Woodpeckers minimize cranial absorption of shocks.

The skull of a woodpecker is hypothesized to serve as a shock absorber that minimizes the harmful deceleration of its brain upon impact into trees1-11 and has inspired the engineering of shock-absorbing materials12-15 and tools, such as helmets.16 However, this hypothesis remains paradoxical since any absorption or dissipation of the head's kinetic energy by the skull would likely impair the bird's hammering performance4 and is therefore unlikely to have evolved by natural selection. In vivo quantification of impact decelerations during pecking in three woodpecker species and biomechanical models now show that their cranial skeleton is used as a stiff hammer to enhance pecking performance, and not as a shock-absorbing system to protect the brain. Numerical simulations of the effect of braincase size and shape on intracranial pressure indicate that the woodpeckers' brains are still safe below the threshold of concussions known for primate brains. These results contradict the currently prevailing conception of the adaptive evolution of cranial function in one of nature's most spectacular behaviors. VIDEO ABSTRACT.

Selective Inner Hair Cell Loss in a Neonate Harbor Seal (Phoca vitulina).

Congenital hearing loss is recognized in humans and other terrestrial species. However, there is a lack of information on its prevalence or pathophysiology in pinnipeds. It is important to have baseline knowledge on marine mammal malformations in the inner ear, to differentiate between congenital and acquired abnormalities, which may be caused by infectious pathogens, age, or anthropogenic interactions, such as noise exposure. Ultrastructural evaluation of the cochlea of a neonate harbor seal (Phoca vitulina) by scanning electron microscopy revealed bilateral loss of inner hair cells with intact outer hair cells. The selective inner hair cell loss was more severe in the basal turn, where high-frequency sounds are encoded. The loss of inner hair cells started around 40% away from the apex or tip of the spiral, reaching a maximum loss of 84.6% of hair cells at 80-85% of the length from the apex. Potential etiologies and consequences are discussed. This is believed to be the first case report of selective inner hair cell loss in a marine mammal neonate, likely congenital.

Anatomical mechanism for protecting the airway in the largest animals on earth.

Separation of respiratory and digestive tracts in the mammalian pharynx is critical for survival. Food must be kept out of the respiratory tract, and air must be directed into the respiratory tract when breathing.1 Cetaceans have the additional problem of feeding while underwater. Lunge-feeding baleen whales (rorquals) open the mouth while swimming at high speeds to engulf a volume of prey-laden water as large as their own body2 and experience tremendous forces as water floods the mouth. How the respiratory tract is protected in the pharynx during engulfment and while swallowing a massive slurry of tiny living prey remains unknown, despite its importance to survival. By dissecting adult and fetal fin whales, we determined that a large musculo-fatty structure passively seals the oropharyngeal channel. This "oral plug" is not observed in other animals, and its position indicates it must be shifted to allow swallowing; it is a part of the soft palate and can only shift posteriorly and dorsally. Elevation of the oral plug allows food transfer to the pharynx and protects the upper airways from food entry. The laryngeal inlet in the floor of the pharynx is sealed by laryngeal cartilages, and the muscular laryngeal sac moves upward into the laryngeal cavity, completely occluding the airway. The pharynx is dedicated to the digestive tract during swallowing, with no connection between upper and lower airways. These adaptations to facilitate swallowing were a critical development in the evolution of large body size in these, the largest animals on earth.

Cochlear apical morphology in toothed whales: Using the pairing hair cell-Deiters' cell as a marker to detect lesions.

