Decapod crustaceans are conspicuous members of marine benthic communities to at least 7,700 m deep. To assess the bathymetric extent of this taxonomic group, baited landers were deployed to across the abyssal-hadal transition zone of 11 subduction trenches spanning the Pacific, Atlantic, Southern, and Indian oceans and additional sites. Decapods were dominated by penaeid shrimps (superfamily Penaeoidea), in particular Benthesicymus Spence Bate, 1881 and Cerataspis Gray, 1828, with the former being found deeper. Benthesicymus cf. crenatus Spence Bate, 1881 was observed in the Kermadec, Mariana, New Hebrides, Puerto Rico, Peru-Chile, Tonga, San Cristobal, and Santa Cruz trenches, plus the South Fiji Basin and the WallabyZenith Fracture Zone. They were not recorded in the Abaco Canyon, Agulhas Fracture Zone, Java Trench, or any of the polar locations. Cerataspis cf. monstrosus Gray, 1828 was present in the Kermadec, Mariana, New Hebrides, Puerto Rico, and Java trenches, the Abaco Canyon, Agulhas Fracture Zone, Wallaby-Zenith Fracture Zone and the South Fiji Basin, but absent from the Tonga, San Cristobal and Santa Cruz trenches. Hymenopenaeus nereus (Faxon, 1893) was only recorded in the Peru-Chile Trench. Unidentified species belonging to superfamily Oplophoroidea were observed to a maximum depth of 6,931 m. Decapods are thus are primarily represented at hadal depths by penaeoid shrimps, consistently present at tropical and temperate latitudes to similar to 7,700 m, while absent from equivalent depths in polar regions. Their maximum depth may be limited due to hydrostatic pressure, while potentially affected by temperature and oxygen in some instances. Muscle samples of three specimens from 6,000 m (Mariana and Kermadec trenches) were found to have high levels of trimethylamine N-oxide (TMAO; 260 mmol kg(-1)), the major piezolyte, a protectant against hydrostatic pressure, in other deep-sea organisms. We speculate that physiological limits to TMAO concentration may prevent them from inhabiting the greatest hadal depths.

Hydrostatic pressure (HP), increasing by 1 atm per 10 m in the ocean, perturbs many cellular processes, for example, by rigidifying membranes and disturbing protein folding and ligand binding. Membranes can be fluidized to work under high HP by increasing unsaturated fatty acids, for example, docosahexaenoic acid. Over generations, some deep-sea proteins have evolved intrinsic resistance to HP, but often incompletely. These may be protected from HP with piezolytes, small organic molecules with pressure-counteracting properties. The key example is the osmolyte trimethylamine N-oxide (TMAO), which marine fishes and crustaceans accumulates linearly with depth. TMAO can effectively counteract many inhibitory effects of HP on numerous proteins. For short-term HP stress, cellular stress (transient) and homeostasis (persistent) responses (CSRs, CHRs) remain poorly characterized, but across different taxa of shallow and terrestrial organisms, they include common CSR/CHR mechanisms known for other stressors-heat shock proteins (HSPs), boosted energy metabolism, antioxidants, cellular repair systems. For vertically migrating marine animals, HP stress responses are even more poorly characterized. Some species (e.g., Anguilla silver eel, king crab Lithodes maja, snubnosed eel Simenchelys parasiticus) cope with HP changes in their habitat range by intrinsic adaptations, lipid desaturase activation, and metabolic adjustments, but perhaps not common CSR mechanisms. Such species may have constitutive stress proteins and/or are able to adjust membrane saturation and/or TMAO rapidly with depth. For permanent deep-sea species, CSR/CHR mechanisms have not been directly tested, but evidence in Mariana Trench amphipods and snailfish suggest that HSP and desaturase genes, and possibly piezolyte synthesis, have undergone habitat-related selection.

