Chela-Flores, J. (1998). Europa: A potential source of parallel evolution for microorganisms. In: Instruments, Methods and Missions for Astrobiology. The International Society for Optical Engineering, Bellingham, Washington USA. (R.B.Hoover, ed.), Proc. SPIE, 3441, pp. 55-66.

Europa: A potential source of parallel evolution for micro-organisms

Julian Chela-Flores

The Abdus Salam International Centre for Theoretical Physics
Office 276, Miramare, P.O.Box 586; Strada Costiera 11; 34136 Trieste, Italy and
Instituto de Estudios Avanzados, (Universidad Simon Bolivar)
Apartado 17606 Parque Central, Caracas 1015A, Venezuela.

ABSTRACT

Europa, the ice-covered satellite of Jupiter, is currently the most favourable site for the search of extraterrestrial life. Hydrothermal vents on the Earth's sea floor have been found to sustain life forms that can live without solar energy. Similar possible volcanic activity on Europa, caused by its interaction with Jupiter and the other Galilean satellites, makes this Jovian moon the best target for identifying a separate evolutionary line. This search addresses the main problem remaining in astrobiology, namely, the quest for discrete, or 'parallel' evolutionary lines in the universe. We explore ideas related to Europa's possible biological activity, particularly its likely degree of evolution. We have conjectured that evolution may have occurred in Europa and that the experimental test of such a conjecture is feasible 1-3: A lander space craft capable of penetrating the Europan surface ice-layer does not seem beyond present technological capabilities. Although difficult instrumentation issues are involved, we have initiated the discussion of what would seem to be a reasonable biological experiment. The possibility of detecting biomolecules on the ice surface of Europa has recently been made by others 4. A possible mechanism for bringing biomolecules to Europa's surface will be critically reviewed.

Keywords: Europa, eukaryogenesis, distribution of life in the universe, astrobiology, solar system exploration, instrumentation.

1. INTRODUCTION

In the present work astrobiology is defined as the science of the origin, evolution and distribution of life in the universe. An inevitable revolution is expected to occur in this new scientific discipline in the next decade. The main reason for this expectation is the large number of space missions that are either at the planning stage, or are currently studying the sun, planets, satellites and small bodies of the solar system. This feeling is reinforced by the ever increasing capability of searching for extraterrestrial intelligence. We feel that some guiding principles are necessary in the selection of possible, and relevant experiments, that may be feasible within the constraints of the payloads that will be available. We focus our attention on certain questions regarding the expected universal validity of the theories that we know are relevant to the Earth biota. We expect that some general guiding principles may emerge from solar system exploration regarding the least known aspect of astrobiology, namely, the distribution of life in the universe.

However, some theories that are expected to be of universal validity have already guided our research in the evolution of life on Earth. The two main ingredients of the theory of evolution are good examples, namely, the theory of common descent and the theory of natural selection. All the Earth biota demonstrates the general validity of both of Darwin's key intuitions. We expect similar guiding principles to hold when we turn our attention to the evolution of life in the universe. In this paper we turn our attention to the question of the distribution of life in the universe. We therefore find ourselves in a subject for which there is no theory now. This total lack of understanding of the distribution of extraterrestrial life is particularly troublesome. Nevertheless, technical ability to search for extraterrestrial intelligence, by means of radioastronomy, has made remarkable technological advances 5. In spite of this success, a theoretical insight in the subject of the distribution of life in the universe is still missing. Consequently, in Sec. 2 we make a preliminary approach of the question of identifying some general principles that may guide us in our search for extraterrestrial life.

2. AN APPROACH TO THE DISTRIBUTION OF LIFE IN THE UNIVERSE

2.1. The genesis of the three domains of life on Earth.
Micro-organisms without a nuclear membrane enveloping its genome are called prokaryotes, since the Greek word karyon means 'nucleus'. When a truly internal membrane is present (with pores and with the presence of the protein lamin), the micro-organism is referred to as a "eukaryote". With the enormous scope of prokaryotic evolutionary data available today from an extensive micropaleontological record, we are certain that the theory of common descent leads back to a single common prokaryotic ancestor, or "progenote".

