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Freeze-tolerance in the subarctic earthworm Eisenia nordenskioeldi (Eisen)
Abstract
The survival of freezing in the Siberian earthworm Eisenia nordenskioeldi was tested under dry conditions and in frozen soil. Worms cooled under dry conditions to -4.5°C for 24 hours had a mean temperature of crystallisation of -3.5°C. The survival rate of animals exposed in frozen soil for 56 days was 50 to 82 % at temperatures between -4 and -10°C. After warming to summer temperatures, animals previously frozen produced viable egg capsules (cocoons). This is the first evidence for freeze-tolerance in an earthworm species.
... Many species also produce cold-tolerant cocoons which may enable populations to persist in cold regions. However, previous studies (Berman and Leirikh, 1985;Holmstrup and Petersen, 1997;Holmstrup et al., 1999) have documented that a Siberian species, Eisenia nordenskioeldi (Eisen), living in areas with permafrost, has developed freeze-tolerance as a mechanism for survival in this extreme habitat, where more than one summer season is required to complete a life cycle (Mazantzeva, 1985;Holmstrup and Petersen, 1997). A recent study has shown that E. nordenskioeldi synthesizes and accumulates glucose as an immediate response to ice formation in extracellular body¯uids (Holmstrup et al., 1999). ...
... Many species also produce cold-tolerant cocoons which may enable populations to persist in cold regions. However, previous studies (Berman and Leirikh, 1985;Holmstrup and Petersen, 1997;Holmstrup et al., 1999) have documented that a Siberian species, Eisenia nordenskioeldi (Eisen), living in areas with permafrost, has developed freeze-tolerance as a mechanism for survival in this extreme habitat, where more than one summer season is required to complete a life cycle (Mazantzeva, 1985;Holmstrup and Petersen, 1997). A recent study has shown that E. nordenskioeldi synthesizes and accumulates glucose as an immediate response to ice formation in extracellular body¯uids (Holmstrup et al., 1999). ...
Two earthworm species, the freeze-intolerant Lumbricus rubellus (Hoffmeister) and the freeze-tolerant Eisenia nordenskioeldi (Eisen), were compared with respect to the effects of temperature on lipid composition. The animals were incubated at either 0°C or 20°C for 28 days, and then analyzed for the composition of free and ester-linked long-chain fatty acids, as well as other lipids with similar properties. Inter-species differences dominated the patterns of lipid composition, and the two species responded similarly to incubation temperature with respect to changes in lipid composition. In particular, polyunsaturated fatty acids were present in higher concentrations at 0°C, while straight-chain saturated fatty acids were more common at 20°C. The results did not implicate specialized lipid adjustments in the freeze-tolerance of E. nordenskioeldi.
... Most earthworms are freeze-intolerant organisms (Lee 1985;Holmstrup and Zachariassen 1996). At present only one species, the Siberian earthworm Eisenia nordenskioeldi Eisen, is known to be freeze-tolerant (Berman and Leirikh 1985;Holmstrup and Petersen 1997). The physiological mechanisms promoting freeze tolerance in E. nordenskioeldi, or any other oligochaete, have not previously been investigated. ...
... It should be stressed that the experiments undertaken in the present study were not designed to assess freeze tolerance in terms of survival. Previous studies show that E. nordenskioeldi is able to survive freezing at temperatures down to A30°C for months when the worms are exposed in soil at natural cooling and thawing rates (Berman and Leirikh 1985;Holmstrup and Petersen 1997). ...
