Dasyatis Sabina Research Articles

Characterization of gonadal and extra-gonadal forms of the cDNA encoding the Atlantic stingray (Dasyatis sabina) cytochrome P450 aromatase (CYP19)

Cytochrome P450 aromatase (P450arom; CYP19) mediates the conversion of androgens to estrogens and its activity has been found in all vertebrates studied to date. This study describes the full-length cDNA encoding the ovarian form of P450arom and the differences in the 5′-untranslated region (5′-UTR) of the extra-gonadal P450arom transcript expressed by the Atlantic stingray (Dasyatis sabina). Elasmobranchs (cartilaginous fishes such as sharks, rays and skates) diverged from the other vertebrates more than 350 million years ago, therefore the stingray P450arom cDNA may represent an ancient form of this gene. Northern blot analysis showed that the ovarian follicle expressed transcripts of 3.1 and 1.7 kb in size which correspond to the clones isolated from a stingray ovarian follicle cDNA library. Both transcripts consisted of an identical 1.5 kb coding region and a 41 bp 5′-UTR, however the 3′-UTRs differed in the use of the most proximate and the most distal of four polyadenylation signals. COS cells transfected with the 1.7 kb cDNA had twice the aromatase activity as cells transfected with the 3.1 kb cDNA. The coding region of the cDNA predicted a 58.5 kDa protein which consisted of 511 residues. Alignment of the stingray protein indicates that the P450arom is equally identical (53–59%) to all other vertebrate forms of P450arom characterized to date, thus indicating a common ancestry. The evolutionary relationship of the stingray form of P450arom clearly predates the other forms and belongs to a unique lineage. Transcripts of P450arom were expressed in ovarian follicles (of all sizes), the testis, the pituitary, in all sections of the brain, and in the kidney. The extra-gonadal transcripts appear to encode a protein identical to the ovarian form, however, the 5′-UTR was 657 bp longer presumably due to the transcription of an untranslated ‘first exon’ as seen in the mammalian form of this gene.

Dermal Bite Wounds as Indicators of Reproductive Seasonality and Behaviour in the Atlantic Stingray, Dasyatis sabina

Elasmobranch fishes exhibit a series of complex courtship and mating behaviours in which males inflict significant bite wounds to the body of female mates. However, the variety and frequency of mating wounds are not known across a full reproductive season for any species. We examined the distribution and abundance of dermal wounds in adult Atlantic stingrays, Dasyatis sabina, which have a protracted and defined mating season to determine (1) if dermal wounds can be used as indicators of mating activity, (2) whether different categories of bite wounds can be associated with specific mating behaviours, and (3) whether the skin thickness in females is sexually dimorphic. Adults of both sexes showed fresh wounds during the full duration of the mating season (October–June) and there was no relationship between ray size and wound density. Females had more total wounds than males in every month with a maximum average of 20.2 wounds per female in April. Mating wounds were categorized into five distinct forms: single track, double track, bite, margin abrasion and excision. Wounds were randomly distributed over the body of males but concentrated on the posterior half of the disc in females. Each wound type occurred in approximately equal proportions among sexes with the exception of the precopulatory and copulatory-induced margin abrasions which accounted for 13.7% of the total wounds in females but only 3.1% in males. We suggest that the pronounced and concurrent appearance of single track, double track and bite wounds among males results from random premating courtship attacks by males because females cannot be visually discriminated. However, the concentration of wounds on the posterior disc of females is consistent with the possible presence of olfactory cues (e.g. pheromones) that are released at the cloaca. The pectoral fin dermis of females was 50% thicker than that of males, which eliminated the excision of margins during male grasping and functions to reduce female injury. The temporal occurrence of wounds from October through June and peak in April is consistent with previous reproductive studies that show fresh sperm in the reproductive tract of females over the protracted mating period and also ovulation in late March or early April. The importance of social reproductive biting is discussed in relation to the reproductive induction hypothesis proposed to possibly explain the protracted mating of this species. Monitoring of dermal wounds provides a useful non-invasive technique to determine reproductive activity and a means for inference of social relationships in elasmobranch populations.

