Embryonic development of the axial column in the little skate,Leucoraja erinacea

The morphological patterns and molecular mechanisms of vertebral column development are well understood in bony fishes (osteichthyans). However, vertebral column morphology in elasmobranch chondrichthyans (e.g., sharks and skates) differs from that of osteichthyans, and its development has not been extensively studied. Here, we characterize vertebral development in an elasmobranch fish, the little skate, Leucoraja erinacea , using microCT, paraffin histology, and whole‐mount skeletal preparations. Vertebral development begins with the condensation of mesenchyme, first around the notochord, and subsequently around the neural tube and caudal artery and vein. Mesenchyme surrounding the notochord differentiates into a continuous sheath of spindle‐shaped cells, which forms the precursor to the mineralized areolar calcification of the centrum. Mesenchyme around the neural tube and caudal artery/vein becomes united by a population of mesenchymal cells that condenses lateral to the sheath of spindle‐shaped cells, with this mesenchymal complex eventually differentiating into the hyaline cartilage of the future neural arches, hemal arches, and outer centrum. The initially continuous layers of areolar tissue and outer hyaline cartilage eventually subdivide into discrete centra and arches, with the notochord constricted in the center of each vertebra by a late‐forming “inner layer” of hyaline cartilage, and by a ring of areolar calcification located medial to the outer vertebral cartilage. The vertebrae of elasmobranchs are distinct among vertebrates, both in terms of their composition (i.e., with centra consisting of up to three tissues layers—an inner cartilage layer, a calcified areolar ring, and an outer layer of hyaline cartilage), and their mode of development (i.e., the subdivision of arch and outer centrum cartilage from an initially continuous layer of hyaline cartilage). Given the evident variation in patterns of vertebral construction, broad taxon sampling, and comparative developmental analyses are required to understand the diversity of mechanisms at work in the developing axial skeleton of vertebrates. J. Morphol. 278:300–320, 2017. © 2017 Wiley Periodicals, Inc.     

Morphological relationships and leukocyte influence on steroid production in the epigonal organ-ovary complex of the skate, Leucoraja erinacea

In elasmobranchs, a unique association exists between an immune tissue, the epigonal organ (EO), and the gonads. In this study, the histological and vascular relationships of the EO and ovarian follicles of the little skate, Leucoraja erinacea, were assessed. Perfusions of Evans blue dye and Batson's monomer showed a shared vascular pathway from the gonadal artery into the epigonal-ovary complex, with blood first entering the EO and then perfusing the ovarian follicles. Histological studies demonstrated direct cellular contact between epigonal leukocytes and the follicle wall (FW), as well as the presence of leukocytes between the steroidogenic theca and granulosa cells. In vitro analyses demonstrated that epigonal cells co-cultured with FW cells cause a dose-dependent inhibition of estrogen (E2) and testosterone (T) production. In contrast, conditioned media from epigonal leukocytes, stimulated or unstimulated with lipopolysaccharide (10 microg/ml), increase the production of E2 and T from FW cells of the ovaries. These studies provide a basis for further investigations of leukocyte secreted factors and cell contact modulation of follicular steroid production.     

Hepatic uptake and biliary excretion of manganese in the little skate,Leucoraja erinacea

The liver is a major organ involved in regulating whole body manganese (Mn) homeostasis; however, the mechanisms of Mn transport across the hepatocyte basolateral and canalicular membranes remain poorly defined. To gain insight into these transport steps, the present study measured hepatic uptake and biliary excretion of Mn in an evolutionarily primitive marine vertebrate, the elasmobranch Leucoraja erinacea, the little skate. Mn was rapidly removed from the recirculating perfusate of isolated perfused skate livers in a dose-dependent fashion; however, only a small fraction was released into bile (< 2% in 6 h). Mn was also rapidly taken up by freshly isolated skate hepatocytes in culture. Mn uptake was inhibited by a variety of divalent metals, but not by cesium. Analysis of the concentration-dependence of Mn uptake revealed of two components, with apparent K_m values 1.1 ± 0.1 µM and 112 ± 29 µM. The K_m value for the high-affinity component was similar to the measured skate blood Mn concentration, 1.9 ± 0.5 µM. Mn uptake was reduced by nearly half when bicarbonate was removed from the culture medium, but was unaffected by a change in pH from 6.5 to 8.5, or by substitution of Na with Li or K. Mn efflux from the hepatocytes was also rapid, and was inhibited when cells were treated with 0.5 mM 2,4-dinitrophenol to deplete ATP levels. These data indicate that skate liver has efficient mechanisms for removing Mn from the sinusoidal circulation, whereas overall biliary excretion is low and appears to be mediated in part by an ATP-sensitive mechanism.     