In this paper we present the results of physiological responses to winter acclimation and tissue freezing in a freeze-tolerant
Siberian earthworm, Eisenia nordenskioeldi, and two freeze-intolerant, temperate earthworm species, Lumbricus rubellus and Aporrectodea caliginosa. By analysing the physiological responses to freezing of both types we sought to identify some key factors promoting freeze
tolerance in earthworms. Winter acclimation was followed by a significant increase in osmolality of body fluids in E. nordenskioeldi, from 197 mosmol kg−1 in 10 °C-acclimated animals to 365 mosmol kg−1 in animals acclimated to 0 °C. Cold acclimation did not cause any change in body fluid osmolality in the two freeze-intolerant
species. As a response to ice formation in the body, the freeze-intolerant species produced copious amounts of slime and expulsion
of coelomic fluids, and thereby lost 10–30% of their total water content. Contrary to this, the freeze-tolerant species did
not lose water upon freezing. At temperatures down to −6.5 °C, the ice content in the freeze-tolerant E. nordenskioeldi was significantly lower than in L. rubellus. At lower temperatures there were no differences in ice content between the two species. Cold acclimated, but unfrozen, specimens
of all three species had low levels of ammonia, urea, lactate, glycerol and glucose. As a response to ice formation, glucose
levels significantly increased within the first 24 h of freezing. This was most pronounced in E. nordenskioeldi where a 153-fold increase of glucose was seen (94 mmol · l−1). In L. rubellus and A. caliginosa a 19-fold and 17-fold increase in glucose was seen. This is the first study on physiological mechanisms promoting freeze
tolerance in E. nordenskioeldi, or any other oligochaete. Our results suggest that the cryoprotective system of this species more closely resembles that
of freeze-tolerant anurans, which synthesize cryoprotectants only after tissues begin to freeze, than that of cold-hardy invertebrates
which exhibit a preparatory accumulation of cryoprotectants during seasonal exposure to low temperature.
... It is possible that other aspects of cold hardiness, e.g., the ability to survive freezing for extended time, would give a different result (Fisker et al. 2014b); however, this aspect was beyond the scope of the present study. The populations of E. albidus studied here displayed remarkable cold hardiness (LLT50 < − 25 °C in Arctic populations) that is comparable to or better than other freeze-tolerant earthworms from Arctic and subarctic areas (Berman and Leirikh 1985;Coulson and Birkemoe 2000;Holmstrup and Petersen 1997). Even in the populations from some temperate regions (e.g., southern Sweden) LLT50 were around − 20 °C. ...
Tolerance to thermal extremes is critical for the geographic distributions of ectotherm species, many of which are probably going to be modified by future climatic changes. To predict species distributions it is important to understand the potential of species to adapt to changing thermal conditions. Here, we tested whether the thermal tolerance traits of a common freeze-tolerant potworm were correlated with climatic conditions and if adaptation to extreme cold constrains the evolutionary potential for high temperature tolerance. Further, we tested if evolution of thermal tolerance traits is associated with costs in other fitness traits (body size and reproduction). Lastly, we tested if slopes of temperature-survival curves (i.e., the sensitivity distribution) are related to tolerance itself. Using 24 populations of the potworm, Enchytraeus albidus Henle (Enchytraeidae), collected from a wide range of climatic conditions, we established a common garden experiment in which we determined high and low temperature tolerance (using survival as endpoint), average reproductive output and adult body size. Heat tolerance was not related to environmental temperatures whereas lower lethal temperature was about 10 °C lower in Arctic populations than in populations from temperate regions. Reproduction was not related to environmental temperature, but was negatively correlated with cold tolerance. One explanation for the trade-off between cold tolerance and reproduction could be that the more cold-hardy populations need to channel energy to large glycogen reserves at the expense of less energy expenditure for reproduction. Adult body size was negatively related to environmental temperature. Finally, the slopes of temperature-survival curves were significantly correlated with critical temperature limits for heat and cold tolerance; i.e., slopes increased with thermal tolerance. Our results suggest that relatively heat-sensitive populations possess genetic variation, leaving room for improved heat tolerance through evolutionary processes, which may alleviate the effects of a warmer future climate in the Arctic. On the other hand, we observed relatively narrow sensitivity distributions (i.e., less variation) in the most heat tolerant populations. Taken together, our results suggest that both cold and heat tolerance can only be selected for (and improved) until a certain limit has been reached.
