Richard M. Dillaman

Precocial development of axial locomotor muscle in bottlenose dolphins (Tursiops truncatus).

ABSTRACT

Distribution and Characterization of Ion Transporting and Respiratory Filaments in the Gills of Procambarus clarkii

Individual gill filaments of the freshwater crayfish Procambarus clarkii were determined to be either predominantly respiratory or transporting. Silver staining revealed that the filaments within the central bed of the gills formed silver deposits whereas filaments at the margins and the entire sixth pleurobranch formed no deposits. Designation of the silver staining gills as predominantly transporting and unstained filaments as predominantly respiratory was substantiated by ultrastructural analyses and measurements of ATPase and transepithelial potentials. Presumptive transporting filaments had an epithelium subjacent to the cuticle that was relatively thick and dominated by abundant mitochondria. Lacunae were delineated by pillar structures and served as collateral pathways for the movement of blood from the afferent to efferent blood channels, which were separated by a thin septum. Presumptive respiratory filaments had an extremely thin epithelium with few organelles, but a relatively thick septum. Present in both types of filaments were nerves and podocytes. The values for Na, K-ATPase were significantly higher in the transporting filaments than in those designated as respiratory. The measurement of transepithelial potentials showed both filaments to be cation selective with the respiratory filaments slightly more positive and the transporting filaments slightly more negative than the diffusion potential for Na.

A THEORETICAL MODEL OF CIRCULATORY INTERSTITIAL FLUID FLOW AND SPECIES TRANSPORT WITHIN POROUS CORTICAL BONE

A three-dimensional model of interstitial fluid flow and passive species transport within mineralized regions surrounding cross-cortical vessel canals is developed. In contrast to earlier studies, the present model applies to circulatory, non-stress-induced interstitial flow in porous cortical bone. Based on previous experimental observations, the canals are modeled as line sources that pass at an oblique angle through the cortex. Cross-cortical interstitial flow from the endosteal surface to the periosteal surface is also taken into account. It is found that model transport characteristics are qualitatively consistent with reported observations. In addition, parametric studies reveal the following: (1) Solute contact with the matrix is maximized when the ratio of canal radius to cortex thickness (R) is near physiological R values. (2) Solute-matrix contact falls to low levels when R falls below the physiological range. (3) Solute-matrix contact is maximized when the cross-cortical velocity is approximately an order of magnitude smaller than the canal outflow velocity. The first and second findings suggest that within porous bone physiological ranges of R promote near optimal species contact with the mineralized matrix. The third finding suggests that relatively impermeable layers of bone within the cortex can effectively promote solute-matrix contact by limiting cross-cortical flow. Finally, the model suggests that intra-canal resorption associated with reduced external loading may effectively compensate for reduced stress-induced interstitial flow by enhancing circulatory interstitial flow and species transport.

A skeletal muscle model of extreme hypertrophic growth reveals the influence of diffusion on cellular design

Muscle fibers that power swimming in the blue crab Callinectes sapidus are <80 microm in diameter in juveniles but grow hypertrophically, exceeding 600 microm in adults. Therefore, intracellular diffusion distances become progressively greater as the animals grow and, in adults, vastly exceed those in most cells. This developmental trajectory makes C. sapidus an excellent model for characterization of the influence of diffusion on fiber structure. The anaerobic light fibers, which power burst swimming, undergo a prominent shift in organelle distribution with growth. Mitochondria, which require O2 and rely on the transport of small, rapidly diffusing metabolites, are evenly distributed throughout the small fibers of juveniles, but in the large fibers of adults they are located almost exclusively at the fiber periphery where O2 concentrations are high. Nuclei, which do not require O2, but rely on the transport of large, slow-moving macromolecules, have the inverse pattern: they are distributed peripherally in small fibers but are evenly distributed across the large fibers, thereby reducing diffusion path lengths for large macromolecules. The aerobic dark fibers, which power endurance swimming, have evolved an intricate network of cytoplasmically isolated, highly perfused subdivisions that create the short diffusion distances needed to meet the high aerobic ATP turnover demands of sustained contraction. However, fiber innervation patterns are the same in the dark and light fibers. Thus the dark fibers appear to have disparate functional units for metabolism (fiber subdivision) and contraction (entire fiber). Reaction-diffusion mathematical models demonstrate that diffusion would greatly constrain the rate of metabolic processes without these developmental changes in fiber structure.