The apex or apical region of the cochlear spiral within the inner ear encodes for low-frequency sounds. The disposition of sensory hair cells on the organ of Corti is largely variable in the apical region of mammals, and it does not necessarily follow the typical three-row pattern of outer hair cells (OHCs). As most underwater noise sources contain low-frequency components, we expect to find most lesions in the apical region of the cochlea of toothed whales, in cases of permanent noise-induced hearing loss. To further understand how man-made noise might affect cetacean hearing, there is a need to describe normal morphological features of the apex and document interspecific anatomic variations in cetaceans. However, distinguishing between apical normal variability and hair cell death is challenging. We describe anatomical features of the organ of Corti of the apex in 23 ears from five species of toothed whales (harbor porpoise Phocoena phocoena, spinner dolphin Stenella longirostris, pantropical spotted dolphin Stenella attenuata, pygmy sperm whale Kogia breviceps, and beluga whale Delphinapterus leucas) by scanning electron microscopy and immunofluorescence. Our results showed an initial region where the lowest frequencies are encoded with two or three rows of OHCs, followed by the typical configuration of three OHC rows and three rows of supporting Deiters' cells. Whenever two rows of OHCs were detected, there were usually only two corresponding rows of supporting Deiters' cells, suggesting that the number of rows of Deiters' cells is a good indicator to distinguish between normal and pathological features.

Evidence of Hearing Loss and Unrelated Toxoplasmosis in a Free-Ranging Harbour Porpoise (Phocoena phocoena).

Evidence of hearing impairment was identified in a harbour porpoise (Phocoena phocoena) on the basis of scanning electron microscopy. In addition, based on histopathology and immunohistochemistry, there were signs of unrelated cerebral toxoplasmosis. The six-year old individual live stranded on the Dutch coast at Domburg in 2016 and died a few hours later. The most significant gross lesion was multifocal necrosis and haemorrhage of the cerebrum. Histopathology of the brain revealed extensive necrosis and haemorrhage in the cerebrum with multifocal accumulations of degenerated neutrophils, lymphocytes and macrophages, and perivascular lymphocytic cuffing. The diagnosis of cerebral toxoplasmosis was confirmed by positive staining of protozoa with anti-Toxoplasma gondii antibodies. Tachyzoites were not observed histologically in any of the examined tissues. Ultrastructural evaluation of the inner ear revealed evidence of scattered loss of outer hair cells in a 290 µm long segment of the apical turn of the cochlea, and in a focal region of ~ 1.5 mm from the apex of the cochlea, which was compatible with noise-induced hearing loss. This is the first case of concurrent presumptive noise-induced hearing loss and toxoplasmosis in a free-ranging harbour porpoise from the North Sea.

Combining Cochlear Analysis and Auditory Evoked Potentials in a Beluga Whale With High-Frequency Hearing Loss.

Correlations between inner ear morphology and auditory sensitivity in the same individual are extremely difficult to obtain for stranded cetaceans. Animals in captivity and rehabilitation offer the opportunity to combine several techniques to study the auditory system and cases of hearing impairment in a controlled environment. Morphologic and auditory findings from two beluga whales (Delphinapterus leucas) in managed care are presented. Cochlear analysis of a 21-year-old beluga whale showed bilateral high-frequency hearing loss. Specifically, scanning electron microscopy of the left ear revealed sensory cell death in the first 4.9 mm of the base of the cochlea with scar formation. Immunofluorescence microscopy of the right ear confirmed the absence of hair cells and type I afferent innervation in the first 6.6 mm of the base of the cochlea, most likely due to an ischemia. Auditory evoked potentials (AEPs) measured 1.5 years prior this beluga's death showed a generalized hearing loss, being more pronounced in the high frequencies. This individual might have had a mixed hearing loss that would explain the generalized hearing impairment. Conversely, based on AEP evaluation, her mother had normal hearing and subsequent cochlear analysis did not feature any apparent sensorineural pathology. This is believed to be the first study to compare two cochlear analysis techniques and hearing sensitivity measurements from AEPs in cetaceans. The ability to combine morphological and auditory data is crucial to validate predictions of cochlear frequency maps based on morphological features. In addition, our study shows that these three complementary analysis techniques lead to comparable results, thus improving our understanding of how hearing impairment can be detected in stranding cases.

Correlating Cochlear Morphometrics from Parnell's Mustached Bat (Pteronotus parnellii) with Hearing.

