Low soil Moisture Slows Uptake and Elimination Rates of Phenanthrene in Springtails.

This study investigated the influence of soil water status on the toxicokinetics of phenanthrene in the springtail Folsomia candida allowing estimation of uptake and elimination rates at two contrasting soil water potentials. Fitting a three-phase model to the observations showed that uptake rate (ku) was almost two times higher in moist soil (-2 kPa) than in dry soil (-360 kPa). During the first days of the exposure, elimination rate (ke) was not significantly different in moist and dry soil, but after eight days ke had increased significantly more in moist soil than in dry soil. Our results confirm the general notion that the exposure route via soil pore water is important. Understanding the significance of soil moisture in exposure and effects of contaminants on soil invertebrates is crucial for assessing the ecological risks associated with soil pollution in a changing climate.

Influence of Soil Moisture on Bioaccumulation, Growth, and Recruitment of Folsomia candida Exposed to Phenanthrene-Polluted Soil.

Climate change has resulted in an increased occurrence of summer droughts in large parts of the world. Low soil moisture has marked impacts on the physiology of soil invertebrates and lowers degradation rates of organic contaminants in soil. Polycyclic aromatic hydrocarbons (PAHs) are hydrophobic contaminants that readily accumulate in the lipids of soil organisms. Here, we exposed springtails (Collembola, small soil living arthropods) to phenanthrene (a common PAH) in combination with a range of soil water contents to investigate the combined effects of these factors on the bioaccumulation, survival, recruitment, and body growth in a full factorial experiment. The results showed that phenanthrene up to 60 mg/kg dry soil had moderate effects on survival (<20%), whereas dry soil (4% soil water content) caused approximately 60% mortality. The bioaccumulation of phenanthrene increased almost 3-fold when soil water content decreased from 22 to 4%. We observed a joint effect of low soil water content and phenanthrene on recruitment, suggesting a synergistic interaction. The recruitment EC50 values of phenanthrene decreased from approximately 40 mg/kg dry soil at 22% soil water content to approximately 10 mg/kg dry soil at 12% soil water content. Our results show that the effects of phenanthrene are more pronounced in dry soil partly because bioaccumulation is enhanced when soils become dry.

Tolerance of high temperature and associated effects on reproduction in euedaphic Collembola.

Studies show that tropical and mid-latitude terrestrial ectotherms are more vulnerable to global warming than species from high latitudes. However, thermal tolerance studies from these regions still lack soil invertebrates. In the present study, we investigated six euedaphic species of Collembola (of the genera Onychiurus and Protaphorura) sampled across latitudes ranging from 31° N to 64° N and determined their upper thermal limit (UTL) by static assays. In another experiment, we submitted springtails to high temperatures for exposure times, causing 5% to 30% mortality within each species. Survivors from this series of increasing heat injuries were used to determine the time-to-first-oviposition and the number of eggs produced following heat exposure. Two hypotheses are tested in this study: 1) heat tolerance of species correlates positively with the environmental temperature of their habitat; 2) the most heat-tolerant species require less time to regain reproduction and produce more eggs than the least heat-tolerant species. Results showed that the UTL positively correlates to the soil temperature of the sampling site. The sequence of UTL60 (the temperature causing 50% mortality after 60 min of exposure) from highest to least was O. yodai > P. fimata > P. armata ≈ P. tricampata > P. macfadyeni > P. pseudovanderdrifti. Heat stress inflicted on springtails can delay reproduction in all species, and two species showed a reduced egg production rate after heat exposure. For heat stress causing up to 30% mortality, the most heat-tolerant species did not have advantages over the least heat-tolerant species for what concerns the recovery of reproduction. The relation between UTL and recovery from heat stress is not linear. Our study provides evidence for a potential long-term effect of high-temperature exposure on euedaphic species of Collembola and highlights the need for further studies on the effects of global warming on soil-living organisms.

Survival and predation rate of wild-caught and commercially produced Orius majusculus (Reuter) (Hemiptera: Anthocoridae).