... When temperatures are low, earthworms employ two main strategies: (1) freeze avoidance either by migration or physiological adaptation, such as the synthesis of cryoprotectants to avoid the formation of ice within the animal; and (2) allowing the formation of extracellular ice (Mazur, 1963;Holmstrup and Zachariassen, 1996). Glucose loading appears to be essential for freezing tolerance in earthworms, but other factors may also be involved (Berman and Leirikh, 1985;Holmstrup and Petersen, 1997;Holmstrup et al., 1999). According to Holmstrup (2014) there seems to be a common response between cold tolerance and desiccation tolerance in soil invertebrates, such as initiating same modifications in membrane composition. ...
Nowadays, extreme weather events caused by climate change are becoming more frequent. This leads to the occurrence of extreme habitats to which species must adapt. This challenge becomes crucial for species living in unstable environments, such as the riparian earthworm Eiseniella tetraedra. Its cosmopolitan distribution exposes it to various environmental changes, such as freezing in subarctic regions or droughts in Mediterranean areas. Transcriptional changes under cold and desiccation conditions could therefore shed light on the adaptive mechanisms of this species. An experiment was performed for each condition. In the cold experiment, the temperature was lowered to −14 °C ± 2 °C (compared to 8 °C for control samples), and in the desiccation treatment, humidity was lowered from 60% to 15%. Comparisons of gene expression levels between earthworms under freezing conditions and control earthworms revealed a total of 84 differentially expressed genes and comparisons between the desiccation experiment and the control yielded 163 differentially expressed genes. However, no common responses were found between the two treatments. The results suggest that E. tetraedra can acclimate to low temperatures due to the upregulation of genes involved in glucose accumulation. However, downregulation of the respiratory chain suggests that this earthworm does not tolerate freezing conditions. Under desiccation conditions, genes involved in cell protection from apoptosis and DNA repair were upregulated. In contrast, lipid metabolism was downregulated, presumably to conserve resources by reducing the rate at which they are consumed.
... It is, therefore, of interest to understand how earthworms can survive winters in these cold areas. Several earthworm species survive sub-zero temperatures using a strategy known as "freeze-tolerance" (Berman and Leirikh, 1985), (Holmstrup and Petersen, 1997), (Rasmussen and Holmstrup, 2002). Freeze-tolerance means that the animal tolerates internal, but only extracellular, freezing of body fluids, which induces cellular dehydration (Zachariassen, 1985). ...
... Different earthworms have their own optimum temperature (Lee, 1985). A Siberian species, Eisenia nordenskioeldi (Eisen) lives in the areas with permafrost and has developed freezetolerance mechanism for survival in the extreme habitat, where more than one summer season is required to complete a lifecycle (Mazantzeva, 1985;Holmstrup and Petersen, 1997). Holmstrup et al. (1999) reported that E. nordenskioeldi synthesized and accumulated glucose as an immediate response to ice formation in extracellular body fluids. ...
The effects of varying temperatures (12 - 44° C) on the specific activity of cytoplasmic malate dehydrogenase ((cMDH), mitochondrial malate dehydrogenase (mMDH) and lactate dehydrogenase (LDH) of some earthworms (Metaphire posthuma, Perionyx sansibaricus and Lampito mauritii) were studied. The effects of different temperatures on supernatant and mitochondrial protein contents were also investigated. The specific activities of cMDH, mMDH and LDH of the earthworms decreased gradually as a function of increasing temperature from 12 to 44°C. Higher metabolic energy was needed to maintain the activity at low temperatures. Hence, the earthworms showed increased enzyme specific activity at low temperatures. However, the protein content increased upto 28°C. Afterwards, with the increase in the temperature from 28 to 42°C, the proteins in the earthworms showed a significant decrease. The temperature-associated changes in the protein content could be explained by the fact that protein synthesizing capacity was hampered above and below the optimum temperature range. The most pronounced effects of varying temperatures were on P. sansibaricus. It might be due to the epigeic nature of the earthworm species. Then minimum effect was on the endogeic earthworm M. posthuma. Virtually, the differences in the enzymes physiology were associated with the differences in the ecological categories of the earthworms. This clearly demonstrate a possible link between the physiology and ecology at aerobic (cMDH, mMDH) and anaerobic (LDH) levels in the tropical earthworms.
