Evidence That Aquaporin 11 (AQP11) in the Spiny Dogfish (Squalus acanthias) May Represent a Pseudogene.

Various attempts to amplify an AQP11 cDNA from tissues of the spiny dogfish (Squalus acanthias) were made. Two pairs of deoxy-inosine-containing degenerate primers were designed based on conserved amino acid sequences from an AQP11 alignment. These primers yielded some faint bands from gill cDNA that were sequenced. Blast searches with the sequences showed they were not AQP11. An elasmobranch AQP11 nucleotide sequence alignment was produced to identify conserved regions to make further degenerate primers. One primer pair produced a short 148 bp fragment showing particularly strong amplification in gill and intestine. It was sequenced and represented a piece of the AQP11 gene. However, as the fragment may have resulted from contaminating genomic DNA (in total RNA used to make cDNA), 5' and 3' RACE were performed to amplify the two ends of the putative cDNA. Furthermore, 5' and 3' RACE amplifications depend on the presence of a 5' cap nucleotide and a poly A tail, respectively on the putative AQP11 mRNA. Hence, successful amplification was only possible from cDNA and not genomic DNA. Nested RACE amplifications were performed using gill and intestinal RACE cDNA, but none of the DNA fragments sequenced were AQP11. Consequently, the spiny dogfish AQP11 gene may represent a pseudogene.

Characteristics of the spiny dogfish (Squalus acanthias) nuclear genome.

Sequenced shark nuclear genomes are underrepresented, with reference genomes available for only four out of nine orders so far. Here, we present the nuclear genome, with annotations, of the spiny dogfish (Squalus acanthias), a shark of interest to biomedical and conservation efforts, and the first representative of the second largest order of sharks (Squaliformes) with nuclear genome annotations available. Using Pacific Biosciences Continuous Long Read data in combination with Illumina paired-end and Hi-C sequencing, we assembled the genome de novo, followed by RNA-Seq-supported annotation. The final chromosome-level assembly is 3.7 Gb in size, has a BUSCO completeness score of 91.6%, and an error rate of less than 0.02%. Annotation predicted 33,283 gene models in the spiny dogfish's genome, of which 31,979 are functionally annotated.

Investigating nitrogen movement in North Pacific spiny dogfish (Squalus acanthias suckleyi), with focus on UT, Rhp2, and Rhbg mRNA abundance.

For ureosmotic marine elasmobranchs, the acquisition and retention of nitrogen is critical for the synthesis of urea. To better understand whole-body nitrogen homeostasis, we investigated mechanisms of nitrogen trafficking in North Pacific spiny dogfish (Squalus acanthias suckleyi). We hypothesized that the presence of nitrogen within the spiral valve lumen would affect both the transport of nitrogen and the mRNA abundance of a urea transporter (UT) and two ammonia transport proteins (Rhp2, Rhbg) within the intestinal epithelium. The in vitro preincubation of intestinal tissues in NH4Cl, intended to simulate dietary nitrogen availability, showed that increased ammonia concentrations did not significantly stimulate the net uptake of total urea or total methylamine. We also examined the mRNA abundance of UT, Rhp2, and Rhbg in the gills, kidney, liver, and spiral valve of fasted, fed, excess urea fed, and antibiotic-treated dogfish. After fasting, hepatic UT mRNA abundance was significantly lower, and Rhp2 mRNA in the gills was significantly higher than the other treatments. Feeding significantly increased Rhp2 mRNA levels in the kidney and mid spiral valve region. Both excess urea and antibiotics significantly reduced Rhbg mRNA levels along all three spiral valve regions. The antibiotic treatment also significantly diminished UT mRNA abundance levels in the anterior and mid spiral valve, and Rhbg mRNA levels in the kidney. In our study, no single treatment had significantly greater influence on the overall transcript abundance of the three transport proteins compared to another treatment, demonstrating the dynamic nature of nitrogen balance in these ancient fish.

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.

Aquaporin (AQP) channels in the spiny dogfish, Squalus acanthias I: Characterization of AQP3 and AQP15 function and expression, and localization of the proteins in gill and spiral valve intestine.

