Index of mitochondrial density with the myosin ATPase assay after acidic preincubation), for example, were SO fibers, then the percentages of slow fibers should equal Directly measuring mitochondrial volume density requires the use of those that stain darkly for SDH and lightly for α-GPD. Likewise, if all transmission electron microscopy (e.g., Kanatous et al., 1999, 2002, fast fibers are glycolytic, then percentages of fast fibers should equal 2008; Velten et al., 2013), and this method requires access to fresh those that stain lightly for SDH and darkly for α-GPD. tissue. This study relied upon an archive of high-quality frozen muscle, Differences in the fiber type percentages based on contractile so an alternative method of assessing mitochondrial density, utilized versus metabolic stains suggest that a population of fibers has both by Kielhorn et al. (2013), was used. Because SDH is a mitochondria- oxidative and glycolytic capacity. If for example, the percentage of bound enzyme, staining sections for SDH and analyzing the relative fast fibers is larger than the percentage of glycolytic fibers, it suggests staining intensities of simultaneously incubated sections provides a that some of those fast fibers likely have enhanced oxidative capacity, measure of relative mitochondrial density (Kielhorn et al., 2013; that is, are fast, oxidative glycolytic fibers (Peter et al., 1972). In con- Nachlas et al., 1957; Peter et al., 1972). For this analysis, additional trast, if the percentage of slow fibers is larger than the percentage of muscle samples, taken from a similar position as those used for fiber oxidative fibers, it suggests that some of those slow fibers are SOGs type analyses, were used. Sections of muscle from a subset of individ- (Velten et al., 2013). Differences in the percentages of fibers, based uals (n = 4) of each species (see Table S1) were stained simultaneously upon contractile and metabolic stains, were used to identify potential for SDH as described above to ensure identical staining conditions. populations of these mixed fiber types. Micrographs were captured using the BX60 light microscope and Muscle fiber diameter was determined using alkaline ATPase sta- SPOT RT camera, and exported to Image-Pro Plus. Within Image-Pro ined fibers. Micrographs were exported to Adobe Photoshop, and Plus, the digital micrographs were converted to an 8-bit grayscale. 10 fibers of each type were digitally outlined. The outlines were Areas of Interest (AOIs) were then drawn within 25 light and 25 dark exported to Image-Pro Plus (Media Cybernetics, Inc.), and the diame- fibers. Since intermediate fibers were not present in all three species, ter was measured using the “mean diameter” tool, which measures they were excluded from this analysis. The pixel density within each the diameter of the fiber at 2 intervals around its circumference and AOI was then measured using the “mean density” tool. The raw den- presents the average of these values. sity values were exported to Microsoft Excel and subtracted from 175 KROEGER ET AL. 255 so that higher values indicated darker fibers, and, thus, higher Stenella frontalis and S. coeruleoalba, ATP percentages within Delphinus mitochondrial densities (Kielhorn et al., 2013). delphis, and mitochondrial index values within S. frontalis, for which the data were analyzed using a Mann–Whitney Rank Sum Test. 2.5 | Statistical analyses 3 RE SU LT S | All statistical analyses were conducted with SigmaPlot (Systat Software, Inc.). If the data met the criteria of a parametric test, the within- 3.1 | Muscle fiber type and diameter species comparisons (excluding SDH fiber type data for S. frontalis and D. delphis, which were analyzed using a one-way analysis of vari- The longissimus muscle of Stenella frontalis exhibited a significantly ance [ANOVA]) were analyzed using a Student's t test, and the across higher percentage of fast than slow fibers (p = .010, df = 10, species comparisons were analyzed using a one-way ANOVA. These t = −3.193) (Figure 1 and Tables 3 and 4). Both SDH (p < .001, df = 2, analyses were followed by Tukey's Honestly Significant Difference F = 78.942) and α-GPD (p = .002, T = 21, MWU = 0) staining demon- Test when necessary. strated a significantly greater proportion of glycolytic than oxidative There were instances in which the data did not meet the equal variance criteria of a parametric test: the average diameter between fibers. SDH staining differentiated a third, intermediate fiber type, which accounted for approximately 14% of the muscle cross-section. and across species, for which the data were analyzed using a Kruskal– Delphinus delphis exhibited a similar percentage of slow and fast Wallis one-way ANOVA, as well as the α-GPD percentages within fibers (p = .180, T = 48, MWU = 9), as well as a similar percentage of F I G U R E 1 Sections of longissimus muscle from S. frontalis stained with (a) ATPase following an alkaline preincubation (S denotes a slow fiber, F denotes a fast fiber), (b) succinate dehydrogenase (O denotes an oxidative fiber, G denotes a glycolytic fiber), and (c) alpha-glycerophosphate dehydrogenase (O denotes an oxidative fiber, G denotes a glycolytic fiber). Note the presence of a slow oxidative glycolytic (SOG) fiber [Color figure can be viewed at wileyonlinelibrary.com] T A B L E 3 Mean (±SE) fiber type composition (% of cross-sectional area) of the longissimus muscle using myosin ATPase following an alkaline preincubation, succinate dehydrogenase (SDH), and alpha-glycerophosphate dehydrogenase (α-GPD) staining techniques ATPase - alkaline SDH α-GPD Species Slow Fast Oxidative Intermediate Glycolytic Oxidative Glycolytic Stenella frontalis 41.3 ± 3.9 58.7 ± 3.9 31.0 ± 3.2 13.5 ± 0.9 55.6 ± 2.3 35.8 ± 4 64.2 ± 4 Delphinus delphis 53.3 ± 3.3 46.7 ± 3.3 42.7 ± 2.1 13.3 ± 1.8 43.8 ± 1.8 48.8 ± 1.9 51.2 ± 1.9 Stenella coeruleoalba 57.3 ± 3.9 42.8 ± 3.9 51.7 ± 2.5 - 48.3 ± 2.5 47.5 ± 4.3 52.5 ± 4.3 TABLE 4 Mean (±SE) fiber diameter, myoglobin concentration, and index of mitochondrial density (SDH staining intensity) (IMD) Fiber diameter (μm) IMD Species Slow Fast Myoglobin (g/100 g wet muscle) Stenella frontalis 48.4 ± 2.0 52.7 ± 4.0 4.7 ± 0.5 162.7 ± 3.4 79.5 ± 10.2 Delphinus delphis 43.8 ± 2.5 51.8 ± 3.7 5.0 ± 0.2 191.0 ± 7.2 94.7 ± 4.3 Stenella coeruleoalba 52.9 ± 5.1 56.1 ± 3.1 8.2 ± 0.7 174.0 ± 13.8 96.15 ± 8.5 Oxidative Glycolytic 176 KROEGER ET AL. oxidative and glycolytic fibers (SDH, p = .902, df = 2, F = 83.696; α-GPD, p = .391, df = 10, t = −0.897). SDH staining differentiated a third, intermediate fiber type, which accounted for approximately 13% of the muscle cross-section. Stenella coeruleoalba exhibited a significantly higher percentage of slow than fast fibers (p = .026, df = 10, t = 2.606). Both SDH (p = .163, df = 10, t = −1.507) and α-GPD (p = .31, t = 46.0 MWU = 11.0) staining demonstrated that S. coeruleoalba exhibited similar percentages of oxidative and glycolytic fibers. Unlike S. frontalis and D. delphis, SDH staining differentiated only two fiber types in this species. There was a significant difference in fiber type composition, based on myosin ATPase staining, across the species (p = .022, df = 2, F = 5.014). The longissimus muscle of S. coeruleoalba was composed of significantly more slow fibers than that of S. frontalis (p = .021). Delphinus delphis exhibited a similar percentage of slow fibers as S. coeruleoalba and S. frontalis (p = .733 and p = .089, respectively). F I G U R E 2 Box and whisker plot of SDH staining intensity, which was used as a proxy for mitochondrial density There was a significant difference in fiber type composition, based on both SDH (p < .001, df = 2, F = 17.289) and α-GPD Across these three species, mitochondrial densities were similar for (p = .017, df = 2, F = 5.368) staining, across the species. Both SDH and oxidative and glycolytic fibers averaged together (p = .192, df = 2, α-GPD staining demonstrated that S. coeruleoalba exhibited signifi- F = 1.997) and for oxidative (p = .147, df = 2, F = 2.396) and glycolytic cantly more oxidative fibers than S. frontalis (SDH, p < .001; α-GPD, (p = .315, df = 2, F = 1.315) fibers analyzed independently. p = .021). SDH staining also demonstrated that D. delphis exhibited significantly more oxidative fibers than S. frontalis (p = .017) and significantly fewer oxidative fibers than S. coeruleoalba (p = .041). α-GPD 4 DI SCU SSION | staining demonstrated similar percentages of oxidative and glycolytic fibers between D. delphis and S. frontalis (p = .052) as well as between The goal of this study was to investigate the histochemical fiber type D. delphis and S. coeruleoalba (p = .892). composition of a locomotor muscle of three similarly sized, closely Within each species, slow and fast muscle fibers were similarly related pelagic delphinids that dive to different depths. Previous stud- sized (S. frontalis, p = .350, df = 10, t = −0.979; D. delphis, p = .101, ies already investigated a phylogenetically broad sample of cetaceans df = 10, t = −1.808; S. coeruleoalba, p = .526, df = 10, t = −0.658). of differing body sizes. These studies demonstrated that deeper divers Additionally, all three of these species possessed similarly sized fibers tend to be large and exhibit enhanced oxygen stores and structural for slow and fast fibers averaged together (p = .366, df = 2, H = 2.012) features of their locomotor muscle that decrease its metabolic rate as as well as for slow (p = .315, df = 2, F = 1.249) and fast (p = .679, df = 2, compared to shallow-diving species (reviewed by Ponganis, 2015; F = 0.397) fibers analyzed independently. Pabst et al., 2016). In concert, these features extend the ADL of deep divers (reviewed by Kooyman & Ponganis, 1998). We hypothesized that Stenella frontalis, Delphinus delphis, and S. coeruleoalba would con- 3.2 | Myoglobin concentration form to this traditional view of shallow and deep divers. Although they generally did so, S. coeruleoalba displayed a combination of mus- There was a significant difference in myoglobin concentration across cle traits not previously described in a deep diver. The results for each the species (p < .001, df = 2, F = 61.595) (Table 4). The longissimus feature of locomotor muscle investigated will first be discussed across muscle of S. coeruleoalba exhibited significantly higher myoglobin con- the three delphinid species that were the focus of this study and will centrations than that of both S. frontalis and D. delphis (p < .001). Del- then be considered within a broader phylogenetic context within phinus delphis and S. frontalis exhibited similar muscle myoglobin odontocetes. concentrations (p = .749). 4.1 3.3 | | Features of locomotor muscle Index of mitochondrial density The hypothesis that the deepest-diving of the three species, S. The intensity of SDH staining was used as an index of mitochondrial coeruleoalba, would exhibit significantly more SO fibers than the density (Table 4 and Figure 2). Within each species, oxidative fibers shallow-diving S. frontalis was supported (see Table 3). Unlike many exhibited significantly higher mitochondrial densities than glycolytic other deep divers investigated to date, though, S. coeruleoalba did not fibers (S. frontalis, p = .029, T = 10, MWU = 0; D. delphis, p < .001, display more oxidative than glycolytic fibers. The metabolic histo- df = 6, t = −11.566; S. coeruleoalba, p = .003, DF = 6, t = −4.802). chemical stains (SDH and α-GPD) revealed similar percentages of 177 KROEGER ET AL. oxidative and glycolytic fibers in this species, which suggests that some slow fibers in this species may have enhanced glycolytic capacity. We also hypothesized that the two deeper divers, D. delphis and S. coeruleoalba, would exhibit SOG fibers, as does the larger, deeperdiving delphinid Globicephala macrorhynchus (Velten et al., 2013). The histochemical staining results, though, suggest that all three species likely possess SOGs. In both, D. delphis and the shallow-diving S. frontalis, three populations of fibers were identified by SDH staining, with 13–14% of the fibers staining intermediately between dark (oxidative) and light (glycolytic). In these two species, SDH staining