The possible phenotype and genotype of the progenote can be studied today through comparison of macromolecules, allowing the recognition of early events that led to the divergent genesis of the highest taxa (domains) among micro-organisms. These domains include: Bacteria (all the eubacteria); Archaea (all the archaebacteria); and Eukarya (the highest taxon gathering all the eukaryotic cells). Of the three domains of life, only the first two are prokaryotic.

Regardless of the exact moment in which the progenote first arose, it is generally agreed that over the course of 2 billion years (Gy), micro-organisms oxygenated the Earth, produced the bulk of the iron ores, and fashioned the eukaryotic cell. This was a transcendental transition, which will be referred to as "eukaryogenesis". This event may be considered the first step in the path to multicellular, intelligent organisms on this planet. If intelligent organisms exist elsewhere in the cosmos, the eukaryogenesis stage may have been part of their own evolution. This remark underlies our motivation for making extraterrestrial eukaryotes the principal aim of our research.

However, a deep question remains, namely the origin of the eukaryotic cell, once the fairly straightforward initiation of prokaryotic life got started. Some aspects of 'eukaryogenesis' are evident. For instance, symbiosis must have played a significant role. (Certain difficulties may require some clarification 6.) Nevertheless, the prokaryotic-eukaryotic transition is very ancient. Microplaeontology suggests that during the last 2 Gy eukaryotic cells have been preserved in the fossil record.

2.2. Eukaryogenesis as a universal phenomenon.
We have suggested in our recent work 7 that eukaryogenesis is also a universal phenomenon. To borrow the phrase of Christian De Duve 8, we may say that the laws of physics and chemistry imply an 'imperative' appearance of life during cosmic evolution. However, with the following conjecture we are going one step beyond:

Life is not only a natural consequence of the laws of physics and chemistry, but once the living process has started, then the cellular plans, or blueprints, are also of universal validity: The simplest cellular blueprint (prokaryotic) will lead to a more complex cellular blueprint (eukaryotic). Eukaryogenesis will occur inexorably because of evolutionary pressures, driven by environmental changes in planets, or satellites, where conditions may be similar to the terrestrial ones.

This is a testable hypothesis, in spite of the intrinsic difficulty of identifying terrestrial-like environments. In Sec. 3.2, we shall comment on the analogy between the Earth and a specific solar system satellite, where it would seem natural to begin our search for extraterrestrial eukaryotes. Many hints suggest the conjecture of the universality of eukaryogenesis. One of them concerns the combined action of natural selection, and the inevitable effect of symbiosis. These two mechanisms of evolution will work on the prokaryotic cellular blueprints. However, the most intriguing question is the general nature of the transition prokaryote-eukaryote in a given planetary, or satellite environment. As mentioned in Sec, 2.1, we dwell on this particular phenomenon, since on Earth eukaryogenesis was the first step towards multicellularity and, subsequently, intelligence.

Insights derived form extensive biogeochemical data 9 suggest that the prokaryotic blueprint will make its first appearance on a planet, or satellite, in a relatively short geological time. Once the period of heavy bombardment was over, some 3.9 to 4 Gy before the present, the appearance of prokaryotic life followed a relatively straightforward chemical pathway. Since the early 50s this simplicity for starting off the living processes at the prokaryotic level has been partially elucidated in the laboratories of organic chemists. We may, therefore, assume that prokaryotes are bound to occur in environments where chemical and geological conditions may be similar to the terrestrial ones. This remark induces us to focus attention on whether eukaryogenesis is a universal phenomenon, which is the bigger and deeper issue, as stated in our conjecture.

The main purpose of the present work is to suggest possible experimental means, within our own solar system, of testing the conjecture (2.2).

