Metabolites, ions, and the mechanisms behind seasonal cold hardening of Pyrochroa coccinea (Pyrochroidae) larvae.

The larvae of the black headed cardinal beetle Pyrochroa coccinea, overwinters under the bark of dead logs in northern European dioecious forests, and are thus exposed to temperatures below the melting point of their bodily fluids. Here we explore the mechanisms behind their seasonal cold hardening by characterising field samples collected monthly throughout the year. Both the lower lethal temperature and supercooling point dropped as much as 10℃ in the second half of November, reaching values around -15℃ by the beginning of December. This change was accompanied by a 320 mosmol/kg increase in hemolymph osmolality, which is a doubling compared to the summer levels. We used NMR metabolomics to identify and measure the absolute concentrations of the responsible cryoprotective C-H containing metabolites in the hemolymph. The largest increase was found to be in either glucose or trehalose, with an average total increase of 120 mM. Proline, alanine, and choline concentrations were found to increase by around 10 mM each. Contrarily, phosphocholine and phosphoethanolamine were halved, resulting in a total decrease of around 50 mM. These measurements were complemented with ion exchange chromatography measurements. This allowed us to account for all the osmotic pressure in the summer hemolymph, and the measured concentration changes explained as much as 40 % of the observed osmolality increase upon cold hardening. Preliminary results indicate that the remainder may be explained by non-colligative protein contributions.

Ice Recrystallization Inhibition Is Insufficient to Explain Cryopreservation Abilities of Antifreeze Proteins.

Antifreeze proteins (AFPs) and glycoproteins (AFGPs) are exemplary at modifying ice crystal growth and at inhibiting ice recrystallization (IRI) in frozen solutions. These properties make them highly attractive for cold storage and cryopreservation applications of biological tissue, food, and other water-based materials. The specific requirements for optimal cryostorage remain unknown, but high IRI activity has been proposed to be crucial. Here, we show that high IRI activity alone is insufficient to explain the beneficial effects of AF(G)Ps on human red blood cell (hRBC) survival. We show that AF(G)Ps with different IRI activities cause similar cell recoveries of hRBCs and that a modified AFGP variant with decreased IRI activity shows increased cell recovery. The AFGP variant was found to have enhanced interactions with a hRBC model membrane, indicating that the capability to stabilize cell membranes is another important factor for increasing the survival of cells after cryostorage. This information should be considered when designing novel synthetic cryoprotectants.

Respiration Measurements of Individual Tardigrades of the Species Richtersius cf coronifer as a Function of Temperature and Salinity and Termination of Anhydrobiosis.

Numerous studies have demonstrated that tardigrades in a resting state (tun state) are very resistant to exceptional stress levels in comparison with the resistance observed in multicellular organisms in general. The types of stress include desiccation and radiation, which are also relevant in astrobiological research, and therefore, tardigrades are used as multicellular model organisms. For example, tardigrades have been investigated in the TARSE, TARDIS, RoTaRad, and TARDIKISS projects; their survival has been evaluated according to stressful conditions that prevail in low earth orbit, including the effects of cosmic radiation and microgravity. Despite this interest, the study of tardigrade biology has been severely hampered by the sparsity of appropriate quantitative techniques that inform at the single-organism level. In this study, we present results on mass-specific respiration rates as a function of termination of anhydrobiosis and variations in temperature and salinity, including Mars-analog perchlorate solutions, by using microsensor technology to measure respiration. Based on our results for Richtersius cf coronifer, we estimated the activation energy (50.8 kJ/mole O2) for its metabolism as well as Q10 for selected temperature intervals. Q10 was constant-∼1.5-between 2°C and 33°C, except for the interval 11-16°C, where Q10 was 5.5. The steady-state mass-specific respiration rate of individuals of Richtersius cf coronifer increased with increasing salinity below the lethal limit, likely representing the energy requirements of its osmoregulatory response. We report the first quantitative data of a tardigrade's metabolic dynamics during the termination of anhydrobiosis, revealing significant variation between individuals. However, we observed a general trend, that is, a high initial metabolic rate after exposure to water. Our approach would allow us to carry out quantitative physiological studies of tardigrades on board of the International Space Station, and thus significantly extend the possibility of studying the response of multicellular organisms in space. Summary statement This article reports on first measurements of mass-specific respiration rates of individual tardigrades of the species Richtersius cf coronifer during termination of anhydrobiosis as well as measurements of the impact of temperature and salinity on oxygen uptake rates.

