Unique osmoregulatory morphology in primitive sharks: an intermediate state between holocephalan and derived shark secretory morphology.

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; R2 = 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.

Betaine in the Brain: Characterization of Betaine Uptake, its Influence on Other Osmolytes and its Potential Role in Neuroprotection from Osmotic Stress.

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.

Thermal history and gape of individual Mytilus californianus correlate with oxidative damage and thermoprotective osmolytes.

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.

Marine fish may be biochemically constrained from inhabiting the deepest ocean depths.

No fish have been found in the deepest 25% of the ocean (8,400-11,000 m). This apparent absence has been attributed to hydrostatic pressure, although direct evidence is wanting because of the lack of deepest-living species to study. The common osmolyte trimethylamine N-oxide (TMAO) stabilizes proteins against pressure and increases with depth, going from 40 to 261 mmol/kg in teleost fishes from 0 to 4,850 m. TMAO accumulation with depth results in increasing internal osmolality (typically 350 mOsmol/kg in shallow species compared with seawater's 1,100 mOsmol/kg). Preliminary extrapolation of osmolalities of predicted isosmotic state at 8,000-8,500 m may indicate a possible physiological limit, as greater depths would require reversal of osmotic gradients and, thus, osmoregulatory systems. We tested this prediction by capturing five of the second-deepest known fish, the hadal snailfish (Notoliparis kermadecensis; Liparidae), from 7,000 m in the Kermadec Trench. We found their muscles to have a TMAO content of 386 ± 18 mmol/kg and osmolality of 991 ± 22 mOsmol/kg. These data fit previous extrapolations and, combined with new osmolalities from bathyal and abyssal fishes, predict isosmotic state at 8,200 m. This is previously unidentified evidence that biochemistry could constrain the depth of a large, complex taxonomic group.

Co-evolution of proteins and solutions: protein adaptation versus cytoprotective micromolecules and their roles in marine organisms.

Organisms experience a wide range of environmental factors such as temperature, salinity and hydrostatic pressure, which pose challenges to biochemical processes. Studies on adaptations to such factors have largely focused on macromolecules, especially intrinsic adaptations in protein structure and function. However, micromolecular cosolutes can act as cytoprotectants in the cellular milieu to affect biochemical function and they are now recognized as important extrinsic adaptations. These solutes, both inorganic and organic, have been best characterized as osmolytes, which accumulate to reduce osmotic water loss. Singly, and in combination, many cosolutes have properties beyond simple osmotic effects, e.g. altering the stability and function of proteins in the face of numerous stressors. A key example is the marine osmolyte trimethylamine oxide (TMAO), which appears to enhance water structure and is excluded from peptide backbones, favoring protein folding and stability and counteracting destabilizers like urea and temperature. Co-evolution of intrinsic and extrinsic adaptations is illustrated with high hydrostatic pressure in deep-living organisms. Cytosolic and membrane proteins and G-protein-coupled signal transduction in fishes under pressure show inhibited function and stability, while revealing a number of intrinsic adaptations in deep species. Yet, intrinsic adaptations are often incomplete, and those fishes accumulate TMAO linearly with depth, suggesting a role for TMAO as an extrinsic 'piezolyte' or pressure cosolute. Indeed, TMAO is able to counteract the inhibitory effects of pressure on the stability and function of many proteins. Other cosolutes are cytoprotective in other ways, such as via antioxidation. Such observations highlight the importance of considering the cellular milieu in biochemical and cellular adaptation.

Long-term health outcomes associated with hydration status.

Body water balance is determined by fundamental homeostatic mechanisms that maintain stable volume, osmolality and the composition of extracellular and intracellular fluids. Water balance is maintained by multiple mechanisms that continuously match water losses through urine, the skin, the gastrointestinal tract and respiration with water gains achieved through drinking, eating and metabolic water production. Hydration status is determined by the state of the water balance. Underhydration occurs when a decrease in body water availability, due to high losses or low gains, stimulates adaptive responses within the water balance network that are aimed at decreasing losses and increasing gains. This stimulation is also accompanied by cardiovascular adjustments. Epidemiological and experimental studies have linked markers of low fluid intake and underhydration - such as increased plasma concentration of vasopressin and sodium, as well as elevated urine osmolality - with an increased risk of new-onset chronic diseases, accelerated aging and premature mortality, suggesting that persistent activation of adaptive responses may be detrimental to long-term health outcomes. The causative nature of these associations is currently being tested in interventional trials. Understanding of the physiological responses to underhydration may help to identify possible mechanisms that underlie potential adverse, long-term effects of underhydration and inform future research to develop preventative and treatment approaches to the optimization of hydration status.