identified approximately 10% fewer fibers as oxidative than were identified as slow fibers by ATPase staining. Additionally, α-GPD staining identified approximately 5% more fibers as glycolytic than were indicated F I G U R E 3 Percentage of longissimus muscle cross-sectional area composed of slow fibers of several odontocetes (data from Ponganis & Pierce, 1978; Williams et al., 2011; Kielhorn et al., 2013; Velten et al., 2013; and current study). S. fr. = Stenella frontalis, L. ob. = Lagenorhynchus obliquidens, T. tr. = Tursiops truncatus, K. br. = Kogia breviceps, D. de. = Delphinus delphis, S. co. = Stenella coeruleoalba, G. ma. = Globicephala macrorhynchus, and M. mo. = Monodon monoceros to be fast fibers based upon ATPase staining. These results suggest that the 13–14% of fibers that stained intermediately for SDH in these two species are likely SOGs (Figure 1; Velten et al., 2013). Based upon the differences between ATPase and α-GPD staining, S. coeruleoalba also likely possesses an intermediate fiber type that was not detected by SDH staining. In this deep diver, α-GPD staining identified approximately 10% more fibers as glycolytic than were indicated to be fast fibers based upon ATPase staining, suggesting these fibers are SOGs. Similarly, approximately 5% more fibers were indicated to be glycolytic when stained for SDH than were indicated to be fast fibers when stained for ATPase. We do not yet understand, though, why only two populations of fibers were identified with the SDH stain in S. coeruleoalba, while three were observed in the two other delphinid species. In total, these results suggest that all three delphinids likely possess at least a small percentage of slow muscle fibers that have both enhanced oxidative and glycolytic capacity. F I G U R E 4 Diameter (μm) of the dominant muscle fiber type of several odontocetes (data from Williams et al., 2011; Kielhorn et al., 2013; Velten et al., 2013; Sierra et al., 2015; and current study). D. de. = Delphinus delphis, S. co. = Stenella coeruleoalba, S. fr. = Stenella frontalis, T. tr. = Tursiops truncatus, M. mo. = Monodon monoceros, G. ma. = Globicephala macrorhynchus, K. br. = Kogia breviceps, Z. ca. = Ziphius cavirostris It has been well established that the fiber type profile, even of adult animals, is plastic (Chin et al., 1998; Holloszy & Coyle, 1984; Hudlicka, Dodd, Renkin, & Gray, 1982; Michel, Ordway, Richardson, & hypothesized that S. coeruleoalba would display larger muscle fibers Williams, 1994; Vrbová, 1963). Thus, the fiber type profile displayed than S. frontalis. There was, though, no difference between the muscle by a species at any given time is indicative of the routine use of the fiber diameters of the three delphinids investigated herein. The deep- muscle being investigated. This plasticity likely contributed to the pat- diving S. coeruleoalba possesses similarly sized fibers as the shallow- tern observed within the three delphinids investigated in this study, diving S. frontalis, despite achieving depths over 10 times greater which is generally what would be predicted based upon the maximum (Davis et al., 1996; Iwasaki, 2003). While numerically the largest, the recorded dive depth of these species. fibers of S. coeruleoalba are not statistically significantly larger, and so While fiber type is plastic, a broader phylogenetic comparison of fiber profiles across multiple families of odontocetes suggests that delphinids display a relatively limited range of compositions with do not appear to facilitate a lower resting metabolic rate than the shallower-diving species investigated here. Across delphinids investigated to date, muscle fiber diameters respect to slow and fast fibers (Figure 3). For example, slow fibers rep- range from 44 μm (D. delphis) to 67 μm (G. macrorhynchus; Figure 4). resent only 15% more of the cross-section of the longissimus muscle Within the delphinids, the deepest known diving species, G. macro- of the deep-diving G. macrorhynchus as compared to the extremely rhynchus, does possess the largest fibers, but there