On dormancy strategies in tardigrades

In this review we analyze the dormancy strategies of metazoans inhabiting “hostile to life” habitats, which have a strong impact on their ecology and in particular on the traits of their life history. Tardigrades are here considered a model animal, being aquatic organisms colonizing terrestrial habitats. Tardigrades evolved a large variety of dormant stages that can be ascribed to diapause (encystment, cyclomorphosis, resting eggs) and cryptobiosis (anhydrobiosis, cryobiosis, anoxibiosis). In tardigrades, diapause and cryptobiosis can occur separately or simultaneously, consequently the adoption of one adaptive strategy is not necessarily an alternative to the adoption of the other. Encystment and cyclomorphosis are characterized by seasonal cyclic changes in morphology and physiology of the animals. They share several common features and their evolution is strictly linked to the molting process. A bet-hedging strategy with different patterns of egg hatching time has been observed in a tardigrade species. Four categories of eggs have been identified: subitaneous, delayed-hatching, abortive and diapause resting eggs, which needs a stimulus to hatch (rehydration after a period of desiccation). Cryptobiotic tardigrades are able to withstand desiccation (anhydrobiosis) and freezing (cryobiosis) at any stage of their life-cycle. This ability involves a complex array of factors working at molecular (bioprotectans), physiological and structural levels. Animal survival and the accumulation of molecular damage are related to the time spent in the cryptobiotic state, to the abiotic parameters during the cryptobiotic state, and to the conditions during initial and final phases of the process. Cryptobiosis evolved independently at least two times in tardigrades, in eutardigrades and in echiniscoids. Within each evolutionary line, the absence of cryptobiotic abilities is more related to selective pressures to local habitat adaptation than to phylogenetic relationships. The selective advantages of cryptobiosis (e.g. persistency in “hostile to life” habitats, reduction of competitors, parasites and predators, escaping in time from stressful conditions) could explain the high tardigrade species diversity and number of specimens found in habitats that dry out compared to freshwater habitats.     

Cryopreservation of Greenshell™ mussel (Perna canaliculus) trochophore larvae

The Greenshell™ mussel (Perna canaliculus) is the main shellfish species farmed in New Zealand. The aim of this study was to evaluate the effects of cryoprotectant concentration, loading and unloading strategy as well as freezing and thawing method in order to develop a protocol for cryopreservation of trochophore larvae (16–20 h old). Toxicity tests showed that levels of 10–15% ethylene glycol (EG) were not toxic to larvae and could be loaded and unloaded in a single step. Through cryopreservation experiments, we designed a cryopreservation protocol that enabled 40–60% of trochophores to develop to D-larvae when normalized to controls. The protocol involved: holding at 0 °C for 5 min, then cooling at 1 °C min⁻¹ to −10 °C, holding for a further 5 min, then cooling at 0.5 °C min⁻¹ to −35 °C followed by a 5 min hold and then plunging into liquid nitrogen. A final larval rearing experiment of 18 days was conducted to assess the ability of these frozen larvae to develop further. Results showed that only 2.8% of the frozen trochophores were able to develop to competent pediveligers.     

Contrasting dynamics of cold resistance traits in field-fresh Myrmica ants during the active season

As a result of acclimation populations of long-lived ectotherms should display lowered ability to counter cold stress in warmer periods of active season, and increased resistance in colder ones. We tested this proposition by investigating dynamics of cold resistance in Myrmica ants during most of the active season in two types of habitats. Resistance of ants to knock-down by cold and their rate of recovery after chill coma were expected to be lower in summer.Cooled at a rate of 0.17 °C min⁻¹, the ants showed lower capability to resist knock-down in summer, and a significant lowering in knock-down temperature in response to colder weather both in spring and autumn as confirmed by linear regression against air temperatures. In a more eurytopic species M. rubra the responses were significantly faster in meadow than in forest habitats. However, times of recovery of the ants after 10 min at −3 °C did not change in parallel to air temperatures. Whereas M. rubra from forest habitats took less time to recover in early summer and early autumn, in their conspecifics from meadow habitats the contrary was the case. Regardless of habitat, recoveries tended to be faster in other investigated species, of which M. ruginodis (a forest stenotopic) recovered faster in early summer than later.According to the knock-down data, in warmer months the ants are indeed less resistant to cold stress, whilst the recovery data do not always support the proposition. The contrasting seasonal dynamics of the two measures of low-temperature resistance in field-fresh Myrmica suggest that knock-down (chill coma onset) is a better index of thermal acclimation, whilst the rate of recovery from chill coma is more indicative of interspecific differences and, possibly, behavioural thermoregulation.     