Seawater acclimation and inositol monophosphatase isoform expression in the European eel (Anguilla anguilla) and Nile tilapia (Orechromis niloticus).

Inositol monophosphatase (IMPA) is responsible for the synthesis of inositol, a polyol that can function as an intracellular osmolyte helping re-establish cell volume when exposed to hypertonic environments. Some epithelial tissues in euryhaline teleosts such as the eel and tilapia encounter considerable hyperosmotic challenge when fish move from freshwater (FW) to seawater (SW) environments; however, the roles played by organic osmolytes, such as inositol, have yet to be determined. Syntenic analysis has indicated that, as a result of whole genome- and tandem-duplication events, up to six IMPA isoforms can exist within teleost genomes. Four isoforms are homologs of the mammalian IMPA1 gene, and two isoforms are homologs of the mammalian IMPA2 gene. Although the tissue-dependent isoform expression profiles of the teleost isoforms appear to be species-specific, it was primarily mRNA for the IMPA1.1 isoform that was upregulated in epithelial tissues after fish were transferred to SW (up to 16-fold in eel and 90-fold in tilapia). Although up-regulation of IMPA1.1 expression was evident in many tissues in the eel, more substantial increases in IMPA1.1 expression were found in tilapia tissues, where SW acclimation resulted in up to 2,000-fold increases in protein expression, 16-fold increases in enzyme activity and 15-fold increases in tissue inositol contents. Immunohistochemical studies indicated that the tissue and cellular distribution of IMPA1.1 protein differed slightly between eels and tilapia; however, in both species the basal epithelial cell layers within the skin and fin, and the branchial epithelium and interstitial cells within the kidney, exhibited high levels of IMPA1.1 protein expression.

Osmoregulation of ceroid neuronal lipofuscinosis type 3 in the renal medulla.

Recessive inheritance of mutations in ceroid neuronal lipofuscinosis type 3 (CLN3) results in juvenile neuronal ceroid lipofuscinosis (JNCL), a childhood neurodegenerative disease with symptoms including loss of vision, seizures, and motor and mental decline. CLN3p is a transmembrane protein with undefined function. Using a Cln3 reporter mouse harboring a nuclear-localized bacterial beta-galactosidase (beta-Gal) gene driven by the native Cln3 promoter, we detected beta-Gal most prominently in epithelial cells of skin, colon, lung, and kidney. In the kidney, beta-Gal-positive nuclei were predominant in medullary collecting duct principal cells, with increased expression along the medullary osmotic gradient. Quantification of Cln3 transcript levels from kidneys of wild-type (Cln3(+/+)) mice corroborated this expression gradient. Reporter mouse-derived renal epithelial cultures demonstrated a tonicity-dependent increase in beta-Gal expression. RT-quantitative PCR determination of Cln3 transcript levels further supported osmoregulation at the Cln3 locus. In vivo, osmoresponsiveness of Cln3 was demonstrated by reduction of medullary Cln3 transcript abundance after furosemide administration. Primary cultures of epithelial cells of the inner medulla from Cln3(lacZ/lacZ) (CLN3p-null) mice showed no defect in osmolyte accumulation or taurine flux, arguing against a requirement for CLN3p in osmolyte import or synthesis. CLN3p-deficient mice with free access to water showed a mild urine-concentrating defect but, upon water deprivation, were able to concentrate their urine normally. Unexpectedly, we found that CLN3p-deficient mice were hyperkalemic and had a low fractional excretion of K(+). Together, these findings suggest an osmoregulated role for CLN3p in renal control of water and K(+) balance.

Cellular responses in marine animals to hydrostatic pressure.

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.

Pressure tolerance of deep-sea enzymes can be evolved through increasing volume changes in protein transitions: a study with lactate dehydrogenases from abyssal and hadal fishes.

