Eukaryotes in extreme environments

by Dave Roberts, Zoology Department

In March 1995, I broadcast an appeal through the Biosci newsgroups for Microbiology and Protista for information about eukaryotes in extreme environments. The scope of the enquiry was intended to cover active growth not survival through some form of encystment. A very warm thank you to all those who contributed to my search. Below is a summary of my findings, originally posted in November 1996 and updated thereafter. I intend to maintain this page and to add further categories as time, opportunity and information permit, so please email me with other observations and citations.

The major headings under which the data are organised are:

Summary

The majority of eukaryotes found living in extreme environments are microbial and a central problem in the study of all microbial eukaryotes is the lack of reliable cultivation methods. Only a tiny fraction of the organisms that can be observed in environmental samples can be cultured in the laboratory, even from mesophilic environments. Extreme environments are generally more difficult to replicate in the laboratory and more difficult to keep stable. The ability to bring these fascinating creatures living into the laboratory is currently the biggest stumbling block to advancing knowledge.

Eukaryotic microbial life may be found actively growing in almost any extreme condition where there is a sufficient energy source to sustain it, with the exception of high temperature (>70ºC). For most eukaryotes, therefore, a central requirement for growth in a habitat is sufficient energy flowing through the biosphere to support a second trophic level, as illustrated by the soda lakes Simi and Nakuru (see below). If it were not for the high productivity of Lake Nakuru, the low diversity in Lake Simi could easily have been attributed to its extreme pH.

Colonisation of extreme habitats is not normally restricted to a single taxonomic group, with the exception of xerophilous habitats which are only tolerated by the fungi. Eukaryotic cells are exceedingly adaptable and not notably less adaptable than the prokaryotes, although most habitats have not been sufficiently well explored for sound generalisations.

Anaerobes

One method of adaptation that eukaryotes exploit to great effect is the outright hijacking of the abilities of other organisms. The endosymbiotic origin of the mitochondrion (Margulis, 1970) is probably the best known example, which is made possible by the ability to phagocytose and to compartmentalise cellular activity. In this way eukaryote lineages can adapt rapidly to novel environmental challenges and opportunities, including the colonisation of extreme habitats. Consider, for instance, anaerobic habitats: free-living ciliates are generally regarded as aerobic and totally reliant on mitochondrial respiration for energy generation. However, several lineages have colonised anaerobic habitats where the role of the mitochondrion has been replaced by the hydrogenosome and obligately anaerobic ciliates now lack mitochondria (Brul & Stumm, 1994; Embley et al., 1995; Embley et al., 1992; Esteban et al., 1993). These anaerobic ciliates are not phylogenetically related, implying that the process allowing their existence was not rare (difficult), and they have strong morphological ties to their aerobic relatives, implying that the process was recent. There are a great number of anaerobic eukaryotes, famously among the yeasts, but also the other protists, for instance in the rumen: see Fenchel, 1996 for a review of the physiological implications of anaerobic life for eukaryotes. Evidence is now accumulating that all known extant eukaryotes have contained a mitochondrion at some time in their evolutionary history (Bui et al., 1996; Germot et al., 1996; Germot et al., 1997; van der Giezen et al., 1997; Hirt et al., 1997; Horner et al., 1996; Roger et al., 1996).

There are no known eukaryotic anaerobic phototrophs (ie capable of anoxygenic photosynthesis) (Fenchel, 1996).

Thermophiles

The best studied high-temperature eukaryote is the acidophilic phototroph Cyanidium caldarium: see (Seckbach, 1994) for review. Its exact taxonomic affiliations are still unclear, but it is generally grouped with the "red algae" (rhodophytes) since its chloroplasts possess chlorophyll-a and C-phycocyanin. Brock (1978) carefully examined the growth and ecology of this organism and determined its optimal growth temperature was 45ºC and the maximum temperature at which growth occurred was 57ºC. Earlier reports of growth at much higher temperatures, egCopeland, (1936) reported 75-80ºC, were attributed by Brock and his colleagues to either the measurement of temperature away from the organisms themselves, or to the temperature having increased and the organisms being observed in the process of dying. It is interesting to note that Brock isolated numerous strains of Cyanidium caldarium growing at various temperatures and found that they all had the same optimum growth temperature: that is, they do not seem to have formed strains adapted to growth at higher temperatures. It is also interesting to note that the niche occupied by Cyanidium, of hot acid conditions, does not seem to have any competition for the available resources in that thermophilic cyanobacteria require alkaline conditions for growth. It is also noteworthy that at the time he was writing (Brock, 1978), all the hot, acid soils and waters in the world were colonised by Cyanidium, which seem identical, save for the acid springs in Hawaii, which seem to be devoid of life. Brock suggests that this might simply be because Hawaii is geologically recent and far distant from other hot springs, so that there has not yet been an opportunity for Cyanidium to colonise this particular habitat. There are a wide variety of other eukaryotes living at somewhat less extreme temperatures, see Tansey & Brock (1978) for review.