3. LOOKING FOR SOURCES OF PARALLEL EVOLUTION IN THE SOLAR SYSTEM

3.1. Where can we search for living organisms analogous, but independent, of the Earth biota?
The search for extraterrestrial eukaryotes can be considered as a first step in the search for exogenous evolution of intelligence. Our first efforts should be in likely places in our own solar system. Such an endeavour is not technologically impossible, and its implementation could be achieved reasonably quickly. The ice-covered surface of Jupiter's moon Europa is currently the most favourable site for this type of search. The fundamental question of whether this satellite is analogous to the Earth, as required by conjecture 2.2, will be discussed in Sec. 3.2. Hydrothermal vents on the Earth's sea floor have been found to sustain life forms that can live without solar energy. Similar possible volcanic activity on Europa, caused by its interaction with Jupiter and the other Galilean satellites, makes this Jovian moon the best target for a possible identification of a living micro-organism beyond our planet 10, 11.

The present work aims at turning the question of distribution of life in the Universe, from the present realm of conjectures, into a well-defined scientific discipline that could be tested in the foreseeable future within our solar system, at a cost that is well within the policy of financing missions that are cheaper, faster and better. The current question of distribution of life in the universe will inevitably be faced with the above-mentioned 'armada' of space missions, which should be constrained to test only clearly formulated hypotheses backed by the proposal of realistic, specific, and unambiguous experiments (cf., Sec. 7). We recall the recent proposal 12 of a mission capable of investigating the possible existence of life in Europa. This mission offers an excellent possibility for testing the eukaryogenesis hypothesis (cf., 2.2). We have maintained that an appropriate experiment to test eukaryogenesis is feasible, in spite of the evident severe payload limitations that the nature of the mission is bound to impose on us. Such experiments will be discussed in Secs. 5 and 6.

3.2. Is the Earth analogous to Europa?
Once we give up the chauvinistic point of view that has been forced upon us due to the multicellular nature of Homo sapiens, the similarity between the environments of Europa and the Earth becomes evident. We are beginning to realise that if life does exist on Europa it will be mainly deep, aquatic, cryophilic and, most likely, unicellular. On the other hand, it is worth underlining that deep, aquatic, low temperature environments for unicellular organisms are also predominant on Earth 13, 14: This may be illustrated with the fact that 70% of the Earth is covered by sea water, of which two-thirds have a temperature of around 2° C. Cryogenic conditions are widespread in our planet: more than 80% of the Earth biosphere, including the polar regions, is permanently cold from the point of view of the mean annual temperature. The deep sea (> 1000m) represents 88% of the Earth area covered by sea water and 75% of the total volume of the oceans; in other words, the deep sea represents 62% of the biosphere.

We can go one step further. Micro-organisms can be subject to extreme temperature fluctuations. In polar and tundra soil these fluctuations can have a lower bound of some -15° C. Finally, in taking advantage of the analogy between the Earth and Europa, we may make use of the wide experience with viable micro-organisms in permafrost form Russia, Canada and Alaska, which may serve as the background against which to test conjectures in astrobiological research.

Some care is needed before exploring some of the virgin biotopes on Earth. In this context, it is useful to remember that planetary protection policies have been developed to inhibit contamination in solar system research. These policies will be useful when the scheduled 2005 Mars sample-return mission retrieves rocks at two rover sites selected two years earlier. Planetary protection is also relevant to Europa. In a Jet Propulsion Laboratory workshop in Pasadena, California 15, the knowledge gained in Martian sample-retrieving studies has been put to good use in planning missions to explore the possible Europan Ocean.

The early stages of a proposed Europan mission may be rehearsed firstly on Earth, in a Europa-like environment. For example, a good location is underneath Vostok Station, the Russian Antarctic base about 1,000 km from the South Pole. A lake, the size of Lake Ontario was discovered beneath the Vostok Station in 1996, after having drilled in that area since 1974. The lake lies under some 4 kilometres of ice. Lake Vostok, as it is known, may harbour unique micro flora 16. The protected retrieval of biota from Lake Vostok will serve as a dress rehearsal for handling an aquatic medium that may be teeming with life. So far the lake itself has not been sampled.

