Cold acclimation and cryoprotectants in a freeze-tolerant Antarctic nematode, Panagrolaimus davidi

Panagrolaimus davidi is a freeze-tolerant Antarctic nematode which survives extensive intracellular freezing. This paper describes the development of culture techniques which provide clean samples, with a high degree of freeze tolerance and in sufficient quantities for the analysis of potential cryoprotectants. Cultures grown at 20 °C survived a short-term freezing stress but survival declined with the time spent frozen. Acclimation of cultures at 5 °C enhanced the long-term survival of freezing. Starvation, however, reduced the nematode's ability to survive short-term freezing. The principal cryoprotectants detected by gas chromatography were trehalose and glycerol. The levels of trehalose, but not those of glycerol, increased significantly after acclimation. Trehalose may stabilise membranes and protect them against the dehydrating effects of the osmotic stresses resulting from freeze concentration effects but other factors, such as recrystallisation inhibition, may be involved in long-term survival. Accepted: 7 March 2000     

The environmental physiology of Antarctic terrestrial nematodes: a review

The environmental physiology of terrestrial Antarctic nematodes is reviewed with an emphasis on their cold-tolerance strategies. These nematodes are living in one of the most extreme environments on Earth and face a variety of stresses, including low temperatures and desiccation. Their diversity is low and declines with latitude. They show resistance adaptation, surviving freezing and desiccation in a dormant state but reproducing when conditions are favourable. At high freezing rates in the surrounding medium the Antarctic nematode Panagrolaimus davidi freezes by inoculative freezing but can survive intracellular freezing. At slow freezing rates this nematode does not freeze but undergoes cryoprotective dehydration. Cold tolerance may be aided by rapid freezing, the production of trehalose and by an ice-active protein that inhibits recrystallisation. P. davidi relies on slow rates of water loss from its habitat, and can survive in a state of anhydrobiosis, perhaps aided by the ability to synthesise trehalose. Teratocephalus tilbrooki and Ditylenchus parcevivens are fast-dehydration strategists. Little is known of the osmoregulatory mechanisms of Antarctic nematodes. Freezing rates are likely to vary with water content in Antarctic soils. Saturated soils may produce slow freezing rates and favour cryoprotective dehydration. As the soil dries freezing rates may become faster, favouring freezing tolerance. When the soil dries completely the nematodes survive anhydrobiotically. Terrestrial Antarctic nematodes thus have a variety of strategies that ensure their survival in a harsh and variable environment. We need to more fully understand the conditions to which they are exposed in Antarctic soils and to apply more natural rates of freezing and desiccation to our studies.Communicated by: I.D. Hume     

Cold tolerance of an Antarctic nematode that survives intracellular freezing: comparisons with other nematode species

Panagrolaimus davidi is an Antarctic nematode with very high levels of cold tolerance. Its survival was compared with that of some other nematodes (P. rigidus, Rhabditophanes sp., Steinernema carpocapsae, Panagrellus redivivus and Ditylenchus dipsaci) in both unacclimated samples and those acclimated at 5°C. Levels of recrystallization inhibition in homogenates were also compared, using the splat-cooling assay. The survival of P. davidi after the freezing of samples was notably higher than that of the other species tested, suggesting that its survival ability is atypical compared to other nematodes. In general, acclimation improved survival. Levels of recrystallization inhibition were not associated with survival but such a relationship may exist for those species that are freezing tolerant.     

Anhydrobiosis and Freezing-Tolerance: Adaptations That Facilitate the Establishment of Panagrolaimus Nematodes in Polar Habitats

