Ultrastructural, material and crystallographic description of endophytic masses - A possible damage response in shark and ray tessellated calcified cartilage.

The cartilaginous endoskeletons of elasmobranchs (sharks and rays) are reinforced superficially by minute, mineralized tiles, called tesserae. Unlike the bony skeletons of other vertebrates, elasmobranch skeletons have limited healing capability and their tissues' mechanisms for avoiding damage or managing it when it does occur are largely unknown. Here we describe an aberrant type of mineralized elasmobranch skeletal tissue called endophytic masses (EPMs), which grow into the uncalcified cartilage of the skeleton, but exhibit a strikingly different morphology compared to tesserae and other elasmobranch calcified tissues. We use materials and biological tissue characterization techniques, including computed tomography, electron and light microscopy, X-ray and Raman spectroscopy and histology to characterize the morphology, ultrastructure and chemical composition of tesserae-associated EPMs in different elasmobranch species. EPMs appear to develop between and in intimate association with tesserae, but lack the lines of periodic growth and varying mineral density characteristic of tesserae. EPMs are mineral-dominated (high mineral and low organic content), comprised of birefringent bundles of large calcium phosphate crystals (likely brushite) aligned end to end in long strings. Both tesserae and EPMs appear to develop in a type-2 collagen-based matrix, but in contrast to tesserae, all chondrocytes embedded or in contact with EPMs are dead and mineralized. The differences outlined between EPMs and tesserae demonstrate them to be distinct tissues. We discuss several possible reasons for EPM development, including tissue reinforcement, repair, and disruptions of mineralization processes, within the context of elasmobranch skeletal biology as well as damage responses of other vertebrate mineralized tissues.

Feeding biomechanics of the cownose ray, Rhinoptera bonasus, over ontogeny.

Growth affects the performance of structure, so the pattern of growth must influence the role of a structure and an organism. Because animal performance is linked to morphological specialization, ontogenetic change in size may influence an organism's biological role. High bite force generation is presumably selected for in durophagous taxa. Therefore, these animals provide an excellent study system for investigating biomechanical consequences of growth on performance. An ontogenetic series of 27 cownose rays (Rhinoptera bonasus) were dissected in order to develop a biomechanical model of the feeding mechanism, which was then compared with bite forces measured from live rays. Mechanical advantage of the feeding apparatus was generally conserved throughout ontogeny, while an increase in the mass and cross-sectional area of the jaw adductors resulted in allometric gains in bite force generation. Of primary importance to forceful biting in this taxon is the use of a fibrocartilaginous tendon associated with the insertion of the primary jaw adductor division. This tendon may serve to redirect muscle forces anteriorly, transmitting them within the plane of biting. Measured bite forces obtained through electrostimulation of the jaw adductors in live rays were higher than predicted, possibly due to differences in specific tension of actual batoid muscle and that used in the model. Mass-specific bite forces in these rays are the highest recorded for elasmobranchs. Cownose rays exemplify a species that, through allometric growth of bite performance and morphological novelties, have expanded their ecological performance over ontogeny.

Feeding performance of king Mackerel, Scomberomorus cavalla.

Feeding performance is an organism's ability to capture and handle prey. Although bite force is a commonly used metric of feeding performance, other factors such as bite pressure and strike speed are also likely to affect prey capture. Therefore, this study investigated static bite force, dynamic speeds, and predator and prey forces resulting from ram strikes, as well as bite pressure of the king mackerel, Scomberomorus cavalla, in order to examine their relative contributions to overall feeding performance. Theoretical posterior bite force ranged from 14.0-318.7 N. Ram speed, recorded with a rod and reel incorporated with a line counter and video camera, ranged from 3.3-15.8B L/s. Impact forces on the prey ranged from 0.1-1.9 N. Bite pressure, estimated using theoretical bite forces at three gape angles and tooth cross-sectional areas, ranged from 1.7-56.9 MPa. Mass-specific bite force for king mackerel is relatively low in comparison with other bony fishes and sharks, with relatively little impact force applied to the prey during the strike. This suggests that king mackerel rely on high velocity chases and high bite pressure generated via sharp, laterally compressed teeth to maximize feeding performance.