Comparative cryopreservation study of trochophore larvae from two species of bivalves: Pacific oyster (Crassostrea gigas) and Blue mussel (Mytilus galloprovincialis)

Oysters and mussels are among the most farmed species in aquaculture industry around the world. The aim of this study was to test the toxicity of cryoprotective agents to trochophore larvae from two different species of bivalves and develop an improved cryopreservation protocol to ensure greater efficiency in the development of cryopreserved trochophores (14 h old oyster larvae and 20 h old mussel larvae) to normal D-larvae for future developments of hatchery spat production. The cryopreservation protocol producing the best results for oyster trochophores (60.0 ± 6.7% normal D-larvae) was obtained by holding at 0 °C for 5 min then cooling at 1 °C min⁻¹ to −10 °C and holding for 5 min before cooling at 0.5 °C to −35 °C, holding 5 min and then plunging into liquid nitrogen (LN), using 10% ethylene glycol. For mussel experiments, no significant differences were found when cooling at 0.5 °C min⁻¹ or at 1 °C min⁻¹ for CPA combinations with 10% ethylene glycol and at 0.5 °C min⁻¹. Using these combinations, around half of trochophores were able to develop to normal D-larvae post-thawing (48.9 ± 7.6% normal D-larvae).     

Tardigrade Fauna of the Region between Oka and Volga Rivers (Russia) and Patterns of their Distribution in Substrates and Habitats

The region between the Oka and Volga rivers has been largely neglected with respect to tardigrade biodiversity. In the present study a total of 21 species from 7 genera, 3 families, 2 orders and only 1 class of tardigrades were present, and some of their ecological preferences were noted. The tardigrades in the studied region were mainly cosmopolitan. Eurytopic, hygrophilic and xerophilic species were common while obligate freshwater species were not found. There is a marked similarity in the tardigrade fauna within similar habitats from different locations within the region.     

A Complex RNA-Cleaving DNAzyme That Can Efficiently Cleave a Pyrimidine-Pyrimidine Junction

Several RNA-cleaving deoxyribozymes (DNAzymes) have been reported for efficient cleavage of purine-containing junctions, but none is able to efficiently cleave pyrimidine-pyrimidine (Pyr-Pyr) junctions. We hypothesize that a stronger Pyr-Pyr cleavage activity requires larger DNAzymes with complex structures that are difficult to isolate directly from a DNA library; one possible way to obtain such DNAzymes is to optimize DNA sequences with weak activities. To test this, we carried out an in vitro selection study to derive DNAzymes capable of cleaving an rC-T junction in a chimeric DNA/RNA substrate from DNA libraries constructed through chemical mutagenesis of five previous DNAzymes with a k_obs of ∼ 0.001 min^− 1 for the rC-T junction. After several rounds of selective amplification, DNAzyme descendants with a k_obs of ∼ 0.1 min^− 1 were obtained from a DNAzyme pool. The most efficient motif, denoted “CT10-3.29,” was found to have a catalytic core of ∼ 50 nt, larger than other known RNA-cleaving DNAzymes, and its secondary structure contains five short duplexes confined by a four-way junction. Several variants of CT10-3.29 exhibit a k_obs of 0.3-1.4 min^− 1 against the rC-T junction. CT10-3.29 also shows strong activity (k_obs  > 0.1 min^− 1) for rU-A and rU-T junctions, medium activity (> 0.01 min^− 1) for rC-A and rA-T junctions, and weak activity (> 0.001 min^− 1) for rA-A, rG-T, and rG-A junctions. Interestingly, a single-point mutation within the catalytic core of CT10-3.29 altered the pattern of junction specificity with a significantly decreased ability to cleave rC-T and rC-A junctions and a substantially increased ability to cleave rA-A, rA-T, rG-A, rG-T, rU-A, and rU-T junctions. This observation illustrates the intricacy and plasticity of this RNA-cleaving DNAzyme in dinucleotide junction selectivity. The current study shows that it is feasible to derive efficient DNAzymes for a difficult chemical task and reveals that DNAzymes require more complex structural solutions for such a task.     

