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Elevated plasma potassium levels (hyperkalemia), reduced plasma pH (acidosis), reduced blood oxygen content, and elevated temperatures are associated with species-specific rates of at-vessel and post-release mortality in elasmobranch fishes. The mechanism linking these physiological disturbances to mortality remains undetermined however, and we hypothesize that the proximate cause is reduced myocardial function. We measured changes in the functional properties of isolated ventricular myocardial strips from clearnose skate (Rostroraja eglanteria), smooth dogfish (Mustelus canis), and sandbar shark (Carcharhinus plumbeus) when subjected to the following stressors (both in isolation and in combination): hyperkalemia (7.4 mM K+), acidosis (from 7.9 to 7.1), and reduced oxygen (to 31% O2 saturation) applied at temperatures 5 °C above and below holding temperatures. We selected these species based on phylogenetic distance, diverse routine activity levels, and their tolerance to capture and transport. Stressors had a few significant species-specific detrimental impacts on myocardial function (e.g., a 33–45% decrease in net force under acidosis + low O2). Net force production of myocardial strips from clearnose skate and smooth dogfish approximately doubled following exposure to isoproterenol, demonstrating that these species possess beta-adrenergic receptors and that their stimulation could provide a mechanism for preservation of cardiac function during stress. Our results suggest that disruption of physiological homeostasis associated with capture may fatally impair cardiac function in some elasmobranch species, although research with more severe stressors is needed.

In teleost fishes, catecholamine-induced increases in the activity of cation exchangers compensate for decreases in hemoglobin oxygen affinity and maximum blood oxygen carrying capacity caused by decreases in plasma pH (i.e., metabolic acidosis). The resultant red blood cell (RBC) swelling has been documented in sandbar (Carcharhinus plumbeus) and epaulette (Hemiscyllium ocellatum) sharks following capture by rod-and-reel or after a 1.5 h exposure to anoxia (respectively), although the underlying mechanisms remain unknown. To determine if RBC swelling could be documented in other elasmobranch fishes, we collected blood samples from clearnose skate (Rostroraja eglanteria), blacktip reef shark (Carcharhinus melanopterus), and sicklefin lemon shark (Negaprion acutidens) subjected to exhaustive exercise or air exposure (or both) and measured hematocrit, hemoglobin concentration, RBC count, RBC volume, and mean corpuscular hemoglobin content. We did likewise with sandbar and epaulette sharks to further explore the mechanisms driving swelling when present. We could not document RBC swelling in any species; although hematocrit increased in all species (presumably due to RBC ejection from the spleen or fluid shifts out of the vascular compartment) except epaulette shark. Our results indicate RBC swelling and associated ion shifts in elasmobranch fishes is not inducible by exercise or hypoxia, thus implying this response maybe of lesser importance for maintaining oxygen delivery during acute acidosis than in teleost fishes.

Understanding how rising temperatures, ocean acidification, and hypoxia affect the performance of coastal fishes is essential to predicting species-specific responses to climate change. Although a population&rsquo, s habitat influences physiological performance, little work has explicitly examined the multi-stressor responses of species from habitats differing in natural variability. Here, clearnose skate (Rostaraja eglanteria) and summer flounder (Paralichthys dentatus) from mid-Atlantic estuaries, and thorny skate (Amblyraja radiata) from the Gulf of Maine, were acutely exposed to current and projected temperatures (20, 24, or 28 &deg, C, 22 or 30 &deg, and 9, 13, or 15 &deg, C, respectively) and acidification conditions (pH 7.8 or 7.4). We tested metabolic rates and hypoxia tolerance using intermittent-flow respirometry. All three species exhibited increases in standard metabolic rate under an 8 &deg, C temperature increase (Q10 of 1.71, 1.07, and 2.56, respectively), although this was most pronounced in the thorny skate. At the lowest test temperature and under the low pH treatment, all three species exhibited significant increases in standard metabolic rate (44&ndash, 105%, p &lt, 0.05) and decreases in hypoxia tolerance (60&ndash, 84% increases in critical oxygen pressure, 0.05). This study demonstrates the interactive effects of increasing temperature and changing ocean carbonate chemistry are species-specific, the implications of which should be considered within the context of habitat.

Evacuation rates were determined for Clearnose Skate (Leucoraja eglanteria), an important predator in the mid-Atlantic United States shelf ecosystem. Male Skates (570–730 mm total length) were fed Sand Lance Ammodytes sp. and allowed to digest from 2–48 hr at two different temperatures. At selected times, fish were removed from tanks, sedated with tricaine methanesulfonate, and subjected to gastric lavage. This procedure was successful at removing the food from the stomachs without injury to the fish. Evacuation rates for the two temperature treatments were fit best by exponential models. The evacuation rates, per hour, were faster at 20°C than at 15°C, resulting in empty stomachs by 24 hr at 20°C, and 48 hr at 15°C. Evacuation rates at these temperatures were estimated as 0.102 and 0.059 proportion stomach contents per hour at 20 o C and 15°C. These evacuation rates and their temperature dependence are similar to that of other species of elasmobranchs and some teleosts. Evacuation rates are often combined with stomach content data to estimate consumption. The results of this study indicate that evacuation rates by some skates may be up to five times higher than currently used in multi-species and ecosystem models of the Northeast U.S. Shelf. The implication is that consumption may also be higher, highlighting the need for more research to increase the accuracy in evacuation rates estimates.

