Hormonal effects on glucose and ketone metabolism in a perfused liver of an elasmobranch, the North Pacific spiny dogfish, Squalus suckleyi.

Hormonal influence on hepatic function is a critical aspect of whole-body energy balance in vertebrates. Catecholamines and corticosteroids both influence hepatic energy balance via metabolite mobilization through glycogenolysis and gluconeogenesis. Elasmobranchs have a metabolic organization that appears to prioritize the mobilization of hepatic lipid as ketone bodies (e.g. 3-hydroxybutyrate [3-HB]), which adds complexity in determining the hormonal impact on hepatic energy balance in this taxon. Here, a liver perfusion was used to investigate catecholamine (epinephrine [E]) and corticosteroid (corticosterone [B] and 11-deoxycorticosterone [DOC]) effects on the regulation of hepatic glucose and 3-HB balance in the North Pacific Spiny dogfish, Squalus suckleyi. Further, hepatic enzyme activity involved in ketogenesis (3-hydroxybutyrate dehydrogenase), glycogenolysis (glycogen phosphorylase), and gluconeogenesis (phosphoenolpyruvate carboxykinase) were assessed in perfused liver tissue following hormonal application to discern effects on hepatic energy flux. mRNA transcript abundance key transporters of glucose (glut1 and glut4) and ketones (mct1 and mct2) and glucocorticoid function (gr, pepck, fkbp5, and 11ßhsd2) were also measured to investigate putative cellular components involved in hepatic responses. There were no changes in the arterial-venous difference of either metabolite in all hormone perfusions. However, perfusion with DOC increased gr transcript abundance and decreased flow rate of perfusions, suggesting a regulatory role for this corticosteroid. Phosphoenolpyruvate carboxykinase activity increased following all hormone treatments, which may suggest gluconeogenic function; E also increased 3-hydroxybutyrate dehydrogenase activity, suggesting a function in ketogenesis, and decreased pepck and fkbp5 transcript abundance, potentially showing some metabolic regulation. Overall, we demonstrate hormonal control of hepatic energy balance using liver perfusions at various levels of biological organization in an elasmobranch.

The chondrichthyan glucagon-like peptide 3 regulates hepatic ketone metabolism in the Pacific spiny dogfish Squalus suckleyi.

Chondrichthyans have a novel proglucagon-derived peptide, glucagon-like peptide (GLP)-3, in addition to GLP-1 and GLP-2 that occur in other vertebrates. Given that the GLPs are important regulators of metabolic homeostasis across vertebrates, we sought to investigate whether GLP-3 displays functional actions on metabolism within a representative chondrichthyan, the Pacific spiny dogfish Squalus suckleyi. There were no observed effects of GLP-3 perfusion (10 nM for 15 min) on the rate of glucose or oleic acid acquisition at the level of the spiral valve nor were there any measured effects on intermediary metabolism within this tissue. Despite no effects on apparent glucose transport or glycolysis in the liver, a significant alteration to ketone metabolism occurred. Firstly, ketone flux through the perfused liver switched from a net endogenous production to consumption following hormone application. Accompanying this change, significant increases in mRNA transcript abundance of putative ketone transporters and in the activity of ß-hydroxybutyrate dehydrogenase (a key enzyme regulating ketone flux in the liver) were observed. Overall, while these results show effects on hepatic metabolism, the physiological actions of GLP are distinct between this chondrichthyan and those of GLP-1 on teleost fishes. Whether this is the result of the particular metabolic dependency on ketone bodies in chondrichthyans or a differential function of a novel GLP remains to be fully elucidated.

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.

Mass Spectrometry Analysis of Shark Skin Proteins.

The mucus layer covering the skin of fish has several roles, including protection against pathogens and mechanical damage in which proteins play a key role. While proteins in the skin mucus layer of various common bony fish species have been explored, the proteins of shark skin mucus remain unexplored. In this pilot study, we examine the protein composition of the skin mucus in spiny dogfish sharks and chain catsharks through mass spectrometry (NanoLC-MS/MS). Overall, we identified 206 and 72 proteins in spiny dogfish (Squalus acanthias) and chain catsharks (Scyliorhinus retifer), respectively. Categorization showed that the proteins belonged to diverse biological processes and that most proteins were cellular albeit a significant minority were secreted, indicative of mucosal immune roles. The secreted proteins are reviewed in detail with emphasis on their immune potentials. Moreover, STRING protein-protein association network analysis showed that proteins of closely related shark species were more similar as compared to a more distantly related shark and a bony fish, although there were also significant overlaps. This study contributes to the growing field of molecular shark studies and provides a foundation for further research into the functional roles and potential human biomedical implications of shark skin mucus proteins.