Brock (Brock, 1978; Tansey & Brock, 1978) stressed the difficulties of estimating growth temperatures from ecological observations. There are many thermophilic fungi which have been isolated from compost and similar environments where temperatures can exceed 80ºC, but, until laboratory cultures demonstrate growth at these temperatures, the observations should be treated with caution (Tansey & Brock, 1978). The upper limit for thermophilic fungi seems to be in the region of 60ºC (Tansey & Brock, 1978).

The polychaete Alvinella pompejana, the Pompeii worm, lives in burrows on hydrothermal vent chimneys with a strong temperature gradient, which averages 68ºC but has frequent peaks exceeding 81ºC (Cary et al.,1998), although they do leave their burrows to feed. It is possible that, like Heteromita (see below), the worm conducts temperature-sensitive biochemical processes during those times when the temperature is more modest. A more interesting question, yet to be addressed, is how and at what temperature the post-larval/juvenile worms begin building their burrows on hot chimneys.

Unlike the situation in Cyanidium, three species of flagellates have been raised to grow at 70ºC by incremental increases in cultivation temperature (Dallinger, 1887). This study reported more than 100 years ago has not been repeated, but Dallinger noted that increasing the temperature in too large steps, or before the cells had fully adapted to the current temperature, killed the cultures. This phenomenon of incremental adaptation to growth temperature has also been noted for psychrophiles (Lee & Fenchel, 1972), (see below).

There is a vigorous debate about whether it is possible for eukaryotic architecture to evolve a true hyperthermophile (Forterre, 1995; Forterre et al., 1995; Miller & Lazcano, 1995). The crux of the debate revolves around the ability of the central biochemical machinery, nucleic acid transcription and translation, to operate at these high temperatures. Clearly the cell's membrane composition must change to retain the required degree of fluidity for proper function (Sprott et al., 1991). Also, all hyperthermophiles contain reverse gyrase, which induces positive super-coiling of DNA which enhances its thermal stability (Bouthier de la Tour et al., 1991; Forterre et al., 1996; Forterre et al., 1995). It seems clear that we do not yet understand all the protective mechanisms operating which allow cells like the archaean Pyrococcus to grow above 100ºC, and indeed what the actual upper limit for life might be (Erauso et al., 1996; Stetter et al., 1990). The half-life of unprotected RNA falls very rapidly with increasing temperature (Forterre et al., 1995) and as a direct consequence eukaryotes face two major problems. First is that following transcription, many eukaryotic genes are subjected to post-transcriptional modification, a process which take a certain amount of time. However, a number of hyperthermophilic archaeans post-transcriptionally modify the product of their tRNA genes in a manner which is reminiscent of the eukaryotes (Edmonds et al., 1991). Furthermore, the essential organisation of the transcriptional apparatus predates the divergence of the Archaea and the Eucarya (Ciaramella et al., 1995). Second, the mRNA has to make its way out of the nuclear membrane in order to be translated by ribosomes. Any hyperthermophile would have to possess a mechanism to protect the mRNA from hydrolysis. From an evolutionary perspective, if the Archaea and the Eucarya are sister taxa then the potential to colonise high-temperature environments presumably existed in their last common ancestor and there is therefore the potential in the eukaryotic lineage to do so too.

Psychrophiles

Water is the solvent for life and must be present in a liquid state for growth to occur. This sets a practical lower limit for growth slightly below 0ºC. Colouring of snow can be caused by a variety of photosynthetic eukaryotes such as Chlamydomonas nivalis, Chloromonas (Scotiella), Ankistrodesmus, Raphionema, Mycanthococcus and certain dinoflagellates (Prescott, 1978) and represents a well-known illustration of cold-adaptation.