Thermal conversion of alkaline lignin and its structured derivatives to porous carbonized materials

Alkaline lignin was thermally converted to microporous carbon in ca. 50% yield by heating up from room temperature to 900 °C without activation process under flowing of an argon gas. The carbonized material prepared by heating up conditions of 1 °C min⁻¹ showed 530 m²/g of the Brunauer-Emmett-Teller (BET) specific surface area, which increased to 740 m²/g after washing with water. Furthermore, alkaline lignin derivatives were structured as micron scale particles by micelle formation and polymer gelation techniques. Carbonization of the structured lignins could afford high porous materials having BET surface areas above 1000 m²/g without surface activation processes.     

Cold tolerance of first-instar nymphs of the Australian plague locust, Chortoicetes terminifera

The cold tolerance of first-instar nymphs of the Australian plague locust, Chortoicetes terminifera, was examined using measures of total body water content, supercooling point and mortality for a range of sub-zero temperature exposure regimes. The supercooling points for starved and fed nymphs were −13.1 ± 0.9 and −12.6 ± 1.6 °C, and freezing caused complete mortality. Above these temperatures, nymphs were cold tolerant to different degrees based on whether they were starved or given access to food and water for 24 h prior to exposure. The rate of cooling also had a significant effect on mortality. Very rapid cooling to −7 °C caused 84 and 87% mortality for starved and fed nymphs respectively, but this significantly decreased for starved nymphs if temperature declined by more ecologically realistic rates of 0.5 and 0.1 °C min⁻¹. These results are indicative of a rapid cold hardening response and are discussed in terms of the likely effects of cold nights and frost on first-instar nymphal survival in the field.     

Diversity and abundance of nematode-trapping fungi from decaying litter in terrestrial, freshwater and mangrove habitats

Nematode-trapping fungi are ubiquitous in terrestrial habitats in dung, soils, litter and woody debris and they also occur in freshwater, but only one species has been found in marine habitats. The purpose of this study was therefore to investigate whether nematode-trapping fungi occurred in mangrove habitats. To achieve this we assessed the diversity of nematode-trapping fungi on decaying litter from mangroves, freshwater and terrestrial habitats (22 sites) in Hong Kong. Composite samples (n = 1,320) of decaying litter (wood and leaves) were examined and a total of 31 species of nematode-trapping fungi belonging to four genera, Arthrobotrys, Monacrosporium, and Dactylella were recorded. Twenty-nine species reported in this study are new records for Hong Kong and 16 species are new records from mangrove habitats worldwide. Nematode trapping fungi are therefore present in marine environments. Commonly encountered taxa were Arthrobotrys oligospora and Monacrosporium thaumasium which are abundant in all habitats. A. oligospora, M. thaumasium and Arthrobotrys musiformis were frequent (F > 10%). Twenty-six species were rare (0.16–9.32%). Species richness and diversity was higher in terrestrial than in freshwater and mangrove habitats (ANOVA, P < 0.001). A higher mean diversity was observed on decaying leaves as compared to decaying wood in all habitats (P < 0.001). Based on Shannon diversity index, it was also observed that taxa characterized by adhesive nets were more frequent in all habitats. This can be explained by the fact that these taxa may have a better competitive saprotrophic ability which would allow them to compete favourably in nutrient limited environments. Abiotic factors that could be linked to differences in species diversity between decaying wood and leaves are also discussed.     

Post-cryopreservation viability of the benthic freshwater diatom Planothidium frequentissimum depends on light levels

Over recent years, several planktonic and benthic freshwater diatom taxa have been established as laboratory model strains. In common with most freshwater diatoms the pennate diatom Planothidium frequentissimum suffers irreversible cell shrinkage on prolonged maintenance by serial transfers, without induction of the sexual cycle. Therefore, alternative strategies are required for the long-term maintenance of this strain. Conventional colligative cryopreservation approaches have previously proven unsuccessful with no regrowth. However, in this study using 5% dimethyl sulfoxide (Me₂SO), controlled cooling at 1 °C min⁻¹, automated ice seeding and cooling to −40 °C with a final plunge into liquid nitrogen, viability levels were enhanced from 0.3 ± 0.4% to 80 ± 3%, by incorporating a 48 h dark-recovery phase after rewarming. Omission, or reduction, of this recovery step resulted in obvious cell damage with photo-bleaching of pigments, indicative of oxidative-stress induced cell damage, with subsequent deterioration of cellular architecture.     