Swimming performance is considered a key trait determining the ability of fish to survive. Hydrodynamic theory predicts that the energetic costs required for fishes to swim should vary with speed according to a U-shaped curve, with an expected energetic minimum at intermediate cruising speeds and increasing expenditure at low and high speeds. However, to date no complete datasets have shown an energetic minimum for swimming fish at intermediate speeds rather than low speeds. To address this knowledge gap, we used a negatively buoyant fish, the clearnose skate Raja eglanteria, and took two approaches: a classic critical swimming speed protocol and a single-speed exercise and recovery procedure. We found an anaerobic component at each velocity tested. The two approaches showed U-shaped, though significantly different, speed-metabolic relationships. These results suggest that (i) postural costs, especially at low speeds, may result in J- or U-shaped metabolism-speed curves; (ii) anaerobic metabolism is involved at all swimming speeds in the clearnose skate; and (iii) critical swimming protocols might misrepresent the true costs of locomotion across speeds, at least in negatively buoyant fish.

Developing models in support of ecosystem-based management requires knowledge of trophic dynamics of ecologically important species. A paucity of data on these dynamics for Long Island finfish is hindering development of ecosystem models required by recent legislation. In this study, we analyzed stomach contents of common fishes collected from Port Jefferson Harbor, Great South Bay, and Shinnecock Bay between May and October of 2007 and 2008. General diet composition was described by percent by number (%N), percent by weight (%W), percent frequency of occurrence (%0), and percent index of relative importance (%IRI) for seven species: Paralichthys dentatus (Summer Flounder), young-of-the-year (YOY) Pomatomus saltatrix (Bluefish), Prionotus evolans (Striped Searobin), Stenotomus chrysops (Scup), Scophthalmus aquosus (Windowpane Flounder), Raja eglanteria (Clearnose Skate), and Morone saxatilis (Striped Bass). Temporal diet composition was estimated for the consistently abundant YOY Bluefish, Summer...

Although the majority of batoid elasmo- branchs, skates and rays, are benthically associated, benthic locomotion has been largely overlooked in this group. Only skates have been previously described to per- form a form of benthic locomotion termed ''punting.'' While keeping the rest of the body motionless, the skate's pelvic fins are planted into the substrate and then retracted caudally, which thrusts the body forward. In this study, we demonstrate that this form of locomotion is not confined to the skates, but is found across a range of phylogenetically and morphologically diverse batoid species. However, only the clearnose skate, Raja eglanteria, and the lesser electric ray, Narcine brasiliensis, performed ''true punting,'' in which only the pelvic fins were engaged. The yellow stingray, Urobatis jamaicensis, and the Atlantic stingray, Dasyatis sabina, performed ''augmented punting,'' in which pectoral fin movement was also used to generate thrust. Despite this supplemental use of pectoral fins, the augmented punters failed to exceed the punting capabilities of the true punters. The urobatid and the true punters all punted approximately half their disc length per punt, whereas the dasyatid punted a significantly shorter dis- tance. The skate punted significantly faster than the other species. Examination of the pelvic fin musculature revealed more specialized muscles in the true punters than in the augmented punters. This concordance of musculature with punting ability provides predictive power regarding the punting kinematics of other elasmobranchs based upon gross muscular examinations. In contrast to previous assumptions, our results suggest that benthic locomotion is widespread among batoids. J. Morphol. 271:1219-1228, 2010. 2010 Wiley-Liss, Inc.

We assessed the functional properties in atrial and ventricular myocardium (using isolated cardiac strips) of smooth dogfish (Mustelus canis), clearnose skate (Raja eglanteria), and sandbar shark (Carcharhinus plumbeus) by blocking Ca2+ release from the sarcoplasmic reticulum (SR) with ryanodine and thapsigargin and measuring the resultant changes in contraction–relaxation parameters and the force–frequency relationship at 20 °C and 30 °C. We also examined ultrastructural differences with electron microscopy. In tissues from smooth dogfish, net force (per cross-sectional area) and measures of the speeds of contraction and relaxation were all higher in atrial than ventricular myocardium at both temperatures. Atrial-ventricular differences were evident in the other two species primarily in measures of the rates of contraction and relaxation. Ryanodine-thapsigargin treatment reduced net force and its maximum positive first derivative (i.e., contractility), and increased time to 50 % relaxation in atrial tissue from smooth dogfish at 30 °C. It also increased times to peak force and half relaxation in clearnose skate atrial and ventricular tissue at both temperatures, but only in atrial tissue from sandbar shark at 30 °C; indicating that SR involvement in excitation–contraction (EC) coupling is species- and temperature-specific in elasmobranch fishes, as it is in teleost fishes. Atrial and ventricular myocardium from all three species displayed a negative force–frequency relationship, but there was no evidence that SR involvement in EC coupling was influenced by heart rate. SR was evident in electron micrographs, generally located in proximity to mitochondria and intercalated discs, and to a lesser extent between the myofibrils; with mitochondria being more numerous in ventricular than atrial myocardium in all three species.