Annual Cycles of Steroid Hormone Production, Gonad Development, and Reproductive Behavior in the Atlantic Stingray

The mating season of the Atlantic stingray (Dasyatis sabina), which begins in August and continues through April, is the longest documented for any elasmobranch fish. Despite this protracted mating period, female stingrays ovulate synchronously at the end of the mating season and there is no evidence for sperm storage by females. Thus, the proximate causal factors and ultimate function of this extended preovulatory mating are unknown. Annual cycles of the gonadal steroids testosterone (T), dihydrotestosterone (DHT), 17ß-estradiol (E2), and progesterone (P4) were measured for 26 months in a wild estuarine population of Atlantic stingrays to test for associations with their reproductive biology, gametogenesis, and sexual behavior. Serum androgen levels in males showed four phases within an annual cycle: (1) androgen suppression between reproductive seasons (April–July), (2) primary androgen increase during the onset of spermatocyte development (August–October), (3) androgen decrease following maximum testis growth and spermatocyte development (November–December), and (4) secondary androgen increase during the peak of sperm maturation (January–March). Increases in male E2 and P4 were correlated with spermatocyte/spermatocyst formation, maximum testis weight, and the primary (but not secondary) androgen surge. We propose that the production of male androgens across the full seven-month preovulatory mating period promotes their aggressive reproductive behavior and drives the protracted mating season of this species. In females, serum T and DHT showed relatively brief increases near ovulation, whereas E2 and P4 showed brief increases near both ovulation and parturition. The increase in female androgens near ovulation may increase female aggression when they are impregnable by courting males and enhance their choice of mates. This estuary sample population shows higher absolute steroid levels and distinct differences in temporal cycles compared to another Florida fresh water lake population, but the cause and significance of these differences are unknown. Experiments are needed to confirm that the aggressive and protracted mating behavior is the result of prolonged male androgen production and to determine whether the sustained preovulatory mating serves some function related to female reproduction.

Differential Distribution of Gonadotropin-Releasing Hormone-Immunoreactive Neurons in the Stingray Brain: Functional and Evolutionary Considerations

Gonadotropin-releasing hormone (GnRH) is a neuropeptide that occurs in multiple structural forms among vertebrate species. Bony fishes, amphibians, reptiles, birds, and mammals express different forms of GnRH in the forebrain and endocrine regions of the hypothalamus which regulate the release of reproductive gonadotropins from the pituitary. In contrast, previous studies on bony fishes and tetrapods have localized the chicken GnRH-II (cGnRH-II) nucleus in the midbrain tegmentum and, combined with cladistic analyses, indicate that cGnRH-II is the most conserved form throughout vertebrate evolution. However, in elasmobranch fishes, the neuroanatomical distribution of cGnRH-II and dogfish GnRH (dfGnRH) cells and their relative projections in the brain are unknown. We used high-performance liquid chromatography and radioimmunoassay to test for differential distributions of various GnRH forms in tissues from the terminal nerve (TN) ganglia, preoptic area, and midbrain of the Atlantic stingray, Dasyatis sabina. These experiments identified major peaks that coelute with cGnRH-II and dfGnRH, minor peaks that coelute with lamprey GnRH-III (lGnRH-III), and unknown forms. Immunocytochemistry experiments on brain sections show that dfGnRH-immunoreactive (-ir) cell bodies are localized in the TN ganglia, the caudal ventral telencephalon, and the preoptic area. Axons of these cells project to regions of the hypothalamus and pituitary, diencephalic centers of sensory and behavioral integration, and the midbrain. A large, discrete, bilateral column of cGnRH-II-ir neurons in the midbrain tegmentum has sparse axonal projections to the hypothalamus and regions of the pituitary but numerous projections to sensory processing centers in the midbrain and hindbrain. Immunocytochemical and chromatographic data are consistent with the presence of lGnRH-III and other GnRH forms in the TN that differ from dfGnRH and cGnRH-II. This is the first study that shows differential distribution of cGnRH-II and dfGnRH in the elasmobranch brain and supports the hypothesis of divergent function of GnRH variants related to gonadotropin control and neuromodulation of sensory function.