is otherwise no shallow, short-duration diver Tursiops truncatus. In contrast, Monodon clear relationship between dive depth or duration and fiber size across monoceros, which dives to similar depths and for similar durations as species. All values for delphinid locomotor muscle fibers are also G. macrorhynchus (reviewed by Ponganis, 2015), possesses a fiber smaller than those displayed by deep-diving kogiids and ziphiids. Spe- type profile composed of almost 90% slow fibers. These patterns may cific comparisons reveal interesting differences between families. reflect phylogeny, differences in body size and therefore the meta- Stenella coeruleoalba dives to similar depths as K. breviceps based on bolic rate, and/or differences in activity during a dive. diet analyses (Staudinger, McAlarney, McLellan, & Pabst, 2014) but Based upon comparative analyses across cetaceans and the “opti- possesses fibers approximately 40% smaller. Similarly, G. macro- mal fiber size” hypothesis (Johnston et al., 2003, 2004), we rhynchus possesses fibers about 20% smaller than those of K. 178 KROEGER ET AL. F I G U R E 5 Myoglobin concentration (g Mb/100 g muscle) in longissimus muscle and maximum recorded/estimated dive depth of several odontocetes, shown with 95% confidence intervals (curved dashed lines) (linear regression, r2 = 0.61) ([Mb] data from Castellini & Somero, 1981; Polasek & Davis, 2001; Williams et al., 2011; Kielhorn et al., 2013; Velten et al., 2013; and current study) (Depth data from Evans, 1971; Heide-Jørgensen & Dietz, 1995; Mate, Rossbach, Nieukirk, Wells, & Irvine, 1995; Westgate, Read, Berggren, Koopman, & Gaskin, 1995; Davis et al., 1996; Baird, Ligon, Hooker, & Gorgone, 2001; Iwasaki, 2003; Tyack et al., 2006; Soto et al., 2008; Perrin, 2009; Baird et al., 2013; Staudinger et al., 2014). Open, downward triangle = Phocoena phocoena, star = Stenella attenuata, circle = Tursiops truncatus, plus sign = Stenella frontalis, hexagon = Delphinus delphis, open triangle = Stenella longirostris, square = Kogia breviceps, open diamond = Pseudorca crassidens, triangle = Globicephala macrorhynchus, diamond = mesoplodont beaked whales, downward triangle = Monodon monoceros, circle enclosing star = Stenella coeruleoalba. *Indicates overlapping species: P. ph. and S. at. P. phocoena and S. attenuata F I G U R E 6 Myoglobin concentration (g Mb/100 g wet muscle) of longissimus muscle of several odontocetes (data from Castellini & Somero, 1981; Dolar et al., 1999; Noren & Williams, 2000; Polasek & Davis, 2001; Williams et al., 2011; Kielhorn et al., 2013; Velten et al., 2013; Noren, Noren, & Gaydos, 2014; and current study). L. bo. = Lissodelphis borealis, S. at. = Stenella attenuata, P. ph. = Phocoena phocoena, T. tr. = Tursiops truncatus, D. le. = Delphinapterus leucas, L. ob. = Lagenorhynchus obliquidens, S. fr. = Stenella frontalis, D. de. = Delphinus delphis, S. lo. = Stenella longirostris, K. br. = Kogia breviceps, P. cr. = Pseudorca crassidens, G. ma. = Globicephala macrorhynchus, L. ho. = Lagenodelphis hosei, M. mo. = Monodon monoceros, S. co. = Stenella coeruleoalba Across odontocete families, [Mb] generally varied as would be predicted based upon maximum reported dive depth and/or duration (Figure 6). However, rather than fall out together along the axis, as was seen for values of muscle fiber type and size, the [Mb] values of delphinids span the entire range of values reported to date for odontocetes, from the lowest [Mb] in the northern right whale dolphin (Lissodelphis borealis) to one of the highest values ever recorded in cetaceans in S. coeruleoalba. The [Mb] value for S. coeruleoalba, for example, is higher than that of most of the deep-diving mesoplodont beaked whales, whose congeners routinely dive to depths greater than 800 m and for durations of approximately 45 min (Tyack et al., 2006). Despite S. coeruleoalba achieving similar depths as K. breviceps, this small deep diver exhibits substantially more myoglobin than does K. breviceps. Stenella coeruleoalba is, though, much smaller than this deep- breviceps