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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.

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.

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

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.

Proteins’ physicochemical properties is a broad term, and cover more than has been investigated for antifreeze proteins. In this chapter the investigations of antifreeze proteins (AFPs) are confined to weight, activity, solubility, hydrophobicity, and stability of the proteins. The weight and activity are presented together and compared, as much research suggests a positive correlation between these properties. This correlation applies for both fish AFPs and hyperactive arthropod AFPs, when comparing activity measurements within each AFP type. Not much research has focused on the solubility of AFPs, most likely due to their high solubility, which has not been inflicting with the main objective of the investigations. The AFPs are generally amphiphilic, having a relatively hydrophobic surface at the flat ice-binding region of the protein, while the rest of the proteins’ solvent-exposed surface is regarded as relatively hydrophilic. The hydrophobicity of the ice-binding site is regarded to be a key feature to bind to ice, explained by the anchored clathrate mechanism.

The primary function of antifreeze proteins is without doubt the inhibition of ice crystal growth in the body fluids and the inhibition of ice recrystallization in various organisms. Modification of the structure of the frozen environment by ice algae has also been reported but also this is a mechanism related to ice. However, other properties, not related to ice but still related to cold, of antifreeze proteins have been observed. These properties include interaction with cell membranes and anti-virulence properties in both animals and plants. In this chapter we present evidence that AFPs interact with both biological and model membrane systems and that these interactions rely both on the type of antifreeze protein as well as the saturation/unsaturation of the membrane lipids in question.

When investigating proteins, the possibility of introducing targeted changes and observing their effect can be a useful tool. In regard to antifreeze proteins, this possibility has given rise to various kinds of studies. The method of observing which specific planes on the ice crystal the different antifreeze proteins bind to has been improved by fusing antifreeze proteins to fluorescent proteins. It has also provided insight into whether the antifreeze proteins bind to ice irreversibly or not, by microfluidic experiments. Using site-directed mutagenesis it has been studied how different amino acid side chains, and more specifically their functional groups, affect the interaction with ice. These studies led to a change in the perception that hydrogen bonds were the key force driving the interaction, to believing that the hydrophobic or van der Waals forces are dominant in this regard. It has also been achieved to increase the activity of antifreeze proteins, and though much is still to be learned about the antifreeze proteins, the future mutagenic studies could very well be focusing on optimising the proteins. This could be relevant, not only in regard to activity, but also in the general tailoring of the protein to specific purposes, in order to make the protein relevant for specific commercial respects in the future.

This chapter describes the consequences of cold exposure and ice formation on both the cellular, tissue and the organismal level in cold tolerant ectothermic organisms. This includes the direct implication of cold “per se” on various parameters such as pH, membrane fluidity and phase transitions, ionic gradients, metabolism, cold denaturation of proteins, ice nucleation, ice growth, freezing-induced cellular dehydration, the role of aquaporins and the mechanical stress caused by ice, while referring to the different aspects described in the previous chapter on ice in general. Defensive/preventive adaptations and mechanisms of the organisms are described as well, along with a description of freeze tolerance and freeze avoidance. The synthesis and mechanisms of action of low molecular weight cryoprotectants as well as their distribution are discussed. High molecular weight cryoprotectants are also described concluding with the introduction of the AFPs.

Despite the fact that much progress of understanding the antifreeze proteins, AFPs, (ice-binding proteins ad recrystallization inhibitor proteins) has been made during the last four decades, many questions are still unanswered and each time one question is answered, more seems to appear.

In the first volume of the book, we present the history of the antifreeze proteins (AFPs), their physiological role in freeze-avoiding or freeze-tolerating species, as well as the evolution and grouping of the antifreeze proteins. In the present volume we delve into the functions and interactions of antifreeze proteins at the molecular level. The volume is divided in three parts (Part I, II, and III of this work) followed by a final summary of both volumes. Part I focuses on the biochemistry of the antifreeze proteins and their interaction with both ice and water. Part II describes the mode of actions for obtaining thermal hysteresis and inhibition of recrystallization and how to measure it. Part III concerns the fields in which the applications of antifreeze proteins have come the furthest, as well as the possibilities of mutating and tailoring the proteins. In this chapter we give a short summary of the chapters that constitutes this second and final volume of the book.