... The downward migration prior to frost, also noted by earlier authors (Dash and Cragg 1972), indicates an avoidance response of the worms to low temperatures. In addition, cocoons were found to be the most important overwintering stage in an earlier study from the sub-Arctic (Klungland 1981), and with few exceptions, are considered the only overwintering stage in sub-Arctic earthworms (Edwards and Bohlen 1996;Holmstrup and Petersen 1997). ...
We report the results of the first study of the population dynamics and life cycles of Arctic enchytraeid populations. Sampling was undertaken in a Salix heath in Adventdalen, Svalbard, during one summer and the succeeding spring. In addition, a Cassiope heath at a more northerly site close to Ny-Alesund, Svalbard, was sampled twice. The Arctic enchytraeids were generally smaller at maturity than their temperate-zone relatives. The three most numerous species in the Salix heath, Henlea perpusilla, Henlea glandulifera, and Bryodrilus parvus, hatched from cocoons in early summer and attained adult size early in their second summer. A few H. perpusilla and H. glandulifera reached mature size in their first summer; since the summer of investigation was unusually cold, these species may have a 1-year life cycle in warmer years. Life cycles were apparently longer in the Cassiope heath than in the Salix heath. Henlea perpusilla, H. glandulifera, and B. parvus produced eggs throughout the summer in the Salix heath, though hatching was restricted to early summer. Therefore, the hypothesis that cocoons require a cold period to hatch was tested in a laboratory experiment. When soil containing cocoons was incubated at -5 degreesC for 3 weeks, a significant increase in juveniles was demonstrated for H. perpusilla and Bryodrilus diverticulatus compared with soils kept at constant summer temperatures. This is the first time that breaking of dormancy by an external stimulus has been demonstrated in enchytraeid cocoons.
... The appearance of large numbers of earthworms under rocks in the winter is unexpected. Earthworms are soft bodied and susceptible to freezing in all but one species studied (Holmstrup & Petersen 1997), and thus may be expected to burrow to a more thermally buffered environment. However, moister conditions in winter may render deeper layers of the soil waterlogged and anoxic, making the under-rock environment preferable. ...
Microhabitat selection is important for invertebrates in both summer and winter, but has not been investigated extensively in alpine terrestrial communities. We investigated the factors affecting microhabitat selection by alpine invertebrates in a system of rock slab pavement on the Rock and Pillar Range, Central Otago, New Zealand. We found fewer individuals of most groups under the 379 rocks re-examined during the winter. Logistic regression was used to model the presence of the cockroach Celatoblatta quinquemaculata (Dictyoptera: Blattidae); the alpine weta Hemideina maori (Orthoptera: Anostostomatidae); and the spider Neoramia childi (Aranea: Agelenidae). We found that rock refuge size and season were extremely important in determining presence of all of these species, and that both abiotic and biotic aspects of rock microhabitat were necessary to describe occupancy. We also found clear evidence of biological interactions between the three species modelled. The marked decrease in under-rock occupancy by C. quinquemaculata in the winter is hypothesised to be a result of selection of alternative (non-rock) microhabitats
... Specific responses to extreme physico-chemical factors are also of interest. Lee (1985 p. 44) reports Ghilarov's claim that Eisenia nordenskioldi revives after long periods of being frozen, with freeze tolerance down to -30°C recorded for E. nordenskioldi (subspecies?) by Holmstrup and Petersen (1997) and Berman and Leirikh (1985). Berman et al. (2002) further report on adaptation to arid conditions. ...