A method for studying the metabolic activity of individual tardigrades by measuring oxygen uptake using microrespirometry.

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 h-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±1.84 and 13.1±2.19 nmol O2 mg-1 h-1, respectively.

Inhibition of Bacterial Ice Nucleators Is Not an Intrinsic Property of Antifreeze Proteins.

Cold-adapted organisms use antifreeze proteins (AFPs) or ice-nucleating proteins (INPs) for the survival in freezing habitats. AFPs have been reported to be able to inhibit the activity of INPs, a property that would be of great physiological relevance. The generality of this effect is not understood, and for the few known examples of INP inhibition by AFPs, the molecular mechanisms remain unclear. Here, we report a comprehensive evaluation of the effects of five different AFPs on the activity of bacterial ice nucleators using a high-throughput ice nucleation assay. We find that bacterial INPs are inhibited by certain AFPs, while others show no effect. Thus, the ability to inhibit the activity of INPs is not an intrinsic property of AFPs, and the interactions of INPs and different AFPs proceed through protein-specific rather than universal molecular mechanisms.

Data from thermal testing of the Open Source Cryostage.

The data presented here is related to the research article "An open source cryostage and software analysis method for detection of antifreeze activity" (Buch and Ramløv, 2016) [1]. The design of the Open Source Cryostage (OSC) is tested in terms of thermal limits, thermal efficiency and electrical efficiency. This article furthermore includes an overview of the electrical circuitry and a flowchart of the software program controlling the temperature of the OSC. The thermal efficiency data is presented here as degrees per volt and maximum cooling capacity.

Anhydrobiosis and freezing-tolerance: adaptations that facilitate the establishment of Panagrolaimus nematodes in polar habitats.

Anhydrobiotic animals can survive the loss of both free and bound water from their cells. While in this state they are also resistant to freezing. This physiology adapts anhydrobiotes to harsh environments and it aids their dispersal. Panagrolaimus davidi, a bacterial feeding anhydrobiotic nematode isolated from Ross Island Antarctica, can survive intracellular ice formation when fully hydrated. A capacity to survive freezing while fully hydrated has also been observed in some other Antarctic nematodes. We experimentally determined the anhydrobiotic and freezing-tolerance phenotypes of 24 Panagrolaimus strains from tropical, temperate, continental and polar habitats and we analysed their phylogenetic relationships. We found that several other Panagrolaimus isolates can also survive freezing when fully hydrated and that tissue extracts from these freezing-tolerant nematodes can inhibit the growth of ice crystals. We show that P. davidi belongs to a clade of anhydrobiotic and freezing-tolerant panagrolaimids containing strains from temperate and continental regions and that P. superbus, an early colonizer at Surtsey island, Iceland after its volcanic formation, is closely related to a species from Pennsylvania, USA. Ancestral state reconstructions show that anhydrobiosis evolved deep in the phylogeny of Panagrolaimus. The early-diverging Panagrolaimus lineages are strongly anhydrobiotic but weakly freezing-tolerant, suggesting that freezing tolerance is most likely a derived trait. The common ancestors of the davidi and the superbus clades were anhydrobiotic and also possessed robust freezing tolerance, along with a capacity to inhibit the growth and recrystallization of ice crystals. Unlike other endemic Antarctic nematodes, the life history traits of P. davidi do not show evidence of an evolved response to polar conditions. Thus we suggest that the colonization of Antarctica by P. davidi and of Surtsey by P. superbus may be examples of recent "ecological fitting" of freezing-tolerant anhydrobiotic propagules to the respective abiotic conditions in Ross Island and Surtsey.

Purification, crystal structure determination and functional characterization of type III antifreeze proteins from the European eelpout Zoarces viviparus.

