resurrecting van leeuwenhoek's rotifers: a reappraisal of the role

Received 19 September 2002Accepted 19 November 2002

Published online 23 January 2003

Resurrecting Van Leeuwenhoekrsquos rotifersa reappraisal of the role of disaccharides in

anhydrobiosis

A Tunnacliffe and J LapinskiInstitute of Biotechnology University of Cambridge Tennis Court Road Cambridge CB2 1QT UK

In 1702 Van Leeuwenhoek was the first to describe the phenomenon of anhydrobiosis in a species ofbdelloid rotifer Philodina roseola It is the purpose of this review to examine what has been learned sincethen about the extreme desiccation tolerance in rotifers and how this compares with our understandingof anhydrobiosis in other organisms Remarkably much of what is known today about the requirementsfor successful anhydrobiosis and the degree of biostability conferred by the dry state was already determ-ined in principle by the time of Spallanzani in the late 18th century Most modern research on anhydro-biosis has emphasized the importance of the non-reducing disaccharides trehalose and sucrose one orother sugar being present at high concentrations during desiccation of anhydrobiotic nematodes brineshrimp cysts bakersrsquo yeast resurrection plants and plant seeds These sugars are proposed to act as waterreplacement molecules and as thermodynamic and kinetic stabilizers of biomolecules and membranesIn apparent contradiction of the prevailing models recent experiments from our laboratory show thatbdelloid rotifers undergo anhydrobiosis without producing trehalose or any analogous molecule This hasprompted us to critically re-examine the association of disaccharides with anhydrobiosis in the literatureSurprisingly current hypotheses are based almost entirely on in vitro data there is very limited informationwhich is more than simply correlative in the literature on living systems In many species disaccharideaccumulation occurs at approximately the same time as desiccation tolerance is acquired However severalstudies indicate that these sugars are not sufficient for anhydrobiosis furthermore there is no conclusiveevidence through mutagenesis or functional knockout experiments for example that sugars are necessaryfor anhydrobiosis Indeed some plant seeds and micro-organisms like the rotifer exhibit excellent des-iccation tolerance in the absence of high intracellular sugar concentrations Accordingly it seems appropri-ate to call for a re-evaluation of our understanding of anhydrobiosis and to embark on new experimentalprogrammes to determine the key molecular mechanisms involved

Keywords desiccation desiccation tolerance trehalose sucrose

1 INTRODUCTION

Three hundred years ago in a letter to Hendrik van Bleys-wijk dated 9 February 1702 the pioneering microscopistAntony van Leeuwenhoek (figure 1) was the first todescribe a remarkable phenomenon He discovered thatwhen dry and apparently lifeless dust from a roof gutterwas rehydrated with clean water in a glass tube manysmall lsquoanimalculesrsquo became active within an hour vari-ously lsquoadhering to the glass some creeping along it andsome swimming aboutrsquo This result observed with hischaracteristic single-lens microscopes was routinelyreproducible even with dust kept dry for many monthsVan Leeuwenhoek was astonished lsquoI confess I neverthought that there could be any living creature in a sub-stance so dried as this wasrsquo (p 209) (Van Leeuwenhoek1702 translated by Hoole 1807 Van Rijnberk amp Palm1999) His astonishment is understandable as water isrecognized as the lsquomatrix of lifersquo (Szent-Gyorgyi 1979Franks 2000) Without water there can be no life and

Author for correspondence (at10004biotechcamacuk)

Phil Trans R Soc Lond B (2003) 358 1755ndash1771 1755 Oacute 2003 The Royal SocietyDOI 101098rstb20021214

yet Van Leeuwenhoek is describing creatures which appar-ently survive desiccation

The drawings included with Van Leeuwenhoekrsquos letter(figure 2a) show that his animalcule was what Baker(1744 1753) later called a lsquowheel animalrsquo in reference tothe coronal cilia whose metachronal beat gives theimpression of two rotating wheels on the animalrsquos headThe term in current use for these creatures was introducedfirst in Italian by Felice Fontana (lsquorotiferorsquo Fontana 1767)and derives from the Latin for lsquowheel bearerrsquo A photo-graph of the rotifer Philodina roseola (figure 2b) illustratesthe resemblance to Van Leeuwenhoekrsquos specimen Indeedgiven that P roseola often appears orange or red (henceroseola lsquorose-colouredrsquo) owing to its diet of the astaxan-thin-containing phytoflagellate of the same colourHaematococcus pluvialis and that it inhabits temporaryaqueous environments (Philodina means lsquolover of poolsrsquo)it is highly likely that Van Leeuwenhoekrsquos red gutter-dwelling animalcule was of the same species (Ford 1982Koste amp Hollowday 1993 Van Rijnberk amp Palm 1999)

Rotifers occupy a separate phylum Rotifera comprisingthree classes with P roseola belonging to the BdelloideaBdelloid (lsquoleech-likersquo) rotifers are swimming or creeping

