Metabolic organization of pregnant freshwater stingray and their offspring.

The elucidation of energetic patterns in adult viviparous elasmobranchs and their offspring can contribute to understanding ecophysiological questions, such as maternal-fetal metabolism and group life-history traits. We characterized the energetic substrates in pregnant individuals and stages of offspring development in the freshwater stingray Potamotrygon amandae. Our results show that the energetic distribution of the yolk is composed of more lipids than proteins, whereas the inverse pattern is observed in the egg and uterus, proving the plasticity of the energy provision of the species. As a novelty, we describe that yolk/intestine transfer occurs in this species.

Progesterone receptors in extratesticular ducts of the Amazonian stingray Potamotrygon wallacei: A potential role in sperm maturation and aggregate formation.

In cururu stingray (Potamotrygon wallacei Carvalho, Rosa and Araújo 2016) males, plasma progesterone (P4) levels appear to be associated with spermiation events. However, the specific contribution of P4 in sperm maturation via extratesticular ducts in this stingray species is unknown. With the aim of filling this knowledge gap, this study examined the morphology and the presence of progesterone receptors (PR) in the ducts, and analyzed the relationship of progesterone (P4) with sperm maturation and formation of aggregates. Morphological analysis showed that a columnar pseudostratified epithelium with stereocilia lined all the attached ducts. In active males, the secretory cells of the epididymis and the Leydig glands presented PR; however, these receptors were not found in the distal region of the epididymis (essential for nurturing and capacitation events) of regressing males. In the seminal vesicles of active males, the spermatozoa are parallelly aligned and embedded in a matrix to form the spermatozeugmata. The matrixes are formed by proteins secreted by the ducts and Sertoli cell cytoplasts. These structures presented PR, which suggests that P4 engages in sperm metabolism during storage. Our findings allude to the potential role of P4 in regulating the development and function of the attached ducts in different reproductive phases. Furthermore, P4 seems to be an essential component for regulating sperm progress, protein secretion, aggregate formation, and maintenance of sperm during storage in this freshwater stingray.

Elemental analysis of vertebrae discerns diadromous movements of threatened non-marine elasmobranchs.

River sharks (Glyphis spp.) and some sawfishes (Pristidae) inhabit riverine environments, although their long-term habitat use patterns are poorly known. We investigated the diadromous movements of the northern river shark (Glyphis garricki), speartooth shark (Glyphis glyphis), narrow sawfish (Anoxypristis cuspidata), and largetooth sawfish (Pristis pristis) using in situ laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) on vertebrae to recover elemental ratios over each individual's lifetime. We also measured elemental ratios for the bull shark (Carcharhinus leucas) and a range of inshore and offshore stenohaline marine species to assist in interpretation of results. Barium (Ba) was found to be an effective indicator of freshwater use, whereas lithium (Li) and strontium (Sr) were effective indicators of marine water use. The relationships between Ba and Li and Ba and Sr were negatively correlated, whereas the relationship between Li and Sr was positively correlated. Both river shark species had elemental signatures indicative of prolonged use of upper-estuarine environments, whereas adults appear to mainly use lower-estuarine environments rather than marine environments. Decreases in Li:Ba and Sr:Ba at the end of the prenatal growth zone of P. pristis samples indicated that parturition likely occurs in fresh water. There was limited evidence of prolonged riverine habitat use for A. cuspidata. The results of this study support elemental-environment relationships observed in teleost otoliths and indicate that in situ LA-ICP-MS elemental characterization is applicable to a wide range of elasmobranch species as a discriminator for use and movement across salinity gradients. A greater understanding of processes that lead to element incorporation in vertebrae, and relative concentrations in vertebrae with respect to the ambient environment, will improve the applicability of elemental analysis to understand movements across the life history of elasmobranchs into the future.

Consequences of prenatal exposure to contaminants in elasmobranchs: Biochemical outcomes during the embryonic development of Pseudobatos horkelii.