Size and age estimates at sexual maturity for the little skate Leucoraja erinacea from the western Gulf of Maine,U.S.A.

Size and age estimates at sexual maturity were determined for 162 male and 273 female little skates Leucoraja erinacea collected from the western Gulf of Maine. Maturity ogives suggest that 50% maturity in females occurs at age 9·5 years and 480 mm total length (L_T), whereas 50% maturity in males occurs at a slightly younger age of 7·7 years and smaller size of 460 mm L_T. Age estimates were made from 389 L. erinacea ranging in size from 93 to 570 mm L_T. The index of average per cent error and age‐bias plots indicated that the ageing methods were precise and non‐biased. Additionally, annual periodicity of band formation was validated with oxytetracycline in eight individuals (three males and five females) ranging in age from 3 to 12 years. In conclusion, results from this study indicate that L. erinacea exhibits characteristics that make other elasmobranch populations highly susceptible to overexploitation.     

An improved method for separation of leucocytes from peripheral blood of the little skate (Leucoraja erinacea)

Cartilaginous fish, especially sharks, rays and skates (elasmobranchs), hold interest as comparative models in immunology because they are thought to be among the organisms most closely related to the ancestor animal that first developed acquired immunity. The aim of this study was to improve methods used for the purification of viable leucocytes from peripheral blood of elasmobranchs. Here we describe modifications of density gradient centrifugation and medium formulation that improve isolation and analysis of highly purified leucocytes from peripheral blood of a model elasmobranch, Leucoraja erinacea, the little skate. These techniques contribute to the preparation of elasmobranch immune cells that can be reliably analyzed by a variety of means, including the study of immune function.     

Seasonal variations in the physiological stress response to discrete bouts of aerial exposure in the little skate, Leucoraja erinacea

Aerial exposure and acute thermal stress have been shown to elicit profound physiological disruptions in obligate water-breathing teleosts. However, no study has investigated these responses in an elasmobranch. To address this, venous blood samples were collected and evaluated from little skates (Leucoraja erinacea) subjected to discrete aerial exposure durations (0, 15, and 50 min) coupled with differing abrupt thermal changes (gradient between seawater and air; winter: ΔT=-3 °C; summer: ΔT=+9 °C) in two distinct laboratory studies. In general, blood acid-base properties (e.g. decline in pH; elevation in PCO(2)) and select metabolites (elevated whole-blood lactate) and electrolytes (elevated plasma K(+)) were significantly disrupted by aerial exposure, and were most disturbed after skates were exposed to air for 50 min. However, the magnitude of the blood acid-base perturbations, metabolic contribution to the resulting blood acidosis, elevations to ionic and metabolic parameters, and delayed mortality were more extreme during the summer study, suggesting that acute thermal stress exacerbates the physiological impairments associated with aerial exposure in little skates. Conversely, a reduced thermal gradient (from seawater to air) may attenuate the magnitude of metabolic and ionic perturbations, resulting in a high physiological threshold for coping with extended aerial exposure.     

Gastro-intestinal handling of water and solutes in three species of elasmobranch fish,the white-spotted bamboo shark,Chiloscyllium plagiosum,little skate,Leucoraja erinacea and the clear nose skate Raja eglanteria