Clearnose skates, Raja eglanteria, are a common species of skate found seasonally along the Atlantic coast of North America from Cape Cod to mid-Florida and in the Gulf of Mexico from mid-Florida to eastern Texas. Clearnose skates inhabit the west central coast of Florida during winter months, when Gulf temperatures are conducive to mating and egg-laying (approximately 16°C–22°C). Specimens collected during this time will breed in captivity and, if maintained at 20°C, mated females will store sperm and continue to lay fertile eggs for up to six months. With fertile eggs available from day of laying until hatching, the first complete description of batoid elasmobranch embryonic development with a timetable has been established and is presented here. If eggs are maintained at 20°C, appearance and progression of embryonic events proceed over a reproducible timeline. The timeline has been divided into discrete time periods into which developmental events have been grouped. These include cleavage and expansion of the blastodisc (day 1–day 4), embryonic axis and neural tube formation (day 4–day 7), pharyngeal pouches and gill filament development (day 10–day 28), expansion of fins and transition from external filaments to internal gills (week 4–week 7), and epidermal pigmentation and yolk absorption (week 8–week 12). Fully formed offspring hatch after an incubation period of approximately 12 weeks (85 ± 6 days). To examine possible mechanisms of transport and storage of sperm in the reproductive tract of females following copulation, experiments resulting in the first artificial insemination for any elasmobranch fish are described. These experiments provide evidence for functional roles of the alkaline gland, whose secretions stimulate sperm motility and may enhance migration of sperm to the oviducal glands, and the clasper gland, whose secretions may provide nutrition during storage of viable sperm in the oviducal gland.

Tissues from the male genital ducts of six specimens of the clearnose skate Raja eglanteria, comprising the Leydig gland, upper and lower epididymis, ductus deferens and seminal vesicle, were fixed and embedded for ultrastructural examination. In the Leydig gland, two types of columnar cells were identified, one bearing microvilli, a basal nucleus and evidence of active secretion with plentiful endoplasmic reticulum and numerous secretory droplets, and the other pyriform with cilia, and swathes of cytofilaments emanating from prominent desmosomes. Occasional crystalloid intramitochondrial inclusions were seen in the first type, with a periodicity of 24 nm. The upper epididymis was composed of cuboidal cells with microvilli and cilia and irregular electron dense granules, some of which were basally situated and extremely large, often within cells resembling intraepithelial leucocytes; such cells were also seen in the stroma underlying the epithelium. The lower epididymis cells also bore microvilli and cilia and were heavily vacuolated with fatty inclusions as well as the granule-laden leucocytes seen previously. In the ductus deferens, cells had masses of long cilia with occasional microvilli; endoplasmic reticulum was well developed, forming complex arrays with sparse secretory droplets and basal mitochondria. In the seminal vesicle there were two cell types, the most common having long cilia and short microvilli and an occasional, paler cell with supranuclear accumulations of small, round mitochondria. These ultrastructural appearances have been related to cell glycosylation and functions including protein secretion, water absorption and waste removal, and illustrate how structure and function vary down the length of the genital tract.

Humoral immunity is effected through the rearrangement of immunoglobulin (Ig) genes in individual somatic cells committed to the B lymphocyte lineage. Haplotype or allelic exclusion restricts B lymphocytes to the expression of a single Ig receptor that can sustain further somatic modification. In most species, a specific Ig chain is encoded at a single genetic locus. However, in cartilaginous fish, hundreds of independent Ig heavy- (IgH) and Ig light-chain (IgL) gene loci are present, many of which are joined in the germ line. Ig gene transcripts have been amplified from single peripheral blood lymphocytes isolated from the clearnose skate (Raja eglanteria) using reverse-transcription PCR, and a single productive IgH transcript was detected in the majority of cells analyzed. Similarly, only a single IgL transcript was detected in over half of the individual cells. Taken together, these findings suggest that a mechanism for haplotype exclusion arose early in the evolution of antibody diversity and is independent of a single genetic locus.

The evolutionary origins of lymphocytes can be traced by phylogenetic comparisons of key features. Homologs of rearranging TCR and Ig (B cell receptor) genes are present in jawed vertebrates, but have not been identified in other animal groups. In contrast, most of the transcription factors that are essential for the development of mammalian T and B lymphocytes belong to multigene families that are represented by members in the majority of the metazoans, providing a potential bridge to prevertebrate ancestral roles. This work investigates the structure and regulation of homologs of specific transcription factors known to regulate mammalian T and B cell development in a representative of the earliest diverging jawed vertebrates, the clearnose skate (Raja eglanteria). Skate orthologs of mammalian GATA-3, GATA-1, EBF-1, Pax-5, Pax-6, Runx2, and Runx3 have been characterized. GATA-3, Pax-5, Runx3, EBF-1, Spi-C, and most members of the Ikaros family are shown throughout ontogeny to be 1) coregulated with TCR or Ig expression, and 2) coexpressed with each other in combinations that for the most part correspond to known mouse T and B cell patterns, supporting conservation of function. These results indicate that multiple components of the gene regulatory networks that operate in mammalian T cell and B cell development were present in the common ancestor of the mammals and the cartilaginous fish. However, certain factors relevant to the B lineage differ in their tissue-specific expression patterns from their mouse counterparts, suggesting expanded or divergent B lineage characteristics or tissue specificity in these animals.