One of the major discoveries in modem biology is the remarkable cellular and molecular similarity of life on Earth in all its diverse forms. However, addition of betaine with high urea partly or fully restored normal growth, in accordance with the counteracting hypothesis. A large number of different carbohydrate osmolytes are known, including monosaccharides, disaccharides such as sucrose, heterosides, linear polyols with three to six carbons, and cyclic polyols. The major evolutionary question is, of course, why osmolytes are constrained by natural selection to a few solute types, mostly metabolically costly organic compounds, with the inorganic ions usually disfavored. Examination of a wide variety of biological processes in vitro have shown a widespread sensitivity to changes in inorganic ion concentration. Independently of biologists working on functional compatibility of cell osmolytes, biochemists had found as early as 1929 that sugars and polyols exhibit another widespread property: the ability to stabilize macromolecular structure under extreme conditions.

We explore the principles of pressure tolerance in enzymes of deep‐sea fishes using lactate dehydrogenases (LDH) as a case study. We compared the effects of pressure on the activities of LDH from hadal snailfishes Notoliparis kermadecensis and Pseudoliparis swirei with those from a shallow‐adapted Liparis florae and an abyssal grenadier Coryphaenoides armatus. We then quantified the LDH content in muscle homogenates using mass‐spectrometric determination of the LDH‐specific conserved peptide LNLVQR. Existing theory suggests that adaptation to high pressure requires a decrease in volume changes in enzymatic catalysis. Accordingly, evolved pressure tolerance must be accompanied with an important reduction in the volume change associated with pressure‐promoted alteration of enzymatic activity ( ). Our results suggest an important revision to this paradigm. Here, we describe an opposite effect of pressure adaptation—a substantial increase in the absolute value of in deep‐living species compared to shallow‐water counterparts. With this change, the enzyme activities in abyssal and hadal species do not substantially decrease their activity with pressure increasing up to 1–2 kbar, well beyond full‐ocean depth pressures. In contrast, the activity of the enzyme from the tidepool snailfish, L. florae, decreases nearly linearly from 1 to 2500 bar. The increased tolerance of LDH activity to pressure comes at the expense of decreased catalytic efficiency, which is compensated with increased enzyme contents in high‐pressure‐adapted species. The newly discovered strategy is presumably used when the enzyme mechanism involves the formation of potentially unstable excited transient states associated with substantial changes in enzyme–solvent interactions.

Discovery of an unusual rectal gland in the Atlantic sixgill shark Hexanchus vitulus led us to examine the rectal glands of 31 species of sharks to study diversity in rectal-gland morphology. Twenty-four of 31 species of sharks had digitiform glands (mean width-length ratio +/- SD = 0.17 +/- 0.04) previously assumed to be characteristic of all elasmobranchs regardless of habitat depth or phylogenetic age. Rectal glands from the family Somniosidae were kidney bean-shaped (mean width: length +/- SD = 0.46 +/- 0.05); whereas those from families Echinorhinidae and Hexanchidae were lobulate (mean width: length +/- SD = 0.55 +/- 0.06). Rectal gland width: length were different among species with digitiform morphology and lobulate morphology (ANOVA; R-2 = 0.9; df = 15, 386; 401, F = 219.24; P < 0.001). Histological and morphological characteristics of the digitiform morphology from deep-sea sharks were similar to those from shallow-water sharks. Histology of lobulate rectal glands from hexanchids were characterised by tubule bundles separated by smooth muscle around a central lumen. Additionally, we examined plasma chemistry of four species of sharks with digitiform rectal glands and two species with lobulate rectal-gland morphology to see if there were differences between morphologies. Plasma chemistry analysis showed that urea and trimethylamine N-oxide (TMAO) followed the piezolyte hypothesis, with TMAO being highest and urea being lowest in deep-sea sharks. Among electrolytes, Na+ was highest in species with lobulate rectal glands. Hexanchids and echinorhinids both have lobulate rectal glands similar to those of holocephalans, despite the more than 400 million years separating these two groups. The morphological similarities between the lobulate rectal-gland anatomy of primitive sharks and the secretory morphology of holocephalans may represent an intermediate state between Holocephali and derived shark species.