The quality of biological control agents used in augmentative releases may be affected by rearing conditions due to inbreeding or laboratory adaptation, or to phenotypic effects of the rearing environment. We hypothesized that individuals from a wild population would be in better body condition and kill more prey than individuals from a commercially produced population. We caught wild Orius majusculus (Reuter) in a maize field and compared their initial body mass, survival, and prey reduction capacity to commercially produced O. majusculus. Predation capacity and survival were compared in short-term Petri dish tests with Frankliniella tenuicornis (Uzel) thrips, Ephestia kuehniella (Zeller) moth eggs, or Rhopalosiphum padi (L.) aphids as prey, and in longer-term outdoor mesocosms containing live seedling wheat grass with thrips or aphids as prey. Wild-caught O. majusculus were typically heavier and overall had higher survival during tests than commercially produced O. majusculus. Females were heavier than males and typically killed more prey. However, we found no difference between wild-caught and commercially produced individuals on prey reduction, neither in Petri dishes nor in mesocosms. Our study suggests that commercially produced O. majusculus have lower body condition than wild O. majusculus due to their lower body mass and survival, but that this does not have any negative effect on the number of pest prey killed over the timelines and conditions of our tests. Commercially produced O. majusculus thus did not have a lower impact on pest prey numbers than wild-caught individuals and therefore had similar biological control value under our study conditions.

Temperature-Dependent Toxicokinetics of Phenanthrene in Enchytraeus albidus (Oligochaeta).

Although the toxicokinetics of organic pollutants in soil invertebrates under optimal and constant temperature has been widely reported, their uptake, elimination, and bioaccumulation under suboptimal temperatures, and especially daily fluctuating temperature (FT) regimes have received only little research attention. In this study, the uptake, elimination, and bioaccumulation of phenanthrene (PHE) in Enchytraeus albidus (Oligochaeta) under different constant temperatures, and an FT regime were investigated in a natural soil. In general, the PHE concentrations in worm tissues reached steady state within 14 days at different temperatures. The uptake (ku) and elimination (ke) rate constants and the bioaccumulation increased with increasing temperature likely because of an increased diffusivity of PHE into the worms and an increased metabolic rate. Interestingly, the bioaccumulation factor of PHE in E. albidus showed a positive relationship with temperature because the slope of the ku-temperature relationship was larger than that of the ke-temperature relationship. Further, the uptake and elimination rate constants were larger under the FT regime than at the constant average of the fluctuating temperature. These findings suggest that, climatic conditions, especially daily fluctuating temperatures, should be considered for the assessment of the toxicokinetics of organic pollutants in terrestrial organisms.

Thermal optimum for mass production of the live feed organism Enchytraeus albidus.

Live feed organisms are essential for the larval stages of many fish species grown in aquaculture, and juvenile fish reared on live feeds often exhibit higher survival and growth than those reared on formulated feed. The terrestrial enchytraeid (white worm), Enchytraeus albidus, has potential as a sustainable source of live feed because it can easily be mass produced, feeds on a wide range of organic waste materials and has high contents of protein and long-chain poly-unsaturated fatty acids. In the present study, we observed the effect of temperature on population growth over five months using soil microcosms. At the outset, each microcosm was supplied with approximately the same number of cocoons. Hatched enchytraeids were given rolled oats ad libitum as feed. We followed the population growth at seven temperatures in the range of 4-25 °C and investigated body composition in order to find optimal temperature for mass production. Results showed that E. albidus has a broad thermal optimum range and displays almost similar biomass production in the range of 15-22 °C with specific growth rates between 6.5 and 6.8%. In this temperature range, protein contents were 40-45%, glycogen contents 20-25% and total fatty acid contents 15-20% of dry weight. The temperature had a highly significant effect on fatty acid composition. In particular, the abundance of omega-3 fatty acids (18:3ω3 and 20:5ω3) was largest at low temperature. For what concerns achievable density of worms in mass cultures, our results surpassed previous results and showed that densities close to 100 g L-1 substrate are realistic. Maximum production of biomass can likely reach 80 g live worms L-1 month-1 at temperatures between 15 and 22 °C.