Complementary DNAs (cDNAs) for two aquaporin water channel genes (AQP3 and AQP15) were amplified cloned and sequenced to initiate this study. Northern blot analysis was carried out to confirm the mRNA sizes of these AQP genes with AQP3 mRNA bands exhibiting sizes of 1.2 and 1.6 k bases and AQP15 had a mRNA band of 2.1 k bases. Northern blot analysis was also performed on kidney and esophagus total RNA samples from fish acclimated to 75%, 100% or 120% seawater (SW). The level of AQP15 mRNA expression was shown to significantly decrease following salinity acclimation from 100 to 120% SW. An opposite but non-significantly different trend was observed for AQP3 mRNA levels. Full length cDNAs were then used to generate AQP3 and AQP15 mRNAs for microinjection into Xenopus oocytes. Both AQP3- and AQP15- microinjected oocytes exhibited significantly elevated apparent water permeability compared to control oocytes at neutral pH. The apparent water permeability was mercury-inhibitable, significantly so in the case of AQP3. AQP3 microinjected oocytes showed pH sensitivity in their apparent water permeability, showing a lack of permeability at acidic pH values. The Carboxyl-terminal derived amino acid sequences of AQP3 and AQP15 were used to generate rabbit affinity-purified polyclonal antibodies. Western blots with the antibodies showed a band of 31.3 kDa for AQP3 in the kidney, with minor bands at 26, 24 and 21 kDa. For AQP15 a band of 26 kDa was seen in gill and kidney. Fainter bands at 28 and 24 kDa were also seen in the kidney. There was also some higher molecular weight banding. None of the bands were seen when the antibodies were pre- blocked with their peptide antigens. Immunohistochemical localization studies were also performed in the gill and spiral valve intestine. In the gill, AQP15 antibody staining was seen sporadically in the membranes of surface epithelial cells of the secondary lamellae. Tyramide amplification of signals was employed in the spiral valve intestine. Tyramide-amplified AQP3 antibody staining was observed in the basal membrane of the invaginated epithelial cell layer of secondary intestinal folds in luminal surface of either the side wall of the spiral valve intestine or in internal valve tissue 'flaps'. For the AQP15 antibody, tyramide-amplified staining was instead found on the apical and to a lesser extent the lateral membranes of the same invaginated epithelial cell layer. The localization of AQP3 and AQP15 in the spiral valve intestine suggests that a trans-cellular water absorption pathway may exist in this tissue.

A nonolfactory shark adenosine receptor activates CFTR with unique pharmacology and structural features.

Adenosine receptors (ADORs) are G protein-coupled purinoceptors that have several functions including regulation of chloride secretion via cystic fibrosis transmembrane conductance regulator (CFTR) in human airway and kidney. We cloned an ADOR from Squalus acanthias (shark) that likely regulates CFTR in the rectal gland. Phylogenic and expression analyses indicate that elasmobranch ADORs are nonolfactory and appear to represent extant predecessors of mammalian ADORs. We therefore designate the shark ADOR as the A0 receptor. We coexpressed A0 with CFTR in Xenopus laevis oocytes and characterized the coupling of A0 to the chloride channel. Two-electrode voltage clamping was performed, and current-voltage (I-V) responses were recorded to monitor CFTR status. Only in A0- and CFTR-coinjected oocytes did adenosine analogs produce a significant concentration-dependent activation of CFTR consistent with its electrophysiological signature. A pharmacological profile for A0 was obtained for ADOR agonists and antagonists that differed markedly from all mammalian ADOR subtypes [agonists: R-phenyl-isopropyl adenosine (R-PIA) > S-phenyl-isopropyl adenosine (S-PIA) > CGS21680 > N6-cyclopentyladenosine (CPA) > 2-chloroadenosine (2ClAdo) > CV1808 = N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine (DPMA) > N-ethyl-carboxyl adenosine (NECA); and antagonists: 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > PD115199 > 1,3-dimethyl-8-phenylxanthine (8PT) > CGS15943]. Structures of human ADORs permitted a high-confidence homology model of the shark A0 core that revealed unique structural features of ancestral receptors. We conclude that 1) A0 is a novel and unique adenosine receptor ancestor by functional and structural criteria; 2) A0 likely activates CFTR in vivo, and this receptor activates CFTR in oocytes, indicating an evolutionary coupling between ADORs and chloride secretion; and 3) A0 appears to be a nonolfactory evolutionary ancestor of all four mammalian ADOR subtypes.

