Counteraction of urea destabilization of protein structure by methylamine osmoregulatory compounds of elasmobranch fishes

Intracellular fluids of marine elasmobranchs (sharks, skates and rays), holocephalans and the coelacanth contain urea at concentrations averaging 0.4m, high enough to significantly affect the structural and functional properties of many proteins. Also present in the cells of these fishes are a family of methylamine compounds, largely trimethylamine N-oxide with some betaine and sarcosine, and certain free amino acids, mainly beta-alanine and taurine, whose total concentration is approx. 0.2m. These methylamine compounds and amino acids have been found to be effective stabilizers of protein structure, and, at a 1:2 molar concentration ratio of these compounds to urea, perturbations of protein structure by urea are largely or fully offset. These counteracting effects of solutes on proteins are seen for: (1) thermal stability of protein secondary and tertiary structure (bovine ribonuclease); (2) the rate and extent of enzyme renaturation after acid denaturation (rabbit and shark lactate dehydrogenases); and (3) the reactivity of thiol groups of an enzyme (bovine glutamate dehydrogenase). Attaining osmotic equilibrium with seawater by these fishes has thus involved the selective accumulation of certain nitrogenous metabolites that individually have significant effects on protein structure, but that have virtually no net effects on proteins when these solutes are present at elasmobranch physiological concentrations. These experiments indicate that evolutionary changes in intracellular solute compositions as well as in protein amino acid sequences can have important roles in intracellular protein function.     

Ventricle morphology in pelagic elasmobranch fishes

Ventricle weights of the warm-bodied great white shark, Atlantic shortfin mako, and the common thresher shark (the latter presumed to be warm-bodied) are similar to those of ectothermic blue sharks, sandbar sharks, dusky sharks, tiger sharks and scalloped hammerhead sharks. Ventricle muscularity, as estimated by the ratio of cortical to spongy layer thickness, is almost twice as great in the former three species than in the latter elasmobranchs. Measurements of ventricular volumes suggest that the ventricles of the great white, Atlantic shortfin mako and common thresher sharks are better adapted to respond to demands for increases in cardiac output via increased heartbeat frequency in comparison with ectothermic species of shark.     

Urea-requiring lactate dehydrogenases of marine elasmobranch fishes

Summary The kinetic properties — apparentK _m of pyruvate, pyruvate inhibition pattern, and maximal velocity — of M₄ (skeletal muscle) lactate dehydrogenases of marine elasmobranch fishes resemble those of the homologous lactate dehydrogenases of non-elasmobranchs only when physiological concentrations of urea (approximately 400 mM) are present in the assay medium. Urea increases the apparentK _m of pyruvate to values typical of other vertebrates (Fig. 2), and reduces pyruvate inhibition to levels seen with other M₄-lactate dehydrogenases (Fig. 3). Urea reduces the activation enthalpy of the reaction, and increasesV _max at physiological temperatures (Fig. 4).The M₄-lactate dehydrogenase of the freshwater elasmobranch,Potamotrygon sp., resembles a teleost lactate dehydrogenase, i.e., although it is sensitive to urea, it does not require the presence of urea for the establishment of optimal kinetic properties.     

The unusual energy metabolism of elasmobranch fishes

The unusual energy metabolism of elasmobranchs is characterized by limited or absent fatty acid oxidation in cardiac and skeletal muscle and a great reliance on ketone bodies and amino acids as oxidative fuels in these tissues. Other extrahepatic tissues in elasmobranchs rely on ketone bodies and amino acids for aerobic energy production but, unlike muscle, also appear to possess a significant capacity to oxidize fatty acids. This organization of energy metabolism is reflected by relatively low plasma levels of non-esterified fatty acids (NEFA) and by plasma levels of the ketone body ß-hydroxybutyrate that are as high as those seen in fasted mammals. The preference for ketone body oxidation rather than fatty acid oxidation in muscle of elasmobranchs under routine conditions is opposite to the situation in teleosts and mammals. Carbohydrates appear to be utilized as a fuel source in elasmobranchs, similar to other vertebrates. Amino acid- and lipid-fueled ketogenesis in the liver, the lipid storage site in elasmobranchs, sustains the demand for ketone bodies as oxidative fuels. The liver also appears to export NEFA and serves a buoyancy role. The regulation of energy metabolism in elasmobranchs and the effects of environmental factors remain poorly understood. The metabolic organization of elasmobranchs was likely present in the common ancestor of the Chondrichthyes ca. 400 million years ago and, speculatively, it may reflect the ancestral metabolism of jawed vertebrates. We assess hypotheses for the evolution of the unusual energy metabolism of elasmobranchs and propose that the need to synthesize urea has influenced the utilization of ketone bodies and amino acids as oxidative fuels.     