Metabolic influences of fiber size in aerobic and anaerobic locomotor muscles of the blue crab, Callinectes sapidus.

SUMMARY Diameters of some white locomotor muscle fibers in the adult blue crab, Callinectes sapidus, exceed 500 μm whereas juvenile white fibers are <100 μm. It was hypothesized that aerobically dependent processes, such as metabolic recovery following burst contractions, will be significantly impeded in the large white fibers. In addition, dark aerobic fibers of adults, which rely on aerobic metabolism for both contraction and recovery, grow as large as the white fibers. These large aerobic fibers are subdivided, however, thus decreasing the effective diameter of each metabolic functional unit and enabling aerobic contraction. The two goals of this study were: (1) to characterize the development of subdivisions in the dark levator muscle fibers and (2) to monitor post-contractile metabolism as a function of fiber size in aerobic and anaerobic levator muscles. Dark levator muscle fibers from crabs ranging from <0.1 g to >190 g were examined with transmission electron microscopy to determine the density of mitochondria and subdivision diameters. Across all size classes, there was a constant mitochondrial fractional area (25% of the total subdivision area) and subdivision size (mean diameter of 36.5±2.7 μm). Thus, blue crab dark levator fibers are unusual in having metabolic functional units (subdivisions) that do not increase in size during development while the contractile functional units (fibers) grow hypertrophically. The body mass scaling of post-contractile lactate dynamics was monitored during recovery from anaerobic, burst exercise in white and dark muscle, and in hemolymph. There were no differences among size classes in lactate accumulation during exercise in either muscle. However, in white fibers from large crabs, lactate continued to increase after exercise, and lactate removal from tissues required a much longer period of time relative to smaller crabs. Differences in lactate removal among size classes were less pronounced in dark fibers, and post-contractile lactate accumulation was significantly higher in white than in dark fibers from large animals. These data suggest that the large white fibers invoke anaerobic metabolism following contraction to accelerate certain phases of metabolic recovery that otherwise would be overly slow. This implies that, in addition to the typical mass-specific decrease in oxidative capacity that accompanies increases in animal mass, aerobic metabolic processes become increasingly limited by surface area to volume and intracellular diffusion constraints in developing white muscle fibers.

Morphology of the Melon and Its Tendinous Connections to the Facial Muscles in Bottlenose Dolphins (Tursiops truncatus)

The melon is a lipid‐rich structure located in the forehead of odontocetes that functions to propagate echolocation sounds into the surrounding aquatic environment. To date, the melons ability to guide and impedance match biosonar sounds to seawater has been attributed to its unique fatty acid composition. However, the melon is also acted upon by complex facial muscles derived from the m. maxillonasolabialis. The goal of this study was to investigate the gross morphology of the melon in bottlenose dolphins (Tursiops truncatus) and to describe how it is tendinously connected to these facial muscles. Standard gross dissection (N = 8 specimens) and serial sectioning (N = 3 specimens) techniques were used to describe the melon and to identify its connections to the surrounding muscles and blubber in three orthogonal body planes. The dolphin forehead was also thin‐sectioned in three body planes (N = 3 specimens), and polarized light was used to reveal the birefringent collagen fibers within and surrounding the melon. This study identified distinct regions of the melon that vary in shape and display locally specific muscle‐tendon morphologies. These regions include the bilaterally symmetric main body and cone and the asymmetric right and left caudal melon. This study is the first to identify that each caudal melon terminates in a lipid cup that envelopes the echolocation sound generators. Facial muscles of the melon have highly organized tendon populations that traverse the melon and insert into either the surrounding blubber, the connective tissue matrix of the nasal plug, or the connective tissue sheath surrounding the sound generators. The facial muscles and tendons also lie within multiple orthogonal body planes, which suggest that the melon is capable of complex shape change. The results of this study suggest that these muscles could function to change the frequency, beam width, and directionality of the emitted sound beam in bottlenose dolphins. The echolocation sound propagation pathway within the dolphin forehead appears to be a tunable system. J. Morphol., 2008.

