Martin Holmstrup

Drought acclimation confers cold tolerance in the soil collembolan Folsomia candida.

It has been noted that both summer drought and sub-zero winter temperatures induce the synthesis of sugars and polyols in invertebrate tissues. This has led several authors to suggest that many of the adaptations, previously viewed as a response to cold, might be part of a more universal desiccation tolerance mechanism. Here we show that acclimation of the soil dwelling collembolan Folsomia candida to a sublethal desiccation stress confers tolerance to cold shock and a significant increase in the molar percent of membrane fatty acids with a mid-chain double bond. These changes in membrane fatty acids are interpreted as conferring a significant reduction in the transition temperature of cell membranes, as would be expected in acclimation to cold, and these changes are therefore interpreted as contributing to the cross-tolerance. Drought acclimation was also shown to trigger the synthesis of the 70kDa family of heat-shock proteins (Hsp70). This group of heat shock proteins is implicated in the reestablishment of the normal three-dimensional structure of partially unfolded proteins and therefore are also likely to contribute to the observed cross-tolerance. This study provides evidence that the stresses exerted by desiccation and cold at the cellular level have sufficient similarities to induce overlapping adaptations.

Effects of acclimation temperature on thermal tolerance and membrane phospholipid composition in the fruit fly Drosophila melanogaster

Adaptative responses of ectothermic organisms to thermal variation typically involve the reorganization of membrane glycerophospholipids (GPLs) to maintain membrane function. We investigated how acclimation at 15, 20 and 25 degrees C during preimaginal development influences the thermal tolerance and the composition of membrane GPLs in adult Drosophila melanogaster. Long-term cold survival was significantly improved by low acclimation temperature. After 60 h at 0 degrees C, more than 80% of the 15 degrees C-acclimated flies survived while none of the 25 degrees C-acclimated flies survived. Cold shock tolerance (1h at subzero temperatures) was also slightly better in the cold acclimated flies. LT50 shifted down by ca 1.5 degrees C in 15 degrees C-acclimated flies in comparison to those acclimated at 25 degrees C. In contrast, heat tolerance was not influenced by acclimation temperature. Low temperature acclimation was associated with the increase in proportion of ethanolamine (from 52.7% to 58.5% in 25 degrees C-acclimated versus 15 degrees C-acclimated flies, respectively) at the expense of choline in GPLs. Relatively small, but statistically significant changes in lipid molecular composition were observed with decreasing acclimation temperature. In particular, the proportions of glycerophosphoethanolamines with linoleic acid (18:2) at the sn-2 position increased. No overall change in the degree of fatty acid unsaturation was observed. Thus, cold tolerance but not heat tolerance was influenced by preimaginal acclimation temperature and correlated with the changes in GPL composition in membranes of adult D. melanogaster.

Dehydration of earthworm cocoons exposed to cold: a novel cold hardiness mechanism

Mechanisms involved in cold hardiness of cocoons of the lumbricid earthworm Dendrobaena octaedra were elucidated by osmometric and calorimetric studies of water relations in cocoons exposed to subzero temperatures. Fully hydrated cocoons contained ca. 3 g water · g dry weight-1; about 15% of this water (0.5 g·g dry weight-1) was osmotically inactive or “bound”. The melting point of the cocoon fluids in fully hydrated cocoons was-0.20°C. Exposure to frozen surroundings initially resulted in supercooling of the cocoons dehydrated (as a result of the vapour pressure difference at a given temperature between supercooled water and ice) to an extent where the vapour pressure of water in the body fluids was in equilibrium with the surrounding ice. This resulted in a profound dehydration of the cocoons, even at mild freezing exposures, and a concomitant slight reduction in the amount of osmotically inactive water. At temperatures around-8°C, which cocoons readily survive, almost all (>97%) osmotically active water had been withdrawn from the cocoons. It is suggested that cold injuries in D. octaedra cocoons observed at still lower temperatures may be related to the degree of dehydration, and possibly to the loss of all osmotically active water. The study indicates that ice formation in the tissues is prevented by equilibrating the body fluid melting point with the exposure temperature. This winter survival mechanism does not conform with the freeze tolerance/freeze avoidance classification generally applied to cold-hardy poikilotherms. Implications of this cold hardiness mechanism for other semi-terrestrial invertebrates are discussed.

