Panagrolaimus Davidi Research Articles

Molecular snapshot of an intracellular freezing event in an Antarctic nematode

The Antarctic nematode, Panagrolaimus sp. DAW1 (formerly called Panagrolaimus davidi), is the best documented example of an organism able to survive intracellular ice formation in all of its compartments. Not only is it able to survive such extreme physiological disruption, but it is able to produce progeny once thawed from such a state. In addition, under slower rates, or less extreme degrees, of cooling, its body remains unfrozen and the vapour pressure difference between the supercooled body fluids and the surrounding ice leads to a process termed cryoprotective dehydration. In contrast to a fairly large body of work in building up our molecular understanding of cryoprotective dehydration, no comparable work has been undertaken on intracellular freezing. This paper describes an experiment subjecting cultures of Panagrolaimus sp. DAW1 to a range of temperatures including a rapid descent to −10 °C, in a medium just prior to, and after, freezing. Through deep sequencing of RNA libraries we have gained a snapshot of which genes are highly abundant when P. sp. DAW1 is undergoing an intracellular freezing event. The onset of freezing correlated with a high production of genes involved in cuticle formation and subsequently, after 24 h in a frozen state, protease production. In addition to the mapping of RNA sequencing, we have focused on a select set of genes arising both from the expression profiles, as well as implicated from other cold tolerance studies, to undertake qPCR. Among the most abundantly represented transcripts in the RNA mapping is the zinc-metalloenzyme, neprilysin, which also shows a particularly strong upregulated signal through qPCR once the nematodes have frozen.

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.

Cold tolerance of the Antarctic nematodes Plectus murrayi and Scottnema lindsayae.

The cold tolerance of the Antarctic nematodes Scottnema lindsayae and Plectus murrayi was determined using material freshly isolated from the field. Both species could survive low temperatures but the survival of S. lindsayae was greater than that of P. murrayi. Field soil temperatures in late spring-early summer indicated a minimum temperature of -19.5°C and a maximum cooling rate of 0.71°C min(-1). In P. murrayi grown in culture, there was no significant effect of acclimation, nor of the two culture media used, on survival after freezing but survival was greater if freezing was seeded at -1°C than at lower temperatures. The freezing survival ability of P. murrayi is much less than that of Panagrolaimus davidi CB1, another Antarctic nematode. Cryomicroscopy indicates that P. murrayi can survive low temperatures by either cryoprotective dehydration or freezing tolerance, but that freezing tolerance is the dominant strategy. Measurable thermal hysteresis was detected only in highly concentrated extracts of the nematodes, indicating the presence of an antifreeze protein, but at the concentrations likely to be found in vivo, the major function of the ice active protein involved is probably recrystallization inhibition.

Molecular analysis of the cold tolerant Antarctic nematode, Panagrolaimus davidi.

Isolated and established in culture from the Antarctic in 1988, the nematode Panagrolaimus davidi has proven to be an ideal model for the study of adaptation to the cold. Not only is it the best-documented example of an organism surviving intracellular freezing but it is also able to undergo cryoprotective dehydration. As part of an ongoing effort to develop a molecular understanding of this remarkable organism, we have assembled both a transcriptome and a set of genomic scaffolds. We provide an overview of the transcriptome and a survey of genes involved in temperature stress. We also explore, in silico, the possibility that P. davidi will be susceptible to an environmental RNAi response, important for further functional studies.

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.

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 2h 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.

Nematodes from the Victoria Land coast, Antarctica and comparisons with culturedPanagrolaimus davidi

AbstractMorphological and genetic information was used to determine the species identity and relationships of four species of nematodes from the Victoria Land coast, Antarctica; and between laboratory and field populations ofPanagrolaimus davidiTimm. The 18S and D3 expansion segments of 28S rRNA were successfully amplified from the four species:Scottnema lindsayaeTimm,Plectus murrayiYeates,Eudorylaimusspp. andPanagrolaimus davidi. The four species from field populations mapped to three different nematode clades, indicating that if they are survivors in, rather than colonizers of, Antarctica they originate from several Gondwanan ancestors. The laboratory culture ofP. davidiis shown to be a different species to that of the field samples ofP. davidi, which were genetically identical to each other. There are several possible explanations for this observation, which require further investigation.