However, it is not only in the submerged lake, where useful information can be retrieved. Indeed, the possibility of detecting biomolecules in Europa, on the ice surface itself, rather than in the possible ocean underneath, was a point made at the recent Trieste Conference on Exobiology 4. A possible mechanism for bringing biomolecules to Europa's surface was subsequently discussed 17. Unfortunately, during this year and throughout 1999, the Galileo Europa Mission (GEM) will continue to be restricted to infrared and ultraviolet spectroscopy. Surface biogenic tests on Europa may have to wait for the further orbital mission to search for traces of putative Europan biochemistry, or signs of extant life.

4. ON THE UBIQUITY OF EUKARYOTES IN ANTARCTICA

The relevance of the information to be retrieved from Lake Vostok can be made more evident by means of the following question (*):

What might be learned regarding eukaryogenesis from the possible study of viable diatoms from permafrost and deep ice?

Since the presence of biochemical traces on the Europan surface has been suggested 4, it becomes imperative to pursue the analogous research in Lake Vostok, particularly concerning eukaryotes. Diatoms, discovered in 1702 by the microscopy pioneer Anton van Leeuwenhoek, are some of the most interesting micro-organisms to consider, given their ubiquity on Earth: In just one litre of sea water one may find as many as ten million diatoms, which may be considered the primary foodstuff of the sea. Marine species often form a brown coating on Arctic ice floes 18.

The ubiquity of diatoms may also be exemplified by what is known in the other Earth analogues of the Europan environment: Permanently frozen lakes in a series of dry valleys were discovered in 1905 by the British explorer Sir Robert Scott. From the point of view of geology and microbiology some of the best studied frozen lakes are in the Taylor Valley (bounded by the Ferrar Glacier and the Asgard Range), namely Lake Fryxel and Lake Hoare; further north, Lake Vanda, in the Wright Valley, is also remarkable. Some species of diatoms (Pennales) are known to dwell under the permanently ice-covered lakes of the Antarctic dry valleys 19. Some further details of the Antarctic lakes were given in our previous San Diego talk20.

Once the planetary protection protocols are duly taken into account, forthcoming knowledge of the micro flora that populates the substantial water volume of Lake Vostok will be of great value for anticipating, and testing the instrumentation requirements that might be needed. Amongst the micro-organisms that are permanently living in the Antarctic lakes there are many examples of eukaryotes, a few of which are illustrated in Table 1.

TABLE 1:
A few examples of eukaryotes present in Antarctica 16, 21-24
__________________________________________________________________________________________________
Organism Domain Habitat
__________________________________________________________________________________________________
Diatom shells Eukarya (Bacillariophyta)
Vostok
(ice core, at depth of 2375m)

Caloneis ventricosa Eukarya(Bacillariophyta) Chad, Fryxell, Hoare and Vanda

Hantzschia amphioxys Eukarya (Bacillariophyta) Fryxell, Hoare and Vanda

Navicula cryptocephala Eukarya (Bacillariophyta) Bonney, Fryxell, Hoare and Vanda

Chlamydomonas subcaudata Eukarya (Chlorophyta) Bonney and Hoare

Chlorococcum sp. Eukarya (Chlorophyta) Hoare

Tetracystis sp. Eukarya(Chlorophyta) Fryxell, Hoare and Vanda

Yeast Eukarya (Ascomycota) Vostok (ice core)
__________________________________________________________________________________________________
(*) We would like to thank Dr. R.B. Hoover for suggesting this question.

We have emphasised the presence of diatoms before mentioning other examples of algae, because diatoms comprise the largest number of algae in the benthic mats of these singular biotopes; our main motivation, however, is to underline the significance of eukaryogenesis in astrobiological research. In the Antarctic biotope, eukaryotes have demonstrated to thrive in Europa-like conditions.

By the mechanism explained by Wharton and co-workers 22, 23, vertical transport of diatoms and other micro-organisms is possible in the permanently frozen lakes. Hence, it is hardly surprising that diatoms have been found recently by our Russian colleagues in the permafrost and deep ice of the Lake Vostok region. It should be kept in mind, however, that the mechanism of vertical transport that applies in the dry valley lakes, where the ice covering the lakes measures a few meters, may not apply in the case of Lake Vostok, where the depth of the ice covering the lake is measured in kilometres.