Towards a unified study of multiple stressors: divisions and common goals across research disciplines.

Anthropogenic environmental changes, or 'stressors', increasingly threaten biodiversity and ecosystem functioning worldwide. Multiple-stressor research is a rapidly expanding field of science that seeks to understand and ultimately predict the interactions between stressors. Reviews and meta-analyses of the primary scientific literature have largely been specific to either freshwater, marine or terrestrial ecology, or ecotoxicology. In this cross-disciplinary study, we review the state of knowledge within and among these disciplines to highlight commonality and division in multiple-stressor research. Our review goes beyond a description of previous research by using quantitative bibliometric analysis to identify the division between disciplines and link previously disconnected research communities. Towards a unified research framework, we discuss the shared goal of increased realism through both ecological and temporal complexity, with the overarching aim of improving predictive power. In a rapidly changing world, advancing our understanding of the cumulative ecological impacts of multiple stressors is critical for biodiversity conservation and ecosystem management. Identifying and overcoming the barriers to interdisciplinary knowledge exchange is necessary in rising to this challenge. Division between ecosystem types and disciplines is largely a human creation. Species and stressors cross these borders and so should the scientists who study them.

Mercury (Hg2+) interferes with physiological adaptations to freezing in the arctic earthworm Enchytraeus albidus.

Freezing temperatures is an important stressor in the arctic regions and has a significant influence on the population dynamics and geographic distribution of terrestrial invertebrates. Toxic metals in the environment can interfere with protective cold-acclimation responses of organisms. It is therefore important to evaluate the combined effects of cold stress and environmental contaminants. Here, we aimed to investigate the effects of Hg (HgCl2) on various physiological aspects of freeze-tolerance in the earthworm (Enchytraeus albidus). We measured the levels of the cryoprotectant glucose, the glycogen content (source of glucose molecules for cryoprotection and fuel for metabolism), and changes in the composition of membrane phospholipid fatty acids (PLFA) as an indicator of lipid peroxidation. Freezing at -6 °C had no effect on survival in uncontaminated soil, however, survival of freezing in Hg contaminated soil was clearly reduced, especially at extended exposure times. Thus, the LC50 value in frozen soil decreased from 8.3 mg Hg kg-1 (when exposed for 17 days) to only 4.2 mg Hg kg-1 after 36 days' exposure indicating that combined effects of Hg and freezing became larger at prolonged exposure times. Hg caused a depletion of glycogen reserves (almost 50% at 12 mg kg-1 dry soil), but despite this effect worms were able to maintain a constant cryoprotectant level (about 0.12 mg glucose mg-1 dry weight) at all Hg concentrations. Hg had clear negative effects on the proportion of unsaturated PLFAs, which could be an indication of lipid peroxidation. Since a high proportion of unsaturated fatty acids in the membrane is important for invertebrate freeze-tolerance, our results suggest that the negative effect of Hg on freeze-tolerance in E. albidus is related to degraded membrane functionality at low temperature.

Fast attrition of springtail communities by experimental drought and richness-decomposition relationships across Europe.

Soil fauna play a fundamental role on key ecosystem functions like organic matter decomposition, although how local assemblages are responding to climate change and whether these changes may have consequences to ecosystem functioning is less clear. Previous studies have revealed that a continued environmental stress may result in poorer communities by filtering out the most sensitive species. However, these experiments have rarely been applied to climate change factors combining multiyear and multisite standardized field treatments across climatically contrasting regions, which has limited drawing general conclusions. Moreover, other facets of biodiversity, such as functional and phylogenetic diversity, potentially more closely linked to ecosystem functioning, have been largely neglected. Here, we report that the abundance, species richness, phylogenetic diversity, and functional richness of springtails (Subclass Collembola), a major group of fungivores and detritivores, decreased within 4 years of experimental drought across six European shrublands. The loss of phylogenetic and functional richness was higher than expected by the loss of species richness, leading to communities of phylogenetically similar species sharing evolutionary conserved traits. Additionally, despite the great climatic differences among study sites, we found that taxonomic, phylogenetic, and functional richness of springtail communities alone were able to explain up to 30% of the variation in annual decomposition rates. Altogether, our results suggest that the forecasted reductions in precipitation associated with climate change may erode springtail communities and likely other drought-sensitive soil invertebrates, thereby retarding litter decomposition and nutrient cycling in ecosystems.