One adaptation to life in the cold has been studied in Heteromita globosa, which is a heterotrophic flagellate growing in Antarctic fellfields whose physical environment is characterised by highly variable moisture and temperature regimens, including short-term freeze-thaw cycles and diurnal temperature fluctuations of up to 20ºC (occasionally up to 40ºC). At least 24 species of protist and some fruticose lichens and mosses grow in these conditions in addition to bacteria (Cowling & Smith, 1987; Smith, 1984). Heteromita is a very common soil microflagellate with a world-wide distribution with an optimum temperature for growth around 23ºC. Under Antarctic conditions it demonstrates adaptations which permit survival of freeze-thaw cycles (by rapid and temperature-sensitive encystment and excystment) and by optimal utilisation of resources during short periods, which allow this temperate species to grow actively at mean temperatures close to zero (Hughes & Smith, 1989).

Adaptation to growth at low temperature has been most extensively studied in the Antarctic sea-ice, where a wide taxonomic range of flagellates have been observed. Here, in contrast to Heteromita, efforts to cultivate these strains, which normally grow at around -2ºC, failed completely if the temperature was raised above +2ºC, even when the cultures were established at lower temperatures and raised in small steps (R. Leakey, British Antarctic Survey, pers. comm.). Lee & Fenchel (1972) observed that Antarctic sea-ice contained some new ciliate species and others which were morphologically identical to well-known marine ciliates. In studies with the ciliate Euplotes, Lee & Fenchel (1972) compared three species isolated from Antarctic sea-ice, temperate waters and tropical waters. They found that the temperature range for the three species overlapped, but the Antarctic species E. antarcticus was unable to survive above 17ºC, although there was no clear cut-off for growth because the range could be enhanced by slow adaptation. The ciliate Holosticha sp. was reported to be unable to divide above -2ºC (Lee & Fenchel, 1972).

Garrison & Buck (1989a) report that in the Weddell Sea, at an unrecorded temperature but close to the edge of the sea-ice, the heterotrophic biomass is dominated by flagellates and ciliates, other protists and micrometazoa making up a small fraction of the heterotrophic biomass. The abundance was greatest in a well-developed ice-edge bloom in the spring. They also report (Garrison & Buck, 1989b) that the sea-ice itself contains a rich and varied population of microbes, which includes phototrophs (diatoms and flagellates) and heterotrophs (flagellates, ciliates and micrometazoa). The presence of the heterotrophs indicates an active food web.

Modifications to low temperature growth must, as in high-temperature systems (see above), involve substantial modification to the cell's lipid or fatty acid composition in order to retain membrane fluidity. Presumably, in the stable, low temperature of the sea, the flagellates have lost the ability to synthesise some components, perhaps the lipids or fatty acids, rendering them unable to grow at higher temperatures. Some low-temperature environments are also characterised by low water availability, such as hypersalinity associated with sea-ice (Garrison et al., 1986) or the lithotrophs associated with fellfields (Cowling & Smith, 1987; Smith, 1984).

Acidophiles

Until recently only four organisms, all eukaryotes, were known to grow near pH 0:Cyanidium caldarium (see above under thermophiles), and three fungi, Acontium cylatium, Cephalosporium spp., and Trichosporon cerebriae (Schleper et al., 1995). It has been observed that Cyanidium maintains its internal milieu at close to neutral pH (Beardall & Entwisle, 1984; Brock, 1978), and the means to do that is presumably a primary adaptation to this niche. Schleper et al. (1995) have recently added two prokaryotic members to this exclusive set, Picrophilus oshimae and P. torridus, thermophilic archaeans from Japanese solfataras. It is an important physiological question to determine how this is achieved, either with a strong proton pump or a low proton membrane permeability?

At more moderate pH values there are, once again, a great profusion of protistan and fungal organisms, for instance in the rumen.

Alkalophiles

Two African soda lakes, with a pH of about 10, have been studied for their microbial populations: Lake Nakuru and Lake Simbi (Curds et al., 1986; Finlay et al., 1987). The former lake supports a very high population of flamingos, counted in millions, feeding largely on cyanobacteria (Brown, 1975) dominated by Spirulina, growing in the lake. The flamingos have been calculated to return about 15.6 tonnes dry weight of faecal and urinary matter to the lake each day, which resulted in a standing crop of non-cyanobacterial prokaryotes in the region of 3x10⁸ cells/ml. In this rich environment, there were at least 20 different heterotrophic species of protist, and three species of rotifers. Lake Simbi, on the other hand, was stratified with an extensive hypolimnion and there were far fewer flamingos, fewer than 20 individuals during the study period. The diversity and abundance of eukaryotic species was much lower than at Lake Nakuru. Samples of these populations (from Lake Nakuru) grew readily in the laboratory in a medium designed to mimic the ionic strength and pH of the lake, where several species not included in the above surveys were observed after enrichment (Roberts, unpublished observation). These figures for abundance and diversity are in the same range as similar studies on non-soda lakes in the same region of Africa (Curds et al., 1986). Alkalophiles, including many eukaryotes, have been reviewed by Kroll (1990).