1756 A Tunnacliffe and J Lapinski Anhydrobiosis in bdelloid rotifers

Figure 1 Antony van Leeuwenhoek at the age of 54 from amezzotint reproduction of an oil painting by JohannesVerkolje 1686 (The Royal Society collection) Reproducedwith permission

unsegmented pseudocoelomates ranging in size from 100to 2000 mm the body (figure 2c) is divided into threeregions the foot with toes the trunk and the head bearingthe characteristic cilia whose action induces a vortex inwater drawing small food particles towards the mouthBdelloids inhabit freshwater ponds lakes temporarypools brackish waters sewage and the interstices of soilmosses and lichens (Ricci 1987) Some of these habitatsdry out frequently and the ability of most bdelloid speciesto withstand desiccation and the dry state (Ricci 1998)must represent a clear selective advantage This abilitymdashsurvival of extreme desiccation and entry into a state ofsuspended animation pending recovery by rehydrationmdashis known as anhydrobiosis a term derived from the Greekfor lsquolife without waterrsquo

Van Leeuwenhoek had described rotifers in earlier cor-respondence with The Royal Society most clearly in hisletter of 17 October 1687 (Van Rijnberk amp Palm 1999)and had sent specimen packages with some letters whichwere rediscovered by Ford (1981) When Ford rehydratedthese specimens some contained animals identifiable asrotifers even allowing a proposed species designationalthough none had apparently survived its three centuriesof dryness (Ford 1982 1991) Nevertheless viability canbe maintained for extended periods in the dry state thecurrent record for (well-documented) survival of a driedanimal being of the order of decades for nematodes andbrine shrimp cysts (Fielding 1951 Clegg 1967 Jonsson ampBertolani 2001) The longest authenticated period sur-vived by a rotifer after desiccation appears to be 9 years(Guidetti amp Jonsson 2002) although there is potential fora much longer phase of suspended animation 1000-year-

Phil Trans R Soc Lond B (2003)

Figure 2 The bdelloid rotifer Philodina roseola (a) Drawingsof the rotifers that Van Leeuwenhoek (1702) observed (b) Aphotograph of P roseola obtained from a bird bath (c) Thestructure of a bdelloid rotifer reproduced from Pierce et al(1987) with permission

old lotus seeds have germinated successfully (Shen et al1995) and there are regular claims of the recovery ofancient bacteria up to 250 million years old (eg Vreeland2000) although these claims are equally regularlydebated (eg Nickle et al 2002)

In the past the tolerance of extreme dehydration andthe dry state that anhydrobiosis represents has challengedsome fundamental biological precepts particularly regard-ing the necessity of water for life and the requirement formetabolic continuity and has contributed to the debateon spontaneous generation (Keilin 1959) Modernresearch places emphasis on unravelling the molecular andcellular mechanisms underlying anhydrobiosis as we arestill a long way from a full understanding of these pro-cesses This review will summarize what is known aboutanhydrobiosis in rotifers with reference to other speciesand include some surprising recent results which maynecessitate modification of prevailing models

Anhydrobiosis in bdelloid rotifers A Tunnacliffe and J Lapinski 1757

2 ANHYDROBIOSIS

(a) Latent lifeThe term anhydrobiosis was introduced by Giard

(1894) and refers specifically to the highly stable state ofsuspended animation that an organism enters by des-iccation Strictly therefore it is distinct from lsquodesiccationtolerancersquo which refers to the ability of a cell or organismto withstand removal of water to a greater or lesser extentalthough the terms are often used interchangeably Anhy-drobiosis is a particular form of cryptobiosismdashmeaninglatent life or suspended animationmdashas defined by Keilin(1959) Other forms of cryptobiosis such as cryobiosisanoxybiosis and osmobiosis are induced by cooling lackof oxygen or high osmolarity in the environment respect-ively

Clegg (2001) has argued that lsquo[a]nhydrobiosis tells ussomething fundamental about the basic nature of livingsystemsrsquo (p 615) In the dry state there is little or noevidence of metabolic activity (Keilin 1959 Clegg 1986)Indeed when a cell has a water content of less than 01 g gdry mass21 (ca 9 ww) an amount typically remainingin dried but viable anhydrobiotic organisms it has beencalculated that there is insufficient water to fully hydrateintracellular proteins and that therefore metabolism isimpossible (Clegg 1978 1986 Potts 1994) Normally ofcourse an organism without metabolism is considereddead and irreversibly so However in cryptobiotic andspecifically here in anhydrobiotic organisms the cessationof metabolism is fully reversible and a kind of resurrectionis routinely performed This has led Clegg (2001) to con-clude that lsquothere are three states of biological organizationalive dead and cryptobioticrsquo (p 615)

Given this we might briefly reflect on nomenclatureKeilin (1959) discussed the diverse expressions in use inthe early literature and pointed out the problems associa-ted with a number of them (lsquoabiosisrsquo lsquoanabiosisrsquo etc)However if dried anhydrobiotic organisms are neitherdead nor alive but in suspended animation his adoptionof Giardrsquos term (ie lsquoanhydrobiosisrsquo) is also unsatisfactoryas is the extension of this construction to other cryptobi-otic states (lsquocryobiosisrsquo etc) While lsquocryptobiosisrsquo is finebecause it tells us that life processes are in a latent con-dition lsquoanhydrobiosisrsquo is not because it does not conveythe idea of latency We are not describing life withoutwater which implies some biology continuing in the drystate but a metabolic stasis (or lsquobiostasisrsquo) where theorganism has shut down completely lsquoAnhydrous crypto-biosisrsquo would be more accurate but too cumbersomeSomething like lsquoanhydrobiostasisrsquo or the shorter lsquoanhyd-rostasisrsquo might be more suitable This argument can betaken a step further because the prefix lsquoanhydro-rsquo meansliterally lsquowithout waterrsquo although dried anhydrobiotesalways retain some water and are therefore never likely tobe truly anhydrous outside the laboratory lsquoDryrsquo howeveris a relative term and could be used correctly in this con-text The Greek prefix lsquoxero-rsquo would then lead to theconstruction lsquoxerobiostasisrsquo or lsquoxerostasisrsquo Unfortunatelyas lsquoanhydrobiosisrsquo is firmly established in the literature itis unlikely that it can now be replaced