Interactions between toxic chemicals and natural environmental factors — A meta-analysis and case studies

The paper addresses problems arising from effects of natural environmental factors on toxicity of pollutants to organisms. Most studies on interactions between toxicants and natural factors, including those completed in the EU project NoMiracle (Novel Methods for Integrated Risk Assessment of Cumulative Stressors in Europe) described herein, showed that effects of toxic chemicals on organisms can differ vastly depending purely on external conditions. We compiled data from 61 studies on effects of temperature, moisture and dissolved oxygen on toxicity of a range of chemicals representing pesticides, polycyclic aromatic hydrocarbons, plant protection products of bacterial origin and trace metals. In 62.3% cases significant interactions (p< or =0.05 or less) between natural factors and chemicals were found, reaching 100% for the effect of dissolved oxygen on toxicity of waterborne chemicals. The meta-analysis of the 61 studies showed that the null hypothesis assuming no interactions between toxic chemicals and natural environmental factors should be rejected at p=2.7 x 10(-82) (truncated product method probability). In a few cases of more complex experimental designs, also second-order interactions were found, indicating that natural factors can modify interactions among chemicals. Such data emphasize the necessity of including information on natural factors and their variation in time and across geographic regions in ecological risk assessment. This can be done only if appropriate ecotoxicological test designs are used, in which test organisms are exposed to toxicants at a range of environmental conditions. We advocate designing such tests for the second-tier ecological risk assessment procedures.

Physiology of cold hardiness in earthworms

Abstract Physiological, biochemical and behavioral mechanisms involved in the winter survival of earthworms and earthworm cocoons are reviewed. Overwintering strategies of cold hardy invertebrates are commonly divided into two groups. In freeze-avoiding animals, ice formation in the tissues is lethal and winter survival is dependent on prolonged and extensive supercooling. Freeze-tolerant animals tolerate extracellular freezing up to a certain fraction of the body water. Hatched earthworms belong to the first group, having a behaviorally-based strategy, escaping frost in the soil by migration. The cold hardiness mechanism of earthworm cocoons is based on a protective dehydration of cocoons occurring when cocoons are exposed to subzero temperatures in a frozen environment. Due to the lower vapour pressure of ice compared to supercooled water at a given temperature, water evaporates from the cocoon surface and condenses onto ice in the surroundings. Dehydration of cocoons continues until vapour pressure equilibrium between cocoon fluids and surrounding ice is attained. At equilibrium the cocoons cannot freeze since the melting point of cocoon fluids equals ambient temperature. Because the melting point of cocoon fluids is high, −0.2 to −0.4°C, the cocoons lose substantial amounts of water, even at mild freezing exposures. As a response to dehydration the embryos of the cocoons accumulate a polyol, probably sorbitol, which may prevent deleterious effects of the extensive water loss coupled with vapour equilibration. Since dehydration is a key event when earthworm cocoons are exposed to low temperatures it is likely that tolerance to drought is important for cold survival. Substantial support for this hypothesis has been found in Dendrobaena octaedra which is far more tolerant to drought and cold than other investigated species. It is suggested that the described “protective dehydration mechanism” of cold hardiness should be considered in cold hardiness studies of other soil animals with high cuticular evaporation rates.

Reorganization of membrane lipids during fast and slow cold hardening in Drosophila melanogaster

Abstract Rapid cold hardening is a naturally occurring phenomenon in insects that is thought to be responsible for increased cold tolerance during diurnal variations in temperature. The underlying physiological mechanisms are still not fully resolved but, in Drosophila melanogaster (Meigen 1830), rapid cold hardening is accompanied by specific changes in the membrane lipid composition. To further understand the link between rapid cold hardening and adjustments in the membrane lipid composition, the present study investigates how different rates of cooling affect thermotolerance and the composition of phospholipid fatty acids. Female Drosophila are cooled gradually from 25 to 0 °C at 0.01, 0.05, 0.1 or 0.5 °C min−1, respectively, and, subsequently, phospholipid fatty acid composition and survival after a 1‐h cold shock at −5 °C is measured. The rapid cold hardening treatments all influence cold tolerance differently so that short and intermediate rapid cold hardening treatments (0.05, 0.1 or 0.5 °C min−1 cooling rates) increase cold shock survival, whereas the slow cooling treatment (0.01 °C min−1) decreases survival relative to an untreated control. The intermediate rapid cold hardening treatments (0.05 or 0.1 °C min−1) induce a similar type of response characterized by an increase in the molar percentage of linoleic acid, 18:2(n‐6), at the expense of 16:0 and 18:1(n‐9), which leads to an increase in the degree of unsaturation. The slowest cooling treatment (0.01 °C min−1) results in a large increase in cis‐16:1(n‐7) and significant reductions in the saturated phospholipid fatty acids 16:0, 18:0 and the unsaturated 16:1(n‐9) and 18:2(n‐6) fatty acids. These changes cause a slight decrease in the average length of the phospholipid fatty acids and an increase in the overall ratio of unsaturated vs. saturated fatty acids. These findings demonstrate that the rate of cooling is important for both the reorganization of membrane lipids, and for the degree of acquired cold tolerance during rapid cold hardening, and they suggest an important role for rapid cold hardening during diurnal rather than seasonal temperature changes.