Hagfish defend themselves from fish predators by producing large volumes of gill-clogging slime when they are attacked. The slime consists of seawater and two major components that are ejected from the slime glands: mucus and threads. The threads are produced within specialized cells and packaged into intricately coiled bundles called skeins. Skeins are kept from unraveling via a protein adhesive that dissolves when the skeins are ejected from the slime glands. Previous work revealed that hagfish slime glands have high concentrations of methylamines including trimethylamine N-oxide (TMAO), trimethylglycine (betaine) and dimethylglycine (DMG); however, the function of these compounds in the slime glands is unknown. We hypothesized that methylamines have stabilizing effects on the skeins that prevent premature unraveling in the gland. To test this hypothesis, we quantified the effect of methylamines on skein unraveling in Pacific hagfish and found that TMAO and betaine have inhibitory effects on skein unraveling in vitro. Furthermore, we found that TMAO is a more effective inhibitor of unraveling than betaine, but the presence of TMAO synergistically boosts the inhibitory action of betaine. Glycine and DMG were far less effective inhibitors of unraveling at natural concentrations. Our results support the hypothesis that high levels of trimethylamines in the slime glands may act to hold the coiled thread skeins together within gland thread cells, and they may do so by stabilizing adhesive proteins. These results advance our knowledge of skein stabilization and deployment and provide yet another example of trimethylamines functioning to stabilize proteins in a marine organism.

Anthropogenic use of high density, toxic elements results in marine pollution which is bio-accumulating throughout marine food webs. While there have been several studies in various locations analyzing such elements in fish, few have investigated patterns in these elements and their isotopes in terms of ocean depth, and none have studied the greatest depth zones. We used a flame atomic absorption spectrophotometer-hydride system and an inductively coupled plasma-mass spectrometer to determine concentrations of the high-density elements arsenic (As), cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), lead (Pb), mercury (Hg), nickel (Ni), selenium (Se), plus the light-metal barium (Ba), in fish ranging from bathyal (1000 m in Monterey Bay) to upper hadal zones (6500-7626 m in the Kermadec and Mariana Trenches) in the Pacific Ocean. Five species of fish-including the Mariana Trench snailfish, the world's deepest known fish newly discovered-were analyzed for patterns in total element concentration, depth of occurrence, Se:Hg ratio, plus mercury isotopes in the deepest species. Co and As levels decreased with depth. In the Mariana Trench, Pb, Hg, Cd, and Cu were higher than in all other samples, and higher in those plus Ba than in the Kermadec Trench. The latter samples had far higher Ni and Cr levels than all others. Mercury relative isotope analysis showed no depth trends in the deepest species. Se:Hg showed a large molar excess of Se in bathyal flatfish species. These patterns indicate that exposures to pollutants differ greatly between habitats including trenches of similar depths.

Deep-sea organisms must cope with hydrostatic pressure, up to - 110 MPa (similar to 1100 atm) at 11 km in the Mariana Trench. Pressure perturbs protein conformations, ligand binding, and can promote damaging protein aggregates. Salinity is another stressor: seawater, averaging similar to 1000 mOsm/kg, is dehydrating towards the similar to 250-400 mOsm/kg of universal cellular solutes. Most marine organisms prevent dehydration by becoming isosmotic with cellular organic osmolytes, which, unlike NaCl, do not perturb proteins and can have chemical chaperone properties, stabilizing proteins against various physicochemical stressors. In shallow-dwelling invertebrates, these osmolytes are typically taurine, glycine, and betaine. Previous work on crustaceans down to 3 km found that "shallow" osmolytes decreased with depth, replaced with trimethylamine N-oxide (TMAO) and other apparent chemical chaperones. In bony fishes, TMAO increases with depth down to 8 km, and is probably a piezolyte (pressure counteractant) as it reverses pressure's inhibitory effects. Here we analyzed amphipods over the full ocean depth range of 0-11 km, from coastal habitats and numerous depths in the Kermadec and Mariana Trenches. We found that "shallow" osmolytes decreased while potential piezolytes, in different combinations in different species, increased with depth. These are TMAO, glycerophosphorylcholine and proline betaine (all methylamine chaperones), glycerophosphorylethanolamine, and scyllo-inositol, a polyol chaperone (being tested for Alzheimer's Disease) that works differently than methylamines by preventing damaging protein aggregations. Though only TMAO has been tested for piezolyte properties, these other solutes may explain how some hadal amphipods live deeper than fish, which appear to be limited to a maximum of 8.4 km.