The present study reports aspects of GI tract physiology in the white-spotted bamboo shark, Chiloscyllium plagiosum, little skate, Leucoraja erinacea and the clear nose skate, Raja eglanteria. Plasma and stomach fluid osmolality and solute values were comparable between species, and stomach pH was low in all species (2.2 to 3.4) suggesting these elasmobranchs may maintain a consistently low stomach pH. Intestinal osmolality, pH and ion values were comparable between species, however, some differences in ion values were observed. In particular Ca²⁺ (19.67 ± 3.65 mM) and Mg²⁺ (43.99 ± 5.11 mM) were high in L. erinacea and Mg²⁺ was high (130.0 ± 39.8 mM) in C. palgiosum which may be an indication of drinking. Furthermore, intestinal fluid HCO₃^? values were low (8.19 ± 2.42 and 8.63 ± 1.48 mM) in both skates but very high in C. plagiosum (73.3 ± 16.3 mM) suggesting ingested seawater may be processed by species-specific mechanisms. Urea values from the intestine to the colon dropped precipitously in all species, with the greatest decrease seen in C. plagiosum (426.0 ± 8.1 to 0 mM). This led to the examination of the molecular expression of both a urea transporter and a Rhesus like ammonia transporter in the intestine, rectal gland and kidney in L. erinacea. Both these transporters were expressed in all tissues; however, expression levels of the Rhesus like ammonia transporter were orders of magnitude higher than the urea transporter in the same tissue. Intestinal flux rates of solutes in L. erinacea were, for the most part, in an inward direction with the notable exception of urea. Colon flux rates of solutes in L. erinacea were all in an outward direction, although absolute rates were considerably lower than the intestine, suggestive of a much tighter epithelia. Results are discussed in the context of the potential role of the GI tract in salt and water, and nitrogen, homeostasis in elasmobranchs.     

Effects of luminal osmolarity on gastric acid secretion in the little skate,Raja erinacea

Summary Isolated gastric mucosa of the skate shows marked changes in acid secretory rate (J _H ), electrical potential difference (PD), and transepithelial resistance (R) with changes in mucosal bathing solution composition and a constant serosal solution. Removal of the 350 mM urea usually present in the mucosal solution reduces acid secretory rate by 25%, while adding urea to 1 M has no significant effect. Complete removal of osmotic solutes (distilled water) inhibits secretion by 78%, isotonic urea (no salts) inhibits by 54%, while isotonic salts alone (no urea) gives control secretory rates. The changes in PD and R are consistent with acid secretory changes. Theory and experience with terrestrial organisms would not predict these changes. The most likely explanation is osmotic swelling and shrinking of the surface cells, and occlusion of the secretory tubules in the swollen condition. Since marine species never encounter hypo- or hyperosmotic conditions due to food ingestion, their surface cells may be water permeable, unlike the situation in terrestrial and fresh water animals.Abbreviations J _H acid secretory rate per square centimetre tissue area - OC oxyntic cell - PBC pit border cell - PD transepithelial electrical potential difference - R transepithelial electrical resistance per square centimetre tissue area - SEC Surface epithelial cell     

Purification, structural characterization, and myotropic activity of a peptide related to des-Arg(9)-bradykinin from an elasmobranch fish, the little skate, Leucoraja erinacea

A bradykinin (BK)-related peptide was isolated from heat-denaturated plasma from an elasmobranch fish, the little skate, Leucoraja erinacea after incubation with porcine pancreatic kallikrein. The primary structure of the peptide (H-Gly-Ile-Thr-Ser-Trp-Leu-Pro-Phe-OH; skate BK) shows limited structural similarity to the mammalian B1 receptor agonist, des-Arg(9)-BK. The myotropic activities of synthetic skate BK, and the analog skate [Arg(9)]BK, were examined in isolated skate vascular and intestinal smooth muscle preparations. Skate BK produced a concentration-dependent constriction of the mesenteric artery (EC(50)=4.37x10(-8)M; maximum response=103.4+/-10.23% of the response to 60mM KCl) but the response to skate [Arg(9)]BK was appreciably weaker (response to 10(-6)M=73.0+/-23.4% of the response to 60mM KCl). Neither the first branchial gill arch nor the ventral aorta responded to either purified peptide. Skate BK also produced a concentration-dependent constriction of intestinal smooth muscle preparations (EC(50)=2.74x10(-7)M; maximum response 31.0+/-12.2% of the response to 10(-5)M acetylcholine). Skate [Arg(9)]BK was without effect on the intestinal preparation. The data provide evidence for the existence of the kallikrein-kinin system in a phylogenetically ancient vertebrate group and the greater potency of skate BK compared with the analog skate [Arg(9)]BK suggests that the receptor mediating vascular responses resembles the mammalian B1 receptor more closely than the B2 receptor.     