despite likely achieving greater depths. However, G. macro- diving kogiid, and, thus, has a higher mass-specific metabolic rate. rhynchus can also be an extremely active diver, sprinting at up to Based upon comparative data, we hypothesized that S. coeruleoalba 9 m/s at the deepest portion of their dive (Soto et al., 2008). Reducing would display lower mitochondrial densities within their locomotor metabolic rate via relatively large muscle fibers may only occur in muscle fibers than would S. frontalis. This hypothesis was not deep-diving species that are not known or thought to be exceedingly supported, as there were no significant differences in the index of mito- active animals (e.g., Heide-Jørgensen & Dietz, 1995; Tyack et al., chondrial density between the three investigated delphinids. This result 2006). Based on quantitative (Soto et al., 2008; reviewed by Fish & was surprising, given that both cetacean and pinniped deep divers tend Rohr, 1999) and anecdotal (reviewed in Fish, 1998; Jefferson et al., to display features that reduce aerobic capacity (i.e., the maximum rate 2008) evidence, delphinids are reported to be more active during of oxygen consumption) and, thus, extend their ADL (e.g., Kanatous dives and while at the surface than most deep divers, including et al., 1999, 2002; Kielhorn et al., 2013; Velten et al., 2013). As dis- beaked whales, narwhals, and kogiids. Thus, fiber size may reflect the cussed below, this relatively high mitochondrial density is potentially activity level as well as phylogeny. Within odontocetes, myoglobin concentration [Mb] is typically reflective of a more active lifestyle for S. coeruleoalba, as compared to other species that dive to comparable depths, such as K. breviceps. positively related to maximum dive depth (Figure 5) and duration (Noren & Williams, 2000). The hypothesis that the locomotor muscle of S. coeruleoalba would conform to that trend and exhibit a significantly higher [Mb] than those of the two shallower-diving species was 4.2 | A previously undescribed model for an active, small-bodied deep diver supported. Interestingly, the [Mb] of D. delphis and S. frontalis were equivalent despite their differences in dive depth and therefore pre- Inherent costs of being a small, diving mammal include a lower abso- sumed differences in dive duration. lute oxygen storage capacity (Noren, Lacave, Wells, & Williams, 2002; 179 KROEGER ET AL. Pabst et al., 2016; Velten et al., 2013) and a higher mass-specific basal fiber number, and therefore size, is under strong selective pressure to metabolic rate (Kleiber, 1932) as compared to larger mammals. For minimize sarcolemmal membrane surface area, presumably to reduce example, based on Kleiber's three-fourth scaling rule, the oxygen con- metabolic costs. This view is supported by the findings of Jimenez et al. sumption rate per unit of body mass of a 160 kg S. coeruleoalba would (2011, 2013), who experimentally determined that larger fibers are be 1.3× higher than that of a 450 kg K. breviceps (Kleiber, 1932; energetically cheaper than smaller fibers due to a lower surface area to Schmidt-Nielsen, 1997). Therefore, the locomotor muscle of a small volume ratio and a reduced cost of membrane ion transport. Thus, the mammal that dives to similar deep depths as a larger mammal would locomotor muscle fibers of S. coeruleoalba, though they do not display be expected to display features that both enhance oxygen storage the large diameters expected of a deep diver that would facilitate a capacity and limit metabolic rate. The locomotor muscle design that lower metabolic rate, are likely as large as they can be, and thus may be reflects this strategy and contributes to an extended ADL is one with “optimized” for the lifestyle exhibited by this species, which we hypoth- high [Mb] (enhanced oxygen storage capacity) and large (low cellular esize to be an active one. resting metabolic rate), predominantly SO (enhanced metabolic effi- Stenella coeruleoalba exhibits a unique muscle design among deep ciency) fibers with low mitochondrial