Coastal elasmobranchs are vulnerable to chemicals mostly due to their k-strategic life history characteristics and high trophic positions. Embryos might be particularly exposed through the maternal offloading of contaminants, possibly leading to disruptions during critical developmental phases. Yet, knowledge on biochemical outcomes of prenatal exposure in elasmobranchs is notably limited. Therefore, we aimed to investigate the effects of prenatal exposure to contaminants in embryos of the critically endangered Brazilian guitarfish, Pseudobatos horkelii. Polybrominated diphenyl ethers (PBDEs), polycyclic aromatic hydrocarbons (PAHs), pharmaceuticals and personal care products, and metals were determined in embryos. Additionally, glutathione S-transferase activity (GST), glutathione (GSH), and metallothionein levels (MT) were analyzed. Finally, lipid peroxidation levels (LPO) and protein carbonyl groups (PCO) were assessed. Embryonic exposure depended on yolk consumption, which was conspicuous in earlier development. We observed a dilution effect of contaminants levels, potentially related to biotransformation of these compounds throughout the embryonic development. Nevertheless, GST was not correlated to contaminant concentrations. The multivariate relationship between antioxidant components (GSH and GST) and LPO and PCO was negative, suggesting the lack of efficient defense of these biomarkers in early development, leading to oxidative damage. In this context, our results indicate that prenatal exposure to contaminants might impact the redox status in embryos of P. horkelii, leading to oxidative damage. Furthermore, metal concentrations influenced MT levels, suggesting this as a potential detoxification pathway in this species.

Increasing the data on elasmobranch plasma protein electrophoresis: electrophoretogram reference values determination in the undulate skate (Raja Undulata) and the nursehound shark (Scyliorhinus stellaris) maintained under human care.

BACKGROUND: This study determined plasma protein electrophoresis (PPE) reference intervals in two elasmobranch species: the undulate skate (Raja undulata) and the nursehound shark (Scyliorhinus stellaris), using a reference population of 48 undulate skates (27 males, 21 females) and 62 nursehounds (32 males, 30 females), considered to be clinically healthy. Plasma samples were analyzed using capillary zone electrophoresis (CZE). RESULTS: The undulate skate electrophoretogram resembled those previously reported in other batoids and could be divided into seven consistent fractions. No statistically significant differences were detected between sexes and developmental stages. The nursehound electrophoretogram was similar to that previously described in other shark species and could be divided into eight consistent fractions. Fraction 5% was significantly higher in juvenile nursehounds when compared to adults, while fraction 6 concentration and percentage were significantly higher in adults. Fraction 4% was higher in males than in females. Albumin band was not detected, and pre-albumin was negligible in both studied species. Alpha-globulins were predominant in the undulate skate, while beta-globulins were predominant in nursehounds. Statistically significant differences were found in all electrophoretogram fraction percentages and concentrations between the two species. CONCLUSION: To the authors knowledge, this is the first study reporting PPE values in undulate skates and nursehounds, and the first study using CZE in elasmobranch plasma. These findings can serve as a primary reference for health monitoring in both species and will add to the limited data available on PPE in elasmobranchs.

The aquaporin 8 (AQP8) membrane channel gene is present in the elasmobranch dogfish (Squalus acanthias) genome and is expressed in brain but not in gill, kidney or intestine.

The transport mechanisms for water, ammonia and urea in elasmobranch gill, kidney and gastrointestinal tract remain to be fully elucidated. Aquaporin 8 (AQP8) is a known water, ammonia and urea channel that is expressed in the kidney and respiratory and gastrointestinal tracts of mammals and teleost fish. However, at the initiation of this study in late 2019, there was no copy of an elasmobranch aquaporin 8 gene identified in the genebank even for closely related holocephalon species such as elephant fish (Callorhinchus milii) or for the elasmobranch little skate (Leucoraja erinacea). A transcriptomic study in spiny dogfish (Squalus acanthias) also failed to identify a copy. Hence this study has remedied this and identified the AQP8 cDNA sequence using degenerate PCR. Agarose electrophoresis of degenerate PCR reactions from dogfish tissues showed a strong band from brain cDNA and faint bands of a similar size in gill and liver. 5' and 3' RACE was used to complete the AQP8 cDNA sequence. Primers were then designed for further PCR reactions to determine the distribution of AQP8 mRNA expression in dogfish tissues. This showed that AQP8 is only expressed in dogfish brain and AQP8 therefore clearly can play no role in water, ammonia and urea transport in the gill, kidney or gastrointestinal tract. The role of AQP8 in dogfish brain remains to be determined.

Physicochemical and Morphological Characteristics of Starch and Flour Obtained from Green Banana cv. Raja (Musa paradisiaca cv. Raja) in West Sumatra, Indonesia.