Anhydrobiotic animals can survive the loss of both free and bound water from their cells. While in this state they are also resistant to freezing. This physiology adapts anhydrobiotes to harsh environments and it aids their dispersal. Panagrolaimus davidi, a bacterial feeding anhydrobiotic nematode isolated from Ross Island Antarctica, can survive intracellular ice formation when fully hydrated. A capacity to survive freezing while fully hydrated has also been observed in some other Antarctic nematodes. We experimentally determined the anhydrobiotic and freezing-tolerance phenotypes of 24 Panagrolaimus strains from tropical, temperate, continental and polar habitats and we analysed their phylogenetic relationships. We found that several other Panagrolaimus isolates can also survive freezing when fully hydrated and that tissue extracts from these freezing-tolerant nematodes can inhibit the growth of ice crystals. We show that P. davidi belongs to a clade of anhydrobiotic and freezing-tolerant panagrolaimids containing strains from temperate and continental regions and that P. superbus, an early colonizer at Surtsey island, Iceland after its volcanic formation, is closely related to a species from Pennsylvania, USA. Ancestral state reconstructions show that anhydrobiosis evolved deep in the phylogeny of Panagrolaimus. The early-diverging Panagrolaimus lineages are strongly anhydrobiotic but weakly freezing-tolerant, suggesting that freezing tolerance is most likely a derived trait. The common ancestors of the davidi and the superbus clades were anhydrobiotic and also possessed robust freezing tolerance, along with a capacity to inhibit the growth and recrystallization of ice crystals. Unlike other endemic Antarctic nematodes, the life history traits of P. davidi do not show evidence of an evolved response to polar conditions. Thus we suggest that the colonization of Antarctica by P. davidi and of Surtsey by P. superbus may be examples of recent “ecological fitting” of freezing-tolerant anhydrobiotic propagules to the respective abiotic conditions in Ross Island and Surtsey.     

Intracellular Freezing in the Infective Juveniles of Steinernema feltiae: An Entomopathogenic Nematode

Taking advantage of their optical transparency, we clearly observed the third stage infective juveniles (IJs) of Steinernema feltiae freezing under a cryo-stage microscope. The IJs froze when the water surrounding them froze at −2°C and below. However, they avoid inoculative freezing at −1°C, suggesting cryoprotective dehydration. Freezing was evident as a sudden darkening and cessation of IJs'' movement. Freeze substitution and transmission electron microscopy confirmed that the IJs of S. feltiae freeze intracellularly. Ice crystals were found in every compartment of the body. IJs frozen at high sub-zero temperatures (−1 and −3°C) survived and had small ice crystals. Those frozen at −10°C had large ice crystals and did not survive. However, the pattern of ice formation was not well-controlled and individual nematodes frozen at −3°C had both small and large ice crystals. IJs frozen by plunging directly into liquid nitrogen had small ice crystals, but did not survive. This study thus presents the evidence that S. feltiae is only the second freeze tolerant animal, after the Antarctic nematode Panagrolaimus davidi, shown to withstand extensive intracellular freezing.     

Osmotic stress effects on the freezing tolerance of the antarctic nematodePanagrolaimus davidi

 The freezing and freezing survival of the Antarctic nematode Panagrolaimus davidi after exposure to solutions of different osmotic concentrations has been examined using a thermoelectric cooling stage and multi-specimen cooling block to see if there is any evidence that freeze-induced desiccation prevents inoculative freezing. The nematodes froze in all the test solutions used (up to 1138 mosmol ⋅ l^-1) and at all cooling rates and nucleation temperatures tested. Freezing survival was at its maximum in 0.1 mol ⋅ l^-1 NaCl in artificial tap water after 1 h exposure to the test solution and in artificial tap water after 24 h exposure. Hyperosmotic and hyposmotic stress adversely affected the nematodes’ ability to survive freezing. In non-frozen controls survival declined with increasing osmolality of the test solution. Measurements of the osmolality of water extracted from a variety of moss samples indicate that the nematodes are exposed to an osmotic concentration of about 9 mosmol ⋅ l^-1 in their natural habitat. This is close to that of artificial tap water. Our experiments, and measurements of freeze concentration effects in the literature, indicate that freeze-induced desiccation is unlikely to prevent inoculative freezing and the survival of nematodes over the winter. Accepted: 5 May 1996     

Adaptations to the Arctic: low-temperature development and cold tolerance in the free-living stages of a parasitic nematode from Svalbard