Structure of the thyroid gland, serum thyroid hormones, and the reproductive cycle of the Atlantic stingray, Dasyatis sabina

This study examines the role of the thyroid gland in the control of reproduction in the viviparous Atlantic stingray, Dasyatis sabina. Thyroid activity in individuals in different reproductive stages was assessed both by microscopic examination of the gland, and by analysis of circulating levels of thyroid hormones from the same individuals. The thyroid gland is a cylindric organ, embedded in a connective tissue capsule, and composed of follicles, i.e., monolayer spheres of thyroid epithelial cells. Stingray follicular cells possess several characteristic features, namely apical cilia and a well-developed endoplasmic reticulum. Cells vary in size and shape, according to the activity of the gland. No structural differences were observed between the thyroid glands of the two sexes. Both thyroid hormones, triiodothyronine, [T3], and thyroxine, [T4], were detected in the serum of all animals examined. Levels ranged from 1.3–2.6 μg/100 ml for total T4, and from 1.2–2.6 ng/ml for total T3. The T4 levels did not vary significantly in any group. Immature individuals and females undergoing oogenesis had the lowest levels of circulating T3 and mature females from ovulation throughout gestation had high thyroid gland activity and high levels of circulating T3. J. Exp. Zool. 284:505–516, 1999. © 1999 Wiley-Liss, Inc.

Structure of the thyroid gland, serum thyroid hormones, and the reproductive cycle of the Atlantic stingray,Dasyatis sabina

This study examines the role of the thyroid gland in the control of reproduction in the viviparous Atlantic stingray, Dasyatis sabina. Thyroid activity in individuals in different reproductive stages was assessed both by microscopic examination of the gland, and by analysis of circulating levels of thyroid hormones from the same individuals. The thyroid gland is a cylindric organ, embedded in a connective tissue capsule, and composed of follicles, i.e., monolayer spheres of thyroid epithelial cells. Stingray follicular cells possess several characteristic features, namely apical cilia and a well-developed endoplasmic reticulum. Cells vary in size and shape, according to the activity of the gland. No structural differences were observed between the thyroid glands of the two sexes. Both thyroid hormones, triiodothyronine, [T(3)], and thyroxine, [T(4)], were detected in the serum of all animals examined. Levels ranged from 1.3-2.6 microg/100 ml for total T(4), and from 1.2-2.6 ng/ml for total T(3). The T(4) levels did not vary significantly in any group. Immature individuals and females undergoing oogenesis had the lowest levels of circulating T(3) and mature females from ovulation throughout gestation had high thyroid gland activity and high levels of circulating T(3). J. Exp. Zool. 284:505-516, 1999.

Regulation of interrenal gland steroidogenesis in the Atlantic stingray (Dasyatis sabina)

The interrenal gland (the homologue of the mammalian adrenal cortex) of elasmobranchs (sharks, skates, and rays) produces 1alpha-hydroxycorticosterone (1alpha-B), which has been reported to function both as a gluco- and mineralocorticosteroid. In vitro synthesis of 1alpha-B by Atlantic stingray (Dasyatis sabina) interrenal glands was stimulated by short-term (2 hr) and long-term (24 hr) treatment with porcine adrenocorticotropic hormone (pACTH). Cycloheximide blocked the pACTH-induced effect on 1alpha-B synthesis, thus demonstrating that the mechanism for the short-term induction of steroidogenesis involved protein synthesis. However, gene transcription did not play a role in the short-term induction of 1alpha-B synthesis, as indicated by the lack of an effect with actinomycin D treatment. Long-term in vitro exposure to pACTH (but not short-term exposure) stimulated the synthesis of another steroid, 11-dehydrocorticosterone (A). This induction was partially blocked by cycloheximide and actinomycin D, which suggests enhanced expression of the 11beta-hydroxysteroid dehydrogenase gene. In addition, the 24-hr treatment with pACTH enhanced the activity of cytochrome P450 side chain cleavage several fold and doubled the activity of 3beta-hydroxysteroid dehydrogenase and cytochrome P450 21-hydroxylase in D. sabina interrenals, again suggesting the induction of steroidogenic genes. In contrast to other elasmobranch species, the salmon and human forms of angiotensin II had no effect on D. sabina interrenal steroidogenesis. J. Exp. Zool. 284:517-525, 1999.