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.

Molecular and pharmacological analysis of the melanocortin-2 receptor and its accessory proteins Mrap1 and Mrap2 in a Squalomorph shark, the Pacific spiny dogfish.

The hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis is a conserved vertebrate neuroendocrine mechanism regulating the stress response. The penultimate step of the HPA/I axis is the exclusive activation of the melanocortin-2 receptor (Mc2r) by adrenocorticotropic hormone (ACTH), requiring an accessory protein, Mrap1 or Mrap2. Limited data for only three cartilaginous fishes support the hypothesis that Mc2r/Mrap1 function in bony vertebrates is a derived trait. Further, Mc2r/Mrap1 functional properties appear to contrast among cartilaginous fishes (i.e., the holocephalans and elasmobranchs). This study sought to determine whether functional properties of Mc2r/Mrap1 are conserved across elasmobranchs and in contrast to holocephalans. The deduced amino acid sequences of Pacific spiny dogfish (Squalus suckleyi; pd) pdMc2r, pdMrap1, and pdMrap2 were obtained from a de novo transcriptome of the interrenal gland and validated against the S. suckleyi genome. pdMc2r showed high primary sequence similarity with elasmobranch and holocephalan Mc2r except at extracellular domains 1 and 2, and transmembrane domain 5. pdMraps showed similarly high sequence similarity with holocephalan and other elasmobranch Mraps, with all cartilaginous fish Mrap1 orthologs lacking an activation motif. cAMP reporter gene assays demonstrated that pdMc2r requires an Mrap for activation, and can be activated by stingray (sr) ACTH(1-24), srACTH(1-13)NH2 (i.e., α-MSH), and γ-melanocyte-stimulating hormone at physiological concentrations. However, pdMc2r was three orders of magnitude more sensitive to srACTH(1-24) than srACTH(1-13)NH2. Further, pdMc2r was two orders of magnitude more sensitive to srACTH(1-24) when expressed with pdMrap1 than with pdMrap2. These data suggest that functional properties of pdMc2r/pdMrap1 reflect other elasmobranchs and contrast what is seen in holocephalans.

Using 15N to determine the metabolic fate of dietary nitrogen in North Pacific spiny dogfish (Squalus acanthias suckleyi).

Marine elasmobranchs are ureosmotic, retaining large concentrations of urea to balance their internal osmotic pressure with that of the external marine environment. The synthesis of urea requires the intake of exogenous nitrogen to maintain whole-body nitrogen balance and satisfy obligatory osmoregulatory and somatic processes. We hypothesized that dietary nitrogen may be directed toward the synthesis of specific nitrogenous molecules in post-fed animals; specifically, we predicted the preferential accumulation and retention of labelled nitrogen would be directed towards the synthesis of urea necessary for osmoregulatory purposes. North Pacific spiny dogfish (Squalus acanthias suckleyi) were fed a single meal of 7 mmol l-1 15NH4Cl in a 2% ration by body mass of herring slurry via gavage. Dietary labelled nitrogen was tracked from ingestion to tissue incorporation and the subsequent synthesis of nitrogenous compounds (urea, glutamine, bulk amino acids, protein) in the intestinal spiral valve, plasma, liver and muscle. Within 20 h post-feeding, we found labelled nitrogen was incorporated into all tissues examined. The highest δ15N values were seen in the anterior region of the spiral valve at 20 h post-feeding, suggesting this region was particularly important in assimilating the dietary labelled nitrogen. In all tissues examined, enrichment of the nitrogenous compounds was sustained throughout the 168 h experimental period, highlighting the ability of these animals to retain and use dietary nitrogen for both osmoregulatory and somatic processes.

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.

Cerebral arterial and ventricular morphology of the dogfish (Squalus acanthias), American bullfrog (Rana catesbeiana), and green iguana (Iguana iguana): Arterial high-resolution micro-CT, dissection, and radiography study.