Two new megadrile earthworms from the steppes, the first species wholly from Outer Mongolia, are ascribed to the partially parthenogenetic Eisenia nordenskioldi (Eisen, 1879) species-complex. Taxonomic justification of sympatric Eisenia nordenskioldi mongol and Eisenia nordenskioldi onon
ssp. n. are supported by mtDNA COI barcodes. The unreliability of molecular differentiation based on voucher names compared to definitive types is again demonstrated, as pertains to the ultimate Eisenia andrei Bouché, 1972 synonym of the Eisenia fetida (Savigny, 1826) sibling species-complex composed of more than a dozen prior names. Similar species described from Northeast China [formerly Manchuria] and North Korea are briefly considered, albeit they are intermittently held in synonymy of cosmopolitan Aporrectodea rosea (Savigny, 1826) along with many other taxa including some exotic lumbricids initially found in India. Japanese and North American lumbricids are also mentioned. Distributions are discussed and an annotated checklist of all nine Siberian/sub-arctic Eisenia nordenskioldi ssp. is appended.
... However, a few species remain in the upper soil layers (Rundgren, 1975) and these may therefore experience freezing of their body fluids, as the intimate contact with ice crystals in the frozen soil makes supercooling unlikely. Indeed, some species like Dendrobaena octaedra and Eisenia nordenskioldi are able to tolerate freezing at low subzero temperatures for extended periods (Berman and Leirikh, 1985;Holmstrup and Petersen, 1997;Holmstrup et al., 1999;Berman et al., 2001;Rasmussen and Holmstrup, 2002). ...
Geographic variation in freeze tolerance, glycogen storage and freeze-induced glucose mobilisation was investigated in the earthworm Dendrobaena octaedra. Specimens from 15 populations collected in Canada, Greenland and Europe were reared in the laboratory in a common-garden experiment to test whether glucose and glycogen concentrations correlated with genetic variation in freeze tolerance among the populations. Populations from Canada, Sweden, Poland and Finland did not differ much in their freeze tolerance and were able to tolerate freezing for 18d down to at least −14°C (lowest temperature tested). Specimens collected in a relatively warm climate (Denmark) were the least freeze tolerant, and also had the lowest concentrations of glucose when frozen at −2°C. However, there was no clear evidence that glucose concentration is a determinant in the degree of freeze tolerance of D. octaedra when considering the whole assemblage of populations. The role of phylogenetic inertia was tested by looking for serial independence and no influence of phylogeny was detected in our findings allowing us to exclude the possibility that phylogenetic relatedness between populations is a major evolutionary factor explaining the observed differences for freeze tolerance and related traits. The size of the glycogen reserve was significantly correlated with the ability to tolerate freezing. Large glycogen reserves may be advantageous in very cold regions in order to maximise cryoprotectant production and/or as a source of energy for the anaerobic metabolism occurring during prolonged freezing.
... The downward migration prior to frost, also noted by earlier authors (Dash and Cragg 1972), indicates an avoidance response of the worms to low temperatures. In addition, cocoons were found to be the most important overwintering stage in an earlier study from the sub-Arctic (Klungland 1981), and with few exceptions, are considered the only overwintering stage in sub-Arctic earthworms (Edwards and Bohlen 1996;Holmstrup and Petersen 1997). ...
We report the results of the first study of the population dynamics and life cycles of Arctic enchytraeid populations. Sampling was undertaken in a Salix heath in Adventdalen, Svalbard, during one summer and the succeeding spring. In addition, a Cassiope heath at a more northerly site close to Ny-Ålesund, Svalbard, was sampled twice. The Arctic enchytraeids were generally smaller at maturity than their temperate-zone relatives. The three most numerous species in the Salix heath, Henlea perpusilla, Henlea glandulifera, and Bryodrilus parvus, hatched from cocoons in early summer and attained adult size early in their second summer. A few H. perpusilla and H. glandulifera reached mature size in their first summer; since the summer of investigation was unusually cold, these species may have a 1-year life cycle in warmer years. Life cycles were apparently longer in the Cassiope heath than in the Salix heath. Henlea perpusilla, H. glandulifera, and B. parvus produced eggs throughout the summer in the Salix heath, though hatching was restricted to early summer. Therefore, the hypothesis that cocoons require a cold period to hatch was tested in a laboratory experiment. When soil containing cocoons was incubated at -5°C for 3 weeks, a significant increase in juveniles was demonstrated for H. perpusilla and Bryodrilus diverticulatus compared with soils kept at constant summer temperatures. This is the first time that breaking of dormancy by an external stimulus has been demonstrated in enchytraeid cocoons.
