An investigation on the effect of photoperiod and temperature on vertebral band deposition in little skate Leucoraja erinacea

An investigation was undertaken to determine whether photoperiod or temperature have an effect on the timing of vertebral opaque–transluscent band‐pair deposition in captive young‐of‐the‐year (YOY) little skate Leucoraja erinacea. The experimental design consisted of a randomized complete block split plot design with two factors: temperature and light. Temperature was nested within light and therefore four variables were tested: 1) constant light, 2) constant temperature, 3) seasonal light and 4) seasonal temperature. For 18 months, L. erinacea experienced accelerated seasonal conditions of temperature and light to mimic 3 years of growth. This study provides primary and supporting evidence that seasonal photoperiod and temperature, respectively, have no effect on timing of vertebral band‐pair deposition in captive L. erinacea. Vertebral analysis of surviving L. erinacea (n = 6, time = 18 months) showed that all produced 1–1·5 band pairs, while centrum edge analysis (n = 56) showed timing of winter and summer band deposition were similar regardless of treatment. The winter band (translucent) appeared in February 2007 and January 2008 while the summer band (opaque) showed up in July for both 2007 and 2008 and mimicked patterns observed in the wild. While temperature and photoperiod appear to have no effect on timing of band‐pair deposition in YOY L. erinacea, other mechanisms which may influence band deposition should be investigated including the effect of food ration and the presence of a circa‐annual rhythm and hormone secretion.     

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.     

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.     

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.     

The hepatic glutathione transferases of the male little skate, Raja erinacea

Five cytosolic glutathione transferases were isolated from the liver of the male little skate, Raja erinacea, a marine elasmobranch. They were designated E-1 through E-5 in order of their elution from a DEAE-cellulose column with a 0 to 100 mM KCl gradient in 0.01 M Tris (pH 8.0). Each eluted peak of glutathione transferase activity, after concentration, was applied to an affinity column prepared by reaction of epoxy-activated Sepharose 6B with glutathione (GSH). Elution of the various glutathione transferases from this column with GSH resulted in the further purification of each enzyme; the major glutathione transferase, E-4 and E-1, were purified to apparent homogeneity by this procedure. Skate glutathione transferase E-4 is dimeric and the subunits are either very similar or identical in molecular weight (about 26 000 daltons). Enzymes E-2 through E-5 were acidic proteins (pI less than 7.0) and had high specific glutathione transferase activity (0.3--12 mumol/min/mg protein) with benzo[a]pyrene 4,5-oxide (BPO) as substrate, whereas the other enzyme (E-1) had low activity (0.01 mumol/min/mg) with BPO and a basic pI (greater than 9.5). Bilirubin and hematin, non-substrate ligands, bound tightly to homogeneous E-4, with dissociation constants in the micromolar range.     

Identification of vitellogenin in the little skate (Raja erinacea).

1. Vitellogenin was isolated from mature female skates by selective precipitation with MgCl2/EDTA followed by chromatography on DEAE-cellulose columns. 2. A single monomer of approximately 205 kDa was identified on 6.0% SDS-PAGE gels. 3. In addition, isolation of yolk proteins with ammonium sulfate yielded proteins of 94 and 38 kDa (putative phosvitins) and putative lipovitellins of ca 105, 91 and 67 kDa. 4. In vivo phosphate incorporation in female and male skates implanted with estradiol indicated that vitellogenin was phosphorylated. 5. Total protein phosphate incorporation was significantly higher in females than male skates. 6. In male skates treated with estradiol, phosphate incorporation increased from 2 days after implantation to a maximum at approximately 11 days after implantation. 7. Determination of the rate of disappearance of 32P-labeled protein suggests a half-life of ca 200 hr in normal female skate plasma.     

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.     

Osteological development of the vertebral column and of the caudal complex in Dentex dentex

Osteological development of the vertebral column and caudal complex in common dentex was described under extensive larval rearing conditions. Generally, the cartilaginous bones developed prior to the membranous bones. The development of the axial skeleton began with the formation of the hypural 1, the neural arches 2 and 3, as well as the haemal arches 1–8 at 4·8, 4·9 and 5·0 mm total length ( L _T, measured in vivo ), respectively. By 7·5 mm L _T, all the cartilaginous elements were formed, except for the ventral ribs, which formed between the range of 8·4–18·0 mm L _T. The caudal lepidotrichia were the first membranous bones to appear (5·3 mm L _T) and attain their full meristic count (7·4 mm L _T), followed by the vertebral centra, which formed between 6·6 and 9·7 mm L _T. By 25·0 mm L _T, all the elements were fully ossified except for the ventral ribs. The developmental direction and order of all the elements were studied with respect to their formation and ossification. The results were discussed in the contexts of ichthyoplankton, ecology and aquaculture. Compared with other Sparidae species, common dentex followed a pattern of relatively rapid rate of osteological development.     

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.     

Captive breeding of the barndoor skate (Dipturus laevis) at the Montreal Biodome,with comparison notes on two other captive‐bred skate species

In 1997, the Montreal Biodome obtained five barndoor skates (Dipturus laevis) from the waters off Boston, Massachusetts. Six years later, those specimens began reproducing, and the first egg case was collected in November 2003. Since then, 73 hatchlings have been born and raised. Egg cases were observed year round, and annual fecundity was measured for the first time: one female laid 69 eggs in 2005, 85 in 2006 and 115 in 2007. Egg incubation was longer than believed previously, ranging from 342 to 494 days. Hatching occurred throughout the year. Hatchlings averaged 193 mm total length and 128 mm disk width and weighed 32 g. They were fed krill and diced fish. All but one survived the first month. A photo identification system was useful in recognizing two groups of 10 specimens during their first year, and transponders could be inserted in the wing muscles of 1‐year‐old skates. Total lengths at birth and at age 2 were similar to the data reported from the wild, suggesting a similar growth pattern in captivity. The reproduction characteristics of the barndoor skate were compared with those of two other skate species currently bred at the Montreal Biodome, the winter skate (Leucoraja ocellata) and the thorny skate (Amblyraja radiata). Zoo Biol 27:145–153, 2008. © 2008 Wiley‐Liss, Inc.     

Evidence of a rudimentary colon in the elasmobranch, Leucoraja erinacea

The transition from aquatic to terrestrial life presented tetrapodamorphs with the challenge of maintaining water homeostasis and preventing desiccation on land. The colon evolved in terrestrial vertebrates to help maintain fluid balance. Although marine elasmobranchs lack a colon, their spiral intestine contains a subregion that histologically appears to be colon-like, possibly representing an evolutionary precursor to terrestrial digestive tracts. The distal-most region of the spiral intestine of elasmobranchs has no villi and a large number of acid mucins: hallmarks of water absorption in the colons of terrestrial animals. To determine if histologically distinct regions of the elasmobranch digestive tract correspond to functional differences, we compared water absorption in different subregions of the skate, Leucoraja erinacea digestive tract. Water absorption in stomach and spiral intestinal sacs was linear with time and not hydrostatic pressure-dependent. The histologically distinct distal portion of the spiral intestine had a threefold higher rate of water absorption than the proximal portion of the spiral intestine. In addition, the water-selective, colon-specific aquaporin 4 is expressed strongly in the distal spiral intestine epithelia, correlating with the region of the spiral intestine exhibiting the greatest rate of water absorption. We demonstrate that the distal spiral intestine is histologically and functionally distinct from the rest of the spiral intestine and represents a rudimentary colon within the vertebrate lineage.     

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.