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Studies of tardigrade biology have been severely limited by the sparsity of appropriate quantitative techniques, informative on a single-organism level. Therefore, many studies rely on motility-based survival scoring and quantifying reproductive success. Measurements of O2 respiration rates, as an integrating expression of the metabolic activity of single tardigrades, would provide a more comprehensive insight into how an individual tardigrade is responding to specific environmental factors or changes in life stages. Here we present and validate a new method for determining the O2 respiration rate (nmol O2 mg−1 hour−1) of single tardigrades under steady state, using O2-microsensors. As an example, we show that the O2 respiration rate determined in MilliQ water for individuals of Richtersius coronifer and of Macrobiotus macrocalix at 22 °C was 10.8±1.8 nmol O2 mg−1 hour−1 and 13.1±2.3 nmol O2 mg−1 hour−1, respectively.

The equilibrium heat stability and the kinetic heat tolerance of a recombinant antifreeze protein (AFP) from the beetle Rhagium mordax (RmAFP1) are studied through differential scanning calorimetry and circular dichroism spectroscopy. In contrast to other insect AFPs studied with this respect, the RmAFP1 has only one disulfide bridge. The melting temperature, Tm, of the protein is determined to be 28.5°C (pH 7.4), which is much lower than most of those reported for AFPs or globular proteins in general. Despite its low melting temperature, both biophysical and activity measurements show that the protein almost completely refolds into the native state after repeated exposure of 70°C. RmAFP1 thus appears to be kinetically stable even far above its melting temperature. Thermodynamically, the insect AFPs seem to be dividable in three groups, relating to their content of disulfide bridges and widths of the ice binding motifs; high melting temperature AFPs (high disulfide content, TxT motifs), low melting temperature but high refolding capability AFPs (one disulfide bridge, TxTxTxT motifs) and irreversibly unfolded AFPs at low temperatures (no disulfide bridges, TxTxTxTxT motifs). The property of being able to cope with high temperature exposures may appear peculiar for proteins which strictly have their effect at subzero temperatures. Different aspects of this are discussed.

The study investigated whether evaporative water loss (EWL) in frogs stems from water diffusing through the skin or fluid secreted by mucous glands. Osmolality of cutaneous surface fluid (CSF) of Rana esculenta (Pelophylax kl. esculentus) subjected to isoproterenol or 30°C-34°C was 191±9.3 and 181±7.5 mosm/kg, respectively, as compared to lymph osmolality of, 249±10 mosm/kg. Cation concentrations of CSF were likewise independent of pre-treatment with averages of, [Na(+)]=65.5±5.1 and [K(+)]=14.9±1.6 mmol/L, and lymph concentrations of 116 mmol Na(+)/L and 5.1 mmol K(+)/L. The relatively high [K(+)] confirms that CSF is produced by submucosal glands. Since the chemical energy of water of CSF was always higher than that of body fluids, diffusion of water would be from CSF to the interstitial fluid and not in the opposite direction. It is concluded that volume and composition of CSF are regulated by subepidermal exocrine gland secretion balanced by EWL into the atmosphere and ion reuptake by the epidermal epithelium. Previously discovered regulatory mechanisms of epithelial ion absorption, hitherto not ascribed a body function, fit well with a role in regulating turnover of CSF. As a regulated external physiological compartment, CSF would be of importance for the immune defenses that amphibians employ in protecting their skin.

Carassius carassius responds to hypoxic conditions by conversion of lactate into ethanol, which is excreted over the gills. However, a closely related species, Cyprinus carpio, does not possess the ability to produce ethanol and would be expected to accumulate lactate during hypoxic exposure. While the increase in oxygen consumption in fish required following strenuous exercise or low environmental oxygen availability has been frequently considered, the primary contributing mechanism remains unknown. This study utilized the close relationship but strongly divergent physiology between C. carpio and C. carassius to examine the possible correlation between excess post-hypoxic oxygen consumption (EPHOC) and lactate accumulation. No difference in the EPHOC:O2 deficit ratio was observed between the two species after 2.5 h anoxia, with ratios of 2.0 ± 0.6 (C. carpio) and 1.3 ± 0.3 (C. carassius). As predicted, lactate accumulation dynamics did significantly differ between the species in both plasma and white muscle following anoxic exposure. Significant lactate accumulation was seen in both plasma and muscle in C. carpio, but there was no accumulation of lactate in white muscle tissue of C. carassius. These findings indicate that lactate accumulated as a consequence of 2.5 h anoxic exposure is not a major determinant of the resulting EPHOC

Larvae of the blackspotted pliers support beetle, Rhagium mordax, were collected monthly, for the duration of 2012 and fixed. The larvae were embedded in paraffin wax and sectioned. Using fluorophore-coupled antibodies specific to the R. mordax antifreeze protein, RmAFP1, sections were visualised with UV reflected light microscopy. An automated software analysis method was developed in order to discard autofluorescence, and quantify fluorescence from bound antibodies. The results show that R. mordax cuticle and gut exhibit a higher degree of fluorophore-bound fluorescence during summer, than in the cold months. It is hypothesised that R. mordax stores RmAFP1 in, or near, the fat body during times when freeze avoidance is not needed.

