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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.

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 (L 50 ) was 659 mm for females and 575 mm for males, corresponding to 7.5 and 5.5 years of age (A 50 ), 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.

Aquaporin 1 is a membrane water channel protein, which was studied here in spiny dogfish (Squalus acanthias) osmoregulatory tissues using a variety of techniques. The cloning of aquaporin 1 (AQP1) in the spiny dogfish identified a splice variant version of the mRNA/protein (AQP1SV1/AQP1SV1). Polymerase chain reaction (PCR) in a range of tissues showed AQP1 to be expressed at very high levels in the rectal gland with ubiquitous mRNA expression at lower levels in other tissues. Northern blotting showed that AQP1 had a mRNA size of 5.3 kb in kidney total RNA. The level of AQP1 mRNA was significantly lower in the rectal glands of fish acclimated to 120% seawater (SW; vs. 75% SW (p = 0.0007) and 100% SW (p = 0.0025)) but was significantly higher in those fish in the kidney (vs. 100% SW (p = 0.0178)) and intestine (vs. 75% SW (p= 0.0355) and 100% SW (p = 0.0285)). Quantitative PCR determined that AQP1SV1 mRNA levels were also significantly lower in the rectal glands of both 120% (p = 0.0134) and 100% SW (p = 0.0343) fish in comparison to 75% SW-acclimated dogfish. Functional expression in Xenopus oocytes showed that AQP1 exhibited significant apparent membrane water permeability (p = 0.000008–0.0158) across a range of pH values, whereas AQP1SV1 showed no similar permeability. Polyclonal antibodies produced against AQP1 (AQP1 and AQP1/2 antibodies) and AQP1SV1 had bands at the expected sizes of 28 kDa and 24 kDa, respectively, as well as some other banding. The weak AQP1 antibody and the stronger AQP1/2 antibody exhibited staining in the apical membranes of rectal gland secretory tubules, particularly towards the periphery of the gland. In the gill, the AQP1/2 antibody in particular showed staining in secondary-lamellar pavement-cell basal membranes, and in blood vessels and connective tissue in the gill arch. In the spiral valve intestine side wall and valve flap, the AQP1/2 antibody stained muscle tissue and blood vessel walls and, after tyramide signal amplification, showed some staining in the apical membranes of epithelial cells at the ends of the luminal surface of epithelial folds. In the rectum/colon, there was also some muscle and blood vessel staining, but the AQP1 and AQP1/2 antibodies both stained a layer of cells at the base of the surface epithelium. In the kidney convoluted bundle zone, all three antibodies stained bundle sheath membranes to variable extents, and the AQP1/2 antibody also showed staining in the straight bundle zone bundle sheath. In the kidney sinus zone, the AQP1/2 antibody stained the apical membranes of late distal tubule (LDT) nephron loop cells most strongly, with the strongest staining in the middle of the LDT loop and in patches towards the start of the LDT loop. There was also a somewhat less strong staining of segments of the first sinus zone nephron loop, particularly in the intermediate I (IS-I) tubule segment. Some tubules appeared to show no or only low levels of staining. The results suggest that AQP1 plays a role in rectal gland fluid secretion, kidney fluid reabsorption and gill pavement-cell volume regulation and probably a minor role in intestinal/rectal/colon fluid absorption.