The ability of the Antarctic nematode Panagrolaimus davidi to survive intracellular freezing is dependent upon nutritional status

The Antarctic nematode Panagrolaimus davidi is the best documented example of an animal surviving intracellular freezing and the only animal so far shown to survive such freezing throughout its tissues. However, a recent study found that after exposure to a freezing stress that produced intracellular freezing in a proportion of nematodes, the resulting survival levels could be explained if those nematodes that froze intracellularly had died. We have thus re-examined the survival of intracellular freezing in this nematode. The ability to survive a freezing exposure that is likely to produce intracellular freezing (freezing at -10 °C) declines with culture age. In cultures that are fed regularly, the ability to survive freezing at -10 °C increases, but in starved cultures freezing survival declines. Survival of intracellular freezing in fed cultures was confirmed using cryomicroscopy, staining of cells with vital dyes and by freeze substitution and transmission electron microscopy. We have thus confirmed that P. davidi can survive intracellular freezing and shown that this ability is dependent upon them being well fed. The effect of culture conditions on the nutrient status of the nematodes should thus be an important factor in the design of experiments.

Osmoregulation in the Antarctic nematodePanagrolaimus davidi

A technique for determining the internal osmotic concentration of a small nematode using a modified nanolitre osmometer is described and used to investigate osmoregulation in the Antarctic nematode Panagrolaimus davidi. This technique enables the osmotic concentration to the measured with an accuracy of +/-12 mmol kg(-1). The pattern of melting in the nematode's different body compartments suggests that it is the osmolality of its pseudocoelomic fluid that is being measured. Panagrolaimus davidi maintains its internal osmotic concentration above that of the external medium and is thus an hyperosmotic regulator. The nematode achieves regulation under hyposmotic stress more rapidly than under hyperosmotic stress.

Changes in soil nematode populations indicate an annual life cycle at Cape Hallett, Antarctica

Soil biological studies have suggested that generations of terrestrial nematodes in continental Antarctica may take many years. We sampled soil nematodes at three sites in the Adélie penguin colony at Cape Hallett on four dates in a two month sampling period (16 November 2002–18 January 2003). The size class distribution of over 3500 nematodes, and the occurrence of adults, indicate an annual life cycle of the bacterial-feeding Panagrolaimus davidi and Plectus murrayi , at each site. Nematode abundance ranged from 2 to 1375/g dry soil. Moderate temperatures and the regular presence of free water underlie this biological activity and related contribution to soil processes.

Surviving the cold: molecular analyses of insect cryoprotective dehydration in the Arctic springtail Megaphorura arctica (Tullberg)

BackgroundInsects provide tractable models for enhancing our understanding of the physiological and cellular processes that enable survival at extreme low temperatures. They possess three main strategies to survive the cold: freeze tolerance, freeze avoidance or cryoprotective dehydration, of which the latter method is exploited by our model species, the Arctic springtail Megaphorura arctica, formerly Onychiurus arcticus (Tullberg 1876). The physiological mechanisms underlying cryoprotective dehydration have been well characterised in M. arctica and to date this process has been described in only a few other species: the Antarctic nematode Panagrolaimus davidi, an enchytraied worm, the larvae of the Antarctic midge Belgica antarctica and the cocoons of the earthworm Dendrobaena octaedra. There are no in-depth molecular studies on the underlying cold survival mechanisms in any species.ResultsA cDNA microarray was generated using 6,912 M. arctica clones printed in duplicate. Analysis of clones up-regulated during dehydration procedures (using both cold- and salt-induced dehydration) has identified a number of significant cellular processes, namely the production and mobilisation of trehalose, protection of cellular systems via small heat shock proteins and tissue/cellular remodelling during the dehydration process. Energy production, initiation of protein translation and cell division, plus potential tissue repair processes dominate genes identified during recovery. Heat map analysis identified a duplication of the trehalose-6-phosphate synthase (TPS) gene in M. arctica and also 53 clones co-regulated with TPS, including a number of membrane associated and cell signalling proteins. Q-PCR on selected candidate genes has also contributed to our understanding with glutathione-S-transferase identified as the major antioxdidant enzyme protecting the cells during these stressful procedures, and a number of protein kinase signalling molecules involved in recovery.ConclusionMicroarray analysis has proved to be a powerful technique for understanding the processes and genes involved in cryoprotective dehydration, beyond the few candidate genes identified in the current literature. Dehydration is associated with the mobilisation of trehalose, cell protection and tissue remodelling. Energy production, leading to protein production, and cell division characterise the recovery process. Novel membrane proteins, along with aquaporins and desaturases, have been identified as promising candidates for future functional analyses to better understand membrane remodelling during cellular dehydration.

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 degrees 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.