Genital ducts of three male Raja eglanteria were fixed and embedded in epoxy and methacrylate resin. Epoxy resin sections from the Leydig gland, upper and lower epididymis, ductus deferens and seminal vesicle were stained with 20 labelled lectins to examine their glycosylation. The Leydig gland consisted of columnar epithelial cells expressing N-linked glycans, N-acetyl galactosamine, glucosamine and lactosamine residues and sialic acid. Interspersed were ciliated cells of a different glycotype. The upper epididymis of cuboidal epithelium had a strongly glycosylated, ciliated apical surface and cytoplasmic granules that stained heavily with many lectins, with increased glycosylation compared to the Leydig gland. In the lower epididymis, tall, vacuolated cells showed some differences and a slight reduction in lectin staining. The ductus deferens contained two cell types and showed increased terminal N-acetyl galactosamine. The ciliated cuboidal epithelium of the seminal vesicle had marked differences from the ductus epithelium, with decreased N-acetyl galactosamine and lactosamine expression but increased subterminal N-acetyl lactosamine and galactosamine expression and sialylation. Spermatozoa were suspended in a glycosylated matrix and, in the seminal vesicle, were embedded in solid masses of matrix forming spermatozeugmata. These data show changes in glycan expression along the male genital tract, probably related to the nurture and maturation of the spermatozoa as they travel towards the seminal vesicle.

Isolated vessels from four elasmobranchs, yellow stingray (Urolophus jamaicensis), clearnose skate (Raja eglanteria), ghost shark (Hydrolagus novaezelandiae), and spiny dogfish (Squalus acanthias), were examined for the presence of spontaneous contractions (SC). SC were observed in otherwise unstimulated dorsal aortas (DA) from stingray and ghost shark, but not in skate DA. Unstimulated ventral aortas (VA) did not exhibit SC. After treatment of VA with a contractile agonist, SC appeared in stingray and skate but not ghost shark or dogfish. SC in stingray VA were subsequently inhibited by either epinephrine (10(-5) M) or indomethacin (10(-4) M). Agonist contraction also elicited strong SC in ductus Cuvier from stingray, but not from ghost shark or dogfish. SC in dogfish hepatic portal veins (HPV) produced a rhythmical oscillation in tension. The frequency of HPV SC was highest (approximately 1 min(-1)) in intact veins and lower (approximately 3 min(-1)) in vein segments, indicative of a dominant pacemaker in the intact vessel. SC in HPV were depressed during the first 30 min of hypoxia, but there was substantial recovery over an additional 30 min of hypoxia and complete recovery upon return to normoxia. Addition of 80 mM KCl completely inhibited HPV SC and lowered resting tone. These results show that SC are a common feature of elasmobranch vessels and there appears to be a correlation between swimming behavior and the propensity for SC. KCl inhibition of SC and tonus in HPV is highly unusual for vascular smooth muscle.

Serum steroid hormones in the peripheral circulation of the clearnose skate, Raja eglanteria, were measured at the time of capture and at various times throughout the year while the animals were maintained as a captive breeding population. These analyses demonstrate interesting correlations between changes in hormone concentrations and annual reproductive events. Animals were sampled once (78 females, 20 males) or multiple times (15 females). For both groups of females, 17β-estradiol was detected throughout the year with significant elevations occurring during October and November when ovarian follicles begin to mature (as determined through necropsy examinations), and January and February when maximum mating activity is observed and egg laying begins. Testosterone and dihydrotestosterone concentrations were significantly elevated in females only during January and February. Testosterone elevations were synchronous with longer-term elevations in 17β-estradiol in females sampled either once or repetitively. Testosterone concentrations in males were significantly elevated during times of maximum breeding activity compared to periods of sexual inactivity. Data from females sampled during five stages of the egg laying process, as defined by the position of palpable egg capsules within the reproductive tract, revealed that 17β-estradiol was highest when egg capsules were forming in the nidamental gland (stage 2) or uterus (stage 3); testosterone and dihydrotestosterone were maximal when eggs were in the uterus (stage 3) or cloaca (stage 4); and progesterone was significantly elevated immediately after oviposition (stage 5), suggesting a possible role for progesterone in the regulation of sequential laying of egg pairs. J. Exp. Zool. 284:575–585, 1999. © 1999 Wiley-Liss, Inc.