Prey-specific impact of cold pre-exposure on kill rate and reproduction.

Temperature influences biological processes of ectotherms including ecological interactions, but interaction strengths may depend on species-specific traits. Furthermore, ectotherms acclimate to prevailing thermal conditions by adjusting physiological parameters, which often implies costs to other fitness-related parameters. Both predators and prey may therefore pay thermal acclimation costs following exposure to suboptimal temperatures. However, these costs may be asymmetrical between predator and prey, and between the predator and different species of concurrent prey. We investigated whether thermal pre-exposure affected subsequent kill rate and predator fitness when foraging on prey that differ in ease of capture, and whether changes were primarily caused by predator or by prey pre-exposure effects. Specifically, we were interested in whether there were interactions between predator pre-exposed temperature and specific prey. Using the mesostigmatid mite Gaeolaelaps aculeifer as a generalist predator and the collembolans Folsomia candida and Protaphorura fimata as prey, we measured the impact of present temperature, predator pre-exposure temperature, prey pre-exposure temperature (all 10 or 20°C), prey species, and all interactions on prey numbers killed, predator eggs produced, and exploitation of killed prey in a full factorial design. Mites killed P. fimata in equal numbers independent of the presence of F. candida, but killed F. candida when P. fimata was absent. Mite kill rate and reproduction were significantly affected by mite pre-exposure temperature and test temperature, but not by prey pre-exposure temperature. Significantly more of the slower prey was killed than of the quicker prey. Importantly, we found significant synergistic negative interaction effects between predator cold pre-exposure and hunting prey of higher agility on predator kill rate and reproduction. Our findings show that the negative effects of cold and cold pre-exposure on kill rate and reproduction may be more severe when predators forage on quick prey. The study implies that predator cold exposure has consequences for specific prey survival following cold due to altered predation pressures, which in nature should influence the specific prey population dynamics and apparent competition outcomes. The findings exemplify how not only current but also preceding conditions affect ecological interactions, and that effect strength depends on the species involved.

Headspace Passive Dosing of Volatile Hydrophobic Organic Chemicals from a Lipid Donor-Linking Their Toxicity to Well-Defined Exposure for an Improved Risk Assessment.

High hydrophobicity and volatility of chemicals often lead to substantial experimental challenges but were here utilized in headspace passive dosing (HS-PD) to establish and maintain exposure: the pure chemical served as a passive dosing donor for controlling exposure at saturation, whereas triglyceride oil containing the chemical was used to control lower exposure levels. These donor solutions were added to glass inserts placed in the closed test systems. Mass balance calculations confirmed a dominant donor capacity for all chemicals except isooctane. This HS-PD method was applied to algal growth inhibition and springtail lethality tests with terpenes, alkanes, and cyclic siloxanes. Headspace concentrations above the lipid donors were measured for three chemicals to determine their chemical activity, using saturated vapor as the analytical standard and thermodynamic reference. Toxicity was related to chemical activity and calculated concentrations in membranes at equilibrium with the lipid donor. For both tests and all chemicals, toxic effects were observed within or above the reported range for baseline toxicity, meaning that no excess toxicity was observed. The toxicity of siloxanes was markedly higher to the terrestrial springtail than the aquatic algae, which is consistent with a more efficient mass transfer of these volatile hydrophobic chemicals in air compared to water.

Increased lipid accumulation but not reduced metabolism explains improved starvation tolerance in cold-acclimated arthropod predators.