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 ( ΔVPP∘ ). 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 ΔVPP∘ 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.

High-density element concentrations in fish from subtidal to hadal zones of the Pacific Ocean.

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.

Osmolyte Adjustments as a Pressure Adaptation in Deep-Sea Chondrichthyan Fishes: An Intraspecific Test in Arctic Skates (Amblyraja hyperborea) along a Depth Gradient.

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.

Correlation of trimethylamine oxide and habitat depth within and among species of teleost fish: an analysis of causation.

Most shallow-water teleosts have moderate levels of trimethylamine N-oxide (TMAO; approximately 50 mmol/kg wet mass), a common osmolyte in many other marine animals. Recently, muscle TMAO contents were found to increase linearly with depth in six families. In one hypothesis, this may be an adaptation to counteract the deleterious effects of pressure on protein function, which TMAO does in vitro. In another hypothesis, TMAO may be accumulated as a by-product of acylglycerol (AG) production, increasing with depth because of elevated lipid metabolisms known to occur in some deep-sea animals. Here we analyze muscle TMAO contents and total body AG (mainly triacyglycerol [TAG]) levels in 15 species of teleosts from a greater variety of depths than sampled previously, including eight individual species caught at two or more depths. Including data of previous studies (total of 17 species, nine families), there is an apparent sigmoidal increase in TMAO contents between 0- and 1.4-km depths, from about 40 to 150 mmol/kg. From 1.4 to 4.8 km, the increase appears to be linear (r2=0.91), rising to 261 mmol/kg at 4.8 km. The trend also occurred within species: in most cases in which a species was caught at two or more depths, TMAO was higher in the deeper-caught specimens (e.g., for Coryphaenoides armatus, TMAO was 173, 229, and 261 mmol/kg at 1.8, 4.1, and 4.8 km, respectively). TMAO contents not only were consistent within species at a given depth but also did not vary with season or over a wide range of body masses or TAG contents. Thus, no clear link between TMAO and lipid was found. However, TMAO contents might correlate with the rate (rather than content) of TAG production, which could not be quantified. Overall, the data strongly support the hypothesis that TMAO is adaptively regulated with depth in deep-sea teleosts. Whether lipid metabolism is the source of that TMAO is a question that remains to be tested fully.

Distribution, composition and functions of gelatinous tissues in deep-sea 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.

The little skate Raja erinacea exhibits an extrahepatic ornithine urea cycle in the muscle and modulates nitrogen metabolism during low-salinity challenge.

Urea synthesis via the hepatic ornithine urea cycle (OUC) has been well described in elasmobranchs, but it is unknown whether OUC enzymes are also present in extrahepatic tissues. Muscle and liver urea, trimethylamine oxide (TMAO), and other organic osmolytes, as well as selected OUC enzymes (carbamoyl phosphate synthetase III, ornithine transcarbamoylase, arginase, and the accessory enzyme glutamine synthetase), were measured in adult little skates (Raja erinacea) exposed to 100% or 75% seawater for 5 d. Activities of all four OUC enzymes were detected in the muscle. There were no changes in muscle OUC activities in skates exposed to 75% seawater; however, arginase activity was significantly lower in the liver, compared to controls. Urea, TMAO, and several other osmolytes were significantly lower in the muscle of little skates exposed to 75% seawater, whereas only glycerophosphorylcholine was significantly lower in the liver. Urea excretion rates were twofold higher in skates exposed to 75% seawater. Taken together, these data suggest that a functional OUC may be present in the skeletal muscle tissues of R. erinacea. As well, enhanced urea excretion rates and the downregulation of the anchor OUC enzyme, arginase, in the liver may be critical in regulating tissue urea content under dilute-seawater stress.

Trehalose is a chemical attractant in the establishment of coral symbiosis.