Morphometric analysis of the inner ear of mammals can provide information for cochlear frequency mapping, a species-specific designation of locations in the cochlea at which different sound frequencies are encoded. Morphometric variation occurs in the hair cells of the organ of Corti along the cochlea, with the base encoding the highest frequency sounds and the apex encoding the lowest frequencies. Changes in cell shape and spacing can yield additional information about the biophysical basis of cochlear tuning mechanisms. Here, we investigate how morphometric analysis of hair cells in mammals can be used to predict the relationship between frequency and cochlear location. We used linear and geometric morphometrics to analyze scanning electron micrographs of the hair cells of the cochleae in Parnell's mustached bat (Pteronotus parnellii) and Wistar rat (Rattus norvegicus) and determined a relationship between cochlear morphometrics and their frequency map. Sixteen of twenty-two of the morphometric parameters analyzed showed a significant change along the cochlea, including the distance between the rows of hair cells, outer hair cell width, and gap width between hair cells. A multiple linear regression model revealed that nine of these parameters are responsible for 86.9 % of the variation in these morphometric data. Determining the most biologically relevant measurements related to frequency detection can give us a greater understanding of the essential biomechanical characteristics for frequency selectivity during sound transduction in a diversity of animals.

Echolocating Whales and Bats Express the Motor Protein Prestin in the Inner Ear: A Potential Marker for Hearing Loss.

Prestin is an integral membrane motor protein located in outer hair cells of the mammalian cochlea. It is responsible for electromotility and required for cochlear amplification. Although prestin works in a cycle-by-cycle mode up to frequencies of at least 79 kHz, it is not known whether or not prestin is required for the extreme high frequencies used by echolocating species. Cetaceans are known to possess a prestin coding gene. However, the expression and distribution pattern of the protein in the cetacean cochlea has not been determined, and the contribution of prestin to echolocation has not yet been resolved. Here we report the expression of the protein prestin in five species of echolocating whales and two species of echolocating bats. Positive labeling in the basolateral membrane of outer hair cells, using three anti-prestin antibodies, was found all along the cochlear spiral in echolocating species. These findings provide morphological evidence that prestin can have a role in cochlear amplification in the basolateral membrane up to 120-180 kHz. In addition, labeling of the cochlea with a combination of anti-prestin, anti-neurofilament, anti-myosin VI and/or phalloidin and DAPI will be useful for detecting potential recent cases of noise-induced hearing loss in stranded cetaceans. This study improves our understanding of the mechanisms involved in sound transduction in echolocating mammals, as well as describing an optimized methodology for detecting cases of hearing loss in stranded marine mammals.

Rorqual whale nasal plugs: protecting the respiratory tract against water entry and barotrauma.

The upper respiratory tract of rorquals, lunge-feeding baleen whales, must be protected against water incursion and the risk of barotrauma at depth, where air-filled spaces like the bony nasal cavities may experience high adverse pressure gradients. We hypothesize these two disparate tasks are accomplished by paired cylindrical nasal plugs that attach on the rostrum and deep inside the nasal cavity. Here, we present evidence that the large size and deep attachment of the plugs is a compromise, allowing them to block the nasal cavities to prevent water entry while also facilitating pressure equilibration between the nasal cavities and ambient hydrostatic pressure (Pamb) at depth. We investigated nasal plug behaviour using videos of rorquals surfacing, plug morphology from dissections, histology and MRI scans, and plug function by mathematically modelling nasal pressures at depth. We found each nasal plug has three structurally distinct regions: a muscular rostral region, a predominantly fatty mid-section and an elastic tendon that attaches the plug caudally. We propose muscle contraction while surfacing pulls the fatty sections rostrally, opening the nasal cavities to air, while the elastic tendons snap the plugs back into place, sealing the cavities after breathing. At depth, we propose Pamb pushes the fatty region deeper into the nasal cavities, decreasing air volume by about half and equilibrating nasal cavity to Pamb, preventing barotrauma. The nasal plugs are a unique innovation in rorquals, which demonstrate their importance and novelty during diving, where pressure becomes as important an issue as the danger of water entry.

Lunge Feeding in Rorqual Whales.

The largest animals are baleen filter feeders that exploit large aggregations of small-bodied plankton. Although this feeding mechanism has evolved multiple times in marine vertebrates, rorqual whales exhibit a distinct lunge filter feeding mode that requires extreme physiological adaptations-most of which remain poorly understood. Here, we review the biomechanics of the lunge feeding mechanism in rorqual whales that underlies their extraordinary foraging performance and gigantic body size.