Predatory arthropods are used for biological control in greenhouses, but there is increasing interest to extend their use to the outdoor environment where temperatures are typically lower. Acclimation at low temperature increases the ability of ectotherms to cope with subsequent more extreme cold, but may involve costs or benefits to other performance traits. A recent study in mesostigmatid mites (Gaeolaelaps aculeifer) showed that starvation tolerance was improved following a period of cold exposure. However, the physiological mechanisms that underlie improved starvation tolerance following cold exposure were not investigated. To examine whether cold acclimation would also improve starvation tolerance in an insect, we repeated the starvation study in another arthropod predator, the pirate bug Orius majusculus, as well as in G. aculeifer. Before tests, the two species were acclimated at 10, 15, or 20 °C for 7 (G. aculeifer) or 16 (O. majusculus) days. We then analyzed the effects of thermal exposure on body composition, consumption, and basal metabolic rate in both species. Our results confirmed that exposure to low temperature improves starvation tolerance in these arthropod predators. Body composition analyses revealed that both species had accumulated larger lipid stores during exposure to colder temperature, which at least in part can explain the larger starvation tolerance following cold exposure. In contrast, consumption and basal metabolic rate were not changed by thermal acclimation. Our study indicates that predatory arthropods exposed to cold increase their physiological robustness and ability to endure environmental challenges, including low temperature and low prey availability.

Comparing Enchytraeus albidus populations from contrasting climatic environments suggest a link between cold tolerance and metabolic activity.

Basal metabolic activity and freezing of body fluids create reactive oxygen species (ROS) in freeze-tolerant organisms. These sources of ROS can have an additive negative effect via oxidative stress. In cells, antioxidant systems are responsible for removing ROS in order to avoid damage due to oxidative stress. Relatively little is known about the importance of metabolic rate for the survival of freezing, despite a good understanding of several cold tolerance related physiological mechanisms. We hypothesized that low basal metabolism would be selected for in freeze-tolerant organisms where winter survival is important for fitness for two reasons. First, avoidance of the additive effect of ROS production from metabolism and freezing, and second, as an energy-saving mechanism under extended periods of freezing where the animal is metabolically active, but unable to feed. We used the terrestrial oligochaete, Enchytraeus albidus, which is widely distributed from Spain to the high Arctic and compared eight populations originating across a broad geographical and climatic gradient after they had been cold acclimated at 5 °C in a common garden experiment. Cold tolerance (lower lethal temperature: LT50) and the potential metabolic activity (PMA, an estimator of the maximal enzymatic potential of the mitochondrial respiration chain) of eight populations were positively correlated amongst each other and correlated negatively with latitude and positively with average yearly temperature and the average temperature of the coldest month. These results indicate that low PMA in cold tolerant populations is important for survival in extremely cold environments.

Screening of cold tolerance in fifteen springtail species.

Springtails (Collembola) are ubiquitous and help ecosystem processes such as the decomposition of dead plant material. Their ability to survive low winter temperatures is an important trait that partly defines their geographic distribution. The cold tolerances of 15 laboratory-reared species of springtails were investigated. Springtails were cold acclimated in the laboratory over two months in order to simulate a seasonal change in temperature during autumn. Springtails were then exposed to decreasing sub-zero temperatures and at the same time simulating the moisture conditions in frozen soil. The cold tolerance of the species reflected well the climate of region of origin. Differential scanning calorimetry of individual springtails showed that melting points of body fluids did not become lower due to long-term cold acclimation (from 20° to 1.5°C). However, both water content and melting point of two arctic species (Hypogastrura viatica and Protaphorura macfadyeni) decreased drastically during exposure to sub-zero temperatures indicating cryoprotective dehydration (CPD). These arctic species survived exposure to - 9 °C for two weeks and - 20 °C for at least one week using CPD. Four other subarctic or cool temperate species also used CPD and survived - 9 °C for weeks, whereas springtails in culture from less cool temperate regions had poor cold tolerance.

Effects of an aged copper contamination on distribution of earthworms, reproduction and cocoon hatchability.