Accumulation of trimethylamine N-oxide (TMAO) by deep-sea animals is proposed to protect proteins against the destabilizing effects of high hydrostatic pressure (the piezolyte hypothesis). Chondrichthyan fishes (sharks, rays, and chimaeras) provide a unique test of this hypothesis because shallow-living species have elevated TMAO levels to counteract the destabilizing effects of high urea levels accumulated for osmoregulation. Limited interspecific studies of chondrichthyans reveal that increasing depth correlates with decreased urea and increased TMAO levels, suggesting a dynamic balance between destabilizing forces on proteins (high urea, hydrostatic pressure) and TMAO to counteract these forces. Indeed, an inability to minimize urea levels or maximize TMAO levels has been proposed to explain why chondrichthyans are absent in the vast abyssal region. An unresolved question is whether the depth-related changes in chondrichthyan osmolytes are a flexible response to depth or whether phylogenetic differences in species-specific physiological set points for osmolytes account for the differences seen with depth. Sampling Arctic skates (Amblyraja hyperborea) across a 1,015-m depth gradient in the Beaufort Sea, we measured organic osmolytes in muscle using spectrophotometry and high-performance liquid chromatography. We found that the urea-to-TMAO ratio decreased linearly with depth, with tighter correlation than that seen in interspecific studies. Minor osmolytes, including betaine, sarcosine, and some α-amino acids, also declined with depth, apparently replaced (as with urea) by TMAO (a stronger piezolyte than those solutes). These data provide the first intraspecific evidence that flexible adjustments of osmolyte combinations are a key response for deep-sea living in individual chondrichthyans, supporting the piezolyte hypothesis.

Betaine (N-trimethylglycine), a common osmolyte, has received attention because of the number of clinical reports associating betaine supplementation with improved cognition, neuroprotection and exercise physiology. However, tissue analyses report little accumulation of betaine in brain tissue despite the presence of betaine/GABA transporters (BGT1) at the blood brain barrier and in nervous tissue, calling into question whether betaine influences neuronal function directly or indirectly. Therefore, the focus of this study was to determine what capacity nervous tissue has to accumulate betaine, specifically in the hippocampus, a region of the brain associated with learning and memory and one that is particularly susceptible to damage (e.g., seizure activity). Here we report that hippocampal slices actively accumulate betaine in a time, dose and osmolality dependent manner, resulting in peak intracellular concentrations four times extracellular concentrations within 8 h. Our data also indicate that betaine uptake differentially influences the accumulation of other osmolytes. Under isosmotic conditions, betaine uptake minimally impacted some osmolytes (e.g., glycerylphosphorylcholine and glutamate) while significantly reducing others (taurine, creatine, and myo-inositol). Under osmotic stress (hyperosmotic) conditions, we observed dramatic changes in osmolytes like glycine and glutamine—key players in inhibitory neurotransmission—and little change in osmolytes such as taurine, creatine and myo-inositol when betaine was available. These data suggest that betaine may influence pathways of inhibitory neurotransmitter production/recycling in addition to serving as an osmolyte and metabolic intermediate. In sum, our data provide detailed characterization of betaine uptake in the hippocampus that implicates betaine in the modulation of hippocampal neurophysiology and neuroprotection.