For nematodes with a direct life cycle, transmission is highly dependent on temperature-related development and survival of the free-living stages. Therefore, in the Arctic, where the winter lasts from October to May, nematode transmission is expected to be focused in the short summer season, yet there is strong evidence that as well as focussing egg output during winter months, the nematode parasite, Marshallagia marshalli, infects Svalbard reindeer during the Arctic winter when temperatures are persistently below freezing. To investigate the potential for development and survival of eggs and infective third-stage larvae in winter and therefore the possibility of for winter transmission, we ran a series of low-temperature laboratory experiments. These provide five key insights into the transmission and survival of the free-living stages of M. marshalli: (1) eggs hatched at temperatures as low as 2 °C, but not below 0 °C, (2) eggs were viable and developed after being exposed to sub-zero temperatures for up to 28 months, (3) infective-stage larvae survived for up to 80 days at 5 °C, (4) infective-stage larvae could survive rapid exposure to temperatures below ?30 °C, and (5) desiccation resistance may be important for long-term larval survival at low temperatures. Together, these results indicate that eggs deposited during the winter are highly tolerant of prevailing environmental conditions and have the potential for rapid development with the onset of spring. It is therefore likely that the parasite remains in the egg stage in the faeces during the winter of deposition, hatch and develop into the infective larval stage in the summer, remaining viable on the tundra until the reindeer host returns to the winter feeding grounds the following winter.     

Ice Nucleation Activity in the Freezing-Tolerant Antarctic NematodePanagrolaimus davidi

Ice nucleation spectrometry was used to look for the presence of ice nucleating agents (INAs), and their inhibitors, in cultures ofPanagrolaimus davidi, an Antarctic nematode which survives intracellular freezing. INA activity was absent in both nematode suspensions and homogenates. The nematodes produce a substance which inhibits the nucleation activity of organic INAs but not of an inorganic INA (AgI). The nucleation inhibitor is both released from the nematode by homogenization and excreted by them into the medium, but the former was more effective at inhibiting nucleation. The inhibitory activity was destroyed by heating. A thermal hysteresis protein, or a similar ice-active substance, may be responsible for the nucleation inhibition.     

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.     

Recrystallization in a Freezing Tolerant Antarctic Nematode,Panagrolaimus davidi,and an Alpine Weta,Hemideina maori(Orthoptera; Stenopelmatidae)

The ability of haemolymph from the freezing tolerant weta,Hemideina maori,and supernatant from homogenates of the freezing tolerant nematodePanagrolaimus davidito inhibit the recrystallization of ice was examined using the “splat freezing” technique and annealing on a cryomicroscope stage. There was no recrystallization inhibition in weta haemolymph or in insect ringer controls. Recrystallization inhibition was present in the nematode supernatant but this was destroyed by heating and was absent in controls.P. davidisurvives intracellular freezing and recrystallization inhibition may be important for the survival of this stress.     

Prey preference and life tables of the predatory mite Parasitus bituberosus (Acari: Parasitidae) when offered various prey combinations

Parasitus bituberosus Karg (Acari: Parasitidae) is one of the predatory mite species inhabiting mushroom houses. It is known to accept a wide range of prey, suggesting that it may be a promising candidate for the biological control of key pests of mushroom culture. In our study it did not show any prey preference among four groups of small organisms often occurring in mushroom growth medium, namely rhabditid nematodes, pygmephorid mites, and sciarid and phorid fly larvae. Nevertheless, the type of food these predators fed on affects their development. The shortest egg-to-adult development time was obtained on a nematode diet. On a diet of phorid larvae, mite development stopped at the deutonymph stage; none reached adulthood. All other diets sufficed to reach the adult phase. Female fecundity when fed nematodes and sciarid larvae did not differ, but it was much lower when fed pygmephorid mites. Other life table parameters confirmed that pygmephorid mites constituted the worst diet for P. bituberosus. The highest intrinsic rate of population increase (r _m  = 0.34) was obtained on the nematode diet; when fed sciarid larvae and pygmephorid mites it was 0.25 and 0.14, respectively. Our study provides good reasons to further test P. bituberosus as biocontrol agent of especially sciarid flies and nematodes, especially when the compost is well colonized by mushroom mycelium (which retards nematode growth).     