<b>Background and Objective:</b> Banana cv. <i>Raja</i> is widely cultivated in West Sumatra, Indonesia. The physicochemical properties of starch and flour were investigated to determine their functional food prospects in industrial food. <b>Materials and Methods:</b> Starch and flour of banana cv. <i>Raja</i> was characterized using proximate analysis, Scanning Electron Microscopy (SEM), X-Ray Diffraction (XRD), Fourier-Transform Infrared (FT-IR), X-Ray Fluorescence (XRF) and Rapid Visco-Analyzer (RVA). <b>Results:</b> Banana cv. <i>Raja</i> starch contains 40.73% starch, 17.49% amylose, 55.5% water, 0.66% ash, 0.83% protein and 0.18% fat. The size of the granules is ranging from 20-30 µm in irregular and ellipsoidal-truncated shapes. The structure of crystallinity belongs to the type B while the gelatinization temperature is 74.9°C. Furthermore, the starch composed of 41.06% potassium, 12.85% phosphorus, 12.74% iron, 9.4% calcium and 7.5% magnesium. <b>Conclusion:</b> The morphological and physicochemical starch characteristics of Banana cv. <i>Raja</i> and has similar characteristics with its flour. Meanwhile the swelling power and the solubility value of the flour were higher than the starch. The gelatinization temperatures of starch and flour were 74.9 and 73.4°C, respectively.

Environmentally-induced osmoregulation in Neotropical freshwater stingrays (Myliobatiformes: Potamotrygoninae) after controlling for phylogeny.

The osmotic physiology of freshwater stingrays was investigated in fifteen species from white (WW), black (BW), and clearwater (CW) rivers of Brazilian hydrographic basins. Regardless of phylogeny, potamotrygonids collected in the BW (Negro, Jutai, Nhamunda, and Manacapuru rivers), and CW (Tapajos, Parana, Mutum, Demeni, and Branco rivers) exhibited lower levels of osmolytes and plasma osmolality than those from WW (Amazon estuary, Solimoes, and Tarauaca rivers). However, the gill and kidney Na+/K+-ATPase activities were higher in the potamotrygonid species from BW and CW than those from WW. These results may be related to the ability of the potamotrygonids to achieve high ion uptake from ion-poor waters, such as those of BW and/or CW. Additionally, the high kidney Na+/K+-ATPase activity may help to minimize ion loss and generate diluted urine. Thus, diffusional losses of salts are balanced by uptake of ions in the gill, and reabsorption by the kidney. The physiological traits showed a weak phylogenetic signal, which indicates a strong evolutionary convergence. Multivariate analyses revealed that variations in physiological traits has a significant association with the type of water, as well as its physical and chemical characteristics such as electric conductivity and pH. Therefore, the South American Neotropical freshwater stingrays adjust their osmoregulatory mechanisms according to the environment in which they live.

Anticancer Activity of Pasak Bumi Root Extract (Eurycoma longifolia Jack) on Raji Cells.

<b>Background and Objective:</b> The use of the roots of the pasak bumi (<i>E. longifolia</i> Jack) to treat cancer has been studied widely, however, the scientific basis of these plants used as an anticancer drug is widely unknown. The purpose of this study was to examine the anticancer activity of ethyl acetate and non-ethyl acetate fractions of pasak bumi roots in Raji cells. <b>Materials and Methods:</b> The cytotoxicity test is using the direct cell count method with trypan blue staining. The growth inhibition is using doubling time analysis of Raji cells. Observation of the apoptotic events of Raji cells used ethidium bromide staining, while observing the expression of p53 protein in Raji cells was done by immunohistochemical staining. <b>Results:</b> The results of the cytotoxicity and doubling time test showed that the activity of the non-ethyl acetate fraction was greater than that of the roots of pasak bumi. The lower concentration of non-ethyl acetate fraction of pasak bumi roots was able to delay the multiplication time of Raji cells which was greater than that of ethyl acetate. The results of the cytotoxicity and doubling time test showed that the activity of the non-ethyl acetate fraction was greater than that of the roots of pasak bumi. <b>Conclusion:</b> It can be concluded that the ethyl acetate and non-ethyl acetate fractions of the roots of pasak bumi have cytotoxic and antiproliferative activity on Raji cells, however they cannot induce apoptosis in Raji cells. The death of Raji cells is through the mechanism of inhibiting Raji cell proliferation as evidenced by an increase in p53 protein expression.