Anhydrobiosis is widespread in nature and in everydayexperience an example being the dried granules of bakersrsquoyeast (Saccharomyces cerevisiae) obtainable from any super-


market (Schebor et al 2000) Other anhydrobiotic micro-organisms include cyanobacteria (Potts 1999) and theradiotolerant Deinococcus radiodurans (Mattimore amp Batti-sta 1996) However Potts (1994) has with justificationrejected the acceptance of bacterial spores and akinetesinto the anhydrobiotic family on the grounds that theycontain too much water and certainly values for theresidual water content in the range 021ndash058 g H2O g dryweight21 could allow some continuation of biological pro-cesses (Clegg 1978 Algie amp Wyatt 1984) In the animalkingdom members of three invertebrate phylamdashtardi-grades (Wright 2001) nematodes (Perry 1999) and roti-fersmdashare capable of anhydrobiosis at all developmentalstages including the adult form Other invertebratesincluding crustaceans such as Artemia spp have anhydro-biotic embryonic cysts (Clegg 2001) The desiccated lar-vae of the fly Polypedium vanderplanki (Diptera) containsome 8 residual water and can be regarded as anhydro-biotic (Hinton 1960) Among plants some algae are cap-able of anhydrobiosis (eg the encysted form ofphytoflagellate H pluvialis a food source for VanLeeuwenhoekrsquos rotifer P roseola) as are lichens and bry-ophytes such as the star moss Tortula ruralis The formof anhydrobiosis found in these primitive plants seems tocomprise constitutively expressed cell protection mech-anisms together with inducible repair systems which areactivated on rehydration (Oliver 1997) Most higherplants have lost vegetative desiccation tolerance but somefor example the lsquoresurrectionrsquo plants Selaginella lepido-phylla and Craterostigma plantagineum reacquired it later inevolution in a modified form as entry into anhydrobiosisrequires metabolic adjustment during a slow dehydrationphase (Ingram amp Bartels 1996 Oliver amp Bewley 1997Oliver et al 2000) implicating inducible protectionmechanisms These examples are anhydrobiotic in thevegetative state but the seeds and to a lesser extent pol-len of many flowering plants are also well known to survivelong periods in the dry state as seen in the example of thelotus seeds (above) It might be argued that anhydrobiosisin a whole plant is more impressive than in its seeds orpollen as the latter are specialized forms whose develop-ment is intrinsically linked to the desiccation processHowever it is likely that anhydrobiosis in seeds and otherpropagules is a later evolutionary adaptation and that thisis one example of multiple reacquisitions of desiccationtolerance throughout the evolution of the plant kingdom(Oliver amp Bewley 1997 Oliver et al 2000) The ability ofmembers of all biological kingdoms to undergo anhydro-biosis could suggest that this is a property developed byancient cell types and it may have been essential forefficient colonization of land masses

(b) Defining anhydrobiosisThe question of which organisms are categorized as

anhydrobiotic is not straightforward at least for single-celled species and relates to the degree of survival of thedehydrationrehydration cycle In this regard we defineviability as the maintenance of reproductive capacity afterdesiccation and rehydration Micro-organisms exhibitvarying survival rates under different conditions (Potts1994) but it is not clear where lsquotruersquo anhydrobiosisbegins For example 60ndash100 of D radiodurans(Mattimore amp Battista 1996 Battista et al 2001) and 05ndash


10 of cyanobacterium Chroococcidiopsis sp (Grilli Caiolaet al 1993) populations survive desiccation but both ofthese micro-organisms are recognized anhydrobiotes Bycontrast the enterobacterium Escherichia coli is not gener-ally considered anhydrobiotic with typical survival levelsimmediately after drying of 2ndash8 (eg Louis et al 1994Leslie et al 1995) However mucoid variants of E colican show 10-fold improvement in recovery rates (Ophir ampGutnick 1994) apparently better than ChroococcidiopsisWith suitable preconditioning of bacteria and adjustmentof drying protocols 50ndash80 viability of non-mucoid Ecoli can be maintained for at least six weeks at above ambi-ent temperatures (Garcga de Castro et al 2000a Tunna-cliffe et al 2001) Is E coli therefore capable ofanhydrobiosis Other factors are important of coursesuch as the ability to survive in a lsquorealrsquo environment out-side the laboratory the degree of desiccation and thelength of time spent in the dry state (the lsquostoragersquo period)Nevertheless the definition of anhydrobiosis in micro-organisms is nebulous and although some species performbetter than others the suspicion is that interspecies differ-ences are quantitative rather than qualitative This mayultimately frustrate the identification of specific adap-tations to water stress in micro-organisms