Hepatic urea biosynthesis in the euryhaline elasmobranch Carcharhinus leucas

Plasma urea levels and hepatic urea production in the euryhaline bull shark, Carcharhinus leucas, acclimated to freshwater and seawater environments were measured. It was found that plasma urea concentration increased with salinity and that this increase was, in part, the result of a significant increase in hepatic production of urea. This study provides direct evidence that hepatic production of urea plays an important role in the osmoregulatory strategy of C. leucas.     

Endocrine cells in the gastric mucosa of elasmobranch fishes. An ultrastructural study

The endocrine cells of gastric mucosa of two elasmobranch species were studied by light and electron microscopy. Five cell types were identified in the fundic mucosa, four of which are of "open type". All of them show pleomorphic granules of variable size, except those of the type V cell which are round in shape and of comparatively small diameter. Six different cell types are found in the pyloric mucosa, all of "open type" except for type XI cells which appear to be "closed". Pyloric types VIII, IX, X and XI cells show similar structural characteristics as fundus types I, V, II and IV respectively. Silver impregnation was also used at both light and electron microscopical levels. No functional classification or analogies with other vertebrate gastric endocrine cells were attempted as these would be too speculative on the basis of ultrastructural characteristics only.     

Morpho-functional aspects of the hypothalanus-pituitary-gonadal axis of elasmobranch fishes

Synopsis The tetrapod hypothalamus-pars distalis axis contains a blood portal system. Contrarily, elasmobranchs appear to lack a direct vascular supply from the hypothalamus to the ventral lobe of the pituitary where gonadotropic activity resides. The hypothalamus contains GnRH immunoreactivity and GnRH causes an increase in plasma gonadal steroids, perhaps via ventral lobe stimulation. Therefore, the question arises as to how GnRH reaches the pituitary. We suggest that the general circulation route might be practicable. Indeed, in the plasma of the electric ray,Torpedo marmorata, a major early eluting form has been detected using high performance liquid chromatography coupled with region specific radioimmunoassay. The presence of GnRH in the blood may allow the molecule to reach the gonads and to act there by direct mechanisms. Intragonadal levels of steroids may have a paracrine and/or autocrine role in the regulation of steroidogenesis in the testis and in the development f specific germinal cell stages. Particularly, the zonated morphology of the testis supports the concept of a diverse environment for different spermatogenic stages. Finally, gonadal steroids may feed back to affect pituitary activity.     

Structure and topographic distribution of oral denticles in elasmobranch fishes

The placoid scales, or denticles, of the external epidermis of elasmobranchs are well known as a hard protective coat over the skin to reduce abrasion or as elements to reduce hydrodynamic drag. However, the structure and function of denticles within the oral cavity is uncertain. Using stereological and scanning electron microscopy, this study examines the structure and distribution of oral denticles in a range of elasmobranchs. Of the batoids analyzed, only members of the Rhinobatidae possessed oral denticles, with no denticles found in the members sampled in the Gymnuridae or Dasyatidae. In contrast, oral denticles were located in all the selachians examined, except for members of the Orectolobidae. Within the selachians, the denticles of the Carcharhinidae have a grooved surface and a central spine, which is angled toward the posterior of the mouth. These denticular adaptations are beneficial to reduce hydrodynamic drag, an advantage for these free-swimming species with ram ventilation. Alternatively, members of the Hemiscyllidae have broad bulbous denticles that often overlap, providing a hard surface to protect the epithelium from abrasion during the consumption of hard-bodied prey. The distribution and high number of oral denticles appears to spatially compromise the capacity for oral (taste) papillae to populate the oropharyngeal cavity but provides increased friction and grip on prey items as they are manipulated within the mouth.     