SUMMARY Tardigrades exhibit a remarkable resilience against environmental extremes. In the present study, we investigate mechanisms of survival and physiological adaptations associated with sub-zero temperatures and severe osmotic stress in two commonly found cyclomorphic stages of the marine eutardigrade Halobiotus crispae. Our results show that only animals in the so-called pseudosimplex 1 stage are freeze tolerant. In pseudosimplex 1, as well as active-stage animals kept at a salinity of 20 ppt, ice formation proceeds rapidly at a crystallization temperature of around –20°C,revealing extensive supercooling in both stages, while excluding the presence of physiologically relevant ice-nucleating agents. Experiments on osmotic stress tolerance show that the active stage tolerates the largest range of salinities. Changes in body volume and hemolymph osmolality of active-stage specimens (350–500 μm) were measured following salinity transfers from 20 ppt. Hemolymph osmolality at 20 ppt was approximately 950 mOsm kg–1. Exposure to hypo-osmotic stress in 2 and 10 ppt caused(1) rapid swelling followed by a regulatory volume decrease, with body volume reaching control levels after 48 h and (2) decrease in hemolymph osmolality followed by a stabilization at significantly lower osmolalities. Exposure to hyperosmotic stress in 40 ppt caused (1) rapid volume reduction, followed by a regulatory increase, but with a new steady-state after 24 h below control values and (2) significant increase in hemolymph osmolality. At any investigated external salinity, active-stage H. crispaehyper-regulate, indicating a high water turnover and excretion of dilute urine. This is likely a general feature of eutardigrades.

We investigated the effect of compatible and non-compatible osmolytes in combination with macromolecular crowding on the kinetics of yeast hexokinase. This was motivated by the fact that almost all studies concerning the osmolyte effects on enzyme activity have been performed in diluted buffer systems, which are far from the physiological conditions within cells, where the cytosol contains several hundred mg protein ml − 1 . Four organic (glycerol, betaine, TMAO and urea) and one inorganic (NaCl) osmolyte were tested. It was concluded that the effect of compatible osmolytes (glycerol, betaine and TMAO) on V max and K M was practically equivalent in pure buffer and in 200–250 mg BSA ml − 1 supporting the view that these small organic osmolytes do minimal perturbance on enzyme function in physiological solutions. The effect of urea on enzyme kinetics was not independent of protein concentration, since the presence of 250 mg BSA ml − 1 partly compensated the perturbing effect of urea. Even though the organic osmolytes glycerol, betaine and TMAO are generally considered compatible with enzyme function, especially glycerol did have a significant effect on hexokinase kinetics, decreasing both k cat , K M and k cat / K M . The osmolytes decreased k cat / K M in the order: NaCl > Urea > TMAO/glycerol > betaine. For the organic osmolytes this order correlates with the degree of exclusion from protein–water interfaces. Thus, the stronger the exclusion the weaker the perturbing effects on k cat / K M .

In response to drought bdelloid and monogonont rotifers undergo anhydrobiosis and are assumed to synthesize protective chemicals, which are commonly sugars. In contrast to most anhydrobionts, bdelloids have earlier been shown to lack trehalose as protective chemical, and more importantly to lack trehalose synthase (tps) genes. It remains to be assessed if the absence of trehalose is a characteristic common to the entire taxon Rotifera, or if it is limited to bdelloids, or is peculiar to the two bdelloid species investigated so far. In this study, anhydrobiotic adults of a bdelloid species (Macrotrachela quadricornifera) and resting eggs of a monogonont species (Brachionus plicatilis) were analysed by thin layer chromatography and gas chromatography to detect the presence of trehalose. No trehalose was detected in the bdelloid, while the anhydrobiotic resting egg of the monogonont rotifer contained about 0.35% trehalose of its dry weight. Although very little, the presence of trehalose in B. plicatilis suggests that the trehalose synthase genes, absent in bdelloid rotifers, are present in non-bdelloid rotifers.

The ice binding motifs of insect antifreeze proteins (AFPs) mainly consist of repetitive TxT motifs aligned on a flat face of the protein. However, these motifs often contain non-threonines that disrupt the TxT pattern. We substituted two such disruptive amino acids located in the ice binding face of an AFP from Rhagium mordax with threonine. Furthermore, a mutant with an extra ice facing TxT motif was constructed. These mutants showed enhanced antifreeze activity compared to the wild type at low concentrations. However, extrapolating the data indicates that the wild type will become the most active at concentrations above 270μmol.