The original UT-1 transporter gene was initially identified in the spiny dogfish (Squalus acanthias), but localization of the UT-1 protein was not determined. Subsequent UT-1 expression was shown to localize to the collecting tubule (CT) of the shark nephron in other shark species, with expression in a closely related chimaera species also located additionally at a lower level in the intermediate-I segment (IS-I) of the nephron. In spiny dogfish, two UT-1 splice variants are known (UT-1 long and short), and there was also a second UT-1 gene described (here termed Brain UT). In this study, a second splice variant of the second Brain UT gene was discovered. Expression profiles (mRNA) of UT-1 long and short and Brain UT were determined in a number of spiny dogfish tissues. Quantitative PCR in kidney samples showed that the level of the short variant of UT-1 was around 100 times higher than the long variant, which was itself expressed around 10 times higher than Brain UT cDNA/mRNA (in kidney). For the long variant, there was a significantly higher level of mRNA abundance in fish acclimatized to 75% seawater. Ultimately, three UT-1 antibodies were made that could bind to both the UT-1 short and long variant proteins. The first two of these showed bands of appropriate sizes on Western blots of around 52.5 and 46 kDa. The second antibody had some additional lower molecular weight bands. The third antibody was mainly bound to the 46 kDa band with faint 52.5 kDa staining. Both the 52.5 and 46 kDa bands were absent when the antibodies were pre-blocked with the peptide antigens used to make them. Across the three antibodies, there were many similarities in localization but differences in subcellular localization. Predominantly, antibody staining was greatest in the intermediate segment 1 (IS-I) and proximal (PIb) segments of the first sinus zone loop of the nephron, with reasonably strong expression also found at the start and middle of the late distal tubule (LDT; second sinus zone loop). While some expression in the collecting tubule (CT) could not be ruled out, the level of staining seemed to be low or non-existent in convoluted bundle zone nephron segments such as the CT. Hence, this suggests that spiny dogfish have a fundamentally different mode of urea absorption in comparison to that found in other shark species, potentially focused more on the nephron sinus zone loops than the CT.

The aquaporin 0 (AQP0)/major intrinsic protein of eye lens (MIP) cDNA was cloned and sequenced. Initial studies of the tissue distribution of mRNA expression proved to be incorrect. Subsequent experiments showed that AQP0 mRNA is expressed strongly in the eye with moderately strong expression in the kidneys and some expression was seen in the brain and muscle tissue, and very low expression in the esophagus/fundic stomach. Another set of PCR reactions with five times the amount of cDNA additionally showed mRNA/cDNA expression in the liver, rectal gland, and a very low level in the intestine. Sporadic expression of different pieces of AQP0 cDNA was seen in various experiments in gill and pyloric stomach. A custom polyclonal antibody was produced against a region near the C-terminal end of the AQP0 protein sequence. The antibody gave a band of around the correct size (for the AQP0 protein) on the Western blot, which also showed a few other higher-molecular-weight bands. The antibody was also used in immunohistochemistry, and in the kidney, it showed staining in the proximal II (PII), intermediate segment I (IS I), and late distal tubule (LDT) parts of the sinus zone region of nephrons as well as some staining in the bundle zone tubule segments, suggesting a role for AQP0 as a water channel. In the rectal gland, the antibody showed weak apical membrane staining in a few secretory tubules near the duct, but also somewhat stronger staining in cells appearing to connect various secretory tubules, suggesting a role in cell–cell adhesion. In the spiral valve intestine side wall and valve flap, after signal amplification, weak antibody staining was seen in the apical and lateral membranes of epithelial cells adjacent to the luminal surface. There was also some staining in the intestinal muscle. In the rectum/colon, staining was seen in a layer of cells underlying the epithelium and in some muscle layers. In the gill, there was very weak staining in secondary lamellae epithelial cells and in connective tissue surrounding blood vessels and blood sinuses. The low level of transcript expression in the rectal gland, gill, and intestinal tissues suggests caution in the interpretation of the immunohistochemical staining in these tissues.

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.

Spiny dogfish (Squalus acanthias) are assumed to be a highly migratory species, making habitual north-south migrations throughout their northwestern Atlantic United States (U.S.) range. Also assumed to be a benthic species, spiny dogfish stock structure is estimated through Northeast Fisheries Science Center (NEFSC) bottom-trawl surveys. Recent anomalies in population trends, including a recent four-fold increase in estimated spawning stock biomass, suggest alternative movement patterns could exist for this shark species. To obtain a better understanding of the horizontal and vertical movement dynamics of this species, Microwave Telemetry pop-up satellite archival X-Tags were attached to forty adult spiny dogfish at the northern (Gulf of Maine) and southern (North Carolina) extents of their core U.S. geographic range. Reconstructed geolocation tracks ranging in lengths from two to 12 months suggest that the seasonal migration patterns appear to be local in nature to each respective northern and southern deployment site, differing from previously published migration paradigms. Differences in distance and direction traveled between seasonal geolocations possibly indicate separate migratory patterns between groups. Kernel utilization distribution models also suggest strong separate core home ranges. Significant differences in seasonal temperature and depths between the two regions further substantiate the possibility of separate regional movement patterns between the two groups. Vertical utilization also suggests distinct diel patterns and that this species may not utilize the benthos as previously thought, potentially decreasing availability to benthic gear.