In this volume of the book, we present background on water and ice formation, the history of the antifreeze proteins (AFPs), their physiological role in freeze avoiding or freeze tolerating species, as well as the evolution and grouping of the antifreeze proteins. In the second volume, we delve into the functions and interactions of antifreeze proteins at the molecular level.

The aim of this study is to provide the reader with a simple setup that can detect antifreeze proteins (AFP) by inhibition of ice recrystallisation in very small sample sizes. This includes an open source cryostage, a method for preparing and loading samples as well as a software analysis method. The entire setup was tested using hyperactive AFP from the cerambycid beetle, Rhagium mordax. Samples containing AFP were compared to buffer samples, and the results are visualised as crystal radius evolution over time and in absolute change over 30 min. Statistical analysis showed that samples containing AFP could reliably be told apart from controls after only two minutes of recrystallisation. The goal of providing a fast, cheap and easy method for detecting antifreeze proteins in solution was met, and further development of the system can be followed at https://github.com/pechano/cryostage.

Antifreeze proteins (AFPs) are characterized by their ability to protect organisms from subfreezing temperatures. They constitute a class of promising candidates as environmentally kinetic hydrate inhibitors (KHIs). In this study, the effectiveness of an insect cell expressed novel monomeric streptavidin fusion protein version of Rhagium mordax RmAFP1 antifreeze protein (mSA-RmAFP1), and four amino acids (histidine, lysine, tyrosine and proline), on CH4 hydrate nucleation, growth and decomposition was investigated using a rocking cell apparatus, then compared with the commercial inhibitors Polyvinylpyrrolidone (PVP) and Luvicap Bio. It was found that CH4 hydrate nucleation and growth exhibited good repeatable results under experimental conditions. The results showed that 2250 ppm mSA-RmAFP1 can inhibit CH4 hydrate nucleation as effectively as PVP at the same concentration. The histidine, lysine, tyrosine and proline exhibited weak inhibition effect on CH4 hydrate nucleation. The mSA-RmAFP1 decreased CH4 hydrate growth rate and production in the fresh and memory solutions. The CH4 hydrate formed in the solutions containing various tested KHIs present slightly lower onset decomposition temperatures than the non-inhibited system under experimental conditions. The promising performance of the insect cell expressed mSA-RmAFP1 could promote the further development of green hydrate inhibitors. The production of this protein through insect cell line fermentation provides a platform for the future production and optimization of AFPs for hydrate inhibition.

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.

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.

Antifreeze proteins (AFPs) are a unique class of proteins that bind to ice crystal surfaces and arrest their growth. The working mechanism of AFPs is not well understood because, as of yet, it was not possible to perform molecular-scale studies of AFPs adsorbed to the surface of ice. Here, we study the structural properties of an AFP from the insect Rhagium mordax (RmAFP) adsorbed to ice with surface specific heterodyne-detected vibrational sum-frequency generation spectroscopy and molecular dynamic simulations. We find that RmAFP, unlike other proteins, retains its hydrating water molecules upon adsorption to the ice surface. This hydration water has an orientation and hydrogen-bond structure different from the ice surface, thereby inhibiting the insertion of water layers in between the protein and the ice surface.

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.

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 β-sheet and low levels of α-helix and random coil. Theoretical predictions of residue-specific secondary structure locate these β-sheets within the putative ice-binding motifs and the central parts of the segments separating them, consistent with an overall β-helical structure with the ice-binding motifs stacked in a β-sheet on one side of the coil. Molecular dynamics models based on these findings show that these AFPs would be energetically stable in a β-helical conformation.