This study's objective was to investigate obtaining high-resolution micro-computed tomography (CT) imaging of the injected arterial circulation of the brains of the dogfish (Squalus acanthias), American bullfrog (Rana catesbeiana), and green iguana (Iguana iguana). No micro-CT images of the arterial morphology of the brains of these vertebrates were previously published. Micro-CT imaging was performed on brains that had the cerebral arterial and ventricular systems injected with a radiopaque barium-gelatin compound in the early 1970s. These specimens were dissected and placed in a preservative fluid for 35 years, until imaged with micro-CT. The obtained micro-CT images were processed with a software program that provided 3D rotational motion rendering, and sequential display of 2D renderings of the micro-CT data. The anatomic information provided by the high-resolution micro-CT is not reproducible by any other radiopaque contrast currently available, without tissue removal corrosion, and enhanced the dissection information. The digital videos of the micro-CT 3D rotational motion rendering and sequential display of 2D renderings of the dogfish, bullfrog, and green iguana, demonstrate the extent of the arterial network within the brain, the arterial segments obscured by overlying structures such as nerves, and identified in the bullfrog the venous cerebral circulation resulting from the centrifugal leptomeningeal arterial capillaries. The rotational 3D images separated superimposed arterial structures, and the sequential display of the 2D renderings clarifies the relationship of cut or overlapped arterial branches. Comparing the brain and arterial morphology of the dogfish, bullfrog, and green iguana demonstrates some of the evolutionary modifications in these vertebrates.

The gut microbiome may influence post-prandial nitrogen handling in an elasmobranch, the Pacific spiny dogfish (Squalus suckleyi).

Nitrogen recycling through the gut microbiome is an important mechanism used throughout vertebrates to reclaim valuable nitrogen trapped in urea. Evidence suggests it may be especially important in nitrogen limited animals, yet little is known about its role in marine elasmobranchs, which are said to be severely nitrogen limited. In the present study we used antibiotics to deplete the gut microbiome of Pacific spiny dogfish and assessed the role of the microbiome in nitrogen handling in both fed and fasted states. In fed animals, antibiotic treatment eliminated the activity of the microbial enzyme urease and reduced cellulase activity by 78%. This reduction in microbial enzyme activity resulted in significantly lower plasma urea levels which then trended upward as urea excretion rates decreased. Ammonia excretion rates were also significantly lower in antibiotic treated fish compared to the control fed. Finally, antibiotic treated fed individuals lost an average of 7.4% of their body mass while the fed controls lost only 1.8% of their body mass. Nitrogen handling in fasted animals was not significantly impacted by a reduction in microbial activity. These results suggest that compromising the gut microbiome significantly influences post-prandial nitrogen handling in spiny dogfish, and that the recycling of urea­nitrogen may be vital to maintaining nitrogen balance in these fish.

Sustained endocrine and exocrine function in the pancreas of the Pacific spiny dogfish post-feeding.

Secretions of the exocrine pancreas contain digestive enzymes integral to the digestive process. The Pacific spiny dogfish (Squalus suckleyi) has a discrete pancreas, divided into two lobes termed the dorsal and ventral lobes. These lobes drain into the anterior intestine via a common duct to enable digestion. Previous studies have identified that the exocrine pancreas produces (co)lipases, chymotrypsin, carboxypeptidase, and low levels of chitinases; however, investigations into other digestive enzymes are limited. We detect the presence of lipase, trypsin, and carbohydrase and show that activities are equivalent between both lobes of the pancreas. Additionally, we sought to investigate the influence of a single feeding event (2% body weight ration of herring by gavage) on enzyme activities over an extended time course (0, 20, 48, 72, 168 h) post-feeding. The results indicate that there are no differences in pancreatic tissue digestive enzyme activities between fed or fasted states. Analysis of acinar cell circumference post-feeding demonstrates a significant increase at 20 and 48 h, that returns to fasting levels by 72 h. No significant changes were observed regarding whole-tissue insulin or glucagon mRNA abundance or with glucose transporter (glut) 1 or 3. Yet, a significant and transient decrease in glut4 and sodium glucose-linked transporter mRNA abundance was found at 48 h post-feeding. We propose that the constant enzyme activity across this relatively large organ, in combination with a relatively slow rate of digestion leads to an evenly distributed, sustained release of digestive enzymes regardless of digestive state.

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 II: Localization of AQP3, AQP4 and AQP15 in the kidney.

