Polyproline type II helical antifreeze proteins are widespread in Collembola and likely originated over 400 million years ago in the Ordovician Period.

Antifreeze proteins (AFPs) bind to ice crystals to prevent organisms from freezing. A diversity of AFP folds has been found in fish and insects, including alpha helices, globular proteins, and several different beta solenoids. But the variety of AFPs in flightless arthropods, like Collembola, has not yet been adequately assessed. Here, antifreeze activity was shown to be present in 18 of the 22 species of Collembola from cold or temperate zones. Several methods were used to characterize these AFPs, including isolation by ice affinity purification, MALDI mass spectrometry, amino acid composition analysis, tandem mass spectrometry sequencing, transcriptome sequencing, and bioinformatic investigations of sequence databases. All of these AFPs had a high glycine content and were predicted to have the same polyproline type II helical bundle fold, a fold unique to Collembola. These Hexapods arose in the Ordovician Period with the two orders known to produce AFPs diverging around 400 million years ago during the Andean-Saharan Ice Age. Therefore, it is likely that the AFP arose then and persisted in many lineages through the following two ice ages and intervening warm periods, unlike the AFPs of fish which arose independently during the Cenozoic Ice Age beginning ~ 30 million years ago.

Roles of carbohydrate reserves for local adaptation to low temperatures in the freeze tolerant oligochaete Enchytraeus albidus.

Geographic variation in cold tolerance and associated physiological adaptations were investigated in the freeze tolerant enchytraeid Enchytraeus albidus (Oligochaeta). Specimens from Svalbard, Greenland (Nuuk), Iceland (Hólar and Mossfellsbær) and continental Europe [Norway (Bergen), Sweden (Kullen) and Germany] were reared in the laboratory in a common-garden experiment. The aim was to test for variations in minimum lethal temperature, freeze duration tolerance, carbohydrate reserves and metabolic rate among the populations. Cold tolerance was related to environmental temperature of the respective location. Populations from the coldest climatic regions were able to tolerate freezing down to at least -15 °C and endured being frozen at -5 °C for 27-48 days, respectively. Populations from milder climates had a lower freeze duration tolerance (about -9 °C) and endured -5 °C for a shorter period (between 9 and 16 days). Glucose accumulation and glycogen reserves varied significantly between populations, but was not related directly to cold tolerance. Metabolic rate varied significantly between populations, but was not significantly related to cold tolerance. The metabolic rates at -2 °C of frozen and unfrozen worms from Germany and Svalbard were tested. The metabolic depression due to freezing of these populations was relatively small (<50 %), suggesting that the large carbohydrate accumulations may also be important as fuel during long-term freezing at moderately low temperatures. Differences in metabolic depression may partly explain the difference in cold tolerance of these two populations, however, the mechanisms behind local adaptation to low winter temperatures in these enchytraeid populations seem more complex than earlier studies have indicated.

Cold tolerance and freeze-induced glucose accumulation in three terrestrial slugs.

Cold tolerance and metabolic responses to freezing of three slug species common in Scandinavia (Arion ater, Arion rufus and Arion lusitanicus) are reported. Autumn collected slugs were cold acclimated in the laboratory and subjected to freezing conditions simulating likely winter temperatures in their habitat. Slugs spontaneously froze at about -4 °C when cooled under dry conditions, but freezing of body fluids was readily induced at -1 °C when in contact with external ice crystals. All three species survived freezing for 2 days at -1 °C, and some A. rufus and A. lusitanicus also survived freezing at -2 °C. (1)H NMR spectroscopy revealed that freezing of body fluids resulted in accumulation of lactate, succinate and glucose. Accumulation of lactate and succinate indicates that ATP production occurred via fermentative pathways, which is likely a result of oxygen depletion in frozen tissues. Glucose increased from about 6 to 22 µg/mg dry tissue upon freezing in A. rufus, but less so in A. ater and A. lusitanicus. Glucose may thus act as a cryoprotectant in these slugs, although the concentrations are not as high as reported for other freeze tolerant invertebrates.

Ecological and molecular consequences of prolonged drought and subsequent rehydration in Folsomia candida (Collembola).

