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Ureotelic elasmobranchs require nitrogen for both protein growth and urea-based osmoregulation, and therefore are probably nitrogen-limited in nature. Mechanisms exist for retaining and/or scavenging nitrogen in the gills, kidney, rectal gland and gut, but as yet, the latter are not well characterized. Intestinal sac preparations of the Pacific spiny dogfish shark ( Squalus acanthias suckleyi ) incubated in vitro strongly reabsorbed urea from the lumen after feeding, but mucosal fluid ammonia concentrations increased with incubation time. Phloretin (0.25 mmol l -1 , which blocked urea reabsorption) greatly increased the rate of ammonia accumulation in the lumen. A sensitive [ 14 C]urea-based assay was developed to examine the potential role of microbial urease in this ammonia production. Urease activity was detected in chyme/intestinal fluid and intestinal epithelial tissue of both fed and fasted sharks. Urease was not present in gall-bladder bile. Urease activities were highly variable among animals, but generally greater in chyme than in epithelia, and greater in fed than in fasted sharks. Comparable urease activities were found in chyme and epithelia of the Pacific spotted ratfish ( Hydrolagus colliei ), a ureotelic holocephalan, but were much lower in ammonotelic teleosts. Urease activity in dogfish chyme was inhibited by acetohydroxamic acid (1 mmol l -1 ) and by boiling. Treatment of dogfish gut sac preparations with acetohydroxamic acid blocked ammonia production, changing net ammonia accumulation into net ammonia absorption. We propose that microbial urease plays an important role in nitrogen handling in the elasmobranch intestine, allowing some urea-N to be converted to ammonia, which is then reabsorbed for amino acid synthesis or reconversion to urea., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The critical O(2) tension of whole-animal O(2) consumption rate (M(O2)), or P(crit), is the water P(O2) (Pw(O(2))) at which an animal transitions from an oxyregulator to an oxyconformer. Although P(crit) is a popular measure of hypoxia tolerance in fishes because it reflects the capacity for O(2) uptake from the environment at low Pw(O(2)), little is known about the interrelationships between P(crit) and blood O(2) transport characteristics and increased use of anaerobic metabolism during hypoxia exposure in fishes, especially elasmobranchs. We addressed this knowledge gap using progressive hypoxia exposures of two elasmobranch species with differing hypoxia tolerance. The P(crit) of the hypoxia-tolerant epaulette shark (Hemiscyllium ocellatum, 5.10±0.37 kPa) was significantly lower than that of the comparatively hypoxia-sensitive shovelnose ray (Aptychotrema rostrata, 7.23±0.40 kPa). Plasma [lactate] was elevated above normoxic values at around P(crit) in epaulette sharks, but increased relative to normoxic values at Pw(O(2)) below P(crit) in shovelnose rays, providing equivocal support for the hypothesis that P(crit) is associated with increased anaerobic metabolism. The M(O2), arterial P(O2) and arterial blood O(2) content (Ca(O(2))) were similar between the two species under normoxia and decreased in both species with progressive hypoxia, but as Pw(O(2)) declined, epaulette sharks had a consistently higher M(O2) and Ca(O(2)) than shovelnose rays, probably due to their significantly greater in vivo haemoglobin (Hb)-O(2) binding affinity (in vivo Hb-O(2) P(50)=4.27±0.57 kPa for epaulette sharks vs 6.35±0.34 kPa for shovelnose rays). However, at Pw(O(2)) values representing the same percentage of each species' P(crit) (up to ∼175% of P(crit)), Hb-O(2) saturation and Ca(O(2)) were similar between species. These data support the hypothesis that Hb-O(2) P(50) is an important determinant of P(crit) and suggest that P(crit) can predict Hb-O(2) saturation and Ca(O(2)) during hypoxia exposure, with a lower P(crit) being associated with greater O(2) supply at a given Pw(O(2)) and consequently better hypoxia tolerance. Thus, P(crit) is a valuable predictor of environmental hypoxia tolerance and hypoxia exposures standardized at a given percentage of P(crit) will yield comparable levels of arterial hypoxaemia, facilitating cross-species comparisons of responses to hypoxia.