Three aquaporin water channel proteins, AQP3, AQP4 and AQP15 were localized to cells within the kidney of the spiny dogfish, Squalus acanthias, using an immunohistochemical approach. Dogfish kidney has two zones, the bundle zone (including five nephron segment bundles) and the sinus zone (with two major loops). In order to discriminate between the two loops, the cilia occurring in the first proximal/intermediate loop were labeled with two antibodies including an anti-acetylated tubulin antibody. The second late distal tubule loop (LDT) was identified, as the nephron in that region has no luminal cilia. Strong staining of the rabbit anti-dogfish AQP3, AQP4 (AQP4/2) or AQP15 polyclonal antibodies localized to LDT tubules. These antibodies were further co-stained with a mouse anti-Na+,K+-ATPase a5 monoclonal antibody, as Na+,K+-ATPase has previously been suggested to localize to the early distal tubule (EDT) and LDT and a mouse anti-NKCC T4 antibody, as NKCC2 was previously suggested to be located in the EDT and the second half of the LDT. In the LDT, strong AQP4/2 and AQP15 antibody staining localized together with the strong Na+,K+-ATPase antibody staining, whereas strong AQP3 antibody staining was largely separate but with an overlapping distribution. Very low levels of AQP4/2 antibody basal membrane staining was also detected in the first proximal /intermediate loop of the sinus zone. There was no mouse anti-NKCC T4 antibody staining apparent in the LDT. In the convoluted part of the bundle zone, the AQP4/2 and Na+,K+-ATPase but not the AQP3 or AQP15 antibodies stained tubule segments, with both AQP4/2 and Na+,K+-ATPase staining the EDT, and with low-level AQP4/2 staining of two other tubules of the bundle, which were most likely to be the proximal 1a (PIa) and intermediate II (IS II) tubules. The AQP4/2 antibody also stained the EDT in the straight bundle zone. The mouse anti-NKCC T4 antibody stained the apical region of EDT tubules in the convoluted bundle zone, suggesting that the antibody was binding to the NKCC2 cotransporter. The AQP15 antibody appeared to bind to the peritubular sheath surrounding bundles in the bundle zone. Due to the AQP4/2 antibody staining in the EDT that immediately proceeds and continues into the LDT, this suggested that the strong AQP4/2, AQP15 and Na+,K+-ATPase antibody staining was located at the beginning of the LDT and therefore the strong AQP3 was located at the end of the LDT. The staining of all three AQP antibodies was blocked by the peptide-antigen used to make each one, suggesting that all the staining is specific to each antibody.

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.

Energy and corticosteroid mobilization following an induced stress response in an elasmobranch fish, the North Pacific spiny dogfish (Squalus acanthias suckleyi).

The dominant corticosteroid in elasmobranchs, 1α-hydroxycorticosterone (1α-OHB), has a described role in mineral regulation but a presumptive role in energy balance. Energy demand in vertebrates following exposure to a stressor typically involves an immediate but transient release of glucocorticoids as a means of mobilizing available energy stores, usually in the form of glucose. Although a glucocorticoid role for 1α-OHB would be expected, direct glucocorticoid function of this steroid has yet to be reported in any elasmobranch. In addition, elasmobranchs also utilize the metabolite ß-hydroxybutyrate (ß-HB), which is thought to replace the role fatty acids play in most vertebrates as a predominant fuel source in extrahepatic tissues. To determine the mobilization of metabolites and corticosteroids during a stress event, North Pacific spiny dogfish, Squalus acanthias suckleyi, were cannulated and held in a darkened isolation box to recover (24-48 h) before being subjected to an acute air exposure or corticosterone injection. Dogfish were then serially blood sampled at nine timepoints over 48 h. Glucose, ß-HB, 1α-OHB, corticosterone, as well as lactate, pH, and osmolality were quantified in plasma samples. All measured variables increased in control and treatment groups within 48 h from the start of experimentation, and ß-HB and 1α-OHB remained elevated for the duration of the experiment. There was no linear correlation between glucose and 1α-OHB, but there was a weak (R2 = 0.230) although significant (p = 0.001), positive correlation between ß-HB and 1α-OHB. Interestingly, there were also significant correlations between increasing circulating glucose and corticosterone (R2 = 0.349; p < 0.001), and decreasing ß-HB and corticosterone concentrations (R2 = 0.180; p = 0.008). Our data suggest that following successive stressors of capture, surgery, and confinement, 1α-OHB was not correlated with circulating glucose, only weakly correlated with circulating ß-HB concentrations (R2 = 0.230; p = 0.001), and that corticosterone may also serve a role in energy mobilization in this species.

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 of mechanically stimulated escape responses in the Pacific spiny dogfish, Squalus suckleyi.

Fast escape responses to a predator threat are fundamental to the survival of mobile marine organisms. However, elasmobranchs are often underrepresented in such studies. Here, we measured the escape latency (time interval between the stimulus and first visible reaction) of mechanically induced escape responses in the Pacific spiny dogfish, Squalus suckleyi, and in two teleosts from the same region, the great sculpin, Myoxocephalus polyacanthocephalus, and the pile perch, Rhacochilus vacca We found that the dogfish had a longer minimum latency (66.7 ms) compared with that for the great sculpin (20.8 ms) and pile perch (16.7 ms). Furthermore, the dogfish had a longer latency than that of 48 different teleosts identified from 35 different studies. We suggest such long latencies in dogfish may be due to the absence of Mauthner cells, the giant neurons that control fast escape responses in fishes.