Urease-catalyzed urea synthesis.

The equilibrium of hydrolytic reactions can be shifted toward condensation by carrying out the reaction at low water concentration. The rate and yield of urease-catalyzed urea synthesis from (NH₄)₂CO₃ or NH₄HCO₃ has been examined as a function of water concentration (in mixtures with organic solvents), substrate and H⁺ concentration, and polarity of the nonaqueous component of the solvent. Similar effects of organic solvents are observed on the reaction rate in both directions; the results suggest that at least in some conditions the reaction proceeds through nonenzymically formed carbamate. The equilibrium concentration of urea, in 50% ($\text{v}\text{v}$) water, varies over 10-fold, depending on the nature of the nonaqueous component of the solvent; nonhydroxylic solvents such as acetone given the highest yield. Solubility measurements suggest that the interactions of the solvent mixtures with (NH₄)₂CO₃ (or carbamate), rather than urea, are responsible for the variations in urea yield. Activities of water and the ionic components of the equilibrium are strongly influenced by the nature of the nonaqueous component of the solvent, as well as its concentration.     

Hemolysis of elasmobranch erythrocytes in urea solutions: effect of temperature

Skate and stingray cells were shown to hemolyze in isosmotic solutions containing urea as the sole solute. The rate of urea penetration into these cells as determined by the rate of hemolysis is highly temperature dependent with a Q₁₀ of 2.50–2.75. A reduced rate of methylurea penetration in the presence of urea was reconfirmed. The present results are consonant with the hypothesis of carrier mediated transport of urea in elasmobranch erythrocytes previously proposed by Murdaugh, Robin and Hearn.     

Digenea and acanthocephala of elasmobranch fishes from the southern coast of Brazil.

New records for helminth species recovered from elasmobranch fishes in Brazil are established. Digenean and acanthocephalan parasites of elasmobranch fishes are reported from the southern coast of Brazil: Otodistomum veliporum (Creplin, 1837) Stafford, 1904 (Digenea: Azygiidae) in the stomach and spiral valve of Dipturus trachydermus and in the spiral valve of Squatina sp. Cystacanths and juveniles of the acanthocephalans Corynosoma australe Johnston, 1937 and Corynosoma sp., in the spiral valve of Squatina sp., Galeorhinus galeus and Hexanchus griseus and in the stomach of Squalus megalops; a juvenile of Gorgorhynchus sp., in the spiral valve of Sphyrna zygaena. Dipturus trachydermus and Squatina sp. are new host records for O. veliporum. Digeneans and acanthocephalans are reported for the first time parasitizing elasmobranch fishes in Brazil.     

Three new genera of trypanorhynch cestodes from Australian elasmobranch fishes

Three new genera of trypanorhynch cestodes from Australian elasmobranch fishes collected in the Arafura Sea, off the Northern Territory, are described. Fossobothrium perplexum n. g., n. sp. (Otobothriidae), from the spiral valves of Anoxypristis cuspidata (Latham) and Pristis zijsron Bleeker, is similar to the otobothriid genera Pseudotobothrium Dollfus, 1942 and Poecilancistrium Dollfus, 1929 in possessing bothrial pits and a band of hooks on the tentacle, but differs from all known otobothriid genera in having the pits joined by a prominent velum. Iobothrium elegans n. g., n. sp. (Otobothriidae), from the spiral valve of Himantura jenkinsi (Annandale), is placed in the Otobothriidae because it possesses bothrial pits, but differs from Otobothrium Linton, 1890 and other genera in lacking intercalary hooks between the principal rows and in possessing a chainette on the external surface of the tentacle in the metabasal region. Oncomegoides celatus n. g., n. sp. (Eutetrarhynchidae), from the spiral valve of Dasyatis microps (Annandale) and Himantura jenkinsi, resembles Oncomegas Dollfus, 1929 in possessing two bothria and a megahook on the bothrial surface of the basal armature, but differs in possessing an extra row consisting of four intercalary hooks formed by the overlapping of two intercalary hooks on the external tentacular surface between each of the opposing principal rows and is therefore an atypical heteroacanth.     

