Hans Ramløv

Survival in extreme environments – on the current knowledge of adaptations in tardigrades

Tardigrades are microscopic animals found worldwide in aquatic as well as terrestrial ecosystems. They belong to the invertebrate superclade Ecdysozoa, as do the two major invertebrate model organisms: Caenorhabditis elegans and Drosophila melanogaster. We present a brief description of the tardigrades and highlight species that are currently used as models for physiological and molecular investigations. Tardigrades are uniquely adapted to a range of environmental extremes. Cryptobiosis, currently referred to as a reversible ametabolic state induced by e.g. desiccation, is common especially among limno‐terrestrial species. It has been shown that the entry and exit of cryptobiosis may involve synthesis of bioprotectants in the form of selective carbohydrates and proteins as well as high levels of antioxidant enzymes and other free radical scavengers. However, at present a general scheme of mechanisms explaining this phenomenon is lacking. Importantly, recent research has shown that tardigrades even in their active states may be extremely tolerant to environmental stress, handling extreme levels of ionizing radiation, large fluctuation in external salinity and avoiding freezing by supercooling to below −20 °C, presumably relying on efficient DNA repair mechanisms and osmoregulation. This review summarizes the current knowledge on adaptations found among tardigrades, and presents new data on tardigrade cell numbers and osmoregulation.

Cryptobiosis in the Eutardigrade Adorybiotus (Richtersius) coronifer: Tolerance to Alcohols, Temperature and de novo Protein Synthesis

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.

Survival of the cryptobiotic eutardigrade Adorybiotus coronifer during cooling to −196 °C: Effect of cooling rate, trehalose level, and short-term acclimation

Abstract The cryptobiotic eutardigrade Adorybiotus cornifer survives cooling to −196 °C in the hydrated state, showing decreasing viability with faster cooling rates. The dissaccharide trehalose may have some cryoprotective effect at low cooling rates during cooling of A. coronifer to −196 °C, whereas no effect of short-term acclimation (16 h at 0.5 °C) was observed. Cooling at 30 °C min −1 to an intermediate temperature greater than the ice crystallization temperature of the sample ( T c ) before shock cooling to −196 °C is lethal, whereas cooling at a rate of 30 °C min −1 to an intermediate temperature less than T c before shock cooling to −196 °C achieved a survival of ca. 55%. This is similar to the survival of animals cooled to −196 °C at a constant rate of 30 °C min −1 . The data support the hypothesis that the freeze tolerance exhibited by A. coronifer is closely related to its ability to be in the state of anhydrobiosis.

Microclimate and variations in haemolymph composition in the freezing-tolerant New Zealand alpine weta Hemideina maori Hutton (Orthoptera: Stenopelmatidae)

Abstract 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.

Cyclomorphosis in Tardigrada: adaptation to environmental constraints.

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

Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor.

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.

Ice-active proteins and cryoprotectants from the New Zealand alpine cockroach, Celatoblatta quinquemaculata.

Celatoblatta quinquemaculata is moderately freezing tolerant. We have investigated low and high molecular weight compounds that may be associated with its survival. Glycerol and trehalose were identified as potential cryoprotectants, with trehalose at the higher concentration. Trehalose was at its highest concentration in late autumn, during the periods sampled. Water contents declined with time and were significantly lower in late autumn than in late summer. No thermal hysteresis activity was detected in haemolymph or in extracts of the head, muscles and the fat body. Extracts of the Malpighian tubules showed an hexagonal crystal growth form, as did those of the gut tissue and gut contents. The gut tissue had high levels of thermal hysteresis (approximately 2 degrees C) and the gut contents somewhat lower levels (approximately 0.6 degrees C). Recrystallization inhibition activity mirrored that of thermal hysteresis, with activity absent in the haemolymph or fat body cells but present in the gut tissues and contents. Activity was reduced by heating and was associated with a molecule >14kDa in size. These findings suggest an antifreeze protein is involved. In fed animals, ice nucleation is likely to start in the gut. Gut cells have a much greater resistance to freezing than do fat body or Malpighian tubule cells. The antifreeze protein may enable this tissue to survive freezing stress by inhibiting recrystallization.

ANTIFREEZE ACTIVITY IN THE CERAMBYCID BEETLE RHAGIUM INQUISITOR

Abstract The present study revealed that hibernating freeze-avoiding Rhagium inquisitor beetles have thermal hysteresis antifreeze agents in the intracellular fluid as well as in the intestinal fluid and the haemolymph. The antifreeze activity in all three compartments increased with diminishing size of the seeding ice crystal, suggesting that all three compartments are well protected against spontaneous ice nucleation at low sub-zero temperatures.

Excess post-hypoxic oxygen consumption is independent from lactate accumulation in two cyprinid fishes

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 2.5h 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 2.5h anoxic exposure is not a major determinant of the resulting EPHOC.

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 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.