Extrapolating from the observation that Cyanidium maintains its internal milieu at close to neutral pH (see above), one might speculate that at pH 10 these cells are able to resist the influence of the external medium on their internal chemistry.

Halophiles

Hypersaline conditions support a wide variety of eukaryotic microbes, including diatoms (Rothschild et al., 1994). There are many salt-adapted flagellates (Ruinen, 1938), including numerous heterotrophs. The African soda-lakes are also hypersaline (Lake Nakuru was reported being 8% Na; see above under alkalophiles), and as discussed above can support a very diverse microbial population if there is a ample energy input (Finlay et al., 1987). Halophilic and halotolerant algae have been reviewed by Gilmour (1990) and although Grant (1991) reports that there are no eukaryotes among the extreme halophiles, this view must depend on an exact definition of "extreme" since Dunaniella salina grows up to saline saturation (see below). There is a broad range of eukaryotic taxonomic groups found in hypersaline environments although few have been studied in culture (Grant, 1991).

The major problem facing halophiles is control of their osmotic pressure, without which they might lose water to the surrounding environment. Dunaniella salina synthesises high concentrations of intracellular glycerol to balance the external osmotic pressure (Avron & Ben-Amotz, 1979). The control of water relations has been reviewed by Smith (1978) and Brown (1990).

Barophiles

High pressure is used as a means of sterilising drugs and foodstuffs (Ludwig et al., 1996). The mechanisms by which pressure affects cellular physiology are many and varied (Markley et al., 1996; Marquis & Matsumura, 1978). Ciliates can certainly withstand rapid, repeated cycling (up and down in unit minutes) from 0.1 to 0.3 MPa without ill-effects (Roberts, unpublished data). The fungus Magnaporthe grisea is capable of producing an internal pressure of 8 MPa during the process of mechanical penetration of its host plant (de Jong et al., 1997) by the synthesis of glycerol (cf Dunaliella above). There are abundant eukaryotic communities on the continental shelf (up to 20 MPa). Indeed, there are metazoa present in the deepest oceans (Bruun, 1977), so there seems to be no fundamental reason why heterotrophic eukaryotic microbes should not be present at great depths given sufficient food. Holothurians (sea Cuccmbers; members of the phylum Echinodermata) are reported to be abundant in the world's deepest location, the Challenger Deep (in the West Pacific, 400 km southwest of Guam; approximately 110 MPa) (Kato, 1997). Surface organisms cannot necessarily survive great pressures; amoebae, for instance, become progressively less able to form pseudopodia with increasing pressure, becoming spherical and motionless at about 40 MPa (Kitching, 1970; Marsland, 1958). The mechanism for this loss of mobility is unknown. Increasing pressure causes acidification of the central vacuole in the yeast Saccharomyces cerevisiae at about 40 MPa (Abe & Horikoshi, 1995).

Xerophiles

The driest regions can support eukaryotic life, for instance lichens which grow on stones, even in the Negev Desert (Palmer & Friedmann, 1990). However, the ability to grow in conditions out of liquid water seems to be restricted in the microbes to the fungi. It is an everyday observation in food spoilage that the first colonisers are normally fungi, especially of foods with reduced water activity as a means of preservation (jams, marmalades and similar conserves). The fungi are also able to utilise the filamentous habit to grow through regions unsuitable for growth including bridging air spaces in the search for suitable habitats. The capacity to distribute nutrients through the filamentous colony is clearly an important adaptation for this niche as the capacity to withstand desiccation while growing out of water.

Other extreme environments

Eukaryotes are now accepted as being the product of symbiotic events, which are normally thought of as between an eukaryote and a prokaryote (as in the mitochondrion or plastid, for instance). Many eukaryotes are themselves involved in a variety of intimate associations and there is some evidence that this close relationship is stressful, or in this context extreme (Douglas, 1996). There are many highly evolved groups of parasites which have adapted to these stresses and now cannot grow outside their hosts and thus can be considered to be living in an extreme habitat.

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