Work loop dynamics of the pigeon (Columba livia) humerotriceps demonstrate potentially diverse roles for active wing morphing.

Control of wing shape is believed to be a key feature that allows most birds to produce aerodynamically efficient flight behaviors and high maneuverability. Anatomical organization of intrinsic wing muscles suggests specific roles for the different motor elements in wing shape modulation, but testing these hypothesized functions requires challenging measurements of muscle activation and strain patterns, and force dynamics. The wing muscles that have been best characterized during flight are the elbow muscles of the pigeon (Columba livia). In vivo studies during different flight modes revealed variation in strain profile, activation timing and duration, and contractile cycle frequency of the humerotriceps, suggesting that this muscle may alter wing shape in diverse ways. To examine the multifunction potential of the humerotriceps, we developed an in situ work loop approach to measure how activation duration and contractile cycle frequency affected muscle work and power across the full range of activation onset times. The humerotriceps produced predominantly net negative power, likely due to relatively long stimulus durations, indicating that it absorbs work, but the work loop shapes also suggest varying degrees of elastic energy storage and release. The humerotriceps consistently exhibited positive and negative instantaneous power within a single contractile cycle, across all treatments. When combined with previous in vivo studies, our results indicate that both within and across contractile cycles, the humerotriceps can dynamically shift among roles of actuator, brake, and stiff or compliant spring, based on activation properties that vary with flight mode.

Slick, Stretchy Fascia Underlies the Sliding Tongue of Rorquals.

The tongue of rorqual (balaenopterid) whales slides far down the throat into the expanded oral pouch as an enormous mouthful of water is engulfed during gulp feeding. As the tongue and adjacent oral floor expands and slides caudoventrally, it glides along a more superficial (outer) layer of ventral body wall musculature, just deep to the accordion-like ventral throat pleats. We hypothesize that this sliding movement of adjacent musculature is facilitated by a slick, stretchy layer of loose areolar connective tissue that binds the muscle fibers and reduces friction: fascia. Gross anatomical examination of the gular region of adult minke, fin, and humpback whales confirms the presence of a discrete, three-layered sublingual fascia interposed between adhering fasciae of the tongue and body wall. Histological analysis of this sublingual fascia reveals collagen and elastin fibers loosely organized in a random feltwork along with numerous fibroblasts in a watery extracellular matrix. Biomechanical testing of tissue samples in the field and laboratory, via machine-controlled or manual stretching, demonstrates expansion of the sublingual fascia and its three layers up to 250% beyond resting dimensions, with slightly more extension observed in anteroposterior (rather than mediolateral or oblique) stretching, and with the most superficial of the fascia's three layers. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc. Anat Rec, 302:735-744, 2019. © 2018 Wiley Periodicals, Inc.

Blood pressure in the Greenland shark as estimated from ventral aortic elasticity.

We conducted in vitro inflations of freshly excised ventral aortas of the Greenland shark, Somniosus microcephalus, and used pressure-diameter data to estimate the point of transition from high to low compliance, which has been shown to occur at the mean blood pressure in other vertebrates including fishes. We also determined the pressure at which the modulus of elasticity of the aorta reached 0.4 MPa, as occurs at the compliance transition in other species. From these analyses, we predict the average ventral aortic blood pressure in S. microcephalus to be about 2.3-2.8 kPa, much lower than reported for other sharks. Our results support the idea that this species is slow moving and has a relatively low aerobic metabolism. Histological investigation of the ventral aorta shows that elastic fibres are present in relatively low abundance and loosely connected, consistent with this aorta having high compliance at a relatively low blood pressure.

The caval sphincter in cetaceans and its predicted role in controlling venous flow during a dive.