Electrosensitive spatial vectors in elasmobranch fishes: implications for source localization

The electrosense of sharks and rays is used to detect weak dipole-like bioelectric fields of prey, mates and predators, and several models propose a use for the detection of streaming ocean currents and swimming-induced fields for geomagnetic orientation. We assessed pore distributions, canal vectors, complementarity and possible evolutionary divergent functions for ampullary clusters in two sharks, the scalloped hammerhead (Sphyrna lewini) and the sandbar shark (Carcharhinus plumbeus), and the brown stingray (Dasyatis lata). Canal projections were determined from measured coordinates of each electrosensory pore and corresponding ampulla relative to the body axis. These species share three ampullary groups: the buccal (BUC), mandibular (MAN) and superficial ophthalmic (SO), which is subdivided into anterior (SOa) and posterior (SOp) in sharks. The stingray also has a hyoid (HYO) cluster. The SOp in both sharks contains the longest (most sensitive) canals with main projections in the posterior-lateral quadrants of the horizontal plane. In contrast, stingray SO canals are few and short with the posterior-lateral projections subsumed by the HYO. There was strong projection coincidence by BUC and SOp canals in the posterior lateral quadrant of the hammerhead shark, and laterally among the stingray BUC and HYO. The shark SOa and stingray SO and BUC contain short canals located anterior to the mouth for detection of prey at close distance. The MAN canals of all species project in anterior or posterior directions behind the mouth and likely coordinate prey capture. Vertical elevation was greatest in the BUC of the sandbar shark, restricted by the hammerhead cephalofoil and extremely limited in the dorsoventrally flattened stingray. These results are consistent with the functional subunit hypothesis that predicts specialized ampullary functions for processing of weak dipole and geomagnetic induced fields, and provides an anatomical basis for future experiments on central processing of different forms of relevant electric stimuli.     

Nematodes of elasmobranch fishes from the southern coast of Brazil

New records for nematode species recovered from elasmobranch fishes in Brazil are established and new systematical arrangements proposed. Parascarophis sphyrnae Campana-Rouget, 1955 from the spiral valve of Sphyrna zygaena is referred for the first time in South America as a new host record. Procamallanus (S.) pereirai Annereaux, 1946, from the spiral valve of Raja castelnaui is reported parasitizing an elasmobranch host. Nematode larvae of the genera Anisakis, Contracaecum, Pseudoterranova and Raphidascaris are listed from the stomach and spiral valves of several hosts. Anisakidae larvae previously referred in Brazil in the genus Phocanema should be reallocated in Pseudoterranova. Nematodes of the genera Anisakis, Contracaecum, Pseudoterranova and Raphidascaris are reported for the first time parasitizing elasmobranchs in Brazil.     

Detection and processing of electromagnetic and near-field acoustic signals in elasmobranch fishes

The acoustic near field of quietly moving underwater objects and the bio-electric field of aquatic animals exhibit great similarity, as both are predominantly governed by Laplace's equation. The acoustic and electrical sensory modalities thus may, in directing fishes to their prey, employ analogous processing algorithms, suggesting a common evolutionary design, founded on the salient physical features shared by the respective stimulus fields. Sharks and rays are capable of orientating to the earth's magnetic field and, hence, have a magnetic sense. The electromagnetic theory of orientation offers strong arguments for the animals using the electric fields induced by ocean currents and by their own motions in the earth's magnetic field. In the animal's frame of reference, in which the sense organs are at rest, the classical concept of motional electricity must be interpreted in relativistic terms. In the ampullae of Lorenzini, weak electric fields cause the ciliated apical receptor-cell membranes to produce graded, negative receptor currents opposite in direction to the fields applied. The observed currents form part of a positive-feedback mechanism, supporting the generation of receptor potentials much larger than the input signal. Acting across the basal cell membranes, the receptor potentials control the process of synaptic transmission.