The degree of survival required for many unicellularorganisms where only a single individual survivor amongbillions desiccated might be sufficient to regenerate thepopulation is low compared with individual metazoanswhere all or most cells must recover The model organismCaenorhabditis elegans a non-anhydrobiotic nematode canremain viable even when a significant percentage of its1000 or so somatic cells have been ablated or caused toapoptose or otherwise damaged Therefore if the same istrue for the anhydrobiotic nematode Aphelenchus avenaefor example a degree of cell death might be tolerated dur-ing entry into and recovery from anhydrobiosis How-ever further rounds of anhydrobiosis would inflictincreasing levels of cell damage and ultimately be fatalbecause there is no or little capacity for tissue regener-ation or cell division in mature nematodes (Wright 1991)as indeed is the case for post-embryonic rotifers(Clement amp Wurdak 1991) Because it is known thatwith suitable recovery periods an anhydrobiotic animalcan repeatedly survive desiccation and rehydration(Spallanzani 1776 Perry 1977 Schramm amp Becker 1987)it is likely that essentially all somatic and germ cells areanhydrobiotic The fact that most rotifer tissues are synci-tial does not significantly alter this argument The situ-ation is different in plants which can tolerate extensivecell damage and where differentiation continues post-embryonically from meristem tissue In the case of the res-urrection plants however significant cell death does notseem to occur during dehydration and rehydration (Gaff1971) In multicellular animals and plants therefore it islikely that a large majority of and possibly all cells withinthe organism must be capable of and survive anhydro-biosis

(c) The biostability of anhydrobiosisIn itself the ability to survive without (much) water is

noteworthy in itself but once in the dry state anhydrobi-otic organisms become extremely resistant to a number ofstresses besides desiccation Dried rotifers have remained


viable after cooling to within a degree of absolute zero(Becquerel 1950) and heating to 151 degC (Rahm 1923)dried tardigrades exhibit similar thermotolerance and inaddition have survived exposure to 6000 atmospheres ofhydrostatic pressure (Seki amp Toyoshima 1998) and to ion-izing radiation (Rahm 1923 1937 May et al 1964) Toa large extent this extreme biostability is due to theabsence of water as most biochemistrymdashincludingdegradative processesmdashproceeds through aqueous reac-tions Otherwise thermolabile proteins which have beenprotected from damage during desiccation becomeextremely resistant to thermal stress in the dry state(Colaco et al 1992) However dried or partially dried bio-logicals are still susceptible to some chemistry such as theMaillard reaction (Maillard 1912 Lea et al 1950) and theeffects of ultraviolet radiation (eg Rahm 1923 Manci-nelli amp Klovstad 2000)

The remarkable characteristics of anhydrobiosis makeit a fascinating area of study and over the past 30 yearsincreasingly sophisticated biochemical biophysical physio-logical and molecular biological techniques have beenemployed to uncover its molecular mechanisms Howeverthe vast majority of such studies have been carried outwith model in vitro systems and there is still much tolearn about anhydrobiosis in the organisms themselves asa review of what is known in the bdelloid rotifer will show

3 ANHYDROBIOSIS IN ROTIFERS A HISTORICALSURVEY

(a) Early studiesVan Leeuwenhoek did not in fact think that the rotifers

he observed were desiccated instead proposing that intheir contracted form their lsquoskins [hellip] are of such a solidtexture that they do not permit the least evaporation andwere it not so [hellip] these creatures in very dry weatherbeing deprived of water must all perishrsquo (Van Leeuwen-hoek 1702 translated by Hoole 1807 (p 213)) Othercommentators and researchers following Van Leeuwen-hoek such as Joblot (1718) Baker (1742 1753) Trem-bley (1747) and Hill (1752) were not specificallyconcerned with whether rotifers were dry or not althoughclearly the idea that they and other anhydrobiotic animalsmight be in a resting state was pervasive at the time Need-ham (1743) had recovered nematodes (lsquoeel-wormsrsquo) byrehydration of blighted wheat but although Spallanzani(1765) initially strongly disputed their animal nature suchthat Needham felt obliged to withdraw his original work(Keilin 1959) he was later vindicated by lrsquoAbbe Roffredi(1775ab 1776) and Fontana (1776) Both workersshowed independently that the lsquofibresrsquo present in theinfected wheat grains were clearly alive and went on todemonstrate the life cycle of the worms

The discussion surrounding the work of Roffredi andFontana which took place in the early 1770s and was onlypublished several years later must have caused Spallan-zani to revisit the phenomenon of anhydrobiosis In 1776Lazzaro Spallanzani (1729ndash1799) an Italian clergymanand distinguished professor of philosophy at the Univer-sity of Modena published his work Opuscoli fisica animaleet vegetabile (lsquotracts on the natural history of animals andplantsrsquo) in which he argued against the churchrsquos positionon spontaneous generation and provided evidence for the


preformation theory This made Spallanzani not only oneof the best known scientists of his time but also one ofthe most controversial Spallanzani devoted one of thechapters of the Opuscoli to the anhydrobiosis of rotifersHe established that to survive rotifers needed to be driedin sand probably indicating a requirement for slow dry-ing and he made observations consistent with loss of bodywater lsquoWhen dried the solids are contracted and dis-torted the whole body of the animal is reduced to a hardshapeless atom of matter pierced by a needle it flies inpieces like a grain of saltrsquo (Spallanzani 1776 translated byDalyell (1803) p 137) Spallanzani furthermore showedthat rotifers can be stored in the dried state for longer than4 years and that warm water revives dried animals quickerthan cold water Dried rotifers were cooled or heated totemperatures the equivalent of at least ndash12 degC and 68 degCrespectively and survived this treatment These experi-ments caused Spallanzani to believe that all life processesstopped when the animals were dried The overall qualityand breadth of Spallanzanirsquos work is astonishing since bythe late eighteenth century he had established the prin-ciples of anhydrobiosis much as we understand themtoday He was the first to propose clearly that rotifers didnot retain their body water as their surroundings driedout and that they became ametabolic in the dry state