Latency transition of plasminogen activator inhibitor type 1 is evolutionarily conserved.

Plasminogen activator inhibitor type 1 (PAI-1) is a central regulator of fibrinolysis and tissue remodelling. PAI-1 belongs to the serpin superfamily and unlike other inhibitory serpins undergoes a spontaneous inactivation process under physiological conditions, termed latency transition. During latency transition the solvent exposed reactive centre loop is inserted into the central ß-sheet A of the molecule, and is no longer accessible to reaction with the protease. More than three decades of research on mammalian PAI-1 has not been able to clarify the evolutionary advantage and physiological relevance of latency transition. In order to study the origin of PAI-1 latency transition, we produced PAI-1 from Spiny dogfish shark (Squalus acanthias) and African lungfish (Protopterus sp.), which represent central species in the evolution of vertebrates. Although human PAI-1 and the non-mammalian PAI-1 variants share only approximately 50 % sequence identity, our results showed that all tested PAI-1 variants undergo latency transition with a similar rate. Since the functional stability of PAI-1 can be greatly increased by substitution of few amino acid residues, we conclude that the ability to undergo latency transition must have been a specific selection criterion for the evolution of PAI-1. It appears that all PAI-1 molecules must harbour latency transition to fulfil their physiological function, stressing the importance to further pursue a complete understanding of this molecular phenomenon with possible implication to pharmacological intervention. Our results provide the next step in understanding how the complete role of this important protease inhibitor evolved along with the fibrinolytic system.

Multi-tissue RNA-seq and transcriptome characterisation of the spiny dogfish shark (Squalus acanthias) provides a molecular tool for biological research and reveals new genes involved in osmoregulation.

The spiny dogfish shark (Squalus acanthias) is one of the most commonly used cartilaginous fishes in biological research, especially in the fields of nitrogen metabolism, ion transporters and osmoregulation. Nonetheless, transcriptomic data for this organism is scarce. In the present study, a multi-tissue RNA-seq experiment and de novo transcriptome assembly was performed in four different spiny dogfish tissues (brain, liver, kidney and ovary), providing an annotated sequence resource. The characterization of the transcriptome greatly increases the scarce sequence information for shark species. Reads were assembled with the Trinity de novo assembler both within each tissue and across all tissues combined resulting in 362,690 transcripts in the combined assembly which represent 289,515 Trinity genes. BUSCO analysis determined a level of 87% completeness for the combined transcriptome. In total, 123,110 proteins were predicted of which 78,679 and 83,164 had significant hits against the SwissProt and Uniref90 protein databases, respectively. Additionally, 61,215 proteins aligned to known protein domains, 7,208 carried a signal peptide and 15,971 possessed at least one transmembrane region. Based on the annotation, 81,582 transcripts were assigned to gene ontology terms and 42,078 belong to known clusters of orthologous groups (eggNOG). To demonstrate the value of our molecular resource, we show that the improved transcriptome data enhances the current possibilities of osmoregulation research in spiny dogfish by utilizing the novel gene and protein annotations to investigate a set of genes involved in urea synthesis and urea, ammonia and water transport, all of them crucial in osmoregulation. We describe the presence of different gene copies and isoforms of key enzymes involved in this process, including arginases and transporters of urea and ammonia, for which sequence information is currently absent in the databases for this model species. The transcriptome assemblies and the derived annotations generated in this study will support the ongoing research for this particular animal model and provides a new molecular tool to assist biological research in cartilaginous fishes.

Coordination of chemical (trimethylamine oxide) and molecular (heat shock protein 70) chaperone responses to heat stress in elasmobranch red blood cells.