Glycan expression as a tool for a deeper understanding of a reproductive gland in a skate of economic importance.

Atlantoraja platana is an oviparous skate endemic to the south-west Atlantic Ocean, and is one of the skate species most exploited by local industrial bottom trawl fisheries. Oviparous elasmobranchs encapsulate their eggs in complex egg cases produced by the oviductal gland (OG). This organ is exclusively present in these fishes and comprises four distinct zones: club, baffle, papillary and terminal. The relative size and structural complexity of these zones correlate with mode of reproduction. Glycans are known to play major roles in reproduction so their distribution in each zone of the OG could explain the functional multiplicity of the gland in skates, but this topic has not been previously investigated. In this study, morphological, histochemical and lectin-histochemical analysis revealed various novel aspects of A. platana's OG. The club, papillary and terminal zones positively stained for periodic acid Schiff's reagent (PAS) and Alcian Blue (AB), indicating the presence of neutral and acid mucopolysaccharides. However, the buffle zone was negative for PAS and AB stains, but was positive for all the lectins used. Each zone of the OG had a characteristic pattern of glycan expression. Finally, we confirmed the presence of sperm but not sperm storage. This is the first lectin-histochemical study of the OG in chondrichtyan fish and it has proven to be an important tool to understand some of the mechanisms of fertility and reproductive success in economic important species such as A. platana.

Visual Opsin Diversity in Sharks and Rays.

The diversity of color vision systems found in extant vertebrates suggests that different evolutionary selection pressures have driven specializations in photoreceptor complement and visual pigment spectral tuning appropriate for an animal's behavior, habitat, and life history. Aquatic vertebrates in particular show high variability in chromatic vision and have become important models for understanding the role of color vision in prey detection, predator avoidance, and social interactions. In this study, we examined the capacity for chromatic vision in elasmobranch fishes, a group that have received relatively little attention to date. We used microspectrophotometry to measure the spectral absorbance of the visual pigments in the outer segments of individual photoreceptors from several ray and shark species, and we sequenced the opsin mRNAs obtained from the retinas of the same species, as well as from additional elasmobranch species. We reveal the phylogenetically widespread occurrence of dichromatic color vision in rays based on two cone opsins, RH2 and LWS. We also confirm that all shark species studied to date appear to be cone monochromats but report that in different species the single cone opsin may be of either the LWS or the RH2 class. From this, we infer that cone monochromacy in sharks has evolved independently on multiple occasions. Together with earlier discoveries in secondarily aquatic marine mammals, this suggests that cone-based color vision may be of little use for large marine predators, such as sharks, pinnipeds, and cetaceans.

Further studies of ion channels in the electroreceptor of the skate through deep sequencing, cloning and cross species comparisons.

Our comparative studies seek to understand the structure and function of ion channels in cartilaginous fish that can detect very low voltage gradients in seawater. The principal channels of the electroreceptor include a calcium activated K channel whose α subunit is Kcnma1, and a voltage-dependent calcium channel, Cacna1d. It has also been suggested based on physiological and pharmacological evidence that a voltage-gated K channel is present in the basal membranes of the receptor cells which modulates synaptic transmitter release. Large conductance calcium-activated K channels (BK) are comprised of four α subunits, encoded by Kcnma1 and modulatory ß subunits of the Kcnmb class. We recently cloned and published the skate Kcnma1 gene and most of Kcnmb4 using purified mRNA of homogenized electroreceptors. Bellono et al. have recently performed RNA sequencing (RNA-seq) on purified mRNA from skate electroreceptors and found several ion channels including Kcnma1. We searched the Bellono et al. RNA-seq repository for additional channels and subunits. Our most significant findings are the presence of two Shaker type voltage dependent K channel sequences which are grouped together as isoforms in the data repository. The larger of these is a skate ortholog of the voltage dependent fast potassium channel Kv1.1, which is expressed at appreciable levels. The second ortholog is similar to Kv1.5 but has fewer N-terminal amino acids than other species. The sequence for Kv1.5 in the skate is very strongly aligned with the recently reported sequence for potassium channels in the electroreceptors of the cat shark, S. retifer, which also modulate synaptic transmission. The latter channel was designated as Kv1.3 in the initial report, but we suggest that these channels are actually orthologs of each other, and that Kv1.5 is the prevailing designation. We also found a beta subunit sequence (Kcnab2) which may co-assemble with one or both of the voltage gated channels. The new channels and subunits were verified by RT-PCR and the Kv1.1 sequence was confirmed by cloning. We also searched the RNA-seq repository for accessory subunits of Kcnma1, and found a computer-generated assembly that contained a complete sequence of its ß subunit, Kcnmb2. Skate Kcnmb2 has a total of 279 amino acids, with 51 novel amino acids at the N-terminus which may play a specific physiological role. This sequence was confirmed by PCR and cloning. However, skate Kcnmb2 is expressed at low levels in the electroreceptor compared to Kcnma1 and skate Kcnmb1 is absent. The evolutionary origin of the newly described K channels and their subunits was studied by alignments with mammalian sequences, including human, and also those in related fish: the whale shark (R. typus), the ghost shark (C.milii), and (S. retifer). There are also orthologous K channels of the lamprey, which has electroreceptors. Tree building and bootstrap programs were used to confirm phylogenetic inferences. Further research should focus on the subcellular locations of these channels, their gating behavior, and the effects of accessory subunits on gating.