Antifreeze proteins (AFPs) are essential components of many organisms adaptation to cold temperatures. Fish type III AFPs are divided into two groups, SP isoforms being much less active than QAE1 isoforms. Two type III AFPs from Zoarces viviparus, a QAE1 (ZvAFP13) and an SP (ZvAFP6) isoform, are here characterized and their crystal structures determined. We conclude that the higher activity of the QAE1 isoforms cannot be attributed to single residues, but rather a combination of structural effects. Furthermore both ZvAFP6 and ZvAFP13 crystal structures have water molecules around T18 equivalent to the tetrahedral-like waters previously identified in a neutron crystal structure. Interestingly, ZvAFP6 forms dimers in the crystal, with a significant dimer interface. The presence of ZvAFP6 dimers was confirmed in solution by native electrophoresis and gel filtration. To our knowledge this is the first report of dimerization of AFP type III proteins.

Antifreeze activity enhancement by site directed mutagenesis on an antifreeze protein from the beetle Rhagium mordax.

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 270 µmol.

Low thermodynamic but high kinetic stability of an antifreeze protein from Rhagium mordax.

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.

Role of cutaneous surface fluid in frog osmoregulation.

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.

Inorganic ion composition in Tardigrada: cryptobionts contain a large fraction of unidentified organic solutes.

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.

Hyperactive antifreeze proteins from longhorn beetles: some structural insights.

This study reports on structural characteristics of hyperactive antifreeze proteins (AFPs) from two species of longhorn beetles. In Rhagium mordax, eight unique mRNAs coding for five different mature AFPs were identified from cold-hardy individuals. These AFPs are apparently homologues to a previously characterized AFP from the closely related species Rhagium inquisitor, and consist of six identifiable repeats of a putative ice binding motif TxTxTxT spaced irregularly apart by segments varying in length from 13 to 20 residues. Circular dichroism spectra show that the AFPs from both species have a high content of ß-sheet and low levels of α-helix and random coil. Theoretical predictions of residue-specific secondary structure locate these ß-sheets within the putative ice-binding motifs and the central parts of the segments separating them, consistent with an overall ß-helical structure with the ice-binding motifs stacked in a ß-sheet on one side of the coil. Molecular dynamics models based on these findings show that these AFPs would be energetically stable in a ß-helical conformation.

Cyclomorphosis in Tardigrada: adaptation to environmental constraints.

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 degrees 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 microm) 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. crispae hyper-regulate, indicating a high water turnover and excretion of dilute urine. This is likely a general feature of eutardigrades.

Freeze tolerance evolution among anurans: Frequency and timing of appearance.

Despite numerous mechanistic studies on physiological responses supporting freeze tolerance in anurans, few have addressed the evolutionary significance of this trait. We thus investigated the phylogenetic relationships among anuran species whose freeze tolerance has been assessed and in combination with new data on freezing tolerance of two closely related species of the European brown frogs (Rana temporaria and Rana dalmatina). The species we studied exhibited short survival times in frozen state (around 8 h for both species). Phylogenetic analysis suggests that freeze tolerance evolved at least two times among Ranidae and one or two times among Hylidae and never in Bufonidae. Furthermore, in order to assess the timing of divergence of this character we used a relaxed molecular clock created, and found that the most recent separation between a freeze tolerant species and a freeze intolerant species dates from 15.9 +/- 7.6 Myr (Rana arvalis and R. temporaria). The comparison between these two species thus represents the best current model to understand freeze tolerance evolution. Addressing the evolution of this trait with such large-scale approaches will not only improve our understanding of cold hardiness strategies, but might also create a framework guiding future comparative studies.

Survival and metabolism of Rana arvalis during freezing.

Freeze tolerance and changes in metabolism during freezing were investigated in the moor frog (Rana arvalis) under laboratory conditions. The data show for the first time a well-developed freeze tolerance in juveniles of a European frog capable of surviving a freezing exposure of about 72 h with a final body temperature of -3 degrees C. A biochemical analysis showed an increase in liver and muscle glucose in response to freezing (respectively, 14-fold and 4-fold between 4 and -1 degrees C). Lactate accumulation was only observed in the liver (4.1+/-0.8 against 16.6+/-2.4 micromol g(-1) fresh weight (FW) between 4 and -1 degrees C). The quantification of the respiratory metabolism of frozen frogs showed that the aerobic metabolism persists under freezing conditions (1.4+/-0.7 microl O(2) g(-1) FW h(-1) at -4 degrees C) and decreases with body temperature. After thawing, the oxygen consumption rose rapidly during the first hour (6-fold to 16-fold) and continued to increase for 24 h, but at a lower rate. In early winter, juvenile R. arvalis held in an outdoor enclosure were observed to emerge from ponds and hibernate in the upper soil and litter layers. Temperature recordings in the substratum of the enclosure suggested that the hibernacula of these juvenile frogs provided sheltering from sub-zero air temperatures and reduced the time spent in a frozen state corresponding well with the observed freeze tolerance of the juveniles. This study strongly suggests that freeze tolerance of R. arvalis is an adaptive trait necessary for winter survival.