At the first San Diego Symposium 25 the Cryobot/Hydrobot (CH) mission was proposed 12. We also maintained, in a separate work 20, an analogy with phenomena observed in the dry valley lakes of Antarctica. In the light of the analogy we suggested simpler tests on the Europan iced surface. These assays are analogous to the work that has been successfully initiated recently by S.S. Abyzov and R. Hoover 26. Indeed, a factor in the lack of uniformity in surface brightness and colour of the Europan surface may be the presence of micro-organisms, or their biomolecules. This hypothesis can be tested, for instance, either directly on the surface itself, or in orbit by spectroscopic search of the Europan surface ice.

As mentioned in Sec. 3.2, the surficial biogenic tests that we propose (cf., Sec. 5.1), will have to wait either for the landing of the Cryobot, or for a new orbiting spacecraft. In the case of an orbital mission microwave spectroscopic facilities are needed to allow a search for biological macromolecules. The Europa Ocean Discovery mission 27, unfortunately, intends to restrict itself to experiments with ice-penetrating radar, gravity, laser altimetry and optical imaging.

Within the limitations of space missions that have already been proposed, we suggest that fluorescence microscopic techniques be used for the detection of both biochemical and biological indicators, as explained in detail in Sec. 5 and 6 below. As stated above, at present we cannot exclude the presence on the Europan ices of one of the following possibilities:

o The presence of biomolecules. The fluorescence techniques that Kobayashi et al 28, proposed for the corresponding case of detecting potential Martian organic compounds, may be appropriate. Nevertheless, a problem of miniaturisation remains to be solved, if the technique of Kobayashi and co-workers is to be applied to the Europan case.

o The presence of micro-organisms. A test involving fluorescence microscopy for recognising micro-organisms on Mars was recently suggested 29. Another fluorescence technique was independently proposed, in the case of Europa, for detecting the degree of evolution of a given micro-organism 1-3. (For further details, we refer the reader to Sec. 6.) Evolution beyond prokaryotes is still a possibility, not ruling out the universality of eukaryogenesis 6.

5. POSSIBLE BIOLOGY EXPERIMENTS ON THE EUROPAN SURFACE

To settle the question whether Europa is a potential source of parallel evolution for micro-organisms, in this section and the next one we shall discuss possible experiments that may be carried out on and below the Europan ice:

o On the ice surface of Europa. To be implemented by means of the shallow Cryobot melter probe 12 [sCmp].

o In the possible Europan ocean. To search for extant life ( the CH mission 12 ).

5.1. The shallow Cryobot melter probe
We have argued above that a factor in the lack of uniformity in surface brightness and colour of the Europan surface may be the presence of micro-organisms, or their biomolecules. In other words, the search for extraterrestrial biochemistry, or biology on the surface of Europa ought to be a possible straightforward and evident aspect of the Europa campaign.

The biogenic hypothesis can be tested, for instance, by spectroscopic search of the Europan surface ice. On the other hand, the sCmp seems the right mechanism to test directly for surface biochemistry, or organisms. The motivation for this proposal is given in Sec. 5.2.

5.2. Growth of algal mats in the dry valley lakes (20)
A phenomenon involving large biomass of benthic algal mats has been observed: Pieces of these mats escape through about five meters of lake ice 23. Buoyant forces are capable of detaching algal mats formed in the benthic regions. This leads to pieces of the mats rising to the bottom of the ice cover and, subsequently, they freeze into the ice. Some pieces of these mats reach the upper lake surface in about one decade through the combined action of two effects: Firstly, the ablation of ice from the upper surface and, secondly, the formation of new ice at the top of the liquid water.

Since we know that hydrothermal vents are not refuges against evolution 30, chemoautolithotrophic micro-organisms may have evolved at hydrothermal vents at the bottom of the Europan Ocean. These evolved micro-organisms do not necessarily have to be analogues of archaea, as previously assumed 10. We have argued that the presence of eukaryotes is a possibility (for instance, analogues of diatoms). For this reason, mats consisting of either prokaryotes or eukaryotes, conceivably may have been lifted up to the visible surface of Europa. This possibility may be tested experimentally by means of the sCmp, as mentioned in Sec. 5.1.