Drought tolerance in water-permeable, soil-living Collembola (e.g. Folsomia candida) is achieved due to a unique water vapour absorption mechanism, where accumulation of sugars and polyols is essential. However, the molecular mechanisms underlying such adaptation as well as the maintenance of this survival strategy and the responses to rehydration after prolonged drought in these soil-living Collembola are unclear. In the present study, the functional relationships between ecological drought responses and expression of related target genes were investigated in F. candida exposed to mild and severe drought for up to 5 weeks by relating survival, moulting and reproduction rate with mRNA-level expression of 7 target genes during drought, dehydration and rehydration. Prolonged drought and subsequent rehydration induced significant changes in gene expression which could be related to the fitness traits studied. In F. candida the ecological and molecular responses to mild drought differed from those of severe drought. From the changes in gene expression, where significantly increased expression of Glucose-6-phosphate-isomerase (gpi) and Heat shock protein 70 (hsp70) was dominating, it is proposed that protection of cellular structure and function during prolonged mild drought (98.2% RH) is partly achieved from a continuous accumulation of compatible osmolytes in F. candida. To achieve protection during and after prolonged severe drought (96.1% RH), components related to cell division and development such as inositol monophosphatase and one of the small heat shock proteins (sHsps), Heat shock protein23 (hsp23), seem to play an important role in F. candida.

Impacts of heavy metals, polyaromatic hydrocarbons, and pesticides on freeze tolerance of the earthworm Dendrobaena octaedra.

Previous studies have shown that the interactions between chemicals and climatic stressors can lead to synergistically increased mortality. In the present study, we investigated the effect of seven common environmental contaminants on survival at -6 and 15°C as well as on reproduction at 15°C in the earthworm Dendrobaena octaedra. Three classes of chemicals were considered: Heavy metals (nickel, lead, and mercury), polycyclic aromatic hydrocarbons (pyrene and phenanthrene), and pesticides (abamectin and carbendazim). Phenanthrene interacted antagonistically with freezing temperatures, whereas no interaction was observed with any of the tested pesticides. Two of the three tested metals (nickel and mercury) reduced the freeze tolerance synergistically (mercury was especially potent). This suggests that traditional laboratory studies, in which organisms are exposed to increasing concentrations of a single compound under otherwise optimal conditions, may underestimate the toxicity of some metals to field populations living in cold areas.

Slow desiccation improves dehydration tolerance and accumulation of compatible osmolytes in earthworm cocoons (Dendrobaena octaedra Savigny).

The earthworm, Dendrobaena octaedra, is a common species in temperate and subarctic regions of the northern hemisphere. The egg capsules ('cocoons') of D. octaedra are deposited in the upper soil layers where they may be exposed to desiccation. Many previous studies on desiccation tolerance in soil invertebrates have examined acute exposure to harsh desiccating conditions, however, these animals are often more likely to be exposed to a gradually increasing drought stress. In the present study we slowly desiccated D. octaedra cocoons to simulate ecologically realistic drought conditions and the results clearly demonstrate that gradually dehydrated cocoons show an increased tolerance of extreme drought compared with acutely dehydrated cocoons. NMR spectroscopic analysis of compatible osmolytes revealed the presence of sorbitol, glucose, betaine, alanine and mannitol in dehydrated embryos. The superior drought survival of gradually desiccated embryos could partly be attributed to a higher accumulation of osmolytes (especially sorbitol). Thus, gradually and acutely desiccated embryos accumulated approximately 2 mol l(-1) and 1 mol l(-1) total osmolytes, respectively. However, in addition to osmolyte accumulation, the gradually desiccated cocoons also tolerated a higher degree of water loss, demonstrating that gradually dehydrated D. octaedra cocoons are able to survive loss of approximately 95% of the original water content. Although D. octaedra embryos can probably not be categorized as a truly anhydrobiotic organism we propose that they belong in a transition zone between the desiccation sensitive and the truly anhydrobiotic organisms. Clearly, these earthworm embryos share many physiological traits with anhydrobiotic organisms.

Multivariate metabolic profiling using 1H nuclear magnetic resonance spectroscopy of freeze-tolerant and freeze-intolerant earthworms exposed to frost.

Individuals of the freeze-tolerant earthworm, Dendrobaena octaedra, and four freeze-intolerant earthworm species (Dendrodrilus rubidus, Aporrectodea icterica, A. caliginosa, and A. longa) were frozen at -2 degree C. Control earthworms were exposed to +2 degree C. 1H nuclear magnetic resonance spectroscopy-based metabolic profiling in combination with multivariate pattern recognition methods (metabonomics) was used to produce a cross-species comparison. Several biochemical changes were detected as a result of freezing in all worm species, including an increase in relative free alanine concentrations, and an apparent conversion of adenosine to inosine. It was also possible to determine a number of biochemical changes that were unique to the freeze-tolerant species, D. octaedra. The most obvious difference was that, although all species showed an increase in glucose concentrations, the increase was largest in D. octaedra, and was coupled with a concomitant decrease in glycogen. This confirms that--like previously studied freeze-tolerant earthworm species--tolerance is effected by rapid glucose production from glycogen reserves. An additional difference noted was that succinate increased in all species on freezing, but the increase was least in D. octaedra. Furthermore there was no lactate accumulation in D. octaedra, whereas three of the other four species accumulated lactate. This indicates that anoxic metabolism was lowest in the freeze-tolerant species.