Differential scanning calorimetry (DSC) was used to determine the thermal events associated with freezing and melting of the cysts of the potato-cyst nematode Globodera rostochiensis. There were no thermal events during the cooling of dry cysts from 5 to –60 °C and warming back to 5 °C, indicating the absence of water freezing in the dry cysts. During heating of dry cysts from 5 to 80 °C, two overlapping endothermic events were observed at 55 °C, indicating the irreversible destruction of the permeability barrier of the eggshell by the melting of the lipids which constitute the lipid layer. The first exothermic event (T=–9 °C) during the cooling of hydrated cysts indicates the presence of an ice-nucleating agent. A broad exotherm at –38 °C is due to the freezing of eggs. DSC thus confirms that the eggshell prevents exogenous ice nucleation and allows the eggs to supercool in the presence of external ice. The enthalpy of the egg exotherm increased during hydration of the cysts. The temperature of the egg exotherm was elevated after heating of the sample to 70 °C. This is thought to be due to the loss of trehalose from the eggs following the destruction of the permeability barrier of the eggshell. During melting, three endothermic events were observed. These became merged after the destruction of the permeability barrier of the eggshell by heating, and only two peaks were observed in isolated eggs. The sample is thus considered to consist of three freezable compartments: (1) the water surrounding the cyst, (2) the solution between the cyst wall and the eggs and (3) the egg contents.

We report measurements of the supercooling point of the hemolymph of the freeze-tolerant New Zealand alpine weta Hemideina maori Hutton (Orthoptera; Stenopelmatidae). Dilution curve and supercooling points following ultrafiltration argue that the ice-nucleation agents present are proteinaceous. We also report dynamic light-scattering measurements leading to hydrodynamic diameters of large molecules found in the weta hemolymph. The measured diameters agree well with theoretical estimates for sizes of ice-nucleating proteins corresponding to the measured supercooling points.

Many species of tardigrades are known to tolerate extreme environmental stress, yet detailed knowledge of the mechanisms underlying the remarkable adaptations of tardigrades is still lacking, as are answers to many questions regarding their basic biology. Here, we present data on the inorganic ion composition and total osmotic concentration of five different species of tardigrades (Echiniscus testudo, Milnesium tardigradum, Richtersius coronifer, Macrobiotus cf. hufelandi and Halobiotus crispae) using high-performance liquid chromatography and nanoliter osmometry. Quantification of the ionic content indicates that Na(+) and Cl(-) are the principal inorganic ions in tardigrade fluids, albeit other ions, i.e. K(+), NH4(+), Ca(2+), Mg(2+), F(-), SO4(2-) and PO4(3-) were also detected. In limno-terrestrial tardigrades, the respective ions are concentrated by a large factor compared with that of the external medium (Na(+), ×70-800; K(+), ×20-90; Ca(2+) and Mg(2+), ×30-200; F(-), ×160-1040, Cl(-), ×20-50; PO4(3-), ×700-2800; SO4(2-), ×30-150). In contrast, in the marine species H. crispae, Na(+), Cl(-) and SO4(2-) are almost in ionic equilibrium with (brackish) salt water, while K(+), Ca(2+), Mg(2+) and F(-) are only slightly concentrated (×2-10). An anion deficit of ~120 mEq l(-1) in M. tardigradum and H. crispae indicates the presence of unidentified ionic components in these species. Body fluid osmolality ranges from 361±49 mOsm kg(-1) in R. coronifer to 961±43 mOsm kg(-1) in H. crispae. Concentrations of most inorganic ions are largely identical between active and dehydrated groups of R. coronifer, suggesting that this tardigrade does not lose large quantities of inorganic ions during dehydration. The large osmotic and ionic gradients maintained by both limno-terrestrial and marine species are indicative of a powerful ion-retentive mechanism in Tardigrada. Moreover, our data indicate that cryptobiotic tardigrades contain a large fraction of unidentified organic osmolytes, the identification of which is expected to provide increased insight into the phenomenon of cryptobiosis.

Antifreeze proteins (AFPs) are characterized by their capacity to inhibit the growth of ice and are produced by a variety of polar fish, terrestrial arthropods and other organisms inhabiting cold environments. This capacity reflects their role as stabilizers of supercooled body fluids. The longhorn beetle Rhagium inquisitor is known to express AFPs in its body fluids. In this work we report on the primary structure and structural characteristics of a 12.8 kDa AFP from this beetle (RiAFP). It has a high capacity to evoke antifreeze activity as compared to other known insect AFPs and it is structurally unique in several aspects. In contrast to the high content of disulfide bond-formation observed in other coleopteran AFPs, RiAFP contains only a single such bond. Six internal repeat segments of a thirteen residue repeat pattern is irregularly spaced apart throughout its sequence. The central part of these repeat segments is preserved as TxTxTxT, which is effectively an expansion of the TxT ice-binding motif found in the AFPs of several known insect AFPs.