Chemical and molecular chaperones are organic compounds that protect and stabilize proteins from damage and aggregation as a result of cellular stress. Using the dogfish (Squalus acanthias) red blood cell (RBC) as a model, we examined whether elasmobranch cells with naturally high concentrations of the chemical chaperone trimethylamine oxide (TMAO) would induce the molecular chaperone heat shock protein 70 (HSP70) when exposed to an acute thermal stress. Our hypothesis was that TMAO is itself capable of preventing damage and preserving cellular function during thermal stress and thus that the heat shock response would be inhibited/diminished. We incubated RBCs in vitro with and without physiologically relevant concentrations of TMAO at 13°C and then exposed cells to a 1-h acute heat shock at 24°C. HSP70 protein expression was elevated in dogfish RBCs after the acute heat stress, but this induction was inhibited by extracellular TMAO. Regardless of the presence of TMAO and/or HSP70, we did not observe any cell damage, as indicated by changes in caspase 3/7 activity, protein carbonyls, membrane viability, or levels of ubiquitin. We also saw no change in RBC cell function, as determined by hemoglobin oxygen affinity or carrying capacity, in cells lacking the heat shock response but protected by TMAO. This study demonstrates that there is cellular coordination between chemical and molecular chaperones in response to an acute thermal stress in dogfish RBCs and suggests that TMAO has a thermoprotective role in these cells, thus eliminating the need for a heat shock response.

Transcriptome responses in the rectal gland of fed and fasted spiny dogfish shark (Squalus acanthias) determined by suppression subtractive hybridization.

Prior studies of the elasmobranch rectal gland have demonstrated that feeding induces profound and rapid up regulation of the gland's ability to secrete concentrated NaCl solutions and the metabolic capacity to support this highly ATP consuming process. We undertook the current study to attempt to determine the degree to which up regulation of mRNA transcription was involved in the gland's activation. cDNA libraries were created from mRNA isolated from rectal glands of fasted (7days post-feeding) and fed (6h and 22h post-feeding) spiny dogfish sharks (Squalus acanthias), and the libraries were subjected to suppression subtractive hybridization (SSH) analysis. Quantitative real time PCR (qPCR) was also used to ascertain the mRNA expression of several genes revealed by the SSH analysis. In total the treatments changed the abundance of 170 transcripts, with 103 up regulated by feeding, and 67 up regulated by fasting. While many of the changes took place in 'expected' Gene Ontology (GO) categories (e.g., metabolism, transport, structural proteins, DNA and RNA turnover, etc.), KEGG analysis revealed a number of categories which identify oxidative stress as a topic of interest for the gland. GO analysis also revealed that branched chain essential amino acids (e.g., valine, leucine, isoleucine) are potential metabolic fuels for the rectal gland. In addition, up regulation of transcripts for many genes in the anticipated GO categories did not agree (i.e., fasting down regulated in feeding treatments) with previously observed increases in their respective proteins/enzyme activities. These results suggest an 'anticipatory' storage of selected mRNAs which presumably supports the rapid translation of proteins upon feeding activation of the gland.

B cell receptor accessory molecule CD79α: characterisation and expression analysis in a cartilaginous fish, the spiny dogfish (Squalus acanthias).

CD79α (also known as Igα) is a component of the B cell antigen receptor complex and plays an important role in B cell signalling. The CD79α protein is present on the surface of B cells throughout their life cycle, and is absent on all other healthy cells, making it a highly reliable marker for B cells in mammals. In this study the spiny dogfish (Squalus acanthias) CD79α (SaCD79α) is described and its expression studied under constitutive and stimulated conditions. The spiny dogfish CD79α cDNA contains an open reading frame of 618 bp, encoding a protein of 205 amino acids. Comparison of the SaCD79α gene with that of other species shows that the gross structure (number of exons, exon/intron boundaries, etc.) is highly conserved across phylogeny. Additionally, analysis of the 5' flanking region shows SaCD79α lacks a TATA box and possesses binding sites for multiple transcription factors implicated in its B cell-specific gene transcription in other species. Spiny dogfish CD79α is most highly expressed in immune tissues, such as spleen, epigonal and Leydig organ, and its transcript level significantly correlates with those of spiny dogfish immunoglobulin heavy chains. Additionally, CD79α transcription is up-regulated, to a small but significant degree, in peripheral blood cells following stimulation with pokeweed mitogen. These results strongly indicate that, as in mammals, spiny dogfish CD79α is expressed by shark B cells where it associates with surface-bound immunoglobulin to form a fully functional BCR, and thus may serve as a pan-B cell marker in future shark immunological studies.