Infective Juveniles of the Entomopathogenic Nematode,Steinernema feltiae Produce Cryoprotectants in Response to Freezing and Cold Acclimation

Steinernema feltiae is a moderately freeze-tolerant entomopathogenic nematode which survives intracellular freezing. We have detected by gas chromatography that infective juveniles of S. feltiae produce cryoprotectants in response to cold acclimation and to freezing. Since the survival of this nematode varies with temperature, we analyzed their cryoprotectant profiles under different acclimation and freezing regimes. The principal cryoprotectants detected were trehalose and glycerol with glucose being the minor component. The amount of cryoprotectants varied with the temperature and duration of exposure. Trehalose was accumulated in higher concentrations when nematodes were acclimated at 5°C for two weeks whereas glycerol level decreased from that of the non-acclimated controls. Nematodes were seeded with a small ice crystal and held at -1°C, a regime that does not produce freezing of the nematodes but their bodies lose water to the surrounding ice (cryoprotective dehydration). This increased the levels of both trehalose and glycerol, with glycerol reaching a higher concentration than trehalose. Nematodes frozen at -3°C, a regime that produces freezing of the nematodes and results in intracellular ice formation, had elevated glycerol levels while trehalose levels did not change. Steinernema feltiae thus has two strategies of cryoprotectant accumulation: one is an acclimation response to low temperature when the body fluids are in a cooled or supercooled state and the infective juveniles produce trehalose before freezing. During this process a portion of the glycerol is converted to trehalose. The second strategy is a rapid response to freezing which induces the production of glycerol but trehalose levels do not change. These low molecular weight compounds are surmised to act as cryoprotectants for this species and to play an important role in its freezing tolerance.     

Effects of temperature on the life-history traits of Sancassania (Caloglyphus) berlesei (Acari: Astigmatina: Acaridae) feeding on root-knot nematodes,Meloidogyne spp. (Nematoda: Meloidogynidae)

Sancassania (Caloglyphus) berlesei (Michael) is a cosmopolitan and free-living mite that inhabits soil as well as laboratory colonies of insects and fungi and may have a role as a biocontrol agent of nematodes. In this study, we investigated the effects of temperature on the development, reproduction, and food consumption of S. berlesei fed egg masses of root-knot nematodes, Meloidogyne spp., an important group of agricultural pests. Mites were reared at 20, 25 or 30 °C in the dark. The mites could feed on the nematode egg masses, and their developmental time decreased at higher temperatures. Time from the egg to adult was similar in females and males reared at the same temperature. Adult females lived longer than males at 25 °C, but not at 20 or 30 °C. Generally, females showed a higher rate of food consumption than males. Females laid the largest number of eggs at 20 and 25 °C (199.7 and 189.8 eggs/female, respectively), but the intrinsic rate of natural increase was highest at 30 °C (r _m = 0.29). In comparing our data with previous reports, we noted that S. berlesei that fed on egg masses of root-knot nematodes showed a longer developmental time and a lower reproductive rate than Sancassania mites that fed on other diets. Nonetheless, the relatively high value of r _m (e.g., at 25 and 30 °C) suggests that this mite may have certain advantages as a biocontrol agent of root-knot nematodes.     

Ionic regulation in the Antarctic nematode Panagrolaimus davidi,measured using electron probe X-ray microanalysis

The element composition of the pseudocoelomic fluid of the Antarctic nematode Panagrolaimus davidi was analysed by electron probe X-ray microanalysis after absorbing the fluid into Sephadex G-25 beads, and after producing calibration curves by analysing various concentrations of elements of interest absorbed into beads. The nematodes maintain higher concentrations of sodium and potassium in their pseudocoelomic fluid than in the external medium but lower concentrations of magnesium and calcium. When external concentrations of specific ions were elevated there was evidence for the regulation of internal concentrations of sodium, potassium, magnesium and chlorine. The time course of changes in response to exposure to elevated levels of KCl shows an increase in internal concentrations of potassium and chlorine up to 2 h after exposure, followed by a decline. This is consistent with a model of ionic regulation proposed for Caenorhabditis elegans which suggests that high concentrations of ionic osmolytes are replaced by compatible organic osmolytes.     

Susceptibility of Spodoptera littoralis caterpillars to entomopathogenic nematode Steinernema feltiae after consumption of non-specific transgenic plants

Spodoptera littoralis caterpillars were transferred from an artificial diet to potato leaves at the start of the third or fifth instar and exposed to the infective juveniles of the nematode Steinernema feltiae since the beginning of the sixth instar until the start of pupation. Leaves were taken from the control potatoes or from genetically modified potatoes expressing either Cry3Aa toxin of Bacillus thuringiensis (Bt) or Galanthus nivalis agglutinin (GNA) which are mainly non-specific to S. littoralis larvae. The nematodes killed all the caterpillars within seven days compared with the starved larvae in the same period of exposure. The average time to death and the number of nematodes successfully invaded the larvae were affected by the period of feeding on potato leaves. In the non-starved caterpillars, which received potato leaves throughout the whole period of exposure to the nematodes, the type of potato leaves had no effect on the number of nematodes inside cadavers (p = 0.352 and F = 1.070) and also on the effect on the length of survival after exposure to the nematodes (p = 0.7892 and F = 0.596). No hazardous effect on the development and survival of entomopathogenic nematode S. feltiae which successfully invaded larvae fed on modified potato (Bt or GNA) was reported.     