Effects of insulin-like growth factor-I (IGF-I), corticosterone, and triiodothyronine (T3) on in vitro growth of vertebral cartilage of the clearnose skate, Raja eglanteria, were investigated. Uptake of [35S]sulfate in cultured vertebrae was used to characterize glycosaminoglycan (GAG) synthesis and cartilage growth. IGF-I significantly enhanced cartilage growth when concentrations of 1.28 and 12.8 nM were present in the culture system. Corticosterone significantly inhibited vertebral GAG synthesis at concentrations of 1, 10, and 100 nM. This effect was markedly pronounced in cartilage exposed to 1 and 10 nM corticosterone, in which GAG synthesis was virtually ceased. In contrast, T3 (0.75, 7.5, and 75.0 nM) had no significant effect on sulfate uptake. These data suggest that IGF-I and corticosteroids may play important roles in regulating skeletal growth of elasmobranchs, as they appear to do in other vertebrates. While T3 does not appear to exert an immediate, direct effect on vertebral growth, it may still influence elasmobranch chondrogenesis over longer culture periods or indirectly through other regulatory pathways. Thus, further information is necessary to characterize the role of thyroid hormones in the skeletal growth of these fishes. The present study is the first in vitro investigation on the hormonal regulation of elasmobranch cartilage growth. As such, the methods described herein provide a useful technique for examining these physiological processes. J. Exp. Zool. 284:549–556, 1999. © 1999 Wiley-Liss, Inc.

Differential screening has been used to identify cDNAs encoding a long form of IgX in Raja eglanteria (clearnose skate). Comparisons of the IgX long form with the previously described short-form IgX cDNAs and the genomic IgX locus indicate that the V and two 5′C regions of the short and long forms of IgX are >90% identical at the nucleotide level. Differences between the V sequences of the long- and short-form IgX genes are concentrated in complementarity determining regions, suggesting that these forms are derived through alternative splicing of the same genomic loci or transcription of highly related loci. The extreme conservation of nucleotide sequence, including third position codons, among different cDNAs as well as the near identity of nucleotide sequence in the intervening sequences of germline IgX, IgX short-form sterile transcripts and IgX long-form sterile transcripts indicate that the multiple IgX loci are recently diverged from one another and/or are under intense gene correction. Phylogenetic analyses of the known cartilaginous fish immunoglobulin loci demonstrate that the long form of IgX is orthologous to IgW/IgNARC (NARC) and is most consistent with: 1) the divergence of the IgX/IgW/NARC and IgM-like loci from a common ancestral locus prior to the divergence of the cartilaginous/bony fish lineages and 2) the divergence of the NAR locus from the IgX/IgW/NARC gene(s) after the cartilaginous/bony fish split but prior to the shark/skate split, approximately 220 million years ago.

Cells isolated from lymphomyeloid tissues and peripheral blood from two species of elasmobranch, the nurse shark ( Ginglymostoma cirratum Bonaterre) and the clearnose skate ( Raja eglanteria Bosc) were assayed for in vitro phagocytic and pinocytic activities at various times during 24 h in culture. The purpose of this research was to begin to characterise functional activities of cells isolated from unique lymphomyeloid tissues found in elasmobranchs. Phagocytosis was evaluated by assessing relative abilities of immune cells to ingest Congo Red stained yeast, and included determination of percentage of active cells and numbers of yeast per cell. Pinocytosis was assayed by spectrophotometric measurement of neutral red dye accumulation by immune cells. Phagocytic activity was greatest in cells isolated from shark epigonal and skate epigonal and Leydig organs, with over 60% of skate Leydig cells containing yeast after 24 h in culture. Phagocytic activity was 2·5–6 times lower in cells from peripheral circulation or spleen. Pinocytosis was 1·5–3-fold greater in cells isolated from Leydig and epigonal organs compared to peripheral blood leucocytes or splenocytes. Documentation of phagocytic and pinocytic activities by cells isolated from epigonal and Leydig organs suggests significant immune functional roles for these unique tissues in the elasmobranch immune system.

A series of products were amplified using a PCR strategy based on short minimally degenerate primers and R. eglanteria (clearnose skate) spleen cDNA as template. These products were used as probes to select corresponding cDNAs from a spleen cDNA library. The cDNA sequences exhibit significant identity with prototypic (alpha, beta, gamma, and delta T cell antigen receptor (TCR) genes. Characterization of cDNAs reveals extensive variable region diversity, putative diversity segments, and varying degrees of junctional diversification. This demonstrates expression of both alpha/beta and gamma/delta TCR genes at an early level of vertebrate phylogeny and indicates that the three major known classes of rearranging antigen receptors were present in the common ancestor of the present-day jawed vertebrates.

Motoneurons that supply the clearnose skate’s hypobranchial musculature, via the occipital nerve and first seven ventral spinal nerve roots, are located within a column that extends from a level just caudal to the obex through the corresponding rostral spinal cord segments. Individual muscle motoneuron pools within the column are considerably intermingled and overlap. Comparisons with tetrapods, particularly mammals, where the hypobranchial musculature is greatly modified, reveal general conserved features. The motor column’s multisegmental organization is retained although, in mammals, the column begins rostrally at medullary levels, where hypobranchial muscle motoneurons are intimately associated with motoneurons to lingual muscles, and it is restricted caudally to fewer spinal cord segments. In addition, despite an intermingling of motoneurons that supply individual hypobranchial muscles there is a shared rostrocaudal sequence of the motor pools. Rostral most hypobranchial motoneurons supply the most ventral and anterior muscles (i.e., m. coracomandibularis, and likely m. coracohyoideus, of skate and the suprahyoid musculature, m. geniohyoideus, of tetrapods). Caudal hypobranchial motoneurons supply the skate’s mm. coracohyomandibularis, coracoarcualis communis and coracobranchialis and the tetra-pod’s entire infrahyoid muscle complex. The intermingling of multisegmental motoneuron populations innervating different hypobranchial muscles might be attributed to intermixing of premuscle mesoderm derived from several postotic somites but the musculotopic organization along the rostrocaudal axis indicates that pre- and posthyoid muscle mesoderm may partially keep its identity during its migration to the floor of the pharynx and oral cavity.