Contaminated soil is a problem throughout the industrialized world, and a significant proportion of these sites are polluted with heavy metals such as copper. Ecological risk assessment of contaminated sites requires ecotoxicological studies with spiked soils as well as in-situ ecological observations. Here, we report laboratory and field assessment of copper toxicity for earthworms at a Danish site (Hygum) exclusively contaminated with an increasing gradient in copper from background to highly toxic levels (>1000mgkg-1 dry soil). More specifically, we report effects on field populations, body contents of copper, hatching of earthworm cocoons and reproduction of the common species Aporrectodea tuberculata. Abundance of earthworms and cocoons decreased significantly from about 400-150m-2 along the gradient as the soil copper concentration increased from ca. 50 to ca. 1000mgkg-1. At lower concentrations, the population was dominated by endogeic species, whereas at high concentrations the population was dominated by epigeic species. At high copper contents the internal concentration of copper was in the range 100-160mgkg-1 dry tissue. Despite the high internal copper contents, hatchability of field collected cocoons was not impaired in any species. The EC50 reproduction value of A. tuberculata was about 220mg copper kg-1 dry soil in the first two exposure periods, but nearly doubled in the third period suggesting that an acclimation response had occurred. Also in the laboratory reproduction test, cocoon hatchability was not reduced, but rather slightly stimulated by copper. Based on these results we discuss the possibility that acute exposure in laboratory experiments is more detrimental than exposure in a field situation, perhaps because increased tolerance may be acquired through natural selection and genetic adaptation through increased use of defense mechanisms such as metallothioneins. Further, we discuss that the rather high tissue copper level of earthworms from the Hygum site may have smaller effects in these free-ranging worms than it would have in acute-exposure laboratory tests because the copper is more efficiently sequestered and detoxified in the field situation where populations have been exposed for many generations.

A replicated climate change field experiment reveals rapid evolutionary response in an ecologically important soil invertebrate.

Whether species can respond evolutionarily to current climate change is crucial for the persistence of many species. Yet, very few studies have examined genetic responses to climate change in manipulated experiments carried out in natural field conditions. We examined the evolutionary response to climate change in a common annelid worm using a controlled replicated experiment where climatic conditions were manipulated in a natural setting. Analyzing the transcribed genome of 15 local populations, we found that about 12% of the genetic polymorphisms exhibit differences in allele frequencies associated to changes in soil temperature and soil moisture. This shows an evolutionary response to realistic climate change happening over short-time scale, and calls for incorporating evolution into models predicting future response of species to climate change. It also shows that designed climate change experiments coupled with genome sequencing offer great potential to test for the occurrence (or lack) of an evolutionary response.

Earthworms accumulate alanine in response to drought.

Earthworms have ecologically significant functions in tropical and temperate ecosystems and it is therefore important to understand how these animals survive during drought. In order to explore the physiological responses to dry conditions, we simulated a natural drought incident in a laboratory trial exposing worms in slowly drying soil for about one month, and then analyzed the whole-body contents of free amino acids (FAAs). We investigated three species forming estivation chambers when soils dry out (Aporrectodea tuberculata, Aporrectodea icterica and Aporrectodea longa) and one species that does not estivate during drought (Lumbricus rubellus). Worms subjected to drought conditions (< -2MPa) substantially increased the concentration of FAAs and in particular alanine that was significantly upregulated in all tested species. Alanine was the most important FAA reaching 250-650µmolg(-1) dry weight in dehydrated Aporrectodea species and 300µmolg(-1) dry weight in L. rubellus. Proline was only weakly upregulated in some species as were a few other FAAs. Species forming estivation chambers (Aporrectodea spp.) did not show a better ability to conserve body water than the non-estivating species (L. rubellus) at the same drought level. These results suggest that the accumulation of alanine is an important adaptive trait in drought tolerance of earthworms in general.

Does acute lead (Pb) contamination influence membrane fatty acid composition and freeze tolerance in intertidal blue mussels in arctic Greenland?