Feeding in billfishes: inferring the role of the rostrum from a biomechanical standpoint.

Perhaps the most striking feature of billfishes is the extreme elongation of the premaxillary bones forming their rostra. Surprisingly, the exact role of this structure in feeding is still controversial. The goal of this study is to investigate the use of the rostrum from a functional, biomechanical and morphological standpoint to ultimately infer its possible role during feeding. Using beam theory, experimental and theoretical loading tests were performed on the rostra from two morphologically different billfish, the blue marlin (Makaira nigricans) and the swordfish (Xiphias gladius). Two loading regimes were applied (dorsoventral and lateral) to simulate possible striking behaviors. Histological samples and material properties of the rostra were obtained along their lengths to further characterize structure and mechanical performance. Intraspecific results show similar stress distributions for most regions of the rostra, suggesting that this structure may be designed to withstand continuous loadings with no particular region of stress concentration. Although material stiffness increased distally, flexural stiffness increased proximally owing to higher second moment of area. The blue marlin rostrum was stiffer and resisted considerably higher loads for both loading planes compared with that of the swordfish. However, when a continuous load along the rostrum was considered, simulating the rostrum swinging through the water, swordfish exhibited lower stress and drag during lateral loading. Our combined results suggest that the swordfish rostrum is suited for lateral swiping to incapacitate their prey, whereas the blue marlin rostrum is better suited to strike prey from a wider variety of directions.

Myological variability in a decoupled skeletal system: batoid cranial anatomy.

Chondrichthyans (sharks, batoids, and chimaeras) have simple feeding mechanisms owing to their relatively few cranial skeletal elements. However, the indirect association of the jaws to the cranium (euhyostylic jaw suspension) has resulted in myriad cranial muscle rearrangements of both the hyoid and mandibular elements. We examined the cranial musculature of an abbreviated phylogenetic representation of batoid fishes, including skates, guitarfishes and with a particular focus on stingrays. We identified homologous muscle groups across these taxa and describe changes in gross morphology across developmental and functional muscle groups, with the goal of exploring how decoupling of the jaws from the skull has effected muscular arrangement. In particular, we focus on the cranial anatomy of durophagous and nondurophagous batoids, as the former display marked differences in morphology compared to the latter. Durophagous stingrays are characterized by hypertrophied jaw adductors, reliance on pennate versus fusiform muscle fiber architecture, tendinous rather than aponeurotic muscle insertions, and an overall reduction in mandibular kinesis. Nondurophagous stingrays have muscles that rely on aponeurotic insertions onto the skeletal structure, and display musculoskeletal specialization for jaw protrusion and independent lower jaw kinesis, relative to durophagous stingrays. We find that among extant chondrichthyans, considerable variation exists in the hyoid and mandibular muscles, slightly less so in hypaxial muscles, whereas branchial muscles are overwhelmingly conserved. As chondrichthyans occupy a position sister to all other living gnathostomes, our understanding of the structure and function of early vertebrate feeding systems rests heavily on understanding chondrichthyan cranial anatomy. Our findings highlight the incredible variation in muscular complexity across chondrichthyans in general and batoids in particular.

Mechanical properties of sand tiger shark (Carcharias taurus) vertebrae in relation to spinal deformity.

Approximately 35% of sand tiger sharks (Carcharias taurus) in public aquaria exhibit spinal deformities ranging from compressed vertebrae and loss of intervertebral space to dislocated spines with vertebral degeneration and massive spondylosis caused by excessive mineralization both within vertebrae and outside the notochordal sheath. To identify the mechanical basis of these deformities, vertebral centra from affected (N=12) and non-affected (N=9) C. taurus were subjected to axial compression tests on an MTS 858 Bionix material testing system, after which mineral content was determined. Vertebral centra from affected sharks had significantly lower mineral content and material behavior in nearly all variables characterizing elasticity, plasticity and failure. These mechanical deficiencies are correlated with size at capture, capture method, vitamin C and zinc deficiency, aquarium size and swimming behavior in public aquaria. Non-affected C. taurus had greater stiffness and toughness even though these properties are generally incompatible in mineralized structures, suggesting that the biphasic (mineralized, unmineralized phases) nature of chondrichthyan vertebrae yields material behavior not otherwise observed in vertebrate skeletons. However, vertebral centra from non-affected sharks had lower mineral content (33%), stiffness (167 MPa), yield strain (14%) and ultimate strength (16 MPa) than other species of sharks and bony vertebrates, indicating that biomechanical precautions must be taken in the husbandry of this species.