density (reduced aerobic capac- divers that does not limit aerobic capacity. This increased aerobic ity; e.g., Kanatous et al., 1999, 2002; Kielhorn et al., 2013). capacity, which facilitates the higher level of activity reported for del- A diving mammal, though, may possess high onboard oxygen phinids both at the surface and while on a dive (e.g., Aoki, Sato, stores either to extend dive duration and/or be more active at depth. Isojunno, Narazaki, & Miller, 2017; Fish & Rohr, 1999; Soto et al., Because delphinids are typically shallower divers, it has been difficult 2008), is interesting when comparing the diet across deep-diving ceta- to decouple the effects of activity level and dive depth when consid- ceans. Throughout most of its range, S. coeruleoalba feeds predomi- ering muscle designs across a broad phylogenetic sample. By examin- nantly on mesopelagic and bathypelagic fishes (reviewed in Archer ing three closely related, active delphinids, this study offers insights II & Perrin, 1999). In contrast, most other deep divers for which data into those muscle features specifically associated with increased are available, including the sperm whale, Physeter macrocephalus diving depth and, thus, duration. While the muscle of S. coeruleoalba (Evans & Hindell, 2004), K. breviceps, and the dwarf sperm whale, K. displays features that would increase oxygen storage capacity, this sima (Staudinger et al., 2014), and most beaked whales (reviewed by deep-diving species did not display features that would limit the rate MacLeod, Santos, & Pierce, 2003), feed predominantly on gelatinous of oxygen consumption, exhibiting similar fiber sizes and mitochon- and muscular squid. Based on the cost of transport data for several drial densities as the shallow-diving S. frontalis. This design, which species of squid and fish, fishes are far more efficient swimmers than supports the hypothesis of increased locomotor muscle aerobic capac- squids (Videler, 1993; Webber & O'Dor, 1986). These data suggest ity in S. coeruleoalba, is in stark contrast to larger-bodied deep divers, that fish prey may be more energetically costly to pursue than squids. including K. breviceps, mesoplodont beaked whales, and L. weddellii, The locomotor muscle morphology of S. coeruleoalba suggests a that exhibit large muscle fibers and low mitochondrial densities previously undescribed muscle design for an active, small-bodied spe- (Kanatous et al., 2002; Velten et al., 2013). cies that dive deeply, where such a lifestyle could be advantageous While the muscle fibers of S. coeruleoalba were not significantly among relatively slow, teuthophagous deep divers. This hypothesis larger than the other two species as hypothesized, this does not mean could be tested in a number of ways. For example, the morphology of that fiber sizes were inconsistent with the optimal fiber size hypothesis other small-bodied, deep-diving delphinids, such as Fraser's dolphin (Johnston et al., 2003, 2004). In this context, a muscle fiber's maximal (Lagenodelphis hosei) could be investigated. Lagenodelphis hosei (2.7 m, size is constrained by the demand for aerobically generated ATP, which 210 kg; Jefferson et al., 2008) is known to feed on bathypelagic fishes, is dependent on adequate oxygen diffusion to mitochondria. Thus, a indicating it can achieve dive depths comparable to those of S. high aerobic activity level will necessarily lead to smaller fibers. Several coeruleoalba, and its epaxial locomotor muscle [Mb] is an impressive lines of evidence suggest that muscle fibers are often as large as they 7.1 g/100 g muscle (Dolar et al., 1999). Examining the fiber type pro- can be while avoiding diffusional constraints. Mathematical reaction– file, fiber size, and mitochondrial density of the locomotor muscle of L. diffusion modeling of aerobic metabolism, coupled with experimental hosei would enhance our understanding of the relationship between measurements of aerobic metabolic processes and fiber sizes in a vari- muscle design and diving lifestyle in these small deep divers. Addition- ety