Occurrence and tissue distribution of perfluoroalkyl substances (PFASs) in sharks and rays from the eastern Mediterranean Sea.

Persistent organic pollutants (POPs), including Perfluoroalkyl substances (PFASs), enter into the marine ecosystem, raising questions on possible adverse effects caused to the health of marine organisms and especially of top predators. Thus, there is an urge to assess the occurrence and the tissue distribution of PFASs in apex predators. To this end, the current study examines concentrations and distribution of 15 PFASs among 85 samples of different tissues from 9 shark and ray species collected in Greece. The results showed a similar PFAS pattern among the different tissues, with long carbon chain PFASs being the most frequently detected compounds. PFTrDA was the most predominant compound in terms of concentration and frequency of detection, followed by PFUnDA and PFOS. PFTrDA concentrations ranged between < LOQ and 27.1 ng/g ww, while PFUnDA and PFOS levels ranged from  heart > liver ≈ gills > muscle. Relative contribution (%) of individual compounds to ΣPFAS concentration varied among the different shark tissues, and also among the different shark species. No correlation between PFASs levels in tissues and sharks' gender, length and geographical origin was observed.

Expression and transcriptional regulation of gsdf in spotted scat (Scatophagus argus).

Gonadal soma-derived factor (Gsdf) is critical for testicular differentiation and early germ cell development in teleosts. The spotted scat (Scatophagus argus), with a stable XX-XY sex-determination system and the candidate sex determination gene dmrt1, provides a good model for understanding the mechanism of sex determination and differentiation in teleosts. In this study, we analyzed spotted scat gsdf tissue distribution and gene expression patterns in gonads, as well as further analysis of transcriptional regulation. Tissue distribution analysis showed that gsdf was only expressed in testis and ovary. Real-time PCR showed that both gsdf and dmrt1 were expressed significantly higher in testes at different phases (phase III, IV and V) compared to ovaries at phase II, III and IV, while gsdf was expressed significantly higher in phase II ovaries than those of phase III and IV. Western blot analysis also showed that Gsdf was more highly expressed in the testis than ovary. Immunohistochemistry analysis showed that Gsdf was expressed in Sertoli cells surrounding spermatogonia in the testis, while it was expressed in the somatic cells surrounding the oogonia of the ovary. Approximately 2.7 kb of the 5' upstream region of gsdf was cloned from the spotted scat genomic DNA and in silico promoter analysis revealed the putative transcription factor binding sites of Dmrt1 and Sf1. The luciferase reporter assay, using the human embryonic kidney cells, demonstrated that Dmrt1 activated gsdf expression in a dose-dependent manner in the presence of Sf1 in spotted scat. These results suggest that Gsdf could play a role in regulating the development of spermatogonia and oogonia, and also participate in male sex differentiation by acting as a downstream gene of Dmrt1 in spotted scat.

Trace metals and persistent organic pollutants contamination in batoids (Chondrichthyes: Batoidea): A systematic review.