(b) Dry or not dryAfter Spallanzani the matter rested for 62 years before

Ehrenberg proclaimed without the encumbrance ofexperiment that rotifers are not truly desiccation tolerantbut that it is rather the eggs hidden in the sand whichhatch after rehydration (Ehrenberg 1838) This belief wassupported by other researchers at the time including BorySt Vincent (Keilin 1959) The opposing view was takenby Doyere (1842) and Gavarret (1859) who publisheddetailed experiments on rotifer anhydrobiosis Doyereconfirmed all of Spallanzanirsquos experiments and establishedthat slow drying was crucial for survival as opposed to aspecific requirement for sand He stored dried rotifers ina vacuum of 5ndash6 mmHg and the animals revived afterrehydration This led him to conclude that they are in astate of lsquolife in potentiarsquo Gavarret supported this viewafter storing dried rotifers for 51 days in a vacuum of4 mmHg and confirming survival These findings werestrongly disputed by Pouchet (1859andashc) however whosimply rejected the idea of lsquolife in potentiarsquo and tried toprove spontaneous generation instead

The discussion in the French literature of the time wasso vigorous that a commission was appointed by the Soci-ete de Biologie in 1859 to resolve the matter The reportby the head of the commission Paul Broca was publishedin 1860 and contained many new experiments The com-mission concludes that lsquoThe resistance of tardigrades androtifers to elevated temperatures seems to increase furtheronce they have been previously desiccated Rotifers canbe revived after remaining in a dry vacuum for 82 daysand being immediately afterwards heated to 100 degC for30 min Hence animals [hellip] brought to the most extremepoint of desiccation that we can achieve in these con-ditions using current scientific knowledge retain theability to revive once in contact with waterrsquo (Broca 1860p 139 translated by the authors) It is worth noting how-ever that no direct measurements of residual moisture


content have ever been made on dried rotifers Strictlytherefore the degree of desiccation in anhydrobiotic roti-fers remains to be determined

Brocarsquos report should have decided the matter once andfor all but the dispute continued for several decadesmany researchers remained unconvinced of the ability ofsome organisms to survive desiccation This is perhaps notsurprising at a time when researchers and philosopherswere still arguing about spontaneous generation prior toPasteurrsquos work in the early 1860s and with the debatesurrounding the DarwinndashWallace theory of natural selec-tion underway after communications to the LinneanSociety in 1858 and the publication of The origin of speciesin 1859 Thus even towards the end of the nineteenthcentury texts were published which denied anhydrobiosis(eg Zacharias 1886)

(c) Phenomena associated with rotiferanhydrobiosis

Overall however acceptance of the phenomenon becamemore widespread and the focus of attention shifted fromwhether to how rotifers survive desiccation It is not anexaggeration to state that we are no nearer to answeringthis question now a century and a half after Broca andthat most proposed mechanisms have ultimately proveddisappointing For example in the 1870s two authorssuggested that rotifers secrete a gelatinous substancewhich protects them during desiccation (Cubitt 1872Davis 1873) This led to some confusion amongresearchers as no such substance had been reported byBroca (1860) and others before him Although this ideawas shown to be incorrect by Jacobs (1909) Bryce (1929)later claimed that two species of bdelloid rotifer Macrotra-chela natans and P de koningi produce a protective cystin response to water loss However technical details arenot clear in this publication and no further work on anhy-drobiosis in these species has been published encystmentis not currently regarded as critical

Janson (1893) observed that rotifers possess colouredcarotenoid-containing particles in the stomach whichapparently disappear on drying They were not present instarved rotifers which did not survive desiccation andJanson therefore assumed that these particles were vitalfor anhydrobiosis Hickernell (1917) reported that in Proseola food granules present in the stomach disappearedat the onset of desiccation and are absent in dried animalsDobers (1915) however claimed that (i) no colourchanges occur during desiccation (ii) the coloured par-ticles are storage compounds which are used during star-vation periods and (iii) there are rotifers which do notproduce storage compounds yet are desiccation tolerantPart of the confusion over these particles may be due tothe fact that many bdelloid rotifer species feed on colouredalgae which result in coloration of the gut It is nowadaysclear that this colour is evenly distributed throughout thestomach and is maintained during desiccation food gran-ules might be present in addition to this colour Jacobs(1909) established that food availability has a strongimpact on survival 82 of well-fed P roseola survived hisdrying protocol whereas starved animals did not surviveat all

Jacobs (1909) also examined other factors which corre-late with successful anhydrobiosis He confirmed the


importance of slow drying because rotifers dried rapidlyin a desiccator exhibited poor survival (12) comparedwith those dried slowly under a cover (75 survival)Temperature was also shown to have an effect especiallyduring storage of dried animals Rotifers dried at 40 degChad a slightly higher survival rate (94) than those driedat 20 degC (82) but survival decreased during storagealthough more slowly at 20 degC than at 40 degC Dry storageconditions were also shown to be better than storage athigh relative humidity