Coral reefs have evolved with a crucial symbiosis between photosynthetic dinoflagellates (genus Symbiodinium) and their cnidarian hosts (Scleractinians). Most coral larvae take up Symbiodinium from their environment; however, the earliest steps in this process have been elusive. Here we demonstrate that the disaccharide trehalose may be an important signal from the symbiont to potential larval hosts. Symbiodinium freshly isolated from Fungia scutaria corals constantly released trehalose (but not sucrose, maltose or glucose) into seawater, and released glycerol only in the presence of coral tissue. Spawning Fungia adults increased symbiont number in their immediate area by excreting pellets of Symbiodinium, and when these naturally discharged Symbiodinium were cultured, they also released trehalose. In Y-maze experiments, coral larvae demonstrated chemoattractant and feeding behaviors only towards a chamber with trehalose or glycerol. Concomitantly, coral larvae and adult tissue, but not symbionts, had significant trehalase enzymatic activities, suggesting the capacity to utilize trehalose. Trehalase activity was developmentally regulated in F. scutaria larvae, rising as the time for symbiont uptake occurs. Consistent with the enzymatic assays, gene finding demonstrated the presence of a trehalase enzyme in the genome of a related coral, Acropora digitifera, and a likely trehalase in the transcriptome of F. scutaria. Taken together, these data suggest that adult F. scutaria seed the reef with Symbiodinium during spawning and the exuded Symbiodinium release trehalose into the environment, which acts as a chemoattractant for F. scutaria larvae and as an initiator of feeding behavior- the first stages toward establishing the coral-Symbiodinium relationship. Because trehalose is a fixed carbon compound, this cue would accurately demonstrate to the cnidarian larvae the photosynthetic ability of the potential symbiont in the ambient environment. To our knowledge, this is the first report of a chemical cue attracting the motile coral larvae to the symbiont.

Compatible and counteracting solutes: protecting cells from the Dead Sea to the deep sea.

Cells of many organisms accumulate certain small organic molecules--called compatible and counteracting solutes, compensatory solutes, or chemical chaperones--in response to certain physical stresses. These solutes include certain carbohydrates, amino acids, methylamine and methylsulphonium zwitterions, and urea. In osmotic dehydrating stress, these solutes serve as cellular osmolytes. Unlike common salt ions and urea (which inhibit proteins), some organic osmolytes are compatible; i.e., they do not perturb macromolecules such as proteins. In addition, some may protect cells through metabolic processes such as antioxidation reactions and sulphide detoxification. Other osmolytes, and identical or similar solutes accumulated in anhydrobiotic, heat and pressure stresses, are termed counteracting solutes or chemical chaperones because they stabilise proteins and counteract protein-destabilising factors such as urea, temperature, salt, and hydrostatic pressure. Stabilisation of proteins, not necessarily beneficial in the absence of a perturbant, may result indirectly from effects on water structure. Osmotic shrinkage of cells activates genes for chaperone proteins and osmolytes by mechanisms still being elucidated. These solutes have applications in agriculture, medicine and biotechnology.

Decreasing urea∶trimethylamine N-oxide ratios with depth in chondrichthyes: a physiological depth limit?

In marine osmoconformers, cells use organic osmolytes to maintain osmotic balance with seawater. High levels of urea are utilized in chondrichthyans (sharks, rays, skates, and chimaeras) for this purpose. Because of urea's perturbing nature, cells also accumulate counteracting methylamines, such as trimethylamine N-oxide (TMAO), at about a 2∶1 urea∶methylamine ratio, the most thermodynamically favorable mixture for protein stabilization, in shallow species. However, previous work on deep-sea teleosts (15 species) and chondrichthyans (three species) found an increase in muscle TMAO content and a decrease in urea content in chondrichthyans with depth. We hypothesized that TMAO counteracts protein destabilization resulting from hydrostatic pressure, as is demonstrated in vitro. Chondrichthyans are almost absent below 3,000 m, and we hypothesized that a limitation in urea excretion and/or TMAO retention might play a role. To test this, we measured the content of major organic osmolytes in white muscle of 13 chondrichthyan species caught with along-contour trawls at depths of 50-3,000 m; the deepest species caught was from 2,165 m. Urea and TMAO contents changed significantly with depth, with urea∶TMAO declining from 2.96 in the shallowest (50-90 m) groups to 0.67 in the deepest (1,911-2,165 m) groups. Urea content was 291-371 mmol/kg in the shallowest group and 170-189 mmol/kg in the deepest group, declining linearly with depth and showing no plateau. TMAO content was 85-168 mmol/kg in the shallowest group and 250-289 mmol/kg in the deepest groups. With data from a previous study for a skate at 2,850 m included, a second-order polynomial fit suggested a plateau at the greatest depths. When data for skates (Rajidae) were analyzed separately, a sigmoidal fit was suggested. Thus, the deepest chondrichthyans may be unable to accumulate sufficient TMAO to counteract pressure; however, deeper-living specimens are needed to fully test this hypothesis.