Biosynthesis of 1α‐hydroxycorticosterone in the winter skate Leucoraja ocellata: evidence to suggest a novel steroidogenic route

The present study explores the ability of intracellular bacteria within the renal‐inter‐renal tissue of the winter skate Leucoraja ocellata to metabolize steroids and contribute to the synthesis of the novel elasmobranch corticosteroid, 1α‐hydroxycorticosterone (1α‐OH‐B). Despite the rarity of C1 hydroxylation noted in the original identification of 1α‐OH‐B, literature provides evidence for steroid C1 hydroxylation by micro‐organisms. Eight ureolytic bacterial isolates were identified in the renal‐inter‐renal tissue of L. ocellata, the latter being the site of 1α‐OH‐B synthesis. From incubations of bacterial isolates with known amounts of potential 1α‐OH‐B precursors, one isolate UM008 of the genus Rhodococcus was seen to metabolize corticosteroids and produce novel products via HPLC analysis. Cations Zn²⁺ and Fe³⁺ altered metabolism of certain steroid precursors, suggesting inhibition of Rhodococcus steroid catabolism. Genome sequencing of UM008 identified strong sequence and structural homology to that of Rhodococcus erythropolis PR4. A complete enzymatic pathway for steroid‐ring oxidation as documented within other Actinobacteria was identified within the UM008 genome. This study highlights the potential role of Rhodococcus bacteria in steroid metabolism and proposes a novel alternative pathway for 1α‐OH‐B synthesis, suggesting a unique form of mutualism between intracellular bacteria and their elasmobranch host.     

Age and size at sexual maturity for the winter skate, <Emphasis Type="Italic">Leucoraja ocellata</Emphasis>, in the western Gulf of Maine based on morphological,histological and steroid hormone analyses

Synopsis We determined age and size at sexual maturity in male and female winter skates, Leucoraja ocellata, from the western Gulf of Maine. Age estimated from vertebral band counts resulted in an Index of Average Percent Error (IAPE) of 5.6%, suggesting that this method represents an accurate approach to the age assessment of L. ocellata. Size at sexual maturity was assessed by evaluating three endpoints: steroid hormone concentrations, and morphological and histological criteria. Our results suggest that 50% maturity in males occurs at a total length of 730 mm and at 11 years of age. For females, our results suggest that 50% maturity occurs at a total length of 760 mm and between 11 and 12 years of age. Collectively, our study suggests that analyzing a combination of reproductive parameters offers an accurate estimation of sexual maturity in the winter skate. Moreover, our results indicate that L. ocellata is a late-maturing and long-lived species, characteristics which make it highly susceptible to over-exploitation by commercial fisheries.     

A role for GABAergic inhibition in electrosensory processing and common mode rejection in the dorsal nucleus of the little skate, Raja erinacea

The electrosensory primary afferents in elasmobranchs are responsive to electric potentials created by the animal's own ventilation, while the second-order neurons (AENs) which receive this afferent input in the medulla suppress responses to ventilatory potentials but retain their extreme sensitivity to electric signals in the environment. Ventilatory potentials are common mode signals in elasmobranchs and a common mode rejection mechanism is one way the AENs suppress ventilatory noise. By pressure injecting the GABA-A receptor antagonist SR95531 while extracellularly recording from AENs, we tested the hypothesis that the subtractive circuitry that selectively reduces common mode signals in AENs utilizes GABA, and that a GAB-Aergic component of the dorsal nucleus commissural pathway mediates crossed inhibition of AENs. Local application of SR95531 increased the spontaneous activity and the responsiveness of AENs to electrosensory stimuli. AEN responses to a common mode stimulus were selectively increased compared to responses to a localized stimulus due to SR95531 application. Contralateral inhibition of AENs was blocked by SR95531, indicating that GABAergic commissural cells may inhibit AENs when the contralateral side of the body is stimulated, as with common mode stimulation. We conclude that GABAergic inhibition contributes significantly to the shaping of AEN responses including common mode rejection.Abbreviations AENs ascending efferent neurons - GABA gamma-aminobutyric acid     

Insights into vertebral band pair deposition rate in the juvenile common thresher shark (Alopias vulpinus) in the northeastern Pacific Ocean

Validation of band pair deposition rates in elasmobranch vertebrae is essential for accurate age estimation using band pair counting techniques. We present a validation study of the vertebral band pair deposition rate for juvenile common thresher sharks Alopias vulpinus in the northeastern Pacific Ocean (NEPO) using tag and recapture with oxytetracycline (OTC) injection. A total of 14 juvenile A. vulpinus marked with OTC from 1998 through 2013 were recaptured with times at liberty ranging from 1.08 to 3.81 years with an average of 2.14 years (±0.97 years standard deviation, SD). Shark size ranged from 80 to 128 cm fork length (LF) at the time of OTC injection and from 112 to 168 cm LF for those measured at recapture. The slopes of the relationships between band pairs post OTC and years at liberty for each reader ranged from 0.84 to 0.95, slightly lower than the 1.0 slope expected from annual band pair formation. These findings preliminarily support previous age and growth assumptions based on a one band pair per year deposition rate. However, high variation in band pair deposition rates between samples, coupled with regression slopes falling just under one band pair per year, indicates that further investigation is needed to refine band pair deposition rate estimates.     