A chemically modified lipase preparation for catalyzing the transesterification reaction in even highly polar organic solvents

Acylation of Pseudomonas cepacia lipase with Pyromellitic dianhydride to modify 72% of total amino groups was carried out. Different organic solvents were screened for precipitation of modified lipase. It was found that 1,2-dimethoxyethane was the best precipitant which precipitated 97% protein and complete activity. PCMC (protein coated microcrystals), CLPCMC (crosslinked protein coated microcrystals), EPROS (enzyme precipitated and rinsed with organic solvents) and pH tuned preparations of modified and unmodified lipase were prepared and used for carrying out transesterification reaction with n-octane and dimethyl formamide (DMF) as reaction medium. In n-octane, among all the preparations, CLPCMC of modified lipase gave highest rate (1970 nmol min⁻¹ mg⁻¹) as compared to unmodified pH tuned lipase (128 nmol min⁻¹ mg⁻¹). In DMF, with both 1% (v/v) and 5% (v/v) water content, CLPCMC showed highest initial rate of 0.72 and 7.2 nmol min⁻¹ mg⁻¹, respectively. Unmodified pH tuned lipase showed no activity at all in DMF with both 1% and 5% (v/v) water content.     

Freezing avoidance of the Antarctic icefishes (Channichthyidae) across thermal gradients in the Southern Ocean

Biogeographic studies separate the Antarctic Notothenioid fish fauna into high- and low-latitude species. Past studies indicate that some species found in the high-latitude freezing waters of the High-Antarctic Zone have low-serum hysteresis freezing points, while other species restricted to the low-latitude seasonal pack ice zone have higher serum hysteresis freezing points above the freezing point of seawater (−1.9°C), but the relationship has not been systematically investigated. Freeze avoidance was quantified in 11 species of Antarctic icefishes by determining the hysteresis freezing points of their blood serum, in addition, the freezing point depression from serum osmolytes, the antifreeze activity from serum antifreeze glycoproteins (AFGPs), and the antifreeze activity from serum antifreeze potentiating protein were measured for each species. Serum hysteresis freezing point, a proxy for organismal freeze avoidance, decreased as species were distributed at increasing latitude (linear regression r ² 0.66, slope −0.046°C °latitude⁻¹), which appeared largely independent of phylogenetic influences. Greater freeze avoidance at high latitudes was largely a result of higher levels of antifreeze activity from serum AFGPs relative to those in species inhabiting the low-latitude waters. The icefish fauna could be separated into a circum High-Antarctic Group of eight species that maintained serum hysteresis freezing points below −1.9°C even when sampled from less severe habitats. The remaining three species with low-latitude ranges restricted to the waters of the northern part of the west Antarctic Peninsula and Scotia Arc Islands had serum hysteresis freezing points at or above −1.9°C due to significantly lower combined activity from all of their serum antifreeze proteins than found in the High-Antarctic Zone icefish.     

Seagrass complexity hierarchies: Influence on fish groups around the coast of Jersey (English Channel)

Recent research has identified a need for seagrass habitat management plans to be based on landscape-level approaches as they offer a more appropriate scale for large mobile fauna than smaller scales. Also, conservation decisions are more likely to be a choice between different seagrass beds rather than parts of individual beds. The present study examined the spatial utilisation of subtidal seagrass beds by fish around the coast of Jersey, English Channel (49°N 02°W) with the aim of identifying influential scales of complexity. A hierarchical-scale of landscape configuration and habitat characteristics was measured for eight seagrass beds using aerial photographic analysis (e.g. core area, contiguity and other landscape metrics), digital echo-sounder data (e.g. depth, canopy height) and diver surveys (e.g. epiphyte load). The contributions of these variables as predictors of functional fish groups were explored using multiple linear regression models. Results indicated that more fragmented seagrass beds supported lower numbers of fish species than more homogenous seagrass landscapes (squared semi-partial correlation coefficient, sr² = − 0.3). Densities of juveniles of larger fish species showed a negative relationship with increased fragmentation of the seagrass (sr² = − 0.34). At smaller scales of structural complexity the densities of cryptic fish were related positively to canopy height (sr² = 0.46). At night, fewer patterns could be explained by the independent variables in the model, which was attributed to the greater movement of fish between the seagrass and adjacent habitats to forage, and a breakdown in the association with seagrass habitat as a refuge from predation.     