Humoral immune response of the small-spotted catshark, Scyliorhinus canicula.

Cartilaginous fishes are the oldest group in which an adaptive immune system based on immunoglobulin-superfamily members is found. This manuscript compares humoral immune function in small-spotted catshark (Scyliorhinus canicula) with that described for spiny dogfish (Squalus acanthias), another member of the Squalomorphi superorder, and nurse shark, the model for humoral immunity in elasmobranchs and a member of the Galeomorphi superorder. Although small-spotted catshark and nurse shark are separated by over 200 million years we found that immunoglobulin isoforms are well conserved between the two species. However, the plasma protein profile of small-spotted catshark was most similar to that of spiny dogfish, with low levels of pentameric IgM, and IgNAR present as a multimer in plasma rather than a monomer. We show that an antigen-specific monomeric IgM response, with a profile similar to that described previously for nurse sharks, can be raised in small-spotted catshark. Lacking polyclonal or monoclonal antibody reagents for detecting catshark IgNAR we investigated phage-display and recombinant Fc-fusion protein expression as alternative methods to look for an antigen-specific response for this isotype. However, we could find no evidence of an antigen-specific IgNAR in the animals tested using either of these techniques. Thus, unlike nurse sharks where antigen-specific monomeric IgM and IgNAR appear together, it seems there may be a temporal or complete 'uncoupling' of these isotypes during a humoral response in the small-spotted catshark.

Cell and molecular biology of the spiny dogfish Squalus acanthias and little skate Leucoraja erinacea: insights from in vitro cultured cells.

Two of the most commonly used elasmobranch experimental model species are the spiny dogfish Squalus acanthias and the little skate Leucoraja erinacea. Comparative biology and genomics with these species have provided useful information in physiology, pharmacology, toxicology, immunology, evolutionary developmental biology and genetics. A wealth of information has been obtained using in vitro approaches to study isolated cells and tissues from these organisms under circumstances in which the extracellular environment can be controlled. In addition to classical work with primary cell cultures, continuously proliferating cell lines have been derived recently, representing the first cell lines from cartilaginous fishes. These lines have proved to be valuable tools with which to explore functional genomic and biological questions and to test hypotheses at the molecular level. In genomic experiments, complementary (c)DNA libraries have been constructed, and c. 8000 unique transcripts identified, with over 3000 representing previously unknown gene sequences. A sub-set of messenger (m)RNAs has been detected for which the 3' untranslated regions show elements that are remarkably well conserved evolutionarily, representing novel, potentially regulatory gene sequences. The cell culture systems provide physiologically valid tools to study functional roles of these sequences and other aspects of elasmobranch molecular cell biology and physiology. Information derived from the use of in vitro cell cultures is valuable in revealing gene diversity and information for genomic sequence assembly, as well as for identification of new genes and molecular markers, construction of gene-array probes and acquisition of full-length cDNA sequences.

Functional expression of Squalus acanthias melanocortin-5 receptor in CHO cells: ligand selectivity and interaction with MRAP.

The melanocortin-5 receptor (MC(5)) of the dogfish Squalus acanthias (SacMC(5) receptor) can be functionally expressed in CHO cells in the absence of the co-expression of an exogenous MRAP cDNA. Both human ACTH(1-24) and dogfish ACTH(1-25) were much better stimulators of the SacMC(5) receptor than any of the mammalian or dogfish MSH ligands that were tested. The order of ligand selectivity for the dogfish melanocortins was ACTH(1-25)>αMSH>γ-MSH=δ-MSH>ß-MSH. Unlike mammalian MC(5) receptors, the functional expression of the SacMC(5) receptor was not negatively impacted when the receptor was co-expressed with a cartilaginous fish (Callorhinchus milii) MRAP2 cDNA. However, co-expression with either mouse mMRAP1 or zebrafish zfMRAP1 increased the sensitivity of SacMC(5) receptor for hACTH(1-24) by at least one order of magnitude. Hence, SacMC(5) receptor has the potential to interact with MRAP1 orthologs and in this regard behaved more like a melanocortin MC(2) receptor ortholog than a melanocortin MC(5) receptor ortholog. These observations are discussed in light of the evolution of the melanocortin receptor gene family in cartilaginous fish, and the physiological implications of these observations are considered.