Maturity,size at age and predator–prey relationships of winter skate Leucoraja ocellata in the southern Gulf of St Lawrence: potentially an undescribed endemic facing extirpation

The goals of this study were to document the size and age structure, size at maturity, ovarian fecundity and diet of the endangered population of winter skate Leucoraja ocellata that resides in the southern Gulf of St Lawrence (SGSL). The maximum size observed for SGSL L. ocellata was 68 cm total length (L_T) but >99% of animals caught were <60 cm L_T. Fifty per cent of male and female L. ocellata were fully mature at 40 and 42 cm L_T, respectively, age c. 5 years. The oldest individual caught was age 11 years, but 98% of the 561 individuals examined were ≤age 8 years, indicating a short reproductive life span. Ovarian fecundity was low; no more than 29 ova >10 mm diameter were ever observed. At 40 cm L_T, the diet changed from one dominated by shrimp Crangon septemspinosa and gammarid amphipods to one dominated by fishes (mainly sand lance Ammodytes spp. and rainbow smelt Osmerus mordax) and Atlantic rock crab Cancer irroratus. Sufficient differences were observed between SGSL L. ocellata and other populations in their size‐at‐maturity pattern and maximum size to propose the taxonomic re‐evaluation of the population.     

Lack of biliary lipid excretion in the little skate, Raja erinacea, indicates the absence of functional Mdr2, Abcg5, and Abcg8 transporters

The ABC transporters bile salt export pump (BSEP; encoded by the ABCB11 gene), MDR3 P-glycoprotein (ABCB4), and sterolin 1 and 2 (ABCG5 and ABCG8) are crucial for the excretion of bile salt, phospholipid, and cholesterol, respectively, into the bile of mammals. The current paradigm is that phospholipid excretion mainly serves to protect membranes of the biliary tree against bile salt micelles. Bile salt composition and cytotoxicity, however, differ greatly between species. We investigated whether biliary phospholipid and cholesterol excretion occurs in a primitive species, the little skate, which almost exclusively excretes the sulphated bile alcohol scymnolsulphate. We observed no phospholipid and very little cholesterol excretion into bile of these animals. Conversely, when scymnolsulphate was added to the perfusate of isolated mouse liver perfusions, it was very well capable of driving biliary phospholipid and cholesterol excretion. Furthermore, in an erythrocyte cytolysis assay, scymnolsulphate was found to be at least as cytotoxic as taurocholate. These results demonstrate that the little skate does not have a system for the excretion of phospholipid and cholesterol and that both the MDR3 and the two half-transporter genes, ABCG5 and ABCG8, have evolved relatively late in evolution to mediate biliary lipid excretion. Little skate plasma membranes may be protected against bile salt micelles mainly by their high sphingomyelin content.     

Age and growth of the tropical oviparous shark,Chiloscyllium punctatum in Indonesian waters

The brown-banded bamboo shark, Chiloscyllium punctatum, is the most common shark caught in coastal commercial fisheries throughout Southeast Asia, yet there is a lack of the life-history information necessary for reliable stock assessments. The authors estimated growth rates and age at maturity using analysis of growth bands in vertebral centra. They trialled four different techniques to enhance the visibility and improve identification of the putative annual growth bands necessary for age estimation. The authors found that the burn method on whole vertebral centra provided the most readable and consistent results for age analysis. The logistic model was chosen as the best-fit growth model for age estimation of 330 individual C. punctatum from Indonesia. Several age verification methods, including marginal increment ratio and length-frequency analysis, were performed with the support of age validation through the use of calcein-labelled vertebrae from two sharks maintained in captivity. This study found that C. punctatum from Indonesian waters is a fast-growing species that can grow up to 18 cm year⁻¹, reach an estimated maximum total length of 1 m, mature at c. 6.5 years and live for up to 14 years.