Morphology and Epithelial Ion Transport of the Alkaline Gland in the Atlantic Stingray (Dasyatis sabina).

The alkaline glands are two fluid-filled sacs that lie on the ventral, posterior surface of each kidney in skates and rays. In this study, the morphology, transepithelial ion transport, fluid constituents, and histochemistry of the alkaline glands of the Atlantic stingray, Dasyatis sabina, were investigated. The duct from each gland joined the corresponding vas deferens and the resulting two common ducts emptied into the cloaca. Dark burgundy, aqueous fluid (pH 8.0-8.2) was secreted into the sacs by a simple columnar epithelium with extensive rough endoplasmic reticulum and large secondary lysosomes containing lipofuscin and membrane fragments. Zonulae occludentes were deep (~22 fibrils), reflecting an electrically tight epithelium (732 ohms/cm2). Carbonic anhydrase activity was localized histochemically within the intercellular spaces and less intensely in the mid-basal cytoplasm. In vitro electrophysiology showed that baseline shortcircuit current (Isc, 29.1 {mu}A/cm2) was reduced 67.0% after Cl- removal from the medium. Cl- removal also completely abolished luminal alkalinization (baseline 4.5 +/- 0.7 {mu}Eq of acid/cm2/h). Luminal exposure to the chloridebicarbonate exchange inhibitor, DIDS, reduced Isc by 38%. Simultaneous administration of DIDS and bumetanide (Na+/K+/Cl- cotransport inhibitor) to the serosal side of the tissue caused the Isc to decrease >100%. Serosal exposure to ouabain (Na-K, ATPase inhibitor) decreased Isc 48%, whereas amiloride (sodium ion channel blocker) and acetazolamide (carbonic anhydrase inhibitor) had no statistically significant effect on Isc or alkalinization rates. Taken together the results suggest the presence of apical epithelial bicarbonate exchangers that are chloride or sodium dependent, basal sodium and HCO3- transport, and an Isc that is not totally dependent on Na+-K+ ATPase.

Morphology of the mechanosensory lateral line system in the Atlantic Stingray, Dasyatissabina: The mechanotactile hypothesis.

The biological function of anatomical specializations in the mechanosensory lateral line of elasmobranch fishes is essentially unknown. The gross and histological features of the lateral line in the Atlantic stingray, Dasyatis sabina, were examined with special reference to its role in the localization and capture of natural invertebrate prey. Superficial neuromasts are arranged in bilateral rows near the dorsal midline from the spiracle to the posterior body disk and in a lateral position along the entire length of the tail. All dorsal lateral line canals are pored, contain sensory neuromasts, and have accessory lateral tubules that most likely function to increase their receptive field. The pored ventral canal system consists of the lateral hyomandibular canal along the disk margin and the short, separate mandibular canal on the lower jaw. The extensive nonpored and relatively compliant ventral infraorbital, supraorbital, and medial hyomandibular canals form a continuous complex on the snout, around the mouth, and along the abdomen. Vesicles of Savi are small mechanosensory subdermal pouches that occur in bilateral rows only along the ventral midline of the rostrum. Superficial neuromasts are best positioned to detect water movements along the transverse body axis such as those produced by tidal currents, conspecifics, or predators. The pored dorsal canal system is positioned to detect water movements created by conspecifics, predators, or possibly distortions in the flow field during swimming. Based upon the stingray lateral line morphology and feeding behavior, we propose the Mechanotactile Hypothesis, which states that the ventral nonpored canals and vesicles of Savi function as specialized tactile mechanoreceptors that facilitate the detection and capture of small benthic invertebrate prey. J. Morphol. 238:1-22, 1998. © 1998 Wiley-Liss, Inc.