Characterization of the immunoglobulin repertoire of the spiny dogfish (Squalus acanthias).

The cartilaginous fish (chimeras, sharks, skates and rays) are the oldest group relative to mammals in which an adaptive immune system founded upon immunoglobulins has been found. In this manuscript we characterize the immunoglobulins of the spiny dogfish (Squalus acanthias) at both the molecular and expressed protein levels. Despite the presence of hundreds of IgM clusters in this species the serum levels of this isotype are comparatively low. However, analysis of cDNA sequences and serum protein suggests microheterogeneity in the IgM heavy chains and supports the proposal that different clusters are preferentially used in the two forms (monomer or pentamer) of this isotype. We also found that the IgNAR isotype in this species exists in a previously unknown multimeric format in serum. Finally, we identified a new form of the IgW isotype (the shark IgD orthologue), in which the leader is spliced directly to the first constant domain, resulting in a molecule lacking an antigen-binding domain.

Body fluid osmolytes and urea and ammonia flux in the colon of two chondrichthyan fishes, the ratfish, Hydrolagus colliei, and spiny dogfish, Squalus acanthias.

The present study has examined the role of the colon in regulating ammonia and urea nitrogen balance in two species of chondrichthyans, the ratfish, Hydrolagus colliei (a holocephalan) and the spiny dogfish, Squalus acanthias (an elasmobranch). Stripped colonic tissue from both the dogfish and ratfish was mounted in an Ussing chamber and in both species bi-directional urea flux was found to be negligible. Urea uptake by the mucosa and serosa of the isolated colonic epithelium through accumulation of (14)C-urea was determined to be 2.8 and 6.2 fold greater in the mucosa of the dogfish compared to the serosa of the dogfish and the mucosa of the ratfish respectively. Furthermore, there was no difference between serosal and mucosal accumulation of (14)C-urea in the ratfish. Through the addition of 2mM NH(4)Cl to the mucosal side of each preparation the potential for ammonia flux was also examined. This was again found to be negligible in both species suggesting that the colon is an extremely tight epithelium to the movement of both urea and ammonia. Plasma, chyme and bile fluid samples were also taken from the agastric ratfish and were compared with solute concentrations of equivalent body fluids in the dogfish. Finally molecular analysis revealed expression of 3 isoforms of the urea transport protein (UT) and an ammonia transport protein (Rhbg) in the gill, intestine, kidney and colon of the ratfish. Partial nucleotide sequences of the UT-1, 2 and 3 isoforms in the ratfish had 95, 95 and 92% identity to the equivalent UT isoforms recently identified in another holocephalan, the elephantfish, Callorhinchus milii. Finally, the nucleotide sequence of the Rhbg identified in the ratfish had 73% identity to the Rhbg protein recently identified in the little skate, Leucoraja erinacea.

Frequency of multiple paternity in the spiny dogfish Squalus acanthias in the western north Atlantic.

Multiple paternity (MP) has been shown to be widespread in elasmobranch fishes although its prevalence and the number of sires per litter vary considerably among species. In the squaloid shark Squalus acanthias, MP has been reported, but whether it is a common feature of the species' reproductive strategy is unknown. In this study, we determined the frequency of MP in 29 litters of S. acanthias sampled from the lower Chesapeake Bay and coastal Virginia waters, using 7 highly polymorphic nuclear DNA microsatellite loci. Only 5 litters (17% of the total) were genetically polyandrous, with at least 2 sires per litter. Litter size increased with female size but was similar between polyandrous and monandrous females.

Analysis and functional annotation of expressed sequence tags from in vitro cell lines of elasmobranchs: Spiny dogfish shark (Squalus acanthias) and little skate (Leucoraja erinacea).