Salinity decreases are experienced by many marine elasmobranchs. To understand how these fishes cope with hyposmotic stress on a cellular level, we used the spiny dogfish shark ( Squalus acanthias ) as a model to test whether a reciprocal relationship exists between the cell’s two primary protein protection mechanisms, the chemical (e.g., trimethylamine oxide, TMAO) and molecular (e.g., heat shock protein 70, HSP70) chaperone systems. This relationship is interesting given that many elasmobranchs are expected to gain water and lose osmolytes, chemical chaperones, and ions as they osmoconform to new, lowered salinity. Dogfish were cannulated for repeated blood sampling and exposed to 70 % seawater (SW) for 48 h. These hyposmotic conditions had no effect on red blood cell (RBC) and white muscle TMAO concentrations, and did not result in HSP70 induction or signs of protein damage (i.e., increased ubiquitin), suggesting that TMAO levels were sufficiently protective in these tissues. However, in the gill, we observed a significant decrease in TMAO concentration and a significant induction of HSP70 as well as signs of protein damage. In the face of this cellular stress response, gill Na + /K + -ATPase (NKA) activity significantly increased during hyposmotic conditions, as expected. We suggest that this functional preservation in the gill is partly the result of HSP70 induction with lowered salinity. We conclude a reciprocal relationship between TMAO and HSP70 in the gills of dogfish as a result of in vivo hyposmotic stress. When osmotically induced protein damage surpasses the protective capacity of remaining TMAO, HSP70 is induced to preserve tissue and organismal function.

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.

Although they are ureotelic, marine elasmobranchs express Rh glycoproteins, putative ammonia channels. To address questions raised by a recent study on high environmental ammonia (HEA) exposure, dogfish were intravascularly infused for 24 h at 3 ml kg −1 h −1 with isosmotic NaCl (500 mmol l −1 , control), NH 4 HCO 3 (500 mmol l −1 ), NH 4 Cl (500 mmol l −1 ), or HCl (as 125 mmol l −1 HCl + 375 mmol l −1 NaCl). While NaCl had no effect on arterial acid–base status, NH 4 HCO 3 caused mild alkalosis, NH 4 Cl caused strong acidosis, and HCl caused lesser acidosis, all predominantly metabolic in nature. Total plasma ammonia (T Amm ) and excretion rates of ammonia ( J Amm ) and urea-N ( J Urea-N ) were unaffected by NaCl or HCl. However, despite equal loading rates, plasma T Amm increased to a greater extent with NH 4 Cl, while J Amm increased to a greater extent with NH 4 HCO 3 due to much greater increases in blood-to-water PNH 3 gradients. As with HEA, both treatments caused large (90 %) elevations of J Urea-N , indicating that urea-N synthesis by the ornithine-urea cycle (OUC) is driven primarily by ammonia rather than HCO 3 − . Branchial mRNA expressions of Rhbg and Rhp2 were unaffected by NH 4 HCO 3 or NH 4 Cl, but v - type H + - ATPase was down-regulated by both treatments, and Rhbg and Na + /H + exchanger NHE2 were up-regulated by HCl. In the kidney, Rhbg was unresponsive to all treatments, but Rhp2 was up-regulated by HCl, and the urea transporter UT was up-regulated by HCl and NH 4 Cl. These responses are discussed in the context of current ideas about branchial, renal, and OUC function in this nitrogen-limited predator.