Osmoregulation of the Atlantic stingray (Dasyatis sabina) from the freshwater Lake Jesup of the St. Johns River, Florida.

The goals of this study were to (1) measure plasma osmolytes and rectal gland weights of a freshwater (FW) Atlantic stingray (Dasyatis sabina) population in the St. Johns River, Florida, and (2) determine how these parameters change after acclimation to seawater (SW). We hypothesized that the FW D. sabina may show physiological divergence from marine D. sabina, because the FW individuals reproduce and complete their life cycle in the St. Johns River. The FW D. sabina hyperregulate their plasma osmolality (621.4 mOsm kg(-1)), with plasma Na+, Cl-, and urea concentrations of 211.9, 207.8, and 195.9 mmol L(-1), respectively. FW D. sabina were exposed to 100% SW for 8 d, and their hematocrit did not change significantly compared to control animals left in FW. However, plasma osmolality increased significantly (953 mOsm kg(-1)), with significant increases in plasma concentrations of Na+, Cl-, and urea to 319.13, 296.1, and 329.76 mmol L(-1), respectively. The plasma of the SW-adapted D. sabina was hypo-osmotic and hypoionic to 100% SW. Rectal gland weight to body weight (RGBW) ratios of FW D. sabina were about 80% lower than RGBW ratios reported for marine D. sabina; the RGBW ratio did not increase significantly after SW acclimation. This may indicate that branchial and renal mechanisms are also involved with ion excretion. We conclude that the FW D. sabina are physiologically euryhaline and have not evolved the osmoregulatory strategy of stenohaline FW Potamotrygonid stingrays.

Increase in gonadotropin-releasing hormone (GnRH) levels in CSF after stimulation of the nervus terminalis in Atlantic stingray, Dasyatis sabina

The nervus terminalis (NT) contains many cells immunoreactive to gonadotropin-releasing hormone (GnRH). The potential of the NT to release GnRH in vivo was investigated by stimulating the peripheral nerve trunk of Atlantic stingrays and collecting cerebrospinal fluid (CSF). The CSF samples from stimulated animals averaged about twice the levels of mGnRH-like peptide as those of unstimulated animals. These results demonstrate that nervus terminalis activity can effect in vivo GnRH levels in the brain.

Serum Concentrations of Steroid Hormones during Reproduction in the Atlantic Stingray,Dasyatis sabina

The Atlantic stingray,Dasyatis sabina,has a well-defined annual reproductive cycle in Florida. We collected adult specimens over 12 months and evaluated reproductive parameters and serum levels of five steroid hormones, 17β-estradiol (E2), progesterone (P4), testosterone (T), dihydrotestosterone (DHT), and corticosterone (CS). Female E2peaked twice, once in mid-March to early April in association with ovulation and again in mid-June to mid-July in association with the enlargement of a second group of ovarian follicles. Female P4peaked in early March and early April, coincident with the peak in E2. Female DHT was variable but exhibited a pattern not clearly associated with known events in the reproductive cycle. Female T and CS levels did not vary significantly through time. In males, T, DHT, and CS increased progressively through winter and spring, peaking in March when females were ovulating and when copulation probably took place. DHT concentrations were usually at least twice T levels. These three hormones peaked long after the November/December peak in gonadosomatic index. E2was measurable in males and was highest during the period of testicular development. Male P4varied in a pattern not clearly associated with known reproductive events.

Identification of a glycosylated relaxin-like molecule from the male Atlantic stingray, Dasyatis sabina.