... Most earthworm species are susceptible to freezing but a few species occurring in temperate and sub-arctic regions are known to be freeze-tolerant. The earthworm Eisenia nordenskioldi from Siberia is reported to tolerate temperatures down to -25°C (1,17). Recently, Siberian populations of the cosmopolitan Dendrobaena octaedra have been reported as freeze tolerant down to -12°C (2). ...
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.
The distribution of soil invertebrates from different taxa and the adaptive potential of all three cold hardiness strategies in the cold climate of northeastern Asia are analyzed. The correlation between the resistance to low temperatures and the overwintering conditions in habitats of some species is studied. The mechanisms and degree of cold hardiness are shown generally to be unrelated to the taxonomic proximity. The effect of the resistance to low wintering temperatures on the habitat distribution and faunogenesis of soil invertebrates in permafrost regions is discussed.
Data on the occurrence and adaptive potential of three mechanisms of cold-hardiness are analyzed in different taxa of soil invertebrates under the conditions of the cold climate of Northeast Asia. The relationship between low temperature resistance and overwintering conditions in selected species is discussed. It is shown that taxonomically close species may have different mechanisms and values of cold-hardiness. The effect of winter low temperature resistance on the biotopical distribution and formation of the soil invertebrate fauna in permafrost regions is assessed.
The aim of this paper is to give a mini-review of the physiological adaptations to frost in earthworms. Low temperature is one of the most important environmental parameters determining the distribution of earthworms. In temperate areas many species avoid frozen soil by migrating to deeper soil layers during winter. Earthworm species of cold regions where the soil is frozen in winter have been shown to be freeze-tolerant, i.e. they endure ice formation in extracellular body fluids. Eisenia nordenskioldi and Dendrobaena octaedra are freeze-tolerant species that rapidly accumulate high concentrations of glucose as a response to freezing. The accumulation of glucose may slow down the freezing process, and even reduce the amount of ice formed. Furthermore, glucose preserves the structure and function of membranes and proteins during the freezing-induced dehydration of tissues. Earthworm cocoons are exposed to frost because they are often deposited in superficial soil layers. Cocoons are not freeze-tolerant but become dehydrated in frozen soil. The dehydration occurs because water potential (water activity) of ice is lower than water potential of supercooled water, if held at the same temperature. These physical properties of ice and supercooled water result in a passive transport of water out of cocoons causing them to become substantially dehydrated. Cocoons Eire well adapted to dehydration. If dehydrated, cocoons will not freeze, even at low sub-zero temperatures, and thus winter survival is ensured.
Coelomocytes (extruded via dorsal pores of earthworms subjected to 5V electric shock) of adult Allolobophora chlorotica, Lumbricus terrestris, Dendrobaena veneta and Eisenia fetida consisted of species-specific proportions of hyaline amoebocytes, granular amoebocytes, and eleocytes. In all species a significant increase of granular amoebocytes with a parallel decrease of hyaline amoebocytes was recorded by the end of Spring and beginning of Summer, both in animals from the natural environment and in those kept in the laboratory at the room temperature. It indicates that coelomocyte proliferation/maturation is dependent mainly on the annual endogenous rhythm, which is relatively stable even at atypical ambient temperatures. During four successive seasons viability and functions (plastic adherence or neutral red pinocytosis) were recorded in coelomocytes of A. chlorotica assayed at various in vitro temperatures. In coelomocytes retrieved from animals freshly collected from their natural environment, coelomocyte activity was better at low temperatures (0 degreesC and 10 degreesC) during the Winter, while during other seasons (especially in the Summer) it was best at 22 degreesC. Such seasonal effects were abolished in coelomocytes from animals tested after one-month acclimation to the room temperature, as in all seasons cell functions were best at 22 degreesC. These observations indicate that earthworm coelomocytes can thermally adapt. This phenomenon was also recorded in L. terrestris, D. veneta and E. fetida. In conclusion, earthworm coelomocyte proliferation/maturation seems to be endogenously controlled while coelomocyte activity seems to be more plastic and subjected to thermal adaptation.