Batoids (Chondrichthyes: Batoidea; e.g. stingrays, skates, and guitarfish) comprise more than 55% of elasmobranch taxa and represent ecologically important predators in benthic and pelagic habitats. Although overexploitation and habitat degradation are the two biggest threats to batoid populations, coastal and oceanic pollution is also a pervasive potential threat. In this systematic review, we compile published scientific literature on trace metals and persistent organic pollutants (POPs) contamination in elasmobranch species of the Batoidea superorder and present contamination patterns, exposure effects, and potential human exposure risks to most reported contaminants. We found batoids to accumulate a wide range of trace metals, including mercury (Hg), arsenic (As), lead (Pb), copper (Cu), cadmium (Cd) and zinc (Zn). Accumulation of POPs is reported for chlordanes, dichlorodiphenyltrichloroethane (DDT), polychlorinated biphenyl (PCB), dieldrin, Heptachlor epoxide, hexachlorobenzene and perfluoroalkyl substances (PFAS). Hg levels in muscle tissue were significantly different among oceanic basins and habitats, consistent with previous global assessments of Hg oceanic background levels. Some batoid species presented Hg levels higher than large pelagic teleost fishes and comparable to sharks. Ecological traits such as, bottom feeding, upper trophic position and elasmobranch-specific physiology and metabolism are discussed as potential factors associated with Hg uptake and accumulation in batoids. Some species exceeded USEPA's maximum contamination safety limits in edible tissues for Hg, As and ΣPCBs. For most trace metals and POPs, there is a lack of studies focusing on contamination levels in batoids. We recommend future research increasing reporting on POPs and trace metals besides Hg in batoids to further investigate the role of Elasmobranch as a bioindicator for marine pollution.

Bone-like features in skate suggest a novel elasmobranch synapomorphy and deep homology of trabecular mineralization patterns.

Bone is a defining characteristic of the vertebrate skeleton, and while chondrichthyans (sharks, skates, and other cartilaginous fishes) are vertebrates, they are hypothesized to have lost the ability to make bone during their evolution. Multiple descriptions of a bone-like tissue in neural arches of vertebrae in various shark species (selachians), however, challenge this hypothesis. Here, we extend this argument by analyzing vertebrae of two members of the batoids (the little skate Leucoraja erinacea and Eaton's skate Bathyraja eatonii), the sister group to selachians within elasmobranchs. Micro-CT images showed a bone-like mineralization pattern in neural arches of each skate species, and histological analyses confirmed that this bone-like tissue surrounded a cartilage core, exactly as described in sharks. Another mineralization pattern identified in skate vertebrae was distinct from the polygonal tesseral and areolar patterns that classically are associated with the chondrichthyan endoskeleton. Many regions of the vertebrae, including the neural spine and transverse processes, showed this perichondral mineralization pattern, termed here trabecular tesseral. Other than the cartilage core of the neural arch, all mineralized tissues in skate vertebrae had flattened cells surrounded by matrix with bone-like histology. Analyses of quantitative microstructural parameters revealed that, compared to rat vertebrae, the bone-like mineralization pattern in the neural arches of skate vertebrae was more similar to compact bone than trabecular bone. In contrast, the thickness of the trabecular tesseral pattern was more similar to trabecular bone than compact bone of rat vertebrae. In conclusion, a bone-like tissue in neural arches of skate vertebrae appears to be a novel elasmobranch synapomorphy. We propose that the trabecular tesseral mineralization pattern in the skate might have deep homology to the mineralization pattern utilized in trabecular bone. STATEMENT OF SIGNIFICANCE: Mineralization patterns of skeletal tissues have not been investigated thoroughly in all vertebrate clades. Despite their designation as 'cartilaginous fish', chondrichthyans clearly evolved from ancestral vertebrates that made bone. The consensus that chondrichthyans lost the ability to make bone during their evolution, however, is challenged by reports of bone and bone-like tissues in the neural arches of vertebrae in extant sharks (selachians). Here, we provide evidence from micro-CT imaging and histological analyses to support our hypothesis that a bone-like tissue is present in the neural arches of batoids (the sister group to selachians within elasmobranchs). These results argue strongly that the neural arch bone-like tissue is a previously unknown synapomorphy of elasmobranchs. In addition to the bone-like mineralization pattern identified in the neural arches, micro-CT images also showed a novel mineralization pattern which we described as trabecular tesseral. Quantitative microstructural features shared between trabecular tesseral pattern and trabecular bone (from homologous rat vertebrae) suggest that both patterns might derive from an ancestral gene network driving trabecular mineralization (i.e., deep homology).