The alkaline gland fluid of the Atlantic stingray (Dasyatis sabina) contains a molecule that cross-reacts weakly to anti-porcine relaxin antibodies. This material was isolated and purified to homogeneity by reversed-phase high-performance liquid chromatography. In SDS gel electrophoresis, the molecule showed an apparent molecular mass of 13 kDa which upon reduction formed two polypeptide chains of 4 and 9 kDa, respectively. Sequence analyses revealed a 27-amino acid residue A chain and a 54-amino acid residue B chain which contained an N-glycosylation site in position B37. The distribution of the six cysteines and possibly the disulfide bonding is identical to that found in insulins and most relaxins. Although the stingray relaxin-like molecule contains the structurally relevant glycine residues within the A chain, in the midregion of the B chain it has only one of the two requisite binding site arginines, which explains the lack of relaxin bioactivity in standard mammalian assays. Stingray relaxin is the first member of the relaxin family identified in a nonhomeotherm male. Carbohydrate analysis of relaxin revealed an N-linked asialo, agalacto, bisected biantennary, and a core-fucosylated oligosaccharide in the position of Asn B37 which makes it the first reported glycosylated relaxin-like molecule.

Anti-Zebrin II immunopositivity in the cerebellum and octavolateral nuclei in two species of stingrays.

Anti-Zebrin II is an antibody directed against a 36kDa aldolase epitope expressed by Purkinje cells. Two patterns of Zebrin II immunolabeling have been described in the cerebellar corpus. In mammalian cerebella, the anti-Zebrin II labels longitudinal zones of Purkinje cells, whereas in teleosts, all Purkinje cells of the cerebellar corpus are Zebrin II immunopositive. An outgroup analysis is required to determine which of these distribution patterns represents the primitive condition for jawed vertebrates. The sister group of the Osteichthyans (rayfinned fishes, amphibians, and amniotes) is the Chondrichthyans (sharks, skates, and rays). In the present study the distribution of Zebrin II immunopositivity was examined in the Atlantic stingray and the Southern stingray. Western-blot analysis demonstrates that the Zebrin II antibody recognizes an antigen of the same molecular weight in stingrays, teleosts, and mammals. In stingrays, anti-Zebrin II immunohistochemistry reveals a staining pattern in which all Purkinje cells are immunopositive, no banding pattern or zonal compartmentation is observed. Purkinje cell axon projections to the cerebellar nucleus and the octaval nuclei are also revealed. Within the octaval nuclei, immunopositive Purkinje cell axon terminals and boutons en passant were found in the anterior, descending, and posterior nuclei. These immunopositive profiles are found throughout these nuclei, but they are most dense in the lateral and ventral portions. Except for the dorsolateralmost portion, the magnocellular nucleus does not appear to receive Purkinje cell inputs. Based on these results it is concluded that the Zebrin II distribution pattern in which all Purkinje cells of the cerebellar corpus are immunopositive is the primitive condition for jawed vertebrates.