Hickernell (1917) was the first to develop a fixation pro-cedure for bdelloid rotifers allowing in-depth cytologicalstudies of rotifers in the dried and hydrated states Hefound no difference in organ positioning between driedand control animals but showed that the integument ofthe mid-trunk is structurally different from that of thehead and the foot It was suggested that this may be rel-evant to desiccation tolerance because the animals pull inhead and foot on dehydration forming a structure some-times referred to as a tun Hickernell also claimed thatchromatin associates with the nuclear membrane in driedanimals a result which was later confirmed by Dickson ampMercer (1967) for P roseola by using the electron micro-scope However it is not known whether this phenom-enon is restricted to anhydrobiotes and in general nostructural modifications have been linked to desiccationtolerance in rotifers

(d) BiostabilityRahm published several articles on bdelloid rotifers

including work on the remarkable biostability conferredby anhydrobiosis (Rahm 1923 1926 1937) He showedthat dried rotifers can be exposed to 151 degC for 35 minand still survive and also reported that a bdelloid rotiferM quadricornifera revived after having been stored in a drymoss sample for 59 years Rahm is cautious however andstated that this may have been due to contamination withcontemporary dust He himself stored rotifers (in moss)at 01 mmHg pressure for 1 year and detected survivorsRahm also claimed that dried animals (rotifers nematodesand tardigrades) stored in a 100 nitrogen atmospheredo not survive the anhydrobiotic state at all and thereforeassumed that small amounts of oxygen were required forminutely slow metabolism A later study was unable todetect signs of metabolism (Umezawa 1958) howeverand Rahmrsquos view is contrary to current opinion (Orstan1998a) His results might be explained if the nitrogen waswet and the rotifers were therefore stored under unfavour-able conditions Rahm also reported on resistance to radi-ation in the dry state exposure to strong ultraviolet lightdamaged dried animals but they survived lsquoreasonablywellrsquo while hydrated animals were killed almost instantlyBy contrast X-ray exposure did not seem to visibly dam-age either hydrated or dehydrated animals Rahm alsocooled rotifers to very low temperatures (2190 degC) andfound survivors Later Becquerel (1950) cooled driedrotifers of species Habrotrocha constricta and P roseola to005 K and demonstrated survival

Gunter Lindau extended the observations of Jacobs onstorage conditions showing that dried rotifers could bestored at 210 degC to 215 degC for 90 days without any dropin viability Furthermore he found that rotifers whichwere initially exposed to 85 relative humidity and which


were covered immediately after tun formation with a layerof gelatin and then dried to completeness maintainedviability After 11ndash12 days of storage probably at roomtemperature they showed 95ndash100 survival (Lindau1958)

(e) Modern timesKeilin (1959) reviewed anhydrobiosis extensively in his

Leeuwenhoek lecture This stimulated the work of CleggCrowe Carpenter and other leading figures in the fieldbut rotifer anhydrobiosis was not widely studied in the1960s and 1970s In the past 20 years it is largely theresearch of Claudia Riccirsquos group in Italy and that ofAydin Orstan in the USA which has added to our knowl-edge on rotifers Ricci amp Melone (1984) studied M quad-ricornifera with a scanning electron microscope but wereunable to detect gross structural changes in dried animalswhich would indicate specific adaptations for desiccationtolerance Ricci et al (1987) reported that the internalclock of rotifers is arrested during anhydrobiosis rotifersdo not age during suspended animation in the sense thataverage lifespan depends on their age prior to desiccationand not on the time spent in the dry state Furthermorefor anhydrobiotic rotifer eggs age was shown to affect sur-vival the older the eggs the higher the survival rate aresult confirmed by Orstan (1995) for Adineta vagaSchramm amp Becker (1987) showed that bdelloids grownin aquaculture required a lsquolag timersquo between desiccationcycles Once a dried rotifer has been successfully rehy-drated the same animal cannot be subjected to a des-iccation regime immediately but requires a recoveryperiod of at least 1 day Orstan (1998a) performed a seriesof desiccation experiments on bdelloid rotifers in lichensamples He disproved Rahmrsquos hypothesis that dried roti-fers cannot survive without oxygen because storage in anargon atmosphere proved better than storage in air Fur-thermore it was shown that at room temperature storageat intermediate humidities is better than very humid ordry conditions As storage temperature is decreased thiseffect becomes less important until at temperatures below0 degC dried rotifers maintain viability over 18 months

Ricci (1998) demonstrated that most bdelloid rotiferspecies can be considered anhydrobiotic although someare clearly not and found that the degree of survival ofdesiccation seems to be species dependent Between 25and 95 of individuals survive a given drying conditionwith the best-surviving species being P rapida A laterstudy showed that some species like M quadricorniferasurvive rapid water removal and others like P roseolarequire a slower desiccation regime for maximum survival(Caprioli amp Ricci 2001) This is reminiscent of obser-vations on anhydrobiotic nematodes which have beencategorized as fast or slow dehydration strategists(Womersley amp Higa 1998) Finally Guidetti amp Jonsson(2002) studied the long-term anhydrobiotic survival ofrotifers They investigated 63 individual moss and lichensamples from public and private collections kept dry forbetween 9 and 138 years Rotifers were identified in manyof these and 13 survivors of the genus Mniobia were foundin one 9-year-old sample making this the longest well-documented period of rotifer anhydrobiosis

An earlier review of rotifer anhydrobiosis is included inGilbert (1974) For reviews covering the evolutionary and


ecological implications of rotifer anhydrobiosis the readeris referred to Ricci (1987 2001) and Orstan (1998b)