Ice-active proteins from the Antarctic nematode Panagrolaimus davidi

The Antarctic nematode Panagrolaimus davidi has an ice-active protein that shows recrystallization inhibition but no thermal hysteresis. It belongs to a class of ice-active proteins found in a variety of freezing-tolerant organisms that display insignificant levels of thermal hysteresis in the context of the environmental temperatures to which they are exposed. The recrystallization inhibition activity of the P. davidi ice-active protein is present at low concentrations, is relatively heat stable, is affected more by acid than by alkaline pH, is not calcium dependant and is not affected by reagents that target carbohydrate residues or sulphydryl linkages. A hexagonal ice crystal growth form also indicates the presence of an ice-active protein. This protein could have important functions in the survival of intracellular freezing by this organism by controlling the stability of ice after its formation.

Freezing and cryoprotective dehydration in an Antarctic nematode ( Panagrolaimus davidi) visualised using a freeze substitution technique

The pattern of ice formation during the freezing of Panagrolaimus davidi, an Antarctic nematode that can survive intracellular ice formation, was visualised using a freeze substitution technique and transmission electron microscopy. Nematodes plunged directly into liquid nitrogen had small ice crystals throughout their tissues, including nuclei and organelles, but did not survive. Those frozen at high subzero temperatures showed three patterns of ice formation: no ice, extracellular ice, and intracellular ice. Nematodes subjected to a slow-freezing regime (at −1 °C) had mainly extracellular ice (70.4%), with the bulk of the ice in the pseudocoel. Some (24.8%) had no ice within their bodies, due to cryoprotective dehydration. Nematodes subjected to a fast-freezing regime (at −4 °C) had intracellular (54%) and extracellular (42%) ice. Intracellular ice was confined to the cytoplasm of cells, with organelles in the spaces in between ice crystals. The survival of nematodes subjected to the fast-freezing regime (53%) was less than those subjected to the slow-freezing regime (92%).

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.

Freezing survival and cryoprotective dehydration as cold tolerance mechanisms in the Antarctic nematode Panagrolaimus davidi.

The relative importance of freezing tolerance and cryoprotective dehydration in the Antarctic nematode Panagrolaimus davidi has been investigated. If nucleation of the medium is initiated at a high subzero temperature (-1 degree C), the nematodes do not freeze but dehydrate. This effect occurs in deionised water, indicating that the loss of water is driven by the difference in vapour pressure of ice and supercooled water at the same temperature. If the nematodes are held above their nucleation temperature for a sufficient time, or are cooled slowly, enough water is lost to prevent freezing (cryoprotective dehydration). However, if the medium is nucleated at lower temperatures or if the sample is cooled at a faster cooling rate, the nematodes freeze and can survive intracellular ice formation. P. davidi thus has a variety of mechanisms that ensure its survival in its harsh terrestrial Antarctic habitat.

NEMATODES FREEZE-DRY TO SURVIVE

Even though 99% of the Antarctic land mass is smothered in ice, it is one of the driest places on earth. Few plants and animals have risen to the challenge of surviving at the continent's bitterly cold temperatures, but even though the continent's temperature can fall forty degrees below freezing, some nooks and crannies manage to reach temperatures that would be pleasing even in temperate zones. However, surviving the daily freeze/thaw cycle adds another challenge to microscopic nematodes that must protect themselves from the extensive damage wreacked by ice crystals. David Wharton is fascinated by how animals survive in the planet's harshest environments, and recently became interested in how Antarctic nematodes survive freezing. Amazingly, Panagrolaimus davidi is one of the few creatures that can survive intracellular freezing, but when Wharton looked at the way that the nematode responds at temperatures close to zero, he found that rather than freezing solid, the nematodes survive by freeze-drying(p. 215).Wharton describes how he found the nematodes in a coastal valley when he visited Antarctica in 1989. The nematodes were living on a patch of algae that were growing in a snow-melt stream. When he returned to his New Zealand lab,he realised that the nematodes were remarkably easy to keep in culture,flourishing at 25°C and producing another generation every week, as opposed to the year that it takes the worms to reproduce in their natural environment.Fascinated by how the worms survived freezing, Wharton had watched ice crystals form in the worm's cells as he dropped the temperature, but he had also realised that some of the worms didn't freeze; they shrivelled up and dried out instead. Keen to know why some worms froze while others desiccated,Wharton began watching how the worms fared as ice formed around them at different temperatures.Wharton suspended thousands of the nematodes in water, and sandwiched the drop between two coverslips so that he could film the worms as the water froze. After lowering the temperature, he initiated freezing by touching the edge of the droplet with an ice crystal, and watched as the ice slowly encased the nematodes. After 30 minutes Wharton recorded the fraction of frozen nematodes versus dehydrated worms, and found that even though the water was completely frozen at -1°C, all of the worms had dehydrated, while at-5°C, all of the worms froze, and only one third had survived when they thawed.He also tested how the nematodes reacted as he cooled the forming ice at different rates and found that when the worms iced up quickly, they couldn't escape freezing, but if he cooled the forming ice slowly, again the worms dehydrated.But how do the worms escape freezing at some temperatures and cooling rates and not others? Wharton explains that as the water surrounding the nematode freezes, the external vapour pressure falls below the vapour pressure of the liquid water still inside the worm's cells. If the worm is cooling slowly, or it is only just below freezing, the water is gently pulled out of the worm. But if the temperature drops too quickly before the dehydrating pressure gradient is established the worm cannot escape, and it freezes.Wharton explains that some earthworms also protect their eggs from subzero temperatures by dehydration, but the Antarctic nematodes have capitalised on both approaches to thrive at a pedestrian rate in the coldest place on earth.