Periodic gonadal activity and protracted mating in elasmobranch fishes

Temporal patterns of gonad development are determined by environmental cues that regulate hormonal cycles and ultimately affect a population's mating system. Annual periodicity of gonad histology is essentially unknown for the more than 450 species of batoid elasmobranchs. Temporal periodicity in spermatogenic activity and ova production in the Atlantic stingray (Dasyatis sabina) were examined by histology over a consecutive 20 month period. Gonadosomatic index (GSI) shows three distinct phases associated with structural changes at the cellular level. Testes in the inactive phase occur from March through July, have a low GSI, and are represented only by germinal (SI) and early spermatocyst (SII) stages. The enlargement phase begins in mid-August, followed by rapid testicular growth that peaks in October. Testicular recrudescence is characterized by a decline in the proportion of early stage spermatocysts (SI, II) and a sequential maturation of cells to the spermatocyte (SIII), spermatid (SIV), immature sperm (SV), and mature spermatozoa (SVI) stages. The measure of absolute spermatogenic production (ASP) is maximum from about August through January. The diminution phase is characterized by a decrease in male GSI from October through April associated with a reduced tissue biomass and predominance of early spermatogenic stages. The annual succession of peaks in sperm formation indicates continuous spermatogenesis through the fall-winter and shows that peak sperm production lags maximum GSI by approximately 3–4 months. Further, seminal vesicle diameter peaks in February, which also lags maximum GSI by 4 months. Egg growth in females is a periodic process of 5–6 months duration that involves vitellogenesis of 2–4 oocytes. Maximum ova diameter increases after mid-September, peaks in March (x¯ = 10.62 mm), and covaries with the increase in female GSI. Despite the brief period of ovulation and fertilization in March-April, fresh mating scars and sperm in the lower reproductive tract of females confirm a protracted mating period from October through April-May. Thus, mating begins in the population at least 7 months prior to ovulation and fertilization. Current evidence indicates this protracted mating period is not explained by female sperm storage or arrested embryonic development. We suggest the protracted mating period serves some currently undetermined function such as induction of steroidogenesis, oocyte growth, or ovulation in females. © 1996 Wiley-Liss, Inc.

Seasonal dynamics of dental sexual dimorphism in the Atlantic stingray Dasyatis sabina

Cartilaginous fishes continuously replace their teeth throughout their life (polyphyodonty) and often show a sexually dimorphic dentition that was previously thought to be an invariant sex character. Radial vector analysis of tooth shape in the polyphyodontic stingray Dasyatis sabina across a consecutive 24 month period shows a stable molariform morphology for females but a periodic shift in male dentition from a female-like molariform to a recurved cuspidate form during the reproductive season. The grip tenacity of the male dentition is greater for the cuspidate form that occurs during the mating season than for the molariform dentition that occurs during the non-mating season. Dental sexual dimorphism and its sex-dependent temporal plasticity probably evolved via polyphyodontic preadaptation under selective pressures on both sexes for increased feeding efficiency and sexual selection in males to maximize mating success. These phenomena are important considerations for the identification and classification of cartilaginous fishes and possibly other polyphyodontic vertebrates in the fossil record.

Observations on Extant Populations of the Softshell Clam, Mya arenaria Linné, 1758 (Bivalvia: Myidae), from Georgia (USA) Estuarine Habitats

The softshell clam, Mya arenaria Linné, 1758, is reported from Georgia (USA) estuarine habitats based on studies conducted between 1969 and 1972. Observations on Georgia estuarine habitats where extant populations of softshell clams occurred are described. On several occasions, fresh shells with periostracum and tissue remnants were found in a brackish drainage system on Sapelo Island. These shells represent the first southern records of extant populations of softshell clams from such a specialized habitat type. Living specimens of M. arenaria from the benthos and specimens in the stomach contents of stingrays (Dasyatis sabina) were collected at four stations during 1969 in the North and South Newport Rivers. mesohaline tidal systems forming part of the southern and northern headwaters of St. Catherines and Sapelo Sounds.The stomach of a stingray collected near the mouth of Little Ogeeche River during another study also contained specimens of M. arenaria. Ephemeral, intertidal, winter populations of juvenile softshell clams are reported from exposed Pleistocene beach faces along tidal rivers in Chatham, Georgia. The associated fauna collected with softshell clams and occurrence of other cold temperate and boreal species from Georgia estuaries are discussed. The distribution of M. arenaria appears to be mainly limited by a critical maximum temperature of 28º. The relatively cooler summer temperatures observed at the Sapelo Island tidal ditch site may enable the species to survive in this restricted habitat. Data from the present study indicate that during winter and spring, softshell clams appear to be bionomically important components of the benthos and the diet of stingrays in some Georgia estuarine habitats. Whether or not reproducing populations of M. arenaria occur year-round in Georgia estuaries still remains an open question.