Feeding biomechanics and theoretical calculations of bite force in bull sharks (Carcharhinus leucas) during ontogeny.

Evaluations of bite force, either measured directly or calculated theoretically, have been used to investigate the maximum feeding performance of a wide variety of vertebrates. However, bite force studies of fishes have focused primarily on small species due to the intractable nature of large apex predators. More massive muscles can generate higher forces and many of these fishes attain immense sizes; it is unclear how much of their biting performance is driven purely by dramatic ontogenetic increases in body size versus size-specific selection for enhanced feeding performance. In this study, we investigated biting performance and feeding biomechanics of immature and mature individuals from an ontogenetic series of an apex predator, the bull shark, Carcharhinus leucas (73-285cm total length). Theoretical bite force ranged from 36 to 2128N at the most anterior bite point, and 170 to 5914N at the most posterior bite point over the ontogenetic series. Scaling patterns differed among the two age groups investigated; immature bull shark bite force scaled with positive allometry, whereas adult bite force scaled isometrically. When the bite force of C. leucas was compared to those of 12 other cartilaginous fishes, bull sharks presented the highest mass-specific bite force, greater than that of the white shark or the great hammerhead shark. A phylogenetic independent contrast analysis of anatomical and dietary variables as determinants of bite force in these 13 species indicated that the evolution of large adult bite forces in cartilaginous fishes is linked predominantly to the evolution of large body size. Multiple regressions based on mass-specific standardized contrasts suggest that the evolution of high bite forces in Chondrichthyes is further correlated with hypertrophication of the jaw adductors, increased leverage for anterior biting, and widening of the head. Lastly, we discuss the ecological significance of positive allometry in bite force as a possible "performance gain" early in the life history of C. leucas.

Correlations of capture, transport, and nutrition with spinal deformities in sandtiger sharks, Carcharias taurus, in public aquaria.

A number of captive sandtiger sharks (Carcharias taurus) in public aquaria have developed spinal deformities over the past decade, ranging in severity from mild curvature to spinal fracture and severe subluxation. To determine the frequency and etiologic basis of this disease, U.S. public aquaria participated in a two-stage epidemiologic study of resident sharks: 1) a history and husbandry survey and 2) hematology, clinical chemistry, and radiography conducted during health exams. Eighteen aquaria submitted data, samples, or both from 73 specimens, including 19 affected sharks (26%). Sharks caught off the Rhode Island coast or by pound net were smaller at capture and demonstrated a higher prevalence of deformity than did larger sharks caught from other areas via hook and line. Relative to healthy sharks, affected sharks were deficient in zinc, potassium, and vitamins C and E. Capture and transport results lead to two likely etiologic hypotheses: 1) that the pound-net capture process induces spinal trauma that becomes exacerbated over time in aquarium environments or 2) that small (and presumably young) sharks caught by pound net are exposed to disease-promoting conditions (including diet or habitat deficiencies) in aquaria during the critical growth phase of their life history. The last hypothesis is further supported by nutrient deficiencies among affected sharks documented in this study; potassium, zinc, and vitamin C play critical roles in proper cartilage-collagen development and maintenance. These correlative findings indicate that public aquaria give careful consideration to choice of collection methods and size at capture and supplement diets to provide nutrients required for proper development and maintenance of cartilaginous tissue.

Bite force and performance in the durophagous bonnethead shark, Sphyrna tiburo.