A sphincter on the inferior vena cava can protect the heart of a diving mammal from overload when elevated abdominal pressures increase venous return, yet sphincters are reported incompetent or absent in some cetacean species. We previously hypothesized that abdominal pressures are elevated and pulsatile in fluking cetaceans, and that collagen is deposited on the diaphragm according to pressure levels to resist deformation. Here, we tested the hypothesis that cetaceans generating high abdominal pressures need a more robust sphincter than those generating low pressures. We examined diaphragm morphology in seven cetacean and five pinniped species. All odontocetes had morphologically similar sphincters despite large differences in collagen content, and mysticetes had muscle that could modulate caval flow. These findings do not support the hypothesis that sphincter structure correlates with abdominal pressures. To understand why a sphincter is needed, we simulated the impact of oscillating abdominal pressures on caval flow. Under low abdominal pressures, simulated flow oscillated with each downstroke. Under elevated pressures, a vascular waterfall formed, greatly smoothing flow. We hypothesize that cetaceans maintain high abdominal pressures to moderate venous return and protect the heart while fluking, and use their sphincters only during low-fluking periods when abdominal pressures are low. We suggest that pinnipeds, which do not fluke, maintain low abdominal pressures. Simulations also showed that retrograde oscillations could be transmitted upstream from the cetacean abdomen and into the extradural veins, with potentially adverse repercussions for the cerebral circulation. We propose that locomotion-generated pressures have influenced multiple aspects of the cetacean vascular system.

Parasites in the inner ear of harbour porpoise: cases from the North and Baltic Seas.

Peribullar sinuses of harbour porpoises Phocoena phocoena are parasitized with high prevalence by the nematode Stenurus minor. The effect of S. minor on the hearing ability of this species is still undetermined. Here, we review the occurrence of S. minor in the inner ear of harbour porpoises recovered from strandings in the North and Baltic Seas. In particular, we present the results from ears collected in German and Danish waters from 2002 to 2016 and from Dutch waters from 2010 to 2016. While the prevalence of S. minor in pterygoid and peribullar sinuses and tympanic cavity was high in harbour porpoises (66.67% in our cases), its prevalence in the cochlea was rare. Only 1 case out of 129 analysed by either histology, electron microscopy or immunofluorescence showed the presence of a nematode parasite morphologically consistent with S. minor at the most basal portion of the right cochlea. This individual also had severe haemorrhage along the right cochlear spiral, which was likely caused by ectopic S. minor migration. Although this animal might have had impaired hearing in the right ear, it was otherwise in good body condition with evidence of recent feeding. These findings highlight the need to study the effect of parasites on hearing, and other pathological changes that might impair appropriate processing of acoustic information.

Structure and Function in the Lunge Feeding Apparatus: Mechanical Properties of the Fin Whale Mandible.

The mandibles of rorqual whales are highly modified to support loads associated with lunge-feeding, a dynamic filter feeding mechanism that is characterized by rapid changes in gape angle and acceleration. Although these structures are the largest ossified elements in animals and an important part of the rorqual engulfment apparatus, details of internal structure are limited and no direct measurements of mechanical properties exist. Likewise, the forces that are sustained by the mandibles are unknown. Here we report on the structure and mechanical behavior of the mandible of an adult fin whale. A series of transverse sections were cut at locations along the entire length of a 3.6-m left mandible recovered post-mortem from a 16-m fin whale, and CT scanned to make density maps. Cored samples 6-8 mm in diameter were tested in compression to determine the Young's modulus and strength. In addition, wet density, dry density and mineral density were measured. Dense cortical bone occupies only a relatively narrow peripheral layer while much less dense and oil-filled trabecular bone occupies the rest. Mineral density of both types is strongly correlated with dry density and CT Hounsfield units. Compressive strength is strongly correlated with Young's modulus, while strength and stiffness are both correlated with mineral density. It appears that the superficial compact layer is the main load bearing element, and that the mandible is reinforced against dorso-vental flexion that would occur during the peak loads while feeding. Anat Rec, 300:1953-1962, 2017. © 2017 Wiley Periodicals, Inc.

The Functional Anatomy of Nerves Innervating the Ventral Grooved Blubber of Fin Whales (Balaenoptera Physalus).