Identification of gastric H,K-ATPase in an early vertebrate, the Atlantic stingray Dasyatis sabina.

Virtually all vertebrates acidify their gastric contents to a pH between 0.8 and 2.0. In mammals, acid secretion is mediated by a K-stimulated proton-translocating adenosine triphosphatase (H,K-ATPase), which establishes a million-fold gradient of protons across the apical membrane of the gastric parietal cell. The earliest phylogenetic appearance of gastric acid secretion is in cartilaginous fish, and we sought to verify in this class (Chondrichthyes) the presence and distribution of H,K-ATPase in gastric epithelial cells. An antibody against a synthetic peptide based on the C-terminus of pig H,K-ATPase alpha-subunit was localized in the gastric glands of the Atlantic stingray Dasyatis sabina. The C-terminal antibody stained all cells with tubulovesicles and the apical membrane domain of mucous neck cells. In proximal stomach, gastric glands showed the strongest immunoreactivity in cells close to the isthmus; in the distal stomach, strongest immunoreactivity was found in cells at the base of the glands. Oxyntic cells were more intensely immunoreactive than oxynticopeptic cells. This antibody labeled a single band of M(r) 100,600 on immunoblots of D. sabina gastric microsomes. These results show the earliest phylogenetic occurrence of a gastric ATPase in putative acid-secreting cells and suggest that this enzyme shares structural features with mammalian H,K-ATPase.

Morphology and density distribution of cone photoreceptor in the retina of the atlantic stingray, Dasyatis sabina.

Light miscroscopy of the retina in the Atlantic stingray, Dasyatis sabina, reveal two distinct photoreceptor types. Histological criteria establish the two cell types as morphological rods and cones. The retina was studied through a sequential series of vertical sections in a protocol designed to evaluate the entire retina. By performing differential counts in multiple regions and expressing cones as a percent of the total photoreceptors in a domain, it was possible to determine relative cone density and distribution. Cone distribution varies throughout the retina in two patterns. First, relative cone density gradually decreases peripherally. Second, a cone-rich band occurs along the horizontal axis of the eye, extending from ora serrata to ora serrata. This structure appears to be a visual streak. Both distribution patterns are statistically significant and are consistent among animals regardless of age. © 1994 Wiley-Liss, Inc.

UREA TRANSPORT BY HEPATOCYTES AND RED BLOOD CELLS OF SELECTED ELASMOBRANCH AND TELEOST FISHES

Although urea transport is receiving increased attention in mammalian systems, very little is known about urea transport in fish tissues. This study examined mechanisms of urea transport in red blood cells and hepatocytes from the lesser spotted dogfish (Scyliorhinus canicula), Atlantic stingray (Dasyatis sabina), turbot (Scopthalmus maximus), redfish (Scianops ocellatus), gulf toadfish (Opsanus beta) and oyster toadfish (Opsanus tau). Urea appeared to be passively distributed in both tissues (i.e. there was no difference between plasma and tissue urea concentrations). Additionally, a number of in vitro experiments examining [14C]urea flux were performed. In red blood cells from all species except redfish, urea transport occurred via simple passive diffusion, but redfish red blood cells showed a small (25 %) phloretin-sensitive uptake component. In hepatocytes of the two elasmobranch species (dogfish and stingray), urea efflux was also by simple passive diffusion. However, urea efflux in toadfish (both O. beta and O. tau) hepatocytes exhibited a marked phloretin-sensitivity, and O. beta hepatocytes were used in further experiments with other inhibitors and treatments. Urea transport in O. beta had a relatively high specificity for urea compared with the urea analogues acetamide, thiourea and N-methylurea, was unaffected by phloridzin and extracellular Na+ removal, and was not inhibited by physiological levels of glucose (0.5­10 mmol l-1). A phloretin-sensitive glucose transport, that was not inhibited by physiological levels of urea, was discovered in O. beta hepatocytes. The results are discussed in terms of patterns of species distribution and similarities between urea and glucose transport.