(f ) SummaryWhat do 300 years of research into rotifer anhydrobiosis

teach us We know that successful entry into anhydro-biosis requires a slow gentle desiccation regime Oncedry storage conditions are crucial with moisture oxygenand temperature affecting survival Anhydrobiosis confersa state of suspended animation in which life processes arehaltedmdashanimals probably do not respire and their internalclock is arrestedmdashand in which they can remain for manyyears The dry state confers resistance to extreme environ-mental stresses such as cooling to almost absolute zeroheating above the boiling point of water and exposure toionizing radiation Finally there is a clear divide betweenspecies regarded as anhydrobiotic and those which are notHowever nothing is known about biochemical adap-tations how do rotifers actually achieve survival in the drystate and how does it relate to what is known in otheranhydrobiotic organisms

4 HOW DO ROTIFERS ACHIEVE ANHYDROBIOSIS

Numerous reviews over the past decade some of whichare referenced here have summarized research on anhyd-robiosis and the various hypotheses for how it works(Crowe amp Crowe 1992 Crowe et al 1992 1998 2001Potts 1994 1999 2001 Bartels et al 1996 Ingram amp Bar-tels 1996 Oliver 1996 1997 Aguilera amp Karel 1997Sun amp Leopold 1997 Oliver et al 2000 2001 Clegg2001 Bartels amp Salamini 2001 Hoekstra et al 2001Wright 2001) The continuous flow of such reviewsreflects the fact that many questions remain to be answ-ered about anhydrobiosis even 300 years after VanLeeuwenhoekrsquos observations and that it is an increasinglyactive field

(a) Non-reducing disaccharides in anhydrobiosisSince the modern era of investigation into anhydrobiosis

began in the late 1960s and the 1970s non-reducingdisaccharides have been linked with eukaryotic desiccationtolerance specifically trehalose (a-d-glucopyranosyl-(1-1)-a9-d-glucopyranoside) and sucrose (a-d-glucopyrano-syl-(1-2)-b-d-fructofuranoside) Trehalose is found inanhydrobiotic animals fungi and some resurrection plants(eg Selaginella spp Quillet amp Soulet 1964 Gaff 1989)whereas predominantly sucrose but also oligosaccharidessuch as raffinose and stachyose is seen in resurrectionplants such as C plantagineum and in orthodox seeds(Oliver et al 2000) While some bacteria can accumulatetrehalose for example in response to increasing extra-cellular osmolarity (Stroslashm 1998) and anhydrobioticcyanobacteria seem to contain small amounts of bothsugars (Hershkovitz et al 1991 and references therein Hillet al 1994) it is not clear to what extent they areimportant for prokaryote anhydrobiosis

Trehalose was first associated with anhydrobiosis in ani-mals in embryonic cysts of the brine shrimp A salina(Clegg 1962 1965 1967) It was shown that in Artemiacysts trehalose is present at concentrations of ca 15 ofthe total dry weight corresponding to ca 150 mM in thehydrated state The biosynthesis of trehalose also corre-


lates with acquisition of desiccation tolerance in nema-todes (Madin amp Crowe 1975 Womersley 1981 Perry1999) In the tardigrade Macrobiotus areolatus trehaloseconcentrations were found to increase in anhydrobioticanimals (Crowe 1975) although it accumulates to onlyca 2 of dry weight (ca 20 mM) in Adorybiotus coroniferduring dehydration (Westh amp Ramloslashv 1991)

A few publications address the role of trehalose in yeastanhydrobiosis As cultures of S cerevisiae approach thestationary phase they begin to accumulate trehalosewhich correlates with an increased ability to withstanddrying (Gadd et al 1987) Trehalose levels increasefurther when stationary phase cells are dried (Payen 1949Marino et al 1989) During the log phase of growth yeastcells contain little trehalose and correspondingly showpoor survival of desiccation However log-phase cells canbe induced to synthesize trehalose by heat stress whenthey become more resistant to drying (Hottiger et al1987) Further experiments indicate that extracellular tre-halose is also important because mutants in a trehalosetransporter gene whose protein normally exports thedisaccharide to the extracellular milieu have markedlyreduced desiccation tolerance (Eleutherio et al 1993)

In plant seeds production of high concentrations ofsucrose coincides with maturation and the acquisition ofdesiccation tolerance (Oliver et al 2000) In the resurrec-tion plant C plantagineum the novel sugar 2-octulose isreplaced by sucrose as leaf tissues dry such that sucroserepresents 90 of total sugar content (Bianchi et al 1991)and accordingly sucrose synthase genes are upregulatedby dehydration (Kleines et al 1999) It therefore seemsthat intracellular accumulation of disaccharides is linkedto anhydrobiosis in a wide range of organisms (Crowe etal 1992 Aguilera amp Karel 1997) However perhaps sur-prisingly there has been no investigation of the sugar con-tent of rotifers undergoing anhydrobiosis We investigatethis below

(b) Multiple roles of disaccharides in theanhydrobiotic cell

Why are trehalose and sucrose thought to be importantfor anhydrobiosis We can address this by consideringsome of the events occurring during preparation for andentry into anhydrobiosis For a vegetative anhydrobioticcell three overlapping stages can be identified duringwhich different stresses operate