Elasmobranchs are the most commonly used experimental models among the jawed, cartilaginous fish (Chondrichthyes). Previously we developed cell lines from embryos of two elasmobranchs, Squalus acanthias the spiny dogfish shark (SAE line), and Leucoraja erinacea the little skate (LEE-1 line). From these lines cDNA libraries were derived and expressed sequence tags (ESTs) generated. From the SAE cell line 4303 unique transcripts were identified, with 1848 of these representing unknown sequences (showing no BLASTX identification). From the LEE-1 cell line, 3660 unique transcripts were identified, and unknown, unique sequences totaled 1333. Gene Ontology (GO) annotation showed that GO assignments for the two cell lines were in general similar. These results suggest that the procedures used to derive the cell lines led to isolation of cell types of the same general embryonic origin from both species. The LEE-1 transcripts included GO categories "envelope" and "oxidoreductase activity" but the SAE transcripts did not. GO analysis of SAE transcripts identified the category "anatomical structure formation" that was not present in LEE-1 cells. Increased organelle compartments may exist within LEE-1 cells compared to SAE cells, and the higher oxidoreductase activity in LEE-1 cells may indicate a role for these cells in responses associated with innate immunity or in steroidogenesis. These EST libraries from elasmobranch cell lines provide information for assembly of genomic sequences and are useful in revealing gene diversity, new genes and molecular markers, as well as in providing means for elucidation of full-length cDNAs and probes for gene array analyses. This is the first study of this type with members of the Chondrichthyes.

beta-adrenergic regulation of a novel isoform of NCX: sequence and expression of shark heart NCX in human kidney cells.

The function, regulation, and molecular structure of the cardiac Na(+)/Ca(2+) exchangers (NCXs) vary significantly among vertebrates. We previously reported that beta-adrenergic suppression of amphibian cardiac NCX1.1 is associated with specific molecular motifs. Here we investigated the bimodal, cAMP-dependent regulation of spiny dogfish shark (Squalus acanthias) cardiac NCX, exploring the effects of molecular structure, host cell environment, and ionic milieu. The shark cardiac NCX sequence (GenBank accession no. DQ 068478) revealed two novel proline/alanine-rich amino acid insertions. Wild-type and mutant shark NCXs were cloned and expressed in mammalian cells (HEK-293 and FlpIn-293), where their activities were measured as Ni(2+)-sensitive Ca(2+) fluxes (fluo 4) and membrane (Na(+)/Ca(2+) exchange) currents evoked by changes in extracellular Na(+) concentration and/or membrane potential. Regardless of Ca(2+) buffering, beta-adrenergic stimulation of cloned wild-type shark NCX consistently produced bimodal regulation (defined as differential regulation of Ca(2+)-efflux and -influx pathways), with suppression of the Ca(2+)-influx mode and either no change or enhancement of the Ca(2+)-efflux mode, closely resembling results from parallel experiments with native shark cardiomyocytes. In contrast, mutant shark NCX, with deletion of the novel region 2 insertion, produced equal suppression of the inward and outward currents and Ca(2+) fluxes, thereby abolishing the bimodal nature of the regulation. Control experiments with nontransfected and dog cardiac NCX-expressing cells showed no cAMP regulation. We conclude that bimodal beta-adrenergic regulation is retained in cloned shark NCX and is dependent on the shark's unique molecular motifs.

Gene structure and functional characterization of growth hormone in dogfish, Squalus acanthias.

Dogfish (Squalus acanthias) growth hormone (GH) was identified by cDNA cloning and protein purification from the pituitary gland. Dogfish GH cDNA encoded a prehormone of 210 amino acids (aa). Sequence analysis of purified GH revealed that the prehormone is composed of a signal peptide of 27 aa and a mature protein of 183 aa. Dogfish GH showed 94% sequence identity with blue shark GH, and also showed 37-66%, 26%, and 48-67% sequence identity with GH from osteichtyes, an agnathan, and tetrapods. The site of production was identified through immunocytochemistry to be cells of the proximal pars distalis of the pituitary gland. Dogfish GH stimulates both insulin-like growth factor-I and II mRNA levels in dogfish liver in vitro. The dogfish GH gene consisted of five exons and four introns, the same as in lamprey, teleosts such as cypriniforms and siluriforms, and tetrapods. The 5'-flanking region within 1082 bp of the transcription start site contained consensus sequences for the TATA box, Pit-1/GHF-1, CRE, TRE, and ERE. These results show that the endocrine mechanism for growth stimulation by the GH-IGF axis was established at an early stage of vertebrate evolution, and that the 5-exon-type gene organization might reflect the structure of the ancestral gene for the GH gene family.