Stabilisation of heat tolerance traits in Heterorhabditis bacteriophora through selective breeding and creation of inbred lines in liquid culture

Entomopathogenic nematodes (EPNs) suffer from trait deterioration, a potential problem when these antagonists are transferred into artificial environments for mass production. In order to improve beneficial traits of EPN genetic selection and hybridization has been successfully carried out. Should these selected strains deteriorate during serial culturing the efforts would be in vain. Inbreeding might offer a possibility to stabilize traits but can also result in inbreeding depression. This study attempted to increase heat tolerance of Heterorhabditis bacteriophora by selective breeding for seven cycles either with nematodes propagated in vivo in Galleria mellonella or with in vitro propagated nematodes which were exposed to heat stress in monoxenic liquid culture. After release of the selection pressure, the tolerance was monitored over 15 additional reproductive cycles to compare the stability of the trait. Virulence of the selected strains was assessed to check for negative tradeoff effects. Heat tolerance was successfully increased in vivo (from 39.03 to 40.85 °C) and in vitro (from 39 to 40 °C) propagated H. bacteriophora, but could only be maintained in populations which were serially reared in liquid culture. When H. bacteriophora is cultured in vivo, reproduction by cross fertilization is possible. In in vitro culture male and female cannot mate and reproduction is solely by self-fertilizing hermaphrodite resulting in homozygous inbred lines. Trait deterioration seems to be restricted to in vivo propagated H. bacteriophora, whereas monoxenic liquid cultures handling large numbers of inbred lines provided genetically stable and virulent nematode populations. Selection using liquid culture technology is thus superior over in vivo propagation to sustain beneficial traits in H. bacteriophora not only for selective breeding but also for mass production.     

A Free-living Panagrolaimus sp. from Armenia Can Survive in Anhydrobiosis for 8.7 Years

Although the ability of plant-parasitic nematodes to survive in a dehydrated or anhydrobiotic state for long periods of time has been well documented, the ability of free-living nematodes has not. Here we report on the survival of a free-living nematode, Panagrolaimus sp., from Armenia in the anhydrobiotic state for 8.7 years. This Panagrolaimus sp. can be cultured and maintained readily and may provide a good system for studying anhydrobiosis in nematodes.     

Freeze-thaw survival of the free-living nematode Caenorhabditis briggsae.

Approximately 75% or more of the L2 and L3 juvenile stages of the free-living nematode Caenorhabditis briggsae survived freezing and thawing without loss of fertility. Optimum survival depended upon a combination of conditions: (1) pretreatment with 5% DMSO at 0 °C for 10 min, (2) 0.2 °C per minute cooling rate from 0 to ?100 °C prior to immersion into liquid nitrogen, and (3) a 27.6 °C per minute warming rate from ?196 °C to ?10 °C. Storage at ?196 °C for more than 100 days was without effect on viability or fertility. Some of the L4 (about 50%) and adult (about 3%) stages survive the routine freeze-thaw treatment. However, there was no recovery of either embryonic stages or embryonated eggs from ?196 °C under these standard conditions. Either very fast cooling (about 545 °C/min) or fast warming (about 858 °C/min) rates diminished survival of the L2 and L3 stages drastically.Scanning electron microscopy revealed that freeze-thaw survivors with aberrant swimming behavior had cuticular defects. In juvenile forms, the altered swimming motion was lost after a molt whereas as abnormal adults grew, sinusoidal movement resumed. In the L4 and adult forms the cuticular abnormalities lowered viability and fertility. It is concluded that survival of nematodes from a freeze-thaw cycle is contingent upon establishing specific cryobiological conditions by varying aspects of the procedure that gave high recoveries of L2 and L3 stages.