In the oil and gas industry there is ample motivation for moving toward greener kinetic inhibitors of gas hydrates as many of those used today suffer from poor biodegradability. In this work, we have investigated experimentally whether ice-structuring proteins (ISPs) found in fish and insect, assumed biodegradable, are capable of inhibiting the growth of methane hydrate (a structure I hydrate). The ISPs investigated were type III HPLC12 (originally identified in ocean pout) and ISP type III found in meal worm (Tenebrio molitor). These were compared to polyvinylpyrrolidone (PVP) a well-known kinetic hydrate inhibitor. The results revealed that adding ISP in sufficient amounts caused the appearance of an initial nonlinear growth period. At a certain point during the growth process the growth pattern changed to linear which is identical to the growth observed for methane hydrate in the absence of inhibitors. The profile of the nonlinear growth was concentration-dependent but also dependent on the stirring rate. ISP type III HPLC12 decreased the growth rate of methane hydrate during the linear growth period by 17-75% at concentrations of 0.01-0.1 wt % (0.014-0.14 mM) while ISP from Tenebrio molitor and PVP decreased the growth rate by 30% and 39% at concentrations of 0.004 wt % (0.005 mM) and 0.1 wt % (0.1 mM), respectively. Considering the low concentration of Tenebrio molitor ISP used, these results indicate that ISP from Tenebrio molitor is the most effective hydrate inhibitor among those investigated. Thermal hysteresis ice formation experiments revealed that ISP from Tenebrio molitor causes higher thermal hysteresis for ice formation compared to type III ISP identified in ocean pout while PVP did not cause thermal hysteresis. This indicates that there might be a direct relationship between ISP performance for ice and hydrate inhibition, and that thermal hysteresis experiments can be used to screen ISPs as kinetic inhibitors.

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 .

Populations of Acartia tonsa (Dana) and Centropages hamatus (Lilljeborg) were monitored in outdoor enclosures imitating the natural estuarine environment in northern Denmark from August 2003 to February 2004. A. tonsa was predominant in summer while C. hamatus dominated from October and onwards. Mean egg production was normally higher in C. hamatus as compared to A. tonsa and continued until late December before a decrease was observed. Hatching success remained high even in February at temperatures below zero degree indicating that no diapause eggs were produced. Further, the egg quality evaluated from the fatty acids (AA, EPA and DHA) and amino acid contents did not change markedly during the study period. From this observation it is strongly suggested that nauplii originated from copepod eggs produced throughout the monitored sampling period constitute an adequate live feed for fish larvae probably even year round.

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.

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.

For most fish species raised in marine aquaculture, the use of live feeds cannot be replaced by formulated diets. Artemia nauplii and rotifers are still the most commonly used live feeds. A good alternative lies in the use of copepods which could lead to the cultivation of new fish species. Cold stored subitaneous eggs from the continuously cultured calanoid copepod Acartia tonsa were used to investigate the effect of storage upon the viability of the eggs, the development of the copepod community originating from the cold stored eggs. Finally a 3 days snapshot of the egg production of the first generation of females was followed. This was done in order to develop a database usable within copepod dependent hatcheries. The viability of cold stored A. tonsa eggs remained high (> 70% hatching rate) for 11 months of storage. Generally, the period of storage was observed to decrease the viability (hatching rate) of the eggs and no hatching was observed after twenty months of cold storage. Hatched populations of copepods experienced increased mortality rate with longer storage of the eggs from which they originated. This mortality ranged from 0.035 to 0.13 d− 1 for non-stored (fresh) and 12 months stored eggs, respectively. However, all copepod communities originating from fresh to 12 months stored eggs reached adulthood. Additionally, the egg production from the stored generation was apparently normal and the viability of their eggs was not statistically different when compared to productions from non-stored communities. Contents of total fatty acids decreased during the storage period. Contents of free amino acids were not statistically different for eggs cold stored up to 12 months, but had decreased severely by 20 months. In conclusion, we consider it safe to store the eggs for up to one year at 2–3 °C during which the eggs retain their viability and biochemical composition. Cold storage of calanoid copepod eggs is relevant for aquaculture as inoculum for culturing live food.

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 eutardigrade Adorybiotus (Richtersius) coronifer survives cryptobiosis for years. During entrance into anhydrobiosis this species accumulates the disaccharide trehalose reaching a maximum content of 2.3% d.w. In the present study we examined the survival of anhydrobiotic A. (R)coronifer during exposure to alcohols of various polarity, and to high temperatures, as well as qualitative changes in protein synthesis during entrance into anhydrobiosis. Results showed that A. (R) coronifer in anhydrobiosis survived exposure to ethanol for less than 10 minutes whereas exposure to 1-butanol only decreased survival to 40% after the first 7 days and 1-hexanol did not change survival from the controls after the first 7 days. A. (R) coronifer survived temperatures up to approximately 70 °C for 60 minutes without any decrease in survival. However, survival decreased rapidly when the exposure temperature was increased to above 70 °C and no animals survived exposure to 100 °C. During the entrance into anhydrobiosis a protein with a molecular weight of approximately 71 kDa appeared on acryl amide gels showing protein bands after the animals had been incubated with 3H-Leucine. This protein may belong to the Heat-shock protein (Hsp) 70 family. The results on the survival of A. (R) coronifer during exposure to alcohols and high temperature are discussed in light of the trehalose content earlier described in this animal during anhydrobiosis.