Studies were undertaken to examine the importance of organic proteoglycan matrices in the calcification of elasmobranch vertebral cartilage. Proteoglycans were extracted from the vertebral cartilage of the clearnose skate, Raja eglanteria Bosc, with a 3M guanidine hydrochloride/10% EDTA solution. Proteoglycan solutions (12 μg ml(-1)) were effective inhibitors of hydroxyapatite formation in vitro from high concentration calcium phosphate solutions. Inhibition of crystal formation appears to occur through the restriction of phase transformation from a calcium phosphate precursor to hydroxyapatite crystals. The concentration and/or degradation of proteoglycans in elasmobranch vertebral cartilage may have a physiological role in the production of alternating mineral-rich and mineral-poor growth zones, currently used in ageing studies.

The clearnose skate (Raja eglanteria) develops in an almost opaque eggcase and lays its eggs in pairs. One sibling from each of eight pairs of skates was removed from its eggcase during embryonic development, while the other sibling developed inside the eggcase. The refractive development of the eyes at hatching was examined to see if ambient light exposure during embryonic development could influence the refractive states of hatchlings. Measurements included refractive states, ocular dimensions and lens focal properties. The differences in measurements between the two groups were not significant, which would indicate that environmental light does not influence the refractive development of the embryonic skate eye.

Tonic immobility (TI), also known as animal hypnosis, has been observed in a wide variety of taxa and reported in all vertebrate classes with the exception of Agnatha. To date, TI has been reported in six species of elasmobranchs. Preliminary results are presented here for five species of galeomorph sharks, one species of squalomorph shark, and six species of batoids. Tonic immobility was induced in the leopard shark. Triakis semifasciata; the whitetip reef shark, Triaenodon obesus; the blacktip reef shark, Carcharhinus melanopterus; the Caribbean reef shark, Carcharhinus perezi; the swellshark, Cephaloscyllium ventriosum; the California round ray, Urolophus halleri; the shovelnose guitarfish, Rhinobatos productus; the clearnose skate. Raja eglanteria; the cownose ray, Rhinoptera bonasus; and the southern stingray, Dasyatis americana. The broadnose sevengill shark, Notorynchus cepedianus and the yellow stingray, Urolophus jamaicensis failed to exhibit the tonic immobility response, but only one individual of each species was tested. The average induction time ranged from 16.7 sec in the shovelnose guitarfish to 45.7 sec in the clearnose skate. Average duration times ranged from 34.3 sec in the clearnose skate to 274.0 sec in the Caribbean reef shark. Depending upon the species involved, TI has been used as an aid in a few husbandry and medical procedures in captive elasmobranches at the National Aquarium in Baltimore. These procedures have included gross examination, measuring lengths, tube feeding, and drawing blood via caudal venipuncture. Using tonic immobility during these procedures is beneficial as once induced, the subject's struggling is minimal and usually requires little restraint. © 1994 Wiley-Liss, Inc.

Flow cytometric DNA analysis was used to determine the DNA content of red blood cells from Ginglymostoma cirratum (Bonaterre) and Raja eglanteria (Bosc). The DNA content/nucleus was calculated to be 7.6 and 7.1 pg/nucleus, respectively. Peripheral red blood cells obtained both from G. cirratum and from R. eglanteria were demonstrated to be actively cell cycling. Percentages of red blood cells in the phases of the cell cycle (G0/G1, S, and G2/M) not only varied considerably between different fishes but also were quite variable in weekly samples obtained from individual animals.

A complex of three muscles (one lateral, one intermediate and one medial in position) in the clearnose skate,Raja eglanteria, is believed to be wholly, or in part, homologous to the cucullaris (trapezius). The retrograde transport of horseradish peroxidase was used to discover the central location of the motoneurons that supply each of these muscles. Motoneurons that project to the lateral muscle occupy the caudal part of the ventral nucleus of X. This nucleus is situated ventrolateral to the dorsal vagal motor column at caudal medullary levels, and lateral to the main ventral motor column of the rostral spinal cord. The axons of these motoneurons exit the medulla within the caudal vagal rootlets and course peripherally within the intestinal (visceral) ramus of the vagus nerve. Motoneurons that innervate the intermediate and medial muscles are located along the ventral border of the ventral column of gray at spinal cord segments 10–15. Their axons course peripherally within the ventral roots of spinal nerves. The caudal ventral nucleus of X, the nerve that supplies the lateral muscle, and the lateral muscle are likely homologues of the accessory nucleus, accessory nerve, and cucullaris (trapezius), respectively, among other fishes and tetrapods. Intermediate and medial muscles, based on the central location of motoneurons that supply them, are part of the longitudinal epaxial musculature and are not part of a trapezius complex.