To test the hypothesis that the preference for ketone bodies rather than lipids as oxidative fuel in elasmobranchs evolved in response to the appearance of urea-based osmoregulation, we measured total non-esterified fatty acids (NEFA) in plasma as well as maximal activities of enzymes of intermediary metabolism in tissues from marine and freshwater elasmobranchs, including: the river stingray Potamotrygon motoro (<1 mmol l(-1) plasma urea); the marine stingray Taeniura lymma, and the marine shark Chiloscyllium punctatum (>300 mmol l(-1) plasma urea); and the euryhaline freshwater stingray Himantura signifer, which possesses intermediate levels of urea. H. signifer also were acclimated to half-strength seawater (15 per thousand) for 2 weeks to ascertain the metabolic effects of the higher urea level that results from salinity acclimation. Our results do not support the urea hypothesis. Enzyme activities and plasma NEFA in salinity-challenged H. signifer were largely unchanged from the freshwater controls, and the freshwater elasmobranchs did not show an enhanced capacity for extrahepatic lipid oxidation relative to the marine species. Importantly, and contrary to previous studies, extrahepatic lipid oxidation does occur in elasmobranchs, based on high carnitine palmitoyl transferase (CPT) activities in kidney and rectal gland. Heart CPT in the stingrays was detectable but low, indicating some capacity for lipid oxidation. CPT was undetectable in red muscle, and almost undetectable in heart, from C. punctatum as well as in white muscle from T. lymma. We propose a revised model of tissue-specific lipid oxidation in elasmobranchs, with high levels in liver, kidney and rectal gland, low or undetectable levels in heart, and none in red or white muscle. Plasma NEFA levels were low in all species, as previously noted in elasmobranchs. D-beta-hydroxybutyrate dehydrogenase (d-beta-HBDH) was high in most tissues confirming the importance of ketone bodies in elasmobranchs. However, very low d-beta-HBDH in kidney from T. lymma indicates that interspecific variability in ketone body utilization occurs. A negative relationship was observed across species between liver glutamate dehydrogenase activity and tissue or plasma urea levels, suggesting that glutamate is preferentially deaminated in freshwater elasmobranchs because it does not need to be shunted to urea production as in marine elasmobranchs.

We compared levels of the major organic osmolytes in the muscle of elasmobranchs, including the methylamines trimethylamine oxide (TMAO), betaine and sarcosine as well as the beta-amino acids taurine and beta-alanine, and the activities of enzymes of methylamine synthesis (betaine and TMAO) in species with a wide range of urea contents. Four marine, a euryhaline in freshwater (Dasyatis sabina), and two freshwater species, one that accumulates urea (Himantura signifer) and one that does not (Potamotrygon motoro), were analyzed. Urea contents in muscle ranged from 229-352 micromol g-1 in marine species to 2.0 micromol g-1 in P. motoro. Marine elasmobranchs preferentially accumulate methylamines, possibly to counteract urea effects on macromolecules, whereas the freshwater species with lower urea levels accumulate the beta-amino acid taurine as the major non-urea osmolyte. A strong correlation (r2=0.84, P<0.001) with a slope of 0.40 was found between muscle urea content and the combined total methylamines plus total beta-amino acids, supporting the hypothesis that ;non-urea' osmolytes are specifically maintained at an approximately 2:1 ratio with urea in the muscle of elasmobranchs. All species examined had measurable synthetic capacity for betaine in the liver but only one species had detectable TMAO synthetic capacity. We propose a phylogenetic explanation for the distribution of TMAO synthesis in elasmobranchs and suggest that activation of liver betaine aldehyde dehydrogenase, relative to choline dehydrogenase, coincides with betaine accumulation in elasmobranchs. The latter relationship may be important in maintaining methylamine levels during periods of low dietary TMAO intake for species lacking TMAO synthesis.