Coelomocytes (extruded via dorsal pores of earthworms subjected to 5V electric shock) of adult Allolobophora chlorotica, Lumbricus terrestris, Dendrobaena veneta and Eisenia fetida consisted of species-specific proportions of hyaline amoebocytes, granular amoebocytes, and eleocytes. In all species a significant increase of granular amoebocytes with a parallel decrease of hyaline amoebocytes was recorded by the end of Spring and beginning of Summer, both in animals from the natural environment and in those kept in the laboratory at the room temperature. It indicates that coelomocyte proliferation/maturation is dependent mainly on the annual endogenous rhythm, which is relatively stable even at atypical ambient temperatures. During four successive seasons viability and functions (plastic adherence or neutral red pinocytosis) were recorded in coelomocytes of A. chlorotica assayed at various in vitro temperatures. In coelomocytes retrieved from animals freshly collected from their natural environment, coelomocyte activity was better at low temperatures (0 °C and 10 °C) during the Winter, while during other seasons (especially in the Summer) it was best at 22 °C. Such seasonal effects were abolished in coelomocytes from animals tested after one-month acclimation to the room temperature, as in all seasons cell functions were best at 22 °C. These observations indicate that earthworm coelomocytes can thermally adapt. This phenomenon was also recorded in L. terrestris, D. veneta and E. fetida. In conclusion, earthworm coelomocyte proliferation/maturation seems to be endogenously controlled while coelomocyte activity seems to be more plastic and subjected to thermal adaptation.
The effect of different culture methods, cold acclimation and desiccation on the supercooling point (SCP), the melting point (MP) of fluids and the quantity of water freezing (osmotically active water) was investigated in individual cocoons of Enchytraeus crypticus and an undescribed Enchytraeus species using differential scanning calorimetry (DSC) techniques. Both species can be easily cultured in the laboratory in agar where the development and hatching of the worms can be observed. Culture methods (agar with nettle leaves or oats as food and wet filter paper without food supply) had a significant effect on fresh weight and SCP of E. crypticus cocoons. The water content (as a proportion of fresh weight) was slightly lower in the cocoons from the wet filter paper cultures. Acclimation at —3 °C did not affect the supercooling ability of E. crypticus cocoons, whereas the SCP of E. sp.1 cocoons was lowered from a mean of —8.7 to —12.4 °C. The supercooling ability of cocoons of E. crypticus was only slightly increased by desiccation and cold acclimation, but the quantity of frozen water was significantly reduced with acclimation and desiccation (2 and 3 min) at room temperature. The MP values of the cocoon fluids reflected these changes in both species. No cocoons of E. crypticus and E. sp.1 survived desiccation and freezing in our experiments and no worms of Enchytraeus albidus, E. crypticus and Henlea perpusilla survived freezing to —10 or —20 °C.
The aim of this paper is to give a mini-review of the physiological adaptations to frost in earthworms. Low temperature is one of the most important environmental parameters determining the distribution of earthworms. In temperate areas many species avoid frozen soil by migrating to deeper soil layers during winter. Earthworm species of cold regions where the soil is frozen in winter have been shown to be freeze-tolerant, i.e. they endure ice formation in extracellular body fluids. Eisenia nordenskioldi and Dendrobaena octaedra are freeze-tolerant species that rapidly accumulate high concentrations of glucose as a response to freezing. The accumulation of glucose may slow down the freezing process, and even reduce the amount of ice formed. Furthermore, glucose preserves the structure and function of membranes and proteins during the freezing-induced dehydration of tissues. Earthworm cocoons are exposed to frost because they are often deposited in superficial soil layers. Cocoons are not freeze-tolerant but become dehydrated in frozen soil. The dehydration occurs because water potential (water activity) of ice is lower than water potential of supercooled water, if held at the same temperature. These physical properties of ice and supercooled water result in a passive transport of water out of cocoons causing them to become substantially dehydrated. Cocoons are well adapted to dehydration. If dehydrated, cocoons will not freeze, even at low sub-zero temperatures, and thus winter survival is ensured.