5.3. The CH mission
In the CH biology experiment presumably the maximum size of the Cryobot would be some 10 to 15 cm diameter and 1.5 - 2 m long. Within the restricted space of the Cryobot and Hydrobot there would be an "in-situ chemistry laboratory" (ISiCL). As stated in our San Diego paper 12, the Hydrobot instrumentation would aim to determine whether the ocean exhibited one set of requirements for "life we would recognise". We believe that the detection of life and its evolutionary stage should inevitably be one of the primary goals of any Europa exploration program.

5.4. Instrumentation for in situ analysis
We wish to define in some detail the minimum ISiCL equipment that is needed in the biology experiment that will be discussed in Sec. 6. The optical system that would be proposed for the Hydrobot depends on the chemical composition of the putative micro-organisms. The technique relies on the material being able to induce luminescence by the application of various dyes. Fluorescent dyes are detected with a fluorescent microscope. If microscopic fluorescence is used to probe for life, some advantages and some challenges are immediately evident.

First, in contrast to the Martian search for life, the typical resolution needed for fluorescence micrographs of chromosomes is 10 mm 31. Such resolution is well within the scope of a light microscope. Martian research is linked to the electron microscope, since the nodules in the Allan Hills meteorite, which are currently under discussion, have been suggested to be nanobacteria, 50-75 nm long and 10-20 nm in diameter 32. The difficulties of going beyond a light microscope in the Hydrobot are evident. We believe that only a light microscope is needed. Hence, it is feasible to think in terms of a robotic biologist'. It would be contained within the Hydrobot and consist of simple optics and sampling arms.

6. A BIOLOGY EXPERIMENT IN THE EUROPAN OCEAN

6.1. Biological aspects of eukaryogenesis
One way of approaching the intricate problem of eukaryogenesis is to extract from the known phenomena of genetics some general aspects of the relations between the main relevant parameters, and to use such knowledge to sketch possible aspects of the earliest chromosomes.

The folding of biomolecules may offer some insights into the earliest aspects of the cell. Some knowledge has been gained in understanding the underlying mechanism that controls the folding of any polypeptide chain into unique three-dimensional protein structures.

We may consider the analogous problem with the DNA that makes up the chromosomes, namely, the folding of the 100-Å nucleosome filament. There is a hierarchy of levels of folding beyond the 100-Å nucleosome filament: The next level of complexity is provided by 300-Å filament, which is arranged into a solenoid-like configuration with about six nucleosomes per turn 33. During interphase in the cell cycle, it is this solenoid-like arrangement that constitutes the most abundant form of chromatin.

However, at later stages in the cell cycle this structure serves as the basis for further folding, ending up at the highest degree of folding observed at the metaphase chromosome. This is an extremely fortunate feature from the experimental point of view. We only need to recall that the ultimate aim of the Europan biology experiment is to develop robotic tests that are compatible with the Hydrobot reduced dimension. Indeed, chromosomes stain easily, in a well-defined manner. The biochemical basis for the difference between heterochromatin (the more compact structure of chromatin) and euchromatin (its less compact form), remains unknown. In both cases the 100-Å nucleosome filament contains approximately the same DNA/histone ratio 34. Models have attempted to account for impeding fork movement at a terminus 35, where repeated sequences may be observed 36. These models give us confidence that we are able to identify unambiguous indicators of eukaryoticity. We shall insist persistently in arguing that heterochromatin is not only a clear hallmark of eukaryogenesis 2, but that heterochromatin is also a unique indicator of eukaryoticity, which is amenable to the tasks that the robotic biologist will have to perform inside the Hydrobot.