The shape and focal properties (spherical aberration) of the ocular lens of hatchling, yearling and adult clearnose skates (Raja eglanteria) were examined. In contrast to the spherical shape of the typical teleost lens, the lens of the clearnose skate is slightly aspherical; equatorial diameter being greater than axial diameter. The asphericity is approximately equal, in absolute measures, at all ages and is thus more obvious in younger skates. Lens spherical aberration is well corrected at all three levels of development. Yearling and adult lenses have relative focal lengths (Matthiessen's Ratio) which are close to the representative value of 2.55:1 expected for the teleost lens. However, the relative focal length of lenses from hatchling lenses is higher (about 2.75:1). This value approaches the upper limit of reports dealing with teleost lens focal characteristics. The difference between hatchling lenses and those of adult and yearling lenses may be due to the absence of extensive compression of central lens tissue by peripheral growth at early stages in development.

The visual fields of elasmobranch fishes are not well characterized even though this is a fundamental element of the visual system. The batoid fishes (skates, rays) form a monophyletic clade within the subclass Elasmobranchii and exhibit a broad range of morphologies and corresponding ecologies. We hypothesized that their visual field characteristics would reflect their diverse morphology and ecology. This was tested by quantifying the monocular, binocular and cyclopean horizontal and vertical visual fields of four batoid species (Raja eglanteria, Urobatis jamaicensis, Dasyatis sabina and Rhinoptera bonasus) that encompassed a range from a basal skate to a more derived ray. The horizontal and vertical visual fields differed significantly among species; however, all species possessed horizontal anterior and dorsal binocular overlaps. Urobatis jamaicensis, a small reef-associated stingray, demonstrated a 360 degrees panoramic visual field in the horizontal plane, and R. bonasus, a schooling benthopelagic ray, a 360 degrees panoramic view in the vertical plane. Large anterior binocular overlaps were measured in D. sabina (72 degrees ) and R. bonasus (46 degrees ) but came at the expense of large posterior blind areas. The anterior binocular overlaps in R. eglanteria (28 degrees ) and U. jamaicensis (34 degrees ) were smaller but were coupled with large monocular fields that provided expansive peripheral views. The most phylogenetically basal species, the clearnose skate (Raja eglanteria), had the most reduced visual field in contrast to the more derived ray species. To our knowledge, this study represents the first comparative assessment of visual fields in basal vertebrates.

Reactive nitrogen intermediates, such as nitric oxide (NO), are important immunomodulators in vertebrate immune systems, but have yet to be identified as mediators of host defence in any member of class Chondrichthyes, the cartilaginous fishes. In the present study, production of NO by nurse shark (Ginglymostoma cirratum) peripheral blood leucocytes (PBL) stimulated with bacterial cell wall lipopolysaccharide (LPS) was investigated. PBL were cultured for 24 to 96 h following stimulation with LPS at concentrations ranging from 0 to 25 μg ml−1, in both serum-supplemented and serum-free culture conditions. Production of NO was measured indirectly using the Griess reaction, with maximal NO production occurring after 72 h using 10% FBS and 10 μg LPS ml−1. Application of these culture conditions to PBL from another cartilaginous fish (clearnose skate, Raja eglanteria) resulted in a similar NO response. Addition of a specific inhibitor of inducible nitric oxide synthase (iNOS), l -N6-(1-iminoethyl)lysine ( l -NIL), resulted in a significant decrease in the production of NO by PBL from both species.

Cartilaginous fish express canonical B and T cell recognition genes, but their lymphoid organs and lymphocyte development have been poorly defined. Here, the expression of Ig, TCR, recombination-activating gene (Rag)-1 and terminal deoxynucleosidase (TdT) genes has been used to identify roles of various lymphoid tissues throughout development in the cartilaginous fish, Raja eglanteria (clearnose skate). In embryogenesis, Ig and TCR genes are sharply up-regulated at 8 weeks of development. At this stage TCR and TdT expression is limited to the thymus; later, TCR gene expression appears in peripheral sites in hatchlings and adults, suggesting that the thymus is a source of T cells as in mammals. B cell gene expression indicates more complex roles for the spleen and two special organs of cartilaginous fish-the Leydig and epigonal (gonad-associated) organs. In the adult, the Leydig organ is the site of the highest IgM and IgX expression. However, the spleen is the first site of IgM expression, while IgX is expressed first in gonad, liver, Leydig and even thymus. Distinctive spatiotemporal patterns of Ig light chain gene expression also are seen. A subset of Ig genes is pre-rearranged in the germline of the cartilaginous fish, making expression possible without rearrangement. To assess whether this allows differential developmental regulation, IgM and IgX heavy chain cDNA sequences from specific tissues and developmental stages have been compared with known germline-joined genomic sequences. Both non-productively rearranged genes and germline-joined genes are transcribed in the embryo and hatchling, but not in the adult.