The tolerance of freezing and associated accumulation of cryoprotectants was studied in an Arctic population of the enchytraeid Fredericia ratzeli. At -3 and -5 degrees C specimens readily froze when slowly cooled in contact with moist soil. A small fraction of the animals (10-20 %) survived internal ice formation under these circumstances. Frozen specimens had elevated glucose concentrations as compared to unfrozen control animals acclimated at 0 degrees C. In a fraction of the animals, equal to the fraction surviving freezing, a high concentration of glucose was detected. The highest values amounted to ca. 150 microg mg(-1) dry weight, corresponding to ca. 270 mmoles L(-1). It is argued that the physiology of freeze tolerance in this enchytraeid resembles the physiology described for freeze tolerant earthworms and frogs.
Freeze-tolerance and some of the underlying biochemical defence mechanisms in the earthworm Dendrobaena octaedra was investigated. Survival after slow cooling to -2 degrees C, -4 degrees C, or -6 degrees C was analysed in D. octaedra from three geographic regions representing large differences in winter temperature (Denmark, Finland and Greenland). A large variation in freeze-tolerance between the three populations of D. octaedra was found. Earthworms from the northern populations (Finland and Greenland) tolerated lower temperatures (-6 degrees C) than earthworms from the Danish population (poor survival at -4 degrees C and -2 degrees C). In the Finnish population, freezing led to the production of high concentrations of glucose, which reached values much higher than controls (94 mg g(-1) vs. 2 mg g(-1) dry weight). Other potential cryoprotectants were not elevated after freezing. The Danish and Greenlandic populations had substantially lower mean glucose levels after freezing than the Finnish population (about 15 mg g(-1)). Danish earthworms rapidly frozen did not accumulate glucose, and did not survive freezing at -2 degrees C. Danish earthworms exposed to osmotic stress in Ringer's solutions, containing different concentrations of glycerol, showed significantly elevated glucose levels, but did not survive rapid freezing. It was determined if freezing had an influence on the reproduction of the earthworms. After warming to summer temperatures (15 degrees C), survivors of freezing produced viable cocoons. In a field experiment it was tested if natural acclimatization during autumn and winter months had an effect on freeze-tolerance in the Danish population. There was a significant increase of post-freeze survival during this period. The results of the freezing experiments are discussed in relation to the general ecology of D. octaedra.
The freeze-tolerant earthworm Dendrobaena octaedra is found in most of the European forest and tundra, Siberia, North America and Greenland where it over-winters in the top soil and encounters winter frost. In response to freezing this earthworm rapidly synthesises glucose which acts as a cryoprotectant. Frost tolerance varies extensively between geographical populations, and of the populations studied so far, the Finnish worms are most and the Danish worms least frost tolerant. Little is known about the determining factors for glucose synthesis and this study therefore investigated possible roles of acclimation and the cues for synthesis of glucose, in Finnish and Danish worms. The Finnish population had significantly larger glycogen reserves than the Danish during acclimation and in all worms, glucose synthesis was the result of an almost stoichemical reduction in glycogen stores. Maximum glucose levels were reached after the onset of freezing and were significantly higher in Finnish worms where the sugar accounted for as much as 5% of the fresh weight. On average, both the total glycogen phosphorylase activity and the active enzyme pool increased during acclimation in the Finnish but not the Danish populations. However, the increase in this enzyme was only significant during the freezing process. In this study, we show contrary to previous theory that glucose synthesis is initiated before the onset of freezing and that in this species, cryoprotectant synthesis is sensitive to very small temperature changes below 0 degrees C without the presence of ice.