6.2. Eukaryotic identity
Many difficulties are inherent in the eventual design of an assay that would intend to identify eukaryotes, robotically, in an extraterrestrial environment. This question begins to be important in view of the decisions that have to be made in selecting which biological experiments should be performed in Europa, for instance, by means of the CH project. Some examples may serve to illustrate the underlying difficulties:

o The taxon Cyanidiophyceae. These rhodophytes, are known as red algae 37. It has been argued that these acido-thermophilic algae may constitute a bridge between cyanobacteria and red algae 38. In any case the point we wish to emphasise here is that the morphology of these micro-organisms is not radically different from prokaryotes, except for the inner membrane. In particular, Cyanidium caldarium may live at temperatures of up to 57o C. This protist shows better rates of growth and photosynthesis when cultured in an 'atmosphere' of pure CO2 39 .

o Silicification experiments. Laboratory fossilisation of micro-organisms has led to the identification of one artefact, which can cause confusion in the identification of micro-organisms. It has been observed that an artificial nucleus formed during the process of fossilisation 40. If such a phenomenon is preserved in the natural fossil record, then it can lead to a confusion with the eukaryotes.

o Many other illuminating examples are enumerated in ref. 6.

For these reasons, we confine our attention to the clearest hallmark for eukaryogenesis: heterochromatic genomes that respond in an unambiguous manner to well-defined dyes, the result of which could be recorded with video equipment for later analysis, after relaying the results to an Earth-bound laboratory.

6.3. Banding techniques when chemical detail of the genome of a micro-organism is uncertain
Quinacrine fluorescent dye inserts itself between base pairs in the DNA helix producing the so-called Q-bands, which for the robotic mission would probably suffice. We suggested earlier 3 the more involved use of Giemsa stain to produce the more permanent R-bands 41. This is probably an unnecessary complication. Adjacent areas stain differently. The bands give a clear indication of slightly different modes of DNA packaging. It is the tightness of the genomic material that would be an indicator of a higher degree of evolution.

The question is not so much what is the chemical detail of the genome, but what is the degree to which it has been packaged. It may be argued that gene activity is correlated with light-staining bands. (For instance, genes that are transcriptionally active are light-staining 42.) This aspect of the proposed experiment is its most important feature, since it does not force upon us the requirement of previous detailed knowledge of the putative Europan biochemistry. The main scope of the experiment is to expose eukaryoticity at the level of gene expression, whose most characteristic indicator, we have argued repeatedly 2, is heterochromaticity (i.e., a tightly packed genome).

6.4. Some challenges that have to be overcome
1. To extract the genetic material for analysis.
Once the appropriate cell has been identified, the usual technique of hypotonic swelling may be used to let the genetic material be extracted form the interior of the cell nucleus.

2. A robotic means of adding the dye.
The dye quinacrine is a possibility for the simplest case of Q-banding. One of the technical problems to be solved is a detailed project of how to implement this routine by the robotic biologist.

3. Retrieval of the information.
The response of the genetic material should be transmitted with the Hydrobot video equipment for eventual analysis of the images back in an Earth laboratory.

7. DISCUSSION

Our thinking is based on the exclusion of refuges against evolution 30. This new insight is based on the remark that Cambrian fauna, such as lamp shells (inarticulate brachiopods) and primitive molluscs (Monoplacophora), were maintained during Silurian times by micro-organisms that lived in hydrothermal vents. In the current Cenozoic Era these hot environments have seen the extinction of such fauna.

Hence, this remark rules out the possibility that these deep-sea environments are refuges against evolutionary pressures. In other words, the evidence so far does not support the idea that there might be environments, where ecosystems might escape biological evolution, not even at the very bottom of deep oceans. It is then reasonable to assume that any micro-organism, in whatever environment on Earth, or elsewhere, would be inexorably subject to evolutionary pressures. On this planet the eukaryotic cellular blueprint seems to have been the consequence of over 2 Gy of evolutionary pressures acting on the prokaryotic blueprint. The first appearance in the fossil record of eukaryotes occurred during the Proterozoic Eon, after prokaryotic communities (stromatolites) were well established in the Archean Eon 43.