Vascular arrangement and ultrastructure of the European eelpout Zoarces viviparus ovary: implications for maternal-embryonic exchange.

The structural basis for exchange between maternal serum and ovarian fluid in the viviparous teleost Zoarces viviparus was investigated. Casts of the ovarian vasculature showed that blood supply to the ovary is initially directed to the follicular appendages lining the ovarian wall through thick-walled muscular arteries running along the ovary wall and within the follicular appendages. The follicles had a rich capillary network with diffusion distances between maternal blood and ovarian fluid comparable to those found for gill epithelia, suggesting this is the primary site of gas exchange between maternal plasma and ovarian fluid. Follicular capillary beds were continuous with those in the ovary wall and were eventually drained by the ovarian and intestinal venous systems. The barrier between ovarian fluid and maternal blood consisted of the endothelial cells of the maternal blood vessels and a layer of epithelial cells lining the ovarian lumen, with an intermittent layer of loose connective fibers. Junctional complexes between cells were predominantly anchoring junctions with the occurrence of occasional occluding junctions, supporting the possibility of paracellular transport from maternal serum to ovarian fluid of small molecular weight compounds. Heavy investment in keratin filaments suggests that follicles are tissues of high structural integrity. Evidence for protein synthesis in the ovarian lining was found in the form of Golgi apparatus and rough endoplasmic reticulum. Although numerous cytoplasmic vacuoles and secretory granules were present in both epithelial and endothelial cells, the fate of synthesized protein remains to be determined.

Respiration rates of subitaneous eggs from a marine calanoid copepod: monitored by nanorespirometry.

The oxygen consumption rate during embryogenesis of Acartia tonsa subitaneous eggs were measured at different temperatures (10, 15, 17, 21, 24 and 28 degrees C) with nanorespirometry. The oxygen consumption was constant during the embryogenesis but increased rapidly at hatching time. The mean +/- SD oxygen consumption rate increased exponentially with temperature and ranged from 0.09 +/- 0.04 (10 degrees C) to 0.54 +/- 0.09 nmol O(2) egg(-1) h(-1) (28 degrees C). The mean +/- SD Q(10)-value was 2.51 +/- 0.15. Calculations of energy consumption during embryogenesis ranged from 1.86 to 18.28 mJ depending on temperature and development time. We conclude that the effect of temperature on oxygen consumption rate was far less important than the prolonged development time when calculating the energy consumed during embryogenesis.

Metabolic changes associated with active water vapour absorption in the mealworm Tenebrio molitor L. (Coleoptera, Tenebrionidae): a microcalorimetric study.

Water vapour absorption (WVA) is an important mechanism for water gain in several xeric insects. Theoretical calculations indicate that the energetic cost of WVA should be small (5-10% of standard metabolic rate) assuming realistic efficiencies. In this study we explored the relationship between WVA, metabolic heat flux (HFmet.) and CO2 release in larvae of Tenebrio molitor using microcalorimetry. By comparing metabolic heat flux with the catabolic rate estimated from VCO2 , we were able to differentiate anabolic and catabolic rates prior to and during WVA, while simultaneously monitoring water exchange. Three to four hours before the onset of WVA, larvae showed clear increases in HFmet. and catabolic flux, and a simultaneous decrease in anabolic flux. Following the onset of WVA, HFmet. decreased again until indistinguishable from control (non-absorbing) values. Possible factors contributing to the "preparatory phase" are discussed, including mobilization of Malpighian tubule transporters and muscular activity in the rectum. Absorbing larvae reduced the water activity of the calorimetric cell to 0.906, agreeing with gravimetric estimates of the critical equilibrium activity. Periods of movement during WVA coincided with decreased uptake fluxes, consistent with the animal's hydrostatic skeleton and the need to close the anus to generate pressure increases in the haemocoel.

Antifreeze activity in the gastrointestinal fluids of Arctogadus glacialis (Peters 1874) is dependent on food type.

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.