Bite force, a measure of performance, can be used to link anatomical form and function. Earlier studies have shown bite force to have a significant influence on dietary constraints and ontogenetic shifts in resource utilization. The bonnethead shark, Sphyrna tiburo, is a durophagous member of the family Sphyrnidae. Its diet in South Florida waters consists almost entirely of blue crabs, which are crushed or ingested whole. This abundant coastal predator's feeding mechanism is specialized for the consumption of hard prey, including a modified biting pattern and molariform teeth. The goals of this research were to (1) characterize the mechanical function of the feeding mechanism of S. tiburo through biomechanical modeling of biting and in vivo bite force measurements; (2) compare the bite force of S. tiburo with those of other fishes; and (3) identify functional constraints on prey capture by comparing the bite force of S. tiburo with the fracture properties of its primary prey item, blue crabs. Maximum theoretical bite force ranged from 25.7 N anteriorly to 107.9 N posteriorly. S. tiburo has the second lowest mass specific bite force for any fish studied to date, and its posterior mechanical advantage of 0.88 is lower than other durophagous chondrichthyans, indicating that this independent evolutionary acquisition of durophagy was not accompanied by the associated morphological changes found in other durophagous cartilaginous fishes. Blue crab fracture forces (30.0-490.0 N) range well above the maximum bite force of S. tiburo, suggesting that prey material properties functionally constrain dietary ecology to some degree.

Scaling of feeding biomechanics in the horn shark Heterodontus francisci: ontogenetic constraints on durophagy.

Organismal performance changes over ontogeny as the musculoskeletal systems underlying animal behavior grow in relative size and shape. As performance is a determinant of feeding ecology, ontogenetic changes in the former can influence the latter. The horn shark Heterodontus francisci consumes hard-shelled benthic invertebrates, which may be problematic for younger animals with lower performance capacities. Scaling of feeding biomechanics was investigated in H. francisci (n=16, 19-59cm standard length (SL)) to determine the biomechanical basis of allometric changes in feeding performance and whether this performance capacity constrains hard-prey consumption over ontogeny. Positive allometry of anterior (8-163N) and posterior (15-382N) theoretical bite force was attributed to positive allometry of cross-sectional area in two jaw adducting muscles and mechanical advantage at the posterior bite point (0.79-1.26). Mechanical advantage for anterior biting scaled isometrically (0.52). Fracture forces for purple sea urchins Strongylocentrotus purpuratus consumed by H. francisci ranged from 24 to 430N. Comparison of these fracture forces to the bite force of H. francisci suggests that H. francisci is unable to consume hard prey early in its life history, but can consume the majority of S. purpuratus by the time it reaches maximum size. Despite this constraint, positive allometry of biting performance appears to facilitate an earlier entry into the durophagous niche than would an isometric ontogenetic trajectory. The posterior gape of H. francisci is significantly smaller than the urchins capable of being crushed by its posterior bite force. Thus, the high posterior bite forces of H. francisci cannot be fully utilized while consuming prey of similar toughness and size to S. purpuratus, and its potential trophic niche is primarily determined by anterior biting capacity.

Is extreme bite performance associated with extreme morphologies in sharks?

As top predators in many oceanic communities, sharks are known to eat large prey and are supposedly able to generate high bite forces. This notion has, however, largely gone untested due to the experimental intractability of these animals. For those species that have been investigated, it remains unclear whether their high bite forces are simply a consequence of their large body size or the result of diet-related adaptation. As aquatic poikilotherms, sharks can grow very large, making them ideal subjects with which to investigate the effects of body size on bite force. Relative bite-force capacity is often associated with changes in head shape because taller or wider heads can, for example, accommodate larger jaw muscles. Constraints on bite force in general may also be released by changes in tooth shape. For example, more pointed teeth may allow a predator to penetrate prey more effectively than blunt, pavementlike teeth. Our analyses show that large sharks do not bite hard for their body size, but they generally have larger heads. Head width is the best predictor of bite force across the species included in our study as indicated by a multiple regression model. Contrary to our predictions, sharks with relatively high bite forces for their body size also have relatively more pointed teeth at the front of the tooth row. Moreover, species including hard prey in their diet are characterized by high bite forces and narrow and pointed teeth at the jaw symphysis.

Functional morphology of the feeding apparatus, feeding constraints, and suction performance in the nurse shark Ginglymostoma cirratum.