Members of the Ikaros multigene family of zinc finger proteins are expressed in a tissue-specific manner and most are critical determinants in the development of both the B and T lymphocytes as well as NK and dendritic APC lineages. A PCR amplification strategy that is based on regions of shared sequence identity in Ikaros multigene family members found in mammals and several other vertebrates has led to the recovery of cDNAs that represent the orthologues of Ikaros, Aiolos, Helios, and Eos in Raja eglanteria (clearnose skate), a cartilaginous fish that is representative of an early divergence event in the phylogenetic diversification of the vertebrates. The tissue-specific patterns of expression for at least two of the four Ikaros family members in skate resemble the patterns observed in mammals, i.e., in hematopoietic tissues. Prominent expression of Ikaros in skate also is found in the lymphoid Leydig organ and epigonal tissues, which are unique to cartilaginous fish. An Ikaros-related gene has been identified in Petromyzon marinus (sea lamprey), a jawless vertebrate species, in which neither Ig nor TCRs have been identified. In addition to establishing a high degree of evolutionary conservation of the Ikaros multigene family from cartilaginous fish through mammals, these studies define a possible link between factors that regulate the differentiation of immune-type cells in the jawed vertebrates and related factors of unknown function in jawless vertebrates.

The olfactory organ of juvenile clearnose skates (Raja eglanteria) was studied with the light and electron microscopes. The organ is ovoid in shape, and its free surface is complicated by the presence of some 20 lamellae. Each lamella has a folded surface lined by a typical neurosensory olfactory epithelium. Bipolar olfactory receptor neurons, ciliated sustentacular cells, and basal cells are the pre-eminent cellular components of the epithelium. Two types of receptor neurons, both bearing microvilli but not cilia, were identified. The type 1 neuron is similar to that previously described in other fishes. The type 2 neuron has a characteristic morphology justifying a separate description. Its dendritic knob is larger than that of type 1, and its microvilli, which are shorter and thicker, are straight and regularly arranged. Tight bundles of filaments provide a skeleton to each microvillus, and these filament bundles reach more than 5 microns down into the dendrite. Type 2 receptor neurons have a well-developed Golgi complex and sparse rough endoplasmic reticulum (rER), whereas type 1 receptor neurons have a less well-developed Golgi complex and a conspicuous system of rER lamellae. The mucous layer on the epithelial surface is provided by the secretion of goblet cells that are situated mostly in the peripheral regions of each lamella. Secretory granules in the sustentacular cells and glands in the lamina propria were not observed.

Embryos of the clearnose skate, Raja eglanteria, develop in sea water at 20-22 degrees C, hatching after 82 +/- 4 days (Luer and Gilbert, Environ. Biol. Fishes, 13:161-171, 1985). Eyes develop as steadily enlarging spheres whose corneas have the same radius of curvature as the sclera. The cornea begins development as a 2-cell thick epithelium beneath which by Day 12 there is only a basal lamina and a wispy matrix separating it from the underlying lens. This matrix, modified by Day 16, is displaced on Day 22 by a few orthogonal plies of fibrillar primary stroma. Ply number increases to at least 13 by Day 30, reaching the final number of 20 +/- 2 by Day 42. Stromal fibroblasts (keratocytes) appear at the corneal periphery by Day 22, and in increased numbers by Day 30, a time at which no keratocytes are seen in the central stroma. However, by Day 40, many fibroblasts are present at the corneal periphery, invading the primary stroma between plies, occasionally reaching even the central cornea. By Day 53, keratocytes are present between all plies, from corneal periphery to center. Thickness of each ply in this secondary stroma increases, but the number of plies remains the same as in the primary stroma. Bowman's layer, non-invaded matrix beneath the epithelial basal lamina, is not evident until Day 53. Sutural fibers, first seen on Day 22, originate in the corneal epithelial basal lamina, traversing perpendicularly the plies of the primary stroma. Sutural fibers persist throughout development of the secondary stroma and into adulthood. In contrast to chicks, skate corneas remain transparent throughout development, and never form an endothelium.

The vagus nerve of the clearnose skate, Raja eglanteria, on the basis of its central and peripheral patterns and in light of the embryonic origin of its innervation fields, is viewed as a collector of separate elements. The peripheral elements include a series of branchial nerves to a segmented pharynx, an intestinal nerve to an unsegmented gut, a nerve or nerves to the heart, and an accessory nerve to a cucullaris muscle. The central elements include a sensory column, a dorsal motor column, and a ventral motor column. The dorsal motor column and sensory column are segmented in register with the branchial and intestinal nerves. Motoneurons that supply the branchial muscles of somitic origin are only located in the rostral segmented portion of the dorsal motor column. Preganglionic parasympathetics to the enteric plexus, presumably derived from circumpharyngeal crest, form the caudal portion of the dorsal motor column and are probably also present in the rostral segmented portion. Cardiac preganglionic parasympathetics to a visceral field of cardiac crest origin occur in the rostral portion of the ventral motor column as well as in the dorsal motor column. Accessory motoneurons that supply the cucullaris, likely a part of the general body musculature, are unrelated to other vagal motoneurons and form a separate nucleus (caudal ventral motor nucleus) located at spinal levels. The central and peripheral vagal nerve patterns of elasmobranchs suggest a highly conserved, ancestral gnathostome condition.