Depth ranges for representative taxa. Ranges taken from FishBase (Froese & Pauly, 2017).

Metabolic enzyme activities of muscle tissue have been useful and widely-applied indicators of whole animal metabolic capacity, particularly in inaccessible systems such as the deep sea. Previous studies have been conducted at atmospheric pressure, regardless of organism habitat depth. However, maximum reaction rates of some of these enzymes are pressure dependent, complicating the use of metabolic enzyme activities as proxies of metabolic rates. Here, we show pressure-related rate changes in lactate and malate dehydrogenase (LDH, MDH) and pyruvate kinase (PK) in six fish species (2 hadal, 2 abyssal, 2 shallow). LDH maximal reaction rates decreased with pressure for the two shallow species, but, in contrast to previous findings, it increased for the four deep species, suggesting evolutionary changes in LDH reaction volumes. MDH maximal reaction rates increased with pressure in all species (up to 51±10% at 60MPa), including the tide pool snailfish, Liparis florae (activity increase at 60MPa 44±9%), suggesting an inherent negative volume change of the reaction. PK was inhibited by pressure in all species tested, including the hadal liparids (up to 34±3% at 60MPa), suggesting a positive volume change during the reaction. The addition of 400mM TMAO counteracted this inhibition at both 0.5 and 2.0mM ADP concentrations for the hadal liparid, Notoliparis kermadecensis. We revisit depth-related trends in metabolic enzyme activities according to these pressure-related rate changes and new data from seven abyssal and hadal species from the Kermadec and Mariana trenches. Results show that, with abyssal and hadal species, pressure-related rate changes are another variable to be considered in the use of enzyme activities as proxies for metabolic rate, in addition to factors such as temperature and body mass. Intraspecific increases in tricarboxylic acid cycle enzymes with depth of capture, independent of body mass, in two hadal snailfishes suggest improved nutritional condition for individuals deeper in the hadal zone, likely related to food availability. These new data inform the discussion of factors controlling metabolism in the deep sea, including the visual interactions hypothesis and extend published trends to the planet's deepest-living fishes.

Many deep-sea fishes have a gelatinous layer, or subdermal extracellular matrix, below the skin or around the spine. We document the distribution of gelatinous tissues across fish families (approx. 200 species in ten orders), then review and investigate their composition and function. Gelatinous tissues from nine species were analysed for water content (96.53 ± 1.78% s.d.), ionic composition, osmolality, protein (0.39 ± 0.23%), lipid (0.69 ± 0.56%) and carbohydrate (0.61 ± 0.28%). Results suggest that gelatinous tissues are mostly extracellular fluid, which may allow animals to grow inexpensively. Further, almost all gelatinous tissues floated in cold seawater, thus their lower density than seawater may contribute to buoyancy in some species. We also propose a new hypothesis: gelatinous tissues, which are inexpensive to grow, may sometimes be a method to increase swimming efficiency by fairing the transition from trunk to tail. Such a layer is particularly prominent in hadal snailfishes (Liparidae); therefore, a robotic snailfish model was designed and constructed to analyse the influence of gelatinous tissues on locomotory performance. The model swam faster with a watery layer, representing gelatinous tissue, around the tail than without. Results suggest that the tissues may, in addition to providing buoyancy and low-cost growth, aid deep-sea fish locomotion.