The nurse shark, Ginglymostoma cirratum, is an obligate suction feeder that preys on benthic invertebrates and fish. Its cranial morphology exhibits a suite of structural and functional modifications that facilitate this mode of prey capture. During suction-feeding, subambient pressure is generated by the ventral expansion of the hyoid apparatus and the floor of its buccopharyngeal cavity. As in suction-feeding bony fishes, the nurse shark exhibits expansive, compressive, and recovery kinematic phases that produce posterior-directed water flow through the buccopharyngeal cavity. However, there is generally neither a preparatory phase nor cranial elevation. Suction is generated by the rapid depression of the buccopharyngeal floor by the coracoarcualis, coracohyoideus, and coracobranchiales muscles. Because the hyoid arch of G. cirratum is loosely connected to the mandible, contraction of the rectus cervicis muscle group can greatly depress the floor of the buccopharyngeal cavity below the depressed mandible, resulting in large volumetric expansion. Suction pressures in the nurse shark vary greatly, but include the greatest subambient pressures reported for an aquatic-feeding vertebrate. Maximum suction pressure does not appear to be related to shark size, but is correlated with the rate of buccopharyngeal expansion. As in suction-feeding bony fishes, suction in the nurse shark is only effective within approximately 3 cm in front of the mouth. The foraging behavior of this shark is most likely constrained to ambushing or stalking due to the exponential decay of effective suction in front of the mouth. Prey capture may be facilitated by foraging within reef confines and close to the substrate, which can enhance the effective suction distance, or by foraging at night when it can more closely approach prey.

Hard prey, soft jaws and the ontogeny of feeding mechanics in the spotted ratfish Hydrolagus colliei.

The spotted ratfish Hydrolagus colliei is a holocephalan fish that consumes hard prey (durophagy) but lacks many morphological characters associated with durophagy in other cartilaginous fishes. We investigated its feeding biomechanics and biting performance to determine whether it can generate bite forces comparable with other durophagous elasmobranchs, how biting performance changes over ontogeny (21-44 cm SL) and whether biomechanical modelling can accurately predict feeding performance in holocephalans. Hydrolagus colliei can generate absolute and mass-specific bite forces comparable with other durophagous elasmobranchs (anterior=104 N, posterior=191 N) and has the highest jaw leverage of any cartilaginous fish studied. Modelling indicated that cranial geometry stabilizes the jaw joint by equitably distributing forces throughout the feeding mechanism and that positive allometry of bite force is due to hyperallometric mechanical advantage. However, bite forces measured through tetanic stimulation of the adductor musculature increased isometrically. The jaw adductors of H. colliei fatigued more rapidly than those of the piscivorous spiny dogfish Squalus acanthias as well. The feeding mechanism of H. colliei is a volume-constrained system in which negative allometry of cranial dimensions leaves relatively less room for musculature. Jaw adductor force, however, is maintained through ontogenetic changes in muscle architecture.

Scaling of bite force in the blacktip shark Carcharhinus limbatus.

Although bite force is a frequently studied performance measure of feeding ecology, changes in bite force over ontogeny have rarely been investigated. Biting by the blacktip shark Carcharhinus limbatus was theoretically modeled over ontogeny to investigate the scaling of bite force, the morphological basis of the observed scaling relationship, the ecological consequences of ontogenetic changes in performance, and whether cranial morphometrics can be used as an accurate proxy for bite force. Theoretical bite force, which was positively allometric with respect to total length (TL), ranged from 32 N (61 cm TL) to 423 N (152 cm TL) at the anterior tips of the jaws and from 107 (61 cm TL) to 1083 N (152 cm TL) at the posterior teeth. This observation is attributed to positive allometry in the mechanical advantage of the jaw-adducting mechanism and the cross-sectional area of all four jaw-adducting muscles. Theoretical bite force was accurately predicted by cranial morphometrics including prebranchial length and head width as well. Although positive allometry of bite force in C. limbatus would seem to indicate an ecological necessity for this phenomenon, dietary analyses do not necessarily indicate any ontogenetic shift in prey types requiring larger bite forces. The positively allometric increase in theoretical bite force may be associated with numerous other selective pressures including maintenance of an apical position within the ecosystem.

Functional morphology of jaw trabeculation in the lesser electric ray Narcine brasiliensis, with comments on the evolution of structural support in the Batoidea.