Analysis of internal osmolality in developing coral larvae, Fungia scutaria.

Coral species throughout the world are facing severe local and global environmental pressures. Because of the pressing conservation need, we are studying the reproduction, physiology, and cryobiology of coral larvae with the future goal of cryopreserving and maintaining these organisms in a genome resource bank. Effective cryopreservation involves several steps, including the loading and unloading of cells with cryoprotectant and the avoidance of osmotic shock. In this study, during the time course of coral larvae development of the mushroom coral Fungia scutaria, we examined several physiologic factors, including internal osmolality, percent osmotically active water, formation of mucus cells, and intracellular organic osmolytes. The osmotically inactive components of the cell, V(b), declined 33% during development from the oocyte to day 5. In contrast, measurements of the internal osmolality of coral larvae indicated that the internal osmolality was increasing from day 1 to day 5, probably as a result of the development of mucus cells that bind ions. Because of this, we conclude that coral larvae are osmoconformers with an internal osmolality of about 1,000 mOsm. Glycine betaine, comprising more than 90% of the organic osmolytes, was found to be the major organic osmolyte in the larvae. Glycerol was found in only small quantities in larvae that had been infected with zooxanthellae, suggesting that this solute did not play a significant role in the osmotic balance of this larval coral. We were interested in changes in cellular characteristics and osmolytes that might suggest solutes to test as cryoprotectants in order to assist in the successful cryopreservation of the larvae. More importantly, these data begin to reveal the basic physiological events that underlie the move from autonomous living to symbiosis.

Betaines and dimethylsulfoniopropionate as major osmolytes in cnidaria with endosymbiotic dinoflagellates.

Most marine invertebrates and algae are osmoconformers whose cells accumulate organic osmolytes that provide half or more of cellular osmotic pressure. These solutes are primarily free amino acids and glycine betaine in most invertebrates and small carbohydrates and dimethylsulfoniopropionate (DMSP) in many algae. Corals with endosymbiotic dinoflagellates (Symbiodinium spp.) have been reported to obtain from the symbionts potential organic osmolytes such as glycerol, amino acids, and DMSP. However, corals and their endosymbionts have not been fully analyzed for osmolytes. We quantified small carbohydrates, free amino acids, methylamines, and DMSP in tissues of the corals Fungia scutaria, Pocillopora damicornis, Pocillopora meandrina, Montipora capitata, Porites compressa, and Porites lobata (all with symbionts) plus Tubastrea aurea (asymbiotic) from Kaneohe Bay, Oahu (Hawaii). Glycine betaine, at 33-69 mmol/kg wet mass, was found to constitute 90% or more of the measured organic solutes in all except the Porites species. Those were dominated by proline betaine and dimethyltaurine. DMSP was found at 0.5-3 mmol/kg in all species with endosymbionts. Freshly isolated Symbiodinium from Fungia, P. damicornis, and P. compressa were also analyzed. DMSP and glycine betaine dominated in the first two; Porites endosymbionts had DMSP, proline betaine, and dimethyltaurine. In all specimens, glycerol and glucose were detected by high-performance liquid chromatography only at 0-1 mmol/kg wet mass. An enzymatic assay for glycerol plus glycerol 3-phosphate and dihydroxyacetone phosphate yielded 1-10 mmol/kg. Cassiopeia andromeda (upside-down jelly; Scyphozoan) and Aiptasia puchella (solitary anemone; Anthozoan) were also analyzed; both have endosymbiotic dinoflagellates. In both, glycine betaine, taurine, and DMSP were the dominant osmolytes. In summary, methylated osmolytes dominate in many Cnidaria; in those with algal symbionts, host and symbiont have similar methylated amino acids, as do congeners. However, little glycerol was present as an osmolyte and was probably metabolized before it could accumulate.