Embryonic transcriptome sequencing of the ocellate spot skate Okamejei kenojei.

Chondrichthyans (cartilaginous fishes) exhibit highly variable reproductive styles, categorized as viviparity and oviparity. Among these, species with oviparity provide an enormous potential of molecular experimentation with stable sample supply which does not demand the sacrifices of live mothers. Cartilaginous fishes are divided into two subclasses, chimaeras (Holocephali) and elasmobranchs (Elasmobranchii), and the latter consists of two monophyletic groups, Batoidea (rays, skates and torpedoes) and Selachimorpha (sharks). Here we report transcriptome assemblies of the ocellate spot skate Okamejei kenojei, produced by strand-specific RNA-seq of its embryonic tissues. We obtained a total of 325 million illumina short reads from libraries prepared using four different tissue domains and assembled them all together. Our assembly result confirmed the species authenticity and high continuity of contig sequences. Also, assessment of its coverage of pre-selected one-to-one orthologs supported high diversity of transcripts in the assemblies. Our products are expected to provide a basis of comparative molecular studies encompassing other chondrichthyan species with emerging genomic and transcriptomic sequence information.

Sex may influence environmental diphenhydramine accumulation in Round Stingrays.

Despite the amount of treated wastewater discharged into the Southern California Bight, few studies have examined pharmaceutical compounds in local biota. The Round Stingray (Urobatis halleri) was selected as a representative elasmobranch species to perform an exploratory study on environmental pharmaceutical exposure. Archived liver samples of males and females from juvenile to adult size classes from several locations (n = 53) were examined for 18 pharmaceutical and illicit drug compounds using isotope-dilution LC-MS/MS. Very few compounds were detected in stingray livers, with diphenhydramine as the only pharmaceutical above quantitation limits. Only stingrays collected from the urban site (mainland California) had detectable levels of diphenhydramine compared to no detections in reference stingrays (offshore island). Sex and sampling location substantially influenced both detection rate and concentrations. Our results suggest that aspects of species' ecology and physiology should be considered for future studies investigating pharmaceutical exposure in elasmobranchs.

Cartilaginous Fishes Provide Insights into the Origin, Diversification, and Sexually Dimorphic Expression of Vertebrate Estrogen Receptor Genes.

Vertebrate estrogen receptors (ERs) perform numerous cell signaling and transcriptional regulatory functions. ERɑ (Esr1) and ERß (Esr2) likely evolved from an ancestral receptor that duplicated and diverged at the protein and cis-regulatory levels, but the evolutionary history of ERs, including the timing of proposed duplications, remains unresolved. Here we report on identification of two distinct ERs in cartilaginous fishes and demonstrate their orthology to ERα and ERß. Phylogenetic analyses place the ERα/ERß duplication near the base of crown gnathostomes (jawed vertebrates). We find that ERα and ERß from little skate (Leucoraja erinacea) and mammals share key subtype-specific residues, indicating conserved protein evolution. In contrast, jawless fishes have multiple non-orthologous Esr genes that arose by parallel duplications. Esr1 and Esr2 are expressed in subtype-specific and sexually dimorphic patterns in skate embryos, suggesting that ERs might have functioned in sexually dimorphic development before the divergence of cartilaginous and bony fishes.

A role for the CXCR4-CXCL12 axis in the little skate, Leucoraja erinacea.

The interaction between C-X-C chemokine receptor type 4 (CXCR4) and its cognate ligand C-X-C motif chemokine ligand 12 (CXCL12) plays a critical role in regulating hematopoietic stem cell activation and subsequent cellular mobilization. Extensive studies of these genes have been conducted in mammals, but much less is known about the expression and function of CXCR4 and CXCL12 in non-mammalian vertebrates. In the present study, we identify simultaneous expression of CXCR4 and CXCL12 orthologs in the epigonal organ (the primary hematopoietic tissue) of the little skate, Leucoraja erinacea. Genetic and phylogenetic analyses were functionally supported by significant mobilization of leukocytes following administration of Plerixafor, a CXCR4 antagonist and clinically important drug. Our results provide evidence that, as in humans, Plerixafor disrupts CXCR4/CXCL12 binding in the little skate, facilitating release of leukocytes into the bloodstream. Our study illustrates the value of the little skate as a model organism, particularly in studies of hematopoiesis and potentially for preclinical research on hematological and vascular disorders.