Nerves that supply the floor of the oral cavity in rorqual whales are extensible to accommodate the dramatic changes in tissue dimensions that occur during "lunge feeding" in this group. We report here that the large nerves innervating the muscle component of the ventral grooved blubber (VGB) in fin whales are branches of cranial nerve VII (facial nerve). Therefore, the muscles of the VGB are homologous to second branchial arch derived muscles, which in humans include the muscles of "facial expression." We speculate, based on the presence of numerous foramina on the dorsolateral surface of the mandibular bones, that general sensation from the VGB likely is carried by branches of the mandibular division (V3) of cranial nerve V (trigeminal nerve), and that these small branches travel in the lipid-rich layer directly underlying the skin. We show that intercostal and phrenic nerves, which are not extensible, have a different wall and nerve core morphology than the large VGB nerves that are branches of VII. Although these VGB nerves are known to have two levels of waviness, the intercostal and phrenic nerves have only one in which the nerve fascicles in the nerve core are moderately wavy. In addition, the VGB nerves have inner and outer parts to their walls with numerous large elastin fibers in the outer part, whereas intercostal and phrenic nerves have single walls formed predominantly of collagen. Our results illustrate that overall nerve morphology depends greatly on location and the forces to which the structures are exposed. Anat Rec, 300:1963-1972, 2017. © 2017 Wiley Periodicals, Inc.

Controlling thoracic pressures in cetaceans during a breath-hold dive: importance of the diaphragm.

Internal pressures change throughout a cetacean's body during swimming or diving, and uneven pressures between the thoracic and abdominal compartments can affect the cardiovascular system. Pressure differentials could arise from ventral compression on each fluke downstroke or by a faster equilibration of the abdominal compartment with changing ambient ocean pressures compared with the thoracic compartment. If significant pressure differentials do develop, we would expect the morphology of the diaphragm to adapt to its in vivo loading. Here, we tested the hypothesis that significant pressure differentials develop between the thoracic and abdominal cavities in diving cetaceans by examining diaphragms from several cetacean and pinniped species. We found that: (1) regions of cetacean diaphragms possess subserosal collagen fibres that would stabilize the diaphragm against craniocaudal stretch; (2) subserosal collagen covers 5-60% of the thoracic diaphragm surface, and area correlates strongly with published values for swimming speed of each cetacean species (P<0.001); and (3) pinnipeds, which do not locomote by vertical fluking, do not possess this subserosal collagen. These results strongly suggest that this collagen is associated with loads experienced during a dive, and they support the hypothesis that diving cetaceans experience periods during which abdominal pressures significantly exceed thoracic pressures. Our results are consistent with the generation of pressure differentials by fluking and by different compartmental equilibration rates. Pressure differentials during diving would affect venous and arterial perfusion and alter transmural pressures in abdominal arteries.

Two Levels of Waviness Are Necessary to Package the Highly Extensible Nerves in Rorqual Whales.

Peripheral nerves are susceptible to stretch injury [1-4] and incorporate structural waviness at the level of the axons, fascicles, and nerve trunk to accommodate physiological increases in length [5, 6]. It is unknown whether there are limits to the amount of deformation that waviness can accommodate. In rorqual whales, a sub-group of baleen whales, nerves running through the ventral groove blubber (VGB) associated with the floor of the mouth routinely experience dramatically large deformations. In fact, some of these nerves more than double their length during lunge feeding and then recoil to a short, compressed state after each lunge [7-9]. It is unknown how these nerves have adapted to operate in both extended and recoiled states. Using micro-CT and mechanics, we have discovered that the VGB nerves from fin whales require two levels of waviness to prevent stretch damage in both extended and recoiled states. The entire nerve core itself is highly folded when recoiled and appears buckled. This folding provides slack for extension but unavoidably generates large stretches at the bends that could damage nerve fascicles within the core. The strain at the bends is minimized by the specific waveform adopted by the core [10, 11], while the existing bending strains are accommodated by a second level of waviness in the individual fascicles that avoids stretch of the fascicle itself. Structural hierarchy partitions the waviness between the two length scales, providing a mechanism to maintain total elongation while preventing the stretching of fascicles at the bends when recoiled.