The design of minimum-weight structures that retain their integrity under dynamic loading regimes has long challenged engineers. One solution to this problem found in both human and biological design is the optimization of weight and strength by hollowing a structure and replacing its inner core with supportive struts. In animals, this design is observed in sand dollar test, avian beak, and the cancellous bone of tetrapod limbs. Additionally, within the elasmobranch fishes, mineralized trabeculae (struts) have been reported in the jaws of durophagous myliobatid stingrays (Elasmobranchii: Batoidea), but were believed to be absent in basal members of the batoid clade. This study, however, presents an additional case of batoid trabeculation in the lesser electric ray, Narcine brasiliensis (Torpediniformes). The trabeculae in these species likely play different functional roles. Stingrays use their reinforced jaws to crush bivalves, yet N. brasiliensis feeds by ballistically protruding its jaws into the sediment to capture polychaetes. In N. brasiliensis, trabeculae are localized to areas likely to experience the highest load: the quadratomandibular jaw joints, hyomandibular-cranial joint, and the thinnest sections of the jaws immediately lateral to the symphyses. However, the supports perform different functions dependent on location. In regions where the jaws are loaded transversely (as in durophagous rays), "load leading" trabeculae distribute compressive forces from the cortex through the lumen of the jaws. In the parasymphyseal regions of the jaws, "truss" trabeculae form cross-braces perpendicular to the long axes of the jaws. At peak protrusion, the jaw arch is medially compressed and the jaw loaded axially such that these trabeculae are positioned to resist buckling associated with excavation forces. "Truss" trabeculae function to maintain the second moment of area in the thinnest regions of the jaws, illustrating a novel function for batoid trabeculation. Thus, this method of structural support appears to have arisen twice independently in batoids and performs strikingly different ecological functions associated with the distribution of extreme loading environments.

Analysis of the bite force and mechanical design of the feeding mechanism of the durophagous horn shark Heterodontus francisci.

Three-dimensional static equilibrium analysis of the forces generated by the jaw musculature of the horn shark Heterodontus francisci was used to theoretically estimate the maximum force distributions and loadings on its jaws and suspensorium during biting. Theoretical maximum bite force was then compared with bite forces measured (1) voluntarily in situ, (2) in restrained animals and (3) during electrical stimulation of the jaw adductor musculature of anesthetized sharks. Maximum theoretical bite force ranged from 128 N at the anteriormost cuspidate teeth to 338 N at the posteriormost molariform teeth. The hyomandibula, which connects the posterior margin of the jaws to the base of the chondrocranium, is loaded in tension during biting. Conversely, the ethmoidal articulation between the palatal region of the upper jaw and the chondrocranium is loaded in compression, even during upper jaw protrusion, because H. francisci's upper jaw does not disarticulate from the chondrocranium during prey capture. Maximum in situ bite force averaged 95 N for free-swimming H. francisci, with a maximum of 133 N. Time to maximum force averaged 322 ms and was significantly longer than time away from maximum force (212 ms). Bite force measurements from restrained individuals (187 N) were significantly greater than those from free-swimming individuals (95 N) but were equivalent to those from both theoretical (128 N) and electrically stimulated measurements (132 N). The mean mass-specific bite of H. francisci was greater than that of many other vertebrates and second highest of the cartilaginous fishes that have been studied. Measuring bite force on restrained sharks appears to be the best indicator of maximum bite force. The large bite forces and robust molariform dentition of H. francisci correspond to its consumption of hard prey.

Differential expression of duplicated opsin genes in two eyetypes of ostracod crustaceans.

In the first molecular study of ostracod (Crustacea) vision, we present partial cDNA sequences of ostracod visual pigment genes (opsins). We found strong support for differential expression of opsins in ostracod median and compound eyes and suggest that photoreceptor specific expression may be a general phenomenon in organisms with multiple receptors. We infer that eye-specific expression predates the divergence of the two species examined, Skogsbergia lerneri and Vargula hilgendorfii, because eye-specific opsin orthologs are present in both species. We found multiple opsin loci in ostracods, estimating that at least eight are present in Skogsbergia lerneri. All opsins from both ostracod species examined are more closely related to each other than to any other known opsin sequences. Because we find no evidence for gene conversion or alternative splicing, we suggest the occurrence of many recent gene duplications. Why ostracods may have retained multiple recent opsin gene duplicates is unknown, but we discuss several possible hypotheses.