Postecdysial changes in the protein and glycoprotein composition of the cuticle of the blue crab Callinectes sapidus

ABSTRACT Postecdysial modifications of the cuticle of Callinectes sapidus were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of soluble protein extracts. Gels stained with Coomassie blue and with silver demonstrated that major changes in protein composition or mobility occurred immediately after ecdysis. Electroblots of SDS-PAGE gels were probed with various biotinylated lectins. The patterns of lectin binding were also markedly different between blots of pre- and postecdysial cuticle extracts, demonstrating a change in glycosylation or glycoprotein mobility in the early postecdysial period. The dramatic alteration in cuticular composition is concurrent with the onset of sclerotization and mineralization of the preexuvially deposited layers of the cuticle.

Lectin Binding by Crustacean Cuticle: The Cuticle of Callinectes Sapidus Throughout the Molt Cycle, and the Intermolt Cuticle of Procambarus Clarkii and Ocypode Quadrata

ABSTRACT Pieces of dorsal carapace from the crayfish Procambarus clarkii, the ghost crab Ocypode quadrata, and the blue crab Callinectes sapidus were fixed in Rossmans fluid or alcoholic Formalin, decalcified, paraffin-embedded, and probed with up to 21 fluorescein-conjugated lectins in order to determine the composition and distribution of their carbohydrate moieties. Unfixed, frozen sections were also employed. Tissue was examined from the crayfish and the ghost crab at intermolt (C4), and from the blue crab at premolt (all D stages), ecdysis, postmolt (all A and B stages, and stage C,) and intermolt. Different lectins showed different binding patterns at intermolt, but patterns of binding for individual lectins were consistent among the different species. A number of lectins showed different binding patterns at different stages of the molt cycle. Lectin binding was influenced by the method of fixation, with Rossmans-fixed tissue binding more lectins and more intensely. The overall pattern of lectin binding indicated that different carbohydrate moieties exist in the different layers of the integument, and that the availability of these lectin-binding sites may change over the molt cycle.

Novel locomotor muscle design in extreme deep-diving whales

SUMMARY Most marine mammals are hypothesized to routinely dive within their aerobic dive limit (ADL). Mammals that regularly perform deep, long-duration dives have locomotor muscles with elevated myoglobin concentrations that are composed of predominantly large, slow-twitch (Type I) fibers with low mitochondrial volume densities (Vmt). These features contribute to extending ADL by increasing oxygen stores and decreasing metabolic rate. Recent tagging studies, however, have challenged the view that two groups of extreme deep-diving cetaceans dive within their ADLs. Beaked whales (including Ziphius cavirostris and Mesoplodon densirostris) routinely perform the deepest and longest average dives of any air-breathing vertebrate, and short-finned pilot whales (Globicephala macrorhynchus) perform high-speed sprints at depth. We investigated the locomotor muscle morphology and estimated total body oxygen stores of several species within these two groups of cetaceans to determine whether they (1) shared muscle design features with other deep divers and (2) performed dives within their calculated ADLs. Muscle of both cetaceans displayed high myoglobin concentrations and large fibers, as predicted, but novel fiber profiles for diving mammals. Beaked whales possessed a sprinters fiber-type profile, composed of ~80% fast-twitch (Type II) fibers with low Vmt. Approximately one-third of the muscle fibers of short-finned pilot whales were slow-twitch, oxidative, glycolytic fibers, a rare fiber type for any mammal. The muscle morphology of beaked whales likely decreases the energetic cost of diving, while that of short-finned pilot whales supports high activity events. Calculated ADLs indicate that, at low metabolic rates, both beaked and short-finned pilot whales carry sufficient onboard oxygen to aerobically support their dives.

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

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