of species, suggest that muscle fibers reach a maximal fiber size that ally, placing multi-sensor tags on these species, and other deep divers, places them on the brink of diffusion limitation (Kinsey, Hardy, & Locke, would provide information on speeds and durations of activity, and 2007; Kinsey, Locke, & Dillaman, 2011). In fact, in cases where muscle would be invaluable in testing this hypothesis further. fibers exceed mathematically determined, diffusion-based limits, they This study examined the locomotor muscle morphology of three display compensatory responses, such as shifts in fiber growth pattern closely related, similarly sized pelagic odontocetes that exhibit differ- from hypertrophy to hyperplasia (Kinsey et al., 2011; Priester, Morton, ent dive behaviors and depth preferences. As hypothesized based Kinsey, Watanabe, & Dillaman, 2010), rearrangement of cellular organ- upon comparative studies, the shallowest-diving species displayed elles, including mitochondria and nuclei, and changes in metabolism predominantly FG muscle fibers and relatively low [Mb], while the (reviewed by Kinsey et al., 2011; Priester et al., 2010), and compartmen- deepest-diving species displayed predominantly slow muscle fibers talization of fibers to limit diffusion distances (Hardy, Dillaman, Locke, & and extremely high [Mb]. Unexpectedly, the muscle fiber size and Kinsey, 2009). Furthermore, Johnston et al. (2004) demonstrated that index of mitochondrial density of the deep diver were similar to those 180 KROEGER ET AL. values of the shallow diver, suggesting no specialization to decrease muscle metabolic costs. These results suggest a previously undescribed muscle design for a small-bodied deep diver that chases prey that is likely relatively costly to pursue. Continuing to learn about the diving lifestyles facilitated by, and the potential physiological and functional limits of, certain muscle designs is critical to understanding important ecological relationships, including those between predators and prey, and may offer insights into how these species may be able to respond to a changing ocean environment (e.g., Williams et al., 2011). ACKNOWLEDGMENTS We thank the NC Wildlife Resources Commission (Karen Clark), the Virginia Aquarium (Susan Barco, Alex Costidis, and Marina Piscitelli), the National Park Service (Paul Doshkov), the NC Aquarium at Fort Fisher, the NC Aquarium at Roanoke Island, the NC Division of Marine Fisheries (Vicky Thayer), and the Marine Mammal Stranding Program at UNC Wilmington for their significant contributions to specimen collection. We also thank Sentiel Rommel for assistance with illustrations and Mark Gay for assistance with histology and imaging. This work was supported in part by NOAA Prescott Grants to UNCW. AUTHOR CONTRIBUTIONS J.P.K., W.A.M., and D.A.P. conceptualized project; D.A.P. and S.T.K. directed lab work; J.P.K., L.H.A., B.P.V., and E.M.S. carried out laboratory studies; J.P.K. wrote manuscript and all authors reviewed; D.A.P. and W.A.M. received funding. DATA AVAI LAB ILITY S TATEMENT Data available upon request to the author. ORCID Jacqueline P. Kroeger William A. McLellan https://orcid.org/0000-0001-7150-487X https://orcid.org/0000-0003-4617-9029 https://orcid.org/0000-0003-0424-7978 Brandy P. Velten Emily M. Singleton https://orcid.org/0000-0001-8085-6391 Stephen T. Kinsey https://orcid.org/0000-0002-2479-9617 D. Ann Pabst https://orcid.org/0000-0001-6985-9866 RE FE R ENC E S Aoki, K., Sato, K., Isojunno, S., Narazaki, T., & Miller, P. J. O. (2017). High diving metabolic rate indicated by high-speed transit to depth in negatively buoyant long-finned pilot whales. 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SUPPORTING INF ORMATION Additional supporting information may be found online in the Supporting Information section at the end of this article. How to cite this article: Kroeger JP, McLellan WA, Arthur LH, et al. Locomotor muscle morphology of three species of pelagic delphinids. Journal of Morphology. 2020;281:170–182. https://doi.org/10.1002/jmor.21089