The effects of digesting a urea-rich meal on North Pacific spiny dogfish (Squalus acanthias suckleyi).

Marine elasmobranchs are nitrogen-limited owing to the requirement of nitrogen for both somatic growth and urea-based osmoregulation, and due to the loss of urea across the gills and kidney as nitrogenous waste. In this study we used in vitro stomach and intestinal gut sacs to investigate the effects of consuming a urea-rich meal (700 mM within a 2% body-mass ration of food-slurry) on nitrogen movement across the gastrointestinal (GI) tract of North Pacific spiny dogfish (Squalus acanthias suckleyi). Plasma urea concentrations did not differ between fasted (359 ± 19 mM), urea-poor fed (340 ± 16 mM), and urea-rich fed (332 ± 24 mM) dogfish. Interestingly, in vitro gut sacs of urea-rich fed dogfish showed no net urea absorption from the lumen over 3 h incubation, which contrasts previously published data on urea-poor fed dogfish that absorb urea from the lumen. In addition, ammonium (NH4+) concentration within the gut sac intestinal lumen significantly increased from 0.62 to 4.35 mM over 3 h. This is likely due to a combination of tissue production and microbial urease activity in the intestine. The overall results highlight the ability of S. a. suckleyi to regulate and maintain internal nitrogen concentrations despite the addition of excess dietary urea.

Impacts of low salinity exposure and antibiotic application on gut transport activity in the Pacific spiny dogfish, Squalus acanthias suckleyi.

The role of the marine elasmobranch gastrointestinal tract in nitrogen-recycling and osmotic homeostasis has become increasingly apparent, with the gut microbial community likely playing a significant role converting urea, an important osmolyte in elasmobranchs, into ammonia. The Pacific spiny dogfish can experience and tolerate reduced environmental salinities, yet how this environmental challenge may affect the microbiome, and consequently nitrogen transport across the gut, is as of yet unknown. In the present study, excised gut sac preparations were made from dogfish acclimated to the following: full-strength seawater (C), low salinity for 7 days (LS), and after acute transfer of LS-acclimated fish to full-strength SW for 6 h (AT). Significantly reduced microbial derived urease activity was observed in the mucosal saline of gut sac preparations from the LS (by 81%) and AT (by 89%) treatments relative to the C treatment. Microbial derived cellulase activity from mucosal saline samples tended to follow similar patterns. To further ensure an effective decrease in the spiral valve microbial population, an antibiotic cocktail was applied to the mucosal saline used for in vitro measurements of ion, water, and nitrogen flux in these gut sac preparations. This caused a further 57-61% decrease in the mucosal saline urease activity of the C and LS treatments. Overall, we observed relatively little flux across the stomach for all measured parameters aside from water movement, which switched from a net efflux in control fish to a net influx in acutely transferred fish, indicative of drinking. While no significant differences were observed in terms of nitrogen flux (urea or ammonia), we tended to see the accumulation of ammonia in the spiral valve lumen and a switch from efflux to influx of urea in control versus acutely transferred fish. The increased ammonia production likely occurs as a result of heightened metabolism in a challenging environment, while the retention and acquisition of urea is suggestive of nitrogen scavenging under nitrogen-limiting conditions.

Life-history traits of the spiny dogfish Squalus acanthias in the Adriatic Sea.

Pivotal life history traits concerning age structure and reproduction of the spiny dogfish (Squalus acanthias, Linnaeus 1758) were investigated in the Adriatic Sea from mid February 2012 to mid July 2013 and in 2016. The whole sample consisted of 176 females and 150 males, ranging between 217-1025 mm and 219-875 mm, respectively. The individual age, which was estimated using a cross-sectioning technique of the second dorsal-fin spine, ranged from 0 to 13+ years for females and from 0 to 9+ years for males. Based on the length-at-age estimates, the Gompertz growth parameters were L∞ = 1130 mm, k = 0.18 and L∞ = 920 mm, k = 0.24 for females and males, respectively. The size at sexual maturity (L50) was 659 mm for females and 575 mm for males, corresponding to 7.5 and 5.5 years of age (A50), respectively. Mean biennial fecundity was approximately 11 embryos/female and 12 ripe oocytes/female. Mature males occurred during much of the sampling period, while mature females with nearly full-term embryos were exclusively recorded in May 2013 and July 2016. Monitoring of catches conducted in a sample port of the north Adriatic (Chioggia) over the past 20 years has shown fluctuating trends in landings, with peaks during the summer reproductive season.