Survival of sub-zero temperatures by two South Georgian beetles (Coleoptera,Perimylopidae)

Summary The ability of adults and larvae of two species of perimylopid beetles (Hydromedion sparsutum, Perimylops antarcticus) to survive sub-zero temperatures was studied at Husvik, South Georgia in summer during October–December 1990. Experiments determined their survival at constant sub-zero temperatures, their lower lethal temperatures and individual supercooling points. The effects of cooling rates (0.015°, 0.5° and 2.0°C min^–1) and starvation on survival were also assessed. Mean supercooling points of field-collected individuals of both species were in the range -3.0° to -5.4°C with Perimylops having a deeper capacity (ca. 1.5°C) for supercooling relative to Hydromedion. The former species also survived freezing temperatures significantly better than the latter and its mean lower lethal temperature was 2.5°C lower. At a constant temperature of -8.5°C, the median survival times for Perimylops adults and larvae were 19 and 26 h respectively, whilst both stages of Hydromedion died within 3 h. The three cooling rates resulted in significantly different median survival temperatures for adult Hydromedion with 0.5°C min^–1 producing maximum survival. Prior starvation did not have a significant influence on the survival of either species at sub-zero temperatures although both adults survived less well. The results support field observations on the habitats and distribution of these insects, and suggest differing degrees of freezing tolerance.     

Survey of the terrestrial and freshwater molluscan fauna of the Pilliga forest area in northern inland New South Wales,Australia

The Pilliga forest in northern inland New South Wales, Australia, is one of the largest surviving remnants of native forest on the western slopes of the Great Dividing Range. The Pilliga landscape is a challenging environment for molluscs, dominated by dry sclerophyll forest and with limited and largely ephemeral aquatic habitats. A field survey of the area in 2006–2012 identified a surprisingly rich and relatively intact aquatic native molluscan fauna with five species of bivalves in three families and nine species of freshwater gastropods (four families), including some rare species and range extensions. The native land snail fauna comprised 18 species (six families), including an unusually rich pupillid fauna with nine species. Some range extensions are recorded and some species are narrow-range endemics. The distributions of many aquatic and terrestrial species were correlated with geology or soil type. Introduced molluscs were predominantly found in anthropogenic habitats and include two freshwater gastropods (two families) and nine terrestrial snails and slugs (eight families). This study provides insight into the original molluscan fauna of the western slopes prior to landscape-scale agricultural development and provides a benchmark for future reference.     

Pleistocene phylogeography and phylogenetic concordance in cold-adapted spring snails (Bythinella spp.)

Previous studies on Pleistocene phylogeography of European taxa are biased towards (i) vertebrates, (ii) terrestrial taxa, (iii) single species, and (iv) taxa that survived the Pleistocene in southern refugia. Relatively little is known about whether evolutionary patterns of vertebrate and terrestrial taxa are also applicable to freshwater invertebrates, whether cold-adapted freshwater species could survive in extensive permafrost areas without retreating into refugia, and whether Pleistocene phylogeographical patterns are influenced by phylogeny. Here, the widespread and species-rich European spring snail genus Bythinella Moquin-Tandon, 1856 is utilized in an attempt to mitigate this bias. These strongly cold-adapted freshwater animals mostly occur in springs — highly isolated habitats that are relatively unaffected by anthropogenic influences. Phylogenetic and phylogeographical analyses based on mitochondrial DNA and nuclear DNA sequence data were conducted in 458 specimens from 142 populations occurring throughout Europe. The study provides evidence that most Bythinella spp. survived the Pleistocene in restricted northern glacial refugia that largely correspond to refugia previously recognized for other European biota. However, survival of Bythinella spp. in extensive permafrost areas outside of refugia can likely be rejected. Low dispersal ability and the isolation and fragmentation of spring habitats, as well as the distribution of perennial springs within permafrost regions, may account for this result. Tests involving a total of 29 nominal species showed that phylogenetically closely related Bythinella species did not occupy similar refugia. This lack of phylogenetic concordance could possibly be explained by the stochasticity of survival and dispersal in spring snails.     