ABSTRACT The ability of animals to cope with environmental stress depends – in part – on past experience, yet knowledge of the factors influencing an individual's physiology in nature remains underdeveloped. We used an individual monitoring system to record body temperature and valve gaping behavior of rocky intertidal zone mussels (Mytilus californianus). Thirty individuals were selected from two mussel beds (wave-exposed and wave-protected) that differ in thermal regime. Instrumented mussels were deployed at two intertidal heights (near the lower and upper edges of the mussel zone) and in a continuously submerged tidepool. Following a 23-day monitoring period, measures of oxidative damage to DNA and lipids, antioxidant capacities (catalase activity and peroxyl radical scavenging) and tissue contents of organic osmolytes were obtained from gill tissue of each individual. Univariate and multivariate analyses indicated that inter-individual variation in cumulative thermal stress is a predominant driver of physiological variation. Thermal history over the outplant period was positively correlated with oxidative DNA damage. Thermal history was also positively correlated with tissue contents of taurine, a thermoprotectant osmolyte, and with activity of the antioxidant enzyme catalase. Origin site differences, possibly indicative of developmental plasticity, were only significant for catalase activity. Gaping behavior was positively correlated with tissue contents of two osmolytes. Overall, these results are some of the first to clearly demonstrate relationships between inter-individual variation in recent experience in the field and inter-individual physiological variation, in this case within mussel beds. Such micro-scale, environmentally mediated physiological differences should be considered in attempts to forecast biological responses to a changing environment.

Background: Post-Pleistocene diversification of threespine stickleback in fresh water offers a valuable opportunity to study how changes in environmental salinity shape physiological evolution in fish. In Alaska, the presence of both ancestral oceanic populations and derived landlocked populations, including recent lake introductions, allows us to examine rates and direction of evolution of osmoregulation following halohabitat transition.Hypotheses: Strong selection for enhanced freshwater tolerance will improve survival of recently lake-introduced stickleback in ion-poor conditions compared with their oceanic ancestors. Trade-offs between osmoregulation in fresh water and seawater will allow members of the ancestral population to survive better in response to seawater challenge, as mediated by upregulating salt-secreting transporters in the gill. Poorer hypo-osmoregulatory performance of derived fish will be marked by higher levels of taurine and other organic osmolytes.Methods: We reared clutches at a common salinity from an anadromous and a descendant population, Scout Lake, which has been landlocked for only two generations. We challenged 6-week-old juveniles with extreme low and high salinity treatments and sampled fish over 10 days to investigate putative molecular mechanisms underlying differences in halotolerance. We measured whole-body organic osmolyte content as well as gill Na+-/K+-ATPase (NKA) activity and Na+/K+/2Cl(-) cotransporter (NKCC) protein abundance. Other juveniles from these populations and also from Cheney Lake, a fourth-generation landlocked descendant, were gradually salt-acclimated to determine maximum halotolerance limits.Results: Scout Lake stickleback exhibited 67% higher survival in fresh water than the ancestral anadromous population, but individuals from both groups exhibited similar seawater tolerance. Likewise, the gradual salinity threshold for each population was equivalent (71 ppt). Gill NKA activity and NKCC abundance were both higher in seawater-challenged fish, but did not differ between populations. Sticklebacks from both populations responded to acute salinity stress by transiently increasing osmolyte levels in seawater and decreasing them in fresh water.Conclusion: Enhanced freshwater tolerance has evolved rapidly in recently landlocked stickleback compared with their anadromous ancestors (0.569 haldanes), but the former have retained ancestral seawater-osmoregulatory function.

Observations and records for fish exceeding 6000m deep are few and often spurious. Recent developments in accessing and sampling the hadal zone 6000–11,000m) have led to an acceleration in new findings in the deep subduction trenches, particularly in the Pacific Ocean. This study describes the discovery of two new species of snailfish (Liparidae) from the Mariana Trench; the ‘Mariana snailfish’ (6198–8076m) and the ‘Ethereal snailfish’ (7939–8145m). These new findings represent respectively the deepest known specimen caught with corroborating depth data, and the deepest fish seen alive. Further specimens and observations of the Kermadec Trench snailfish, Notoliparis kermadecensis, are also presented, as well as the first hadal records of Synaphobranchidae and Zoarcidae (6068 and 6145m respectively) and a depth extension for the Macrouridae (maximum depth now 7012m). Details of these new snailfish specimens caught by baited trap and behaviour observations filmed by baited cameras are presented. An updated assessment of fishes from hadal depths is also reported.