In their natural habitats, organisms are exposed to multiple stressors. Heavy metal contamination stresses the cell membrane due to increased peroxidation of lipids. Likewise, sub-zero air temperatures potentially reduce membrane functionality in ectothermal animals. We tested if acute lead (Pb) exposure for 7 days would influence survival in intertidal blue mussels (Mytilus edulis) after exposure to realistic sub-zero air temperatures. A full factorial experiment with five tissue Pb concentrations between 0 and 3500 µg Pb/g and six sub-zero temperatures from 0 to -17 °C were used to test the hypothesis that sub-lethal effects of Pb may increase the lethality caused by freezing in blue mussels exposed to temperatures simulating Greenland winter conditions. We found a significant effect of temperature on mortality. However, the short-term exposure to Pb did not result in any effects of Pb, nor did we find interactions between Pb and temperature. We analysed the relative abundance of major phospholipid fatty acids (PLFAs) in the gill tissue, but we found no significant effect of Pb tissue concentration on PLFA composition. Results suggest that Pb accumulation has limited effects on freeze tolerance and does not induce membrane damage in terms of persistent lipid peroxidation.

Reprint of: The ins and outs of water dynamics in cold tolerant soil invertebrates.

Many soil invertebrates have physiological characteristics in common with freshwater animals and represent an evolutionary transition from aquatic to terrestrial life forms. Their high cuticular permeability and ability to tolerate large modifications of internal osmolality are of particular importance for their cold tolerance. A number of cold region species that spend some or most of their life-time in soil are in more or less intimate contact with soil ice during overwintering. Unless such species have effective barriers against cuticular water-transport, they have only two options for survival: tolerate internal freezing or dehydrate. The risk of internal ice formation may be substantial due to inoculative freezing and many species rely on freeze-tolerance for overwintering. If freezing does not occur, the desiccating power of external ice will cause the animal to dehydrate until vapor pressure equilibrium between body fluids and external ice has been reached. This cold tolerance mechanism is termed cryoprotective dehydration (CPD) and requires that the animal must be able to tolerate substantial dehydration. Even though CPD is essentially a freeze-avoidance strategy the associated physiological traits are more or less the same as those found in freeze tolerant species. The most well-known are accumulation of compatible osmolytes and molecular chaperones reducing or protecting against the stress caused by cellular dehydration. Environmental moisture levels of the habitat are important for which type of cold tolerance is employed, not only in an evolutionary context, but also within a single population. Some species use CPD under relatively dry conditions, but freeze tolerance when soil moisture is high.

Effect of repeated freeze-thaw cycles on geographically different populations of the freeze-tolerant worm Enchytraeus albidus (Oligochaeta).

Freeze-tolerant organisms survive internal ice formation; however, the adaptations to repeated freeze-thaw cycles are often not well investigated. Here we report how three geographically different populations of Enchytraeus albidus (Germany, Iceland and Svalbard) respond to three temperature treatments - constant thawed (0°C), constant freezing (-5°C) and fluctuating temperature (0 to -5°C) - over a period of 42 days. Survival varied between treatments and populations such that enchytraeids from arctic locations had a higher survival following prolonged freeze periods compared with temperate populations. However, enchytraeids from temperate locations had the same survival rate as arctic populations when exposed to repeated freeze-thaw events. Across all populations, metabolic rate decreased markedly in frozen animals (-5°C) compared with thawed controls (0°C). This decrease is likely due to the lower temperature of frozen animals, but also to the transition to the frozen state per se. Animals exposed to repeated freeze-thaw events had an intermediate metabolic rate and freeze-thaw events were not associated with pronounced excess energetic costs. Overwintering under either condition was not associated with a decrease in lipid content; however, during exposure to constant freezing and repeated freeze-thaw events there was a noticeable decrease in carbohydrate stores over time. Thus, animals exposed to constant freezing showed a decrease in glycogen stores, while both glucose and glycogen content decreased over time when the organisms were exposed to repeated freezing. The results therefore suggest that carbohydrate resources are important as a fuel for E. albidus during freezing whereas lipid resources are of marginal importance.