The microclimate in the habitat of the New Zealand alpine weta Hemideina maori is very variable with winter temperatures down to −6 °C under the rocks where the insects are found. Subfreezing temperatures may in winter prevail for up to 17 days but diurnal cycles of freezing and thawing are common, as is also the case in summer. Rates of temperature change can be very high and up to −7.20 °C/h. During winter, humidity was high for extended periods ranging from 70% to 100% relative humidity (RH). In the summer, humidity ranged from 30% RH during the day to 100% RH at night. The supercooling point of the haemolymph was approximately −8 °C year round, caused by a heat labile substance. The supercooling point of the haemolymph of an insect of the same genus, Hemideina femorata not regularly exposed to subfreezing temperatures, was ca. −16.5 °C. Thermal hysteresis was not detected in the haemolymph of H. maori. Haemolymph osmolality varied from 380 mOsm (summer) to 700 mOsm (winter). Body water content was ca. 75% all year round. Total concentrations of sodium, potassium and chloride in haemolymph varied from 170 mM (winter) to 250 mM (summer). The total concentration of free amino acids varied from 58 mM (summer) to 263 mM (winter). This variation was mostly due to proline which varied from ca. 15 mM (summer) to ca. 100 mM (winter). The freeze-tolerant weta H. maori is exposed to a highly variable and cold environment all year round and several properties of its haemolymph composition can be attributed to these climatic conditions, e.g. the presence of ice-nucleating agents and an increase in the concentration of proline during cold hardening in the autumn.

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.

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.

Antifreeze proteins (AFPs) are a relatively recently discovered group of proteins found in cold tolerant, cold-blooded organisms across many taxa. The proteins have apparently evolved independently multiple times as they have a wide range of primary, secondary, and tertiary structures. However, they all share the ability to recognize, bind to, and inhibit the growth of ice crystal surfaces. In the present chapter we discuss the properties of water related to freezing, the structure and physics of ice, nucleation, and recrystallization as a background for understanding the actions of antifreeze proteins, both within cold-tolerant organisms, and in the context of applications. As an inspiration and background the chapter also deals with the problems of ice formation in cold-tolerant organisms. This leads to an overview of the types of AFPs found in various organisms, with emphasis on those found in fish, insects, and plants. Antifreeze activity is defined and the antifreeze mechanism is explained, as is the binding of the AFPs to ice crystals. Antifreeze activity can be enhanced in various ways: by interaction with organic and inorganic molecules as well as by mutations of the native AFP. These enhancements may be of great importance for applying AFPs in the food industry. A section gives an overview of our present knowledge of the use of AFPs in food preservation. The physical-chemical properties of AFPs are important when dealing with applications, and as these can be quite different from other proteins a section is devoted to a discussion of these. With many new methods, both technical and bio-molecular, the future of applying AFPs in the food industry seems bright. It is our hope that by reading the present chapter the reader will feel inspired to consider AFPs as an interesting addition to old and well-proved techniques within the technologies of food processing.

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.

Publisher Summary Gas hydrates are crystalline compounds formed when water and suitably sized gas molecules are combined at high pressure and low temperature. They consist of polyhedral cavities formed from networks of hydrogen-bonded water molecules in which small gas molecules can enter. Depending on the type of gas molecules present gas hydrates form different structures, known as structure I (sI), structure II (sII) and in special cases structure H (sH). The gases that form hydrates are normally small molecules, many of which are encountered in natural gas. Traditionally the formation of hydrates has been prevented by addition of thermodynamic hydrate inhibitors such as methanol and glycol. However the amounts needed to avoid hydrate formation may reach 50 wt% in the water rich phase. Since water production from fields can be quite severe, especially in cases where water injection has been used to enhance the oil recovery, large amounts of hydrate inhibitor are required.