It still remains to be confirmed, or rejected, whether the Europan environment may have had liquid water over geologic time. In such a favourable environment a primordial archaea community would have had sufficient time for evolutionary pressures have modelled a primordial archaea community. If these conditions occurred on Europa, then, according to our conjecture (cf., Sec. 2.2), eukaryogenesis would have been inevitable. The most recent observational evidence remarkably suggests the presence of an ocean 44.

To sum up, two ingredients militate in favour of attempting to identify indicators of the degree of evolution of putative Europan biota:

o The possibility of testing the onset of eukaryogenesis in the outer solar system. This may be achieved by means of space missions specifically designed for this purpose, and

o The complete lack of refuges against evolutionary pressures. We have argued that the Earth biota has not escaped evolutionary pressures in any environment for almost 4 Gy of Darwinian evolution.

Hence, we have pointed out the possibility that the eukaryotic transition may be a general consequence of geological changes on an Earth-like planet, or satellite, coupled to the effect of natural selection (cf., Sec. 2.2). In other words, eukaryogenesis may have occurred independent of the fact that the micro-organisms we are familiar with are confined to the Earth's biosphere. We have argued that evolution should have occurred in Europa and that the experimental test of this conjecture is feasible through a CH type of space mission 12. Difficult instrumentation issues are involved. The preparation of a package to search for life either on Mars or Europa is a formidable task. The method elaborated for the exploration of Mars29, which is based on fluorescence microscopy, would still require further miniaturisation before it could be adapted for the case of Europa.

It is over a decade before the CH final mission will be ready to undertake the drilling task on the Europan iced surface. In view of the lack of a precedent for our proposal of a biology experiment, we should rationalise the discussion of such an experiment at this early date. In order to do so, we would like to recall a precedent set by the search for life on Mars.The case in question concerns the Viking mission and, in particular, the Labelled Release (LR) Experiment. This aspect of a very successful mission produced results, which are still being debated, more than two decades after the LR Experiment was concluded. At the First San Diego Conference, a discussion of the LR results, provided by the Mars 76 mission, led to the following comment:

"No non-biological approach published, or known to the author, has duplicated the Mars data...The biological interpretation of the Mars LR results is left standing alone...Application of the scientific principle leads to a conclusion: the Viking LR experiment detected living micro-organisms in the soil of Mars" .

The sentence in italics agrees with the printed version 45. For a life detection experiment in Europa, we may benefit from the interesting debate regarding the LR biology experiment. We learn that extreme care is needed in the definition of an experiment. Consequently, the arguments provided in Sec. 6 aimed at pointing out an unambiguous experiment, namely an assay by means of which the degree of evolution of the putative Europan biota would be probed. These arguments also underline the relevance of the main indicator for eukaryoticity: We argued in Sec. 6 that such an indicator is heterochromaticity, which may be tested with the application of fluorescent dyes.

8. CONCLUSION

We have discussed a theoretical conjecture of the likely course that biological evolution would take in cases when it is disconnected form the Earth biota (cf., Sec. 2.2). Our conjecture is subject to an experimental test with a CH type of mission capable of testing, within a decade, the degree of evolution of putative Europan micro-organisms.

Two aspects of astrobiology, as defined at the beginning of this work (cf., Sec. 1), have already sound scientific approaches. Firstly, the study of the origin of life, the first aspect of astrobiology, is based on the theory of chemical evolution, which is a time-honoured scientific discipline 46-50.

The second aspect of astrobiology, the evolution of life, has scientific bases provided by the two well-established insights of Darwin 51: "the theory of common descent", and "natural selection" as a mechanism for biological evolution.

In the present work we have defended the thesis that if biological experiments testing for evolutionary trends (cf., Secs. 5 and 6) were to be successful, they would bridge the remaining gap in astrobiology. In other words, the conjecture (cf., Sec. 2.2) would serve as a firm scientific basis on which to develop eventually the science of the distribution of life in the universe.

9. ACKNOWLEDGEMENTS

The author would like to thank the Abdus Salam International Centre for Theoretical Physics, Trieste, its Director, Professor M.A. Virasoro, and UNESCO for financial support to attend the international conference Instruments, Methods and Missions for Astrobiology, 19-22 July 1998, San Diego, California.

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