Enhanced drought tolerance of a soil-dwelling springtail by pre-acclimation to a mild drought stress

The springtail Folsomia candida has a highly permeable cuticle, but is able to survive several weeks at 98.2%RH. This corresponds to a water potential deficit of about 17bars between the environment and the normal osmotic pressure of the body fluids of this animal. Recent studies have shown a water vapour absorption mechanism by accumulation of sugars and polyols (SP) in F. candida, which explains how this species can survive dehydrating conditions. In the present study, adult F. candida were pre-acclimated at 98.2%RH to induce the accumulation of SP, and were subsequently exposed for additional desiccating conditions from 98 to 94%RH. Activity level, water content, osmotic pressure of body fluids and SP composition were investigated. After the desiccation period, the animals were rehydrated at 100%RH and survival was assessed. The results showed that F. candida survived a more severe drought stress when it had been pre-acclimated to 98.2%RH before exposure to lower humidity. This species was able to maintain hyperosmosity to the surroundings at 95.5%RH, suggesting that it can absorb water vapour down to this limit. Below this limit, trehalose levels increased while myo-inositol levels decreased. We propose that this is a change of survival strategy where F. candida at mild desiccation levels seek to retain water by colligative means (remain hyperosmotic), but at severe desiccation levels switches to an anhydrobiotic strategy.

Rearing of flounder (Platichthys flesus) juveniles in semiextensive systems

A low-technology rearing system was implemented for rearing juvenile flounder for stock enhancement in a Danish fjord, the Limfjord. Each year during 1996–2002, between 13,000 and 153,000 juveniles were reared from the yolk-sac stage until metamorphosis in outdoor ponds relying on phyto- and zooplankton blooms as their main food source. In contrast to other similar systems, the blooms in this system are closely monitored and, to a certain extent, regulated. The zooplankton blooms consisted mainly of calanoid copepods, dominated by the species Temora longicornis and Centropages hamatus. Most juveniles produced (>99.5%) were normally pigmented with average yearly survival rates from hatch to metamorphosis varying from 7 ± 9% to 48 ± 18%, lowest in the first years of production.

Body metal concentrations and glycogen reserves in earthworms (Dendrobaena octaedra) from contaminated and uncontaminated forest soil.

Stress originating from toxicants such as heavy metals can induce compensatory changes in the energy metabolism of organisms due to increased energy expenses associated with detoxification and excretion processes. These energy expenses may be reflected in the available energy reserves such as glycogen. In a field study the earthworm, Dendrobaena octaedra, was collected from polluted areas, and from unpolluted reference areas. If present in the environment, cadmium, lead and copper accumulated to high concentrations in D. octaedra. In contrast, other toxic metals such as aluminium, nickel and zinc appeared to be regulated and kept at low internal concentrations compared to soil concentrations. Lead, cadmium and copper accumulation did not correlate with glycogen reserves of individual worms. In contrast, aluminium, nickel and zinc were negatively correlated with glycogen reserves. These results suggest that coping with different metals in earthworms is associated with differential energy demands depending on the associated detoxification strategy.

Bioinformatics and protein expression analyses implicate LEA proteins in the drought response of Collembola

Humidity has a large impact on the distribution and abundance of terrestrial invertebrates, but the molecular mechanisms governing drought resistance are not fully understood. Some attention has been given to the role of the heat shock response as a component of desiccation tolerance, but recent focus has been on the chaperone-like LEA (late embryogenesis abundant) proteins in anhydrobiotic animals. This study investigates the expression of putative LEA proteins as well as the heat shock protein Hsp70 during drought stress in soil and surface dwelling species of Collembola (springtails). In silico analysis of four EST candidates from two species of Collembola showed the presence of a Group 3 LEA protein in Megaphorura arctica. In common with other Group 3 LEA proteins, the new sequence is predicted to be 100% natively unfolded, with a strong degree of lysine and alanine periodicity and with a negative average hydrophobicity of -1.273. The sequence clusters with members of the Group 3 LEA in plants. Furthermore, cross-species Western blotting showed drought-induced expression of putative LEA proteins in six species of Collembola. In the surface dwelling species, Orchesella cincta, degree of dehydration and length of exposure correlated with level of putative LEA protein. Hsp70 was also found to increase in individuals of O. cincta and Folsomia candida that had been exposed to drought conditions for 6 days. These results show the presence of a LEA protein-coding region in Collembola, but also indicate that several proteins are involved in response to dehydration stress, including Hsp70.