The influence of two food types, Boreogadus saida (Bs) and crustaceans (Cr), on the osmolality, ion concentrations, antifreeze activity and antifreeze glycoprotein (AFGP) distribution in the gastrointestinal fluids of the Arctic gadoid Arctogadus glacialis was determined. The gastrointestinal fluids were hyperosmotic to serum but no significant differences in osmolality were found between the two food types. The food type significantly affected the antifreeze activity of the mid-gut fluids. The hysteresis freezing points, -3.27+/-0.30 degrees C and -2.44+/-0.11 degrees C for B. saida and crustaceans, respectively, were significantly lower than that of serum (-1.99+/-0.07 degrees C). Furthermore, an exceptionally large thermal hysteresis ranging from 1.47+/-0.19 degrees C to 2.04+/-0.30 degrees C was observed in the intestinal fluids of fish feeding on B. saida. Native gel electrophoresis revealed that the gastrointestinal fluids contained AFGPs in all the different size groups. However, differences in band intensities for the two food types suggest that the ingested food has an influence on the concentration of the different AFGP-sizes in these fluids. A decrease in band intensities combined with a drop in thermal hysteresis from mid-gut to hind-gut fluid suggests that absorption of AFGP or possibly degradation occur during digestion.

We describe a simple and effective procedure to isolate antifreeze proteins (AFPs) from the hemolymph of larvae of the longhorn beetle Rhagium inquisitor , and present some characteristics of their structures. Several AFPs were isolated from the hemolymph of this species by heat and acid extraction followed by cation exchange. The hemolymph contains at least six AFPs ranging in size from 12.5 to 12.8 kDa. Of these, three were separated to purity by the ion exchange step, as indicated by mass spectrometry. The remaining three forms were further separated by size exclusion chromatography, but could not be isolated to purity. All AFPs in the hemolymph of this species appears to have isoelectric points above 8.00. The dominant form, RiAFP H4 , was purified by the ion exchange step. Its amino acid composition reveals a lower level of cysteine and a higher level of threonine, arginine, alanine and glycine than seen in other insect AFPs. Its trypsin fingerprint does not match that of any known protein. It interacts with ice both in the anionic and cationic state.

SUMMARYThis work describes a new calorimetric method in which the metabolic heat production and water exchange rates of an insect larva are measured simultaneously and in real time. The experimental set-up is based on two independent calorimetric cells, which are perfused by a stream of air at controlled relative humidity (RH). The resolution for metabolic heat flow and water flux is 1 μW and 5 μg h-1, respectively. The method was used to investigate water vapour absorption (WVA) in drought-stressed larvae of the common mealworm Tenebrio molitor. It was found that during exposure to a linear increment in RH of 3% per hour, the larvae initiated WVA upon passing a threshold value of 92.7±0.6%RH. The rate of water absorption subsequently increased to reach a maximal level of 86±6μg h-1, 10-15 h after passing the threshold value. Concomitantly, the RH in the calorimetric cell was reduced to 88.6±0.5%.The metabolic heat production of the larvae was 5-6 J h-1g-1 wet mass in the initial part of the experiment. However, this value doubled 2-3 h prior to the onset of WVA, when the RH had reached 88%. This increase in metabolic heat production gradually tapered off over the following 24 h of WVA, during which time WVA remained high. Animals exposed to RH protocols that did not induce WVA showed no such anomalies in metabolic heat flow. This may suggest that the increased metabolism reflects the preparation of the WVA apparatus. Finally, the method was used to quantify water losses in the microgram range associated with wriggling and tracheal ventilation.

The eelpout Zoarces viviparus is a common inhabitant in the shallow waters along the Danish coastline. Specimens were caught in the brackish (12-16 per thousand) Roskilde fjord where water temperatures range from20 degrees C during summer to subzero in winter. The serum melting points found in Z. viviparus varied between -0.76 (September) to -0.94 degrees C (January). Eighty to 97% of the serum melting points could be attributed to sodium, chloride and potassium. Hysteresis freezing points showed seasonal variation varying from -0.83 (September) to -2.08 degrees C (February). Serum antifreeze activity showed a seasonal variation with high levels (1.2 degrees C) in winter and low levels (0.1 degrees C) during summer and autumn. Antifreeze proteins are responsible for this antifreeze activity. Antifreeze activity was also found in Z. viviparus during their embryological development in the female ovary. Embryo thermal hysteresis reached the maximum level (approx. 0.6 degrees C) during December and maintained this level until parturition in January. Antifreeze activity seems unaffected by diminishing ice crystal fractions at ice fractions below 0.1 whereas ice fractions above 0.1 caused a decline in antifreeze activity.