Effects of suspended mussel culture on benthic-pelagic coupling in a coastal upwelling system (Ría de Vigo, NW Iberian Peninsula)

The influence of suspended mussel culture on the benthic-pelagic coupling was evaluated in the Ría de Vigo, in the coastal upwelling system of the NW Iberian Peninsula, during the month of July 2004. Measurements of water column properties were carried out at three stations in the Ría de Vigo: under a mussel raft (1), and at two reference sites in the main channel (2) and in the inner part (3) of the Ría. Dissolved nutrients, dissolved oxygen and inorganic carbon benthic fluxes were measured by means of a benthic chamber at stations 1 and 3. Sediment traps were deployed at 6 locations to estimate vertical fluxes. The water column structure at the three study stations was very similar, characterized by upwelling conditions during the second week of July and a posterior stratification showing a strong thermocline, with a depth fluctuation mainly modulated by the shelf wind regime. Vertical POC fluxes underneath the mussel raft (3 g C m⁻² d^− 1) were 3 times higher than those obtained between rafts and 10 times higher than in the main channel reference site. Dissolved oxygen, ammonium, silicate and phosphate benthic fluxes were significantly higher under the raft than at the inner Ría reference site. A 1D carbon budget showed the importance of benthic metabolism under the raft (2.3 ± 0.8 g C m^− 2 d^− 1), being higher than the organic carbon produced at the photic layer (0.7 ± 0.3 g C m^− 2 d^− 1), as a result of higher organic loading compared to the inner Ría reference site. The results show for the first time the important role that suspended mussel cultures play in the benthic-pelagic coupling in this coastal upwelling ecosystem.     

Temporal and spatial dynamics of macroalgal communities along an anthropogenic salinity gradient in Biscayne Bay (Florida,USA)

The seasonal and spatial dynamics of two groups of macroalgae, drift algae and rhizophytes, commonly found in tropical seagrass meadows were studied. The aim of this study was to provide insight into how freshwater discharges may be altering seagrass-dominated nearshore tropical habitats. Species composition, biomass, and percent cover of macroalgae were collected at six Thalassia testudinum König dominated sites within Biscayne Bay, Florida, representing three salinity regimes: canal-influenced, natural sheet-flow, and oceanic conditions. Mean annual salinities in these three regimes correspond to 10, 25 and 35 psu, respectively, with much greater variability in the canal and sheet-flow regimes, than in the oceanic condition. There were distinct changes in the composition of the macroalgal community along this salinity gradient. Drift algae (Chondria spp., Laurencia spp.) were most commonly found at canal-disturbed sites (10–85 g m⁻²), while rhizophytic calcareous green algae (Halimeda spp., Penicillus spp.) were most abundant at the higher salinity oceanic sites (20–105 g m⁻²). Seasonal patterns exhibited by the two groups differed also, with drift algae being more abundant in the cooler dry-season months, while rhizophytic algae were more abundant during the warmer wet-season months. These periods of higher abundance correlated with higher growth rates (drift = 2.3% day⁻¹, rhizophytes = 0.85% day⁻¹) measured in representative species for each group. Grazing rates on drift algae were found to be low for tropical habitats and did not differ much between canal (0.44% h⁻¹) and oceanic sites (0.42% h⁻¹).     

The oatmeal nematode <Emphasis Type="Italic">Panagrellus redivivus</Emphasis> survives moderately low temperatures by freezing tolerance and cryoprotective dehydration

The cold tolerance abilities of only a few nematode species have been determined. This study shows that the oatmeal nematode, Panagrellus redivivus, has modest cold tolerance with a 50% survival temperature (S ₅₀) of −2.5°C after cooling at 0.5°C min⁻¹ and freezing for 1 h. It can survive low temperatures by freezing tolerance and cryoprotective dehydration; although freezing tolerance appears to be the dominant strategy. Freezing survival is enhanced by low temperature acclimation (7 days at 5°C), with the S ₅₀ being lowered by a small but significant amount (0.42°C). There is no cold shock or rapid cold hardening response under the conditions tested. Cryoprotective dehydration enhances the ability to survive freezing (the S ₅₀ is lowered by 0.55°C, compared to the control, after 4 h freezing at −1°C) and this effect is in addition to that produced by acclimation. Breeding from survivors of a freezing stress did not enhance the ability to survive freezing. The cold tolerance abilities of this nematode are modest, but sufficient to enable it to survive in the cold temperate environments it inhabits.