Invertebrates in ornithogenic soils on Ross Island, Antarctica

A habitat suitability model developed for soil biotic communities in the McMurdo Sound region, Antarctica predicts that soil moisture, organic carbon, and salinity exert control on the abundance and complexity of soil food chains. The model has been intensively tested in dry and carbon-poor soils of the Dry Valleys. To determine the influence of moisture and soil organic mater in wetter soils with high C content, invertebrates (nematodes, rotifers, and tardigrades) from soil samples collected in and near penguin rookeries on Ross Island (Cape Bird, Cape Crozier, and Cape Royds) were examined. Invertebrates were present in less than 50% of all collected soil samples. Although four nematode species were identified (Eudorylaimus antarcticus, Panagrolaimus davidi, Plectus antarcticus, Scottnema lindsayae), only populations of Panagrolaimus davidi were observed in soils collected from within penguin rookeries. Abundances of Panagrolaimus davidi and rotifers differed among rookeries, and year of sampling had a significant effect only on the populations of Panagrolaimus davidi. There were no temporal differences in soil moisture and soil chlorophyll a concentrations within each rookery or across rookeries. No invertebrates were correlated with soil moisture or chlorophyll a at the time of collection. Counter to our expectations, higher nutrient, organic matter, and moisture levels did not result in more abundant and diverse invertebrate communities in the rookery soils. It appears that excessive accumulations of nutrients, creating high soil salinity, may limit soil invertebrate presence within active rookeries.

On the distribution of terrestrial invertebrates at Cape Bird, Ross Island, Antarctica

Terrestrial invertebrate distribution was surveyed over 12 km2 of the ice-free area at Cape Bird, Ross Island, Antarctica. Gomphiocephalus hodgsoni (Collembola: Hypogastruridae), Stereotydeus mollis and Nanorchestes antarcticus (Acari: Prostigmata), Panagrolaimus davidi, Plectus sp. and Scottnema lindsayae (Nematoda), Tardigrada, Rotifera and Protozoa were all recorded. Invertebrates were found at 47 of 103 locations sampled. Logistic regression analysis suggested that the presence of mites and Collembola was strongly related to chlorophyll-a content of soil; but only Stereotydeus mollis and N. antarcticus were related to the presence of macroscopic vegetation, suggesting that current methods of assessing areas for protection may not adequately allow for invertebrate communities. Invertebrate communities were not dependent on ornithogenic carbon input. A better understanding of dispersal mechanisms is necessary to understand distributions of invertebrates in ice-free areas, particularly in light of potential habitat changes as a result of climate change.

Terrestrial invertebrate abundance across a habitat transect in Keble Valley, Ross Island, Antarctica

Summary The abundance and distribution of soil invertebrates was examined along a transect in Keble Valley, Cape Bird, Ross Island, Antarctica during and after snowmelt. During snowmelt, streams were flowing across the transect, and the moisture environment was highly heterogeneous, whereas after the cessation of stream flow the moisture environment became more homogeneous. Of the seven groups of invertebrates examined, only the prostigmatid mite Stereotydeus mollis showed any redistribution between the two sampling points. The streams had higher water availability, higher soil chlorophyll-a content, higher organic content and lower surface salinity than the areas beside the streams. Nematode ( Panagrolaimus davidi ), soil rotifer and soil protozoan abundance was significantly associated with this area of high productivity, and the cryptostigmatid mites S. mollis and Nanorchestes antarcticus were found near the stream centres. By contrast, the collembolan Gomphiocephalus hodgsoni was found at riparian margins, and it is hypothesised that this could be due to habitat and food preferences.