In the first dehydration stage bulk water is lost pro-gressively throughout the cell although macromoleculesand phospholipids initially remain fully hydrated Thisleads to the concentration of intracellular solutes changesin pH and increased viscosity all of which can destabilizenative protein conformations by disturbing hydrogenbonds Van der Waals forces and hydrophobic interactions(Stryer 1999) The cell eventually enters a state wheremetabolism is restricted and biochemical networks beginto break down (Clegg 1978 Womersley 1981) Duringthis period when the cell is experiencing stress but is stillmetabolically active and can mount a stress response anyadaptations required for survival of desiccation shouldappear if they are not present constitutively In anhydro-biotic nematodes and tardigrades trehalose is accumu-lated at this time in maturing plant seeds and the tissuesof resurrection plants sucrose becomes abundant and it


is the solution properties of these molecules which arethought to be important during the dehydration stage

Trehalose is one of the so-called compatible soluteswhich are used by plants animals and micro-organisms tocounteract osmotic stress (Yancey et al 1982 Galinski etal 1997 Stroslashm 1998) Because it is non-toxic and rela-tively highly soluble it can be accumulated in largeamounts inside the cell to help counterbalance elevatedextracellular osmolarity Compatible solutes may also beimportant during the early stages of drying where a single-celled organism is drying from an aqueous environmentor where tissue fluids are becoming increasingly concen-trated in a multicellular organism However the effect ofthe sugar is not purely osmotic trehalose (and sucrosewould behave similarly although it is less commonly usedas a compatible solute) is partially excluded from thehydration shell of biopolymers a situation which increasesorder in the system The degree of this entropy decreaseis proportional to the surface area of proteins and otherbiopolymers within the cell but is minimized when thesemacromolecules retain their native conformation becausea larger surface area is exposed when they unfold This isthe principle of preferential exclusion (Arakawa amp Tima-sheff 1982 Timasheff 1992) and it is thought to be themechanism whereby disaccharides and other compatiblesolutes protect biopolymers against denaturing stressescaused by elevated osmolarity and similarly by dehy-dration This idea has been developed further by Bolenand colleagues who have shown that the preferentialexclusion is due to an unfavourable interaction betweenthe compatible solute and the peptide backbone of pro-teins the so-called osmophobic effect (Bolen amp Baskakov2001) In this respect disaccharides act as thermodynamicstabilizers because structural changes become more ener-getically unfavourable in their presence

The second desiccation stage is reached when watercontent in the cell dips below 03 g g dry weight21 andparticularly when it reaches 01 g g dry weight21 wheremetabolism effectively ceases This is what Clegg calls thelsquoametabolic domainrsquo where water is in such short supplythat macromolecules are not fully hydrated which wouldnormally lead to structural instability In anhydrobiotes itis proposed that disaccharides behave as water replace-ment molecules thereby preventing irreparable structuraldamage to biopolymers and membranes as they dry andenter the solid phase The water replacement hypothesisdeveloped by Clegg (Clegg 1974 1986 Clegg et al 1982)and Crowe (Crowe amp Madin 1974 Crowe et al 1992)from earlier discussions (Clegg 1967 Crowe 1971Crowe amp Clegg 1973) and with reference to Warner(1962) and Webb (1965) indicates that these polyolsform hydrogen bonds as does water with the head groupsof membrane phospholipids and with the surfaces ofnucleic acids and proteins In this way the sugars arethought to take waterrsquos part in maintaining the structureof macromolecules and membranes A number of thereviews quoted earlier have summarized this hypothesis

It is worth commenting that moving from the dehy-dration stage where disaccharides are partially excludedfrom biopolymer surfaces to the desiccation stage wheresugars are intimately associated by hydrogen bonding withsuch surfaces defines a transition state within the anhyd-robiotic cell Clegg et al (1982) reported nuclear magnetic


resonance (NMR) data which are interpreted as providingevidence for such a transition as Artemia cysts are pro-gressively dried the self-diffusion coefficient (D) andrelaxation times (T1 and T2) for water protons decreaseaccordingly However when the ametabolic domain isentered below 03 g H2O g dry weight21 minima arereached in all three curves and values suddenly increaseagain to a threshold It was argued that this demonstratesrelease of residual water from macromolecule hydrationshells allowing increased rotation and translation Ther-mal analyses and NMR experiments in dried yeast sug-gested that trehalose levels of 2ndash3 of dry weight (20ndash30 mM) are sufficient to effect this water replacementalthough survival of desiccation continued to improve athigher trehalose levels (Sano et al 1999) Other data insupport of the water replacement hypothesis come largelyfrom in vitro work with protein and membrane systemsdried with or without sugars (Crowe et al 1998) How-ever there is little evidence particularly in whole cells ororganisms which demonstrates the transition between thethermodynamic effect of protective disaccharides in sol-ution and their role as water replacement molecules inthe solid dry phase

For the third dry stage representing anhydrobiosisproper which can potentially extend for long periods oftime there is increasing information on the nature of thesolid phase where the cytoplasm is thought to form anamorphous organic glass during desiccation (Burke 1986)This drastically alters the reaction kinetics of the systemso that even though they might be thermodynamicallyfavourable any structural changes in biopolymers andmembranes occur at a rate which is slow in comparisonto the duration of the stress It is proposed that disacchar-ides are instrumental in the vitrification process owing totheir high intracellular concentrations sugars like trehal-ose and sucrose act as kinetic stabilizers increasing cyto-plasmic viscosity ultimately leading to vitrification of thecytoplasm the so-called vitrification hypothesis (Sun ampLeopold 1997 Crowe et al 1998)

Molecules embedded in a sugar glass would be trappedin space and time as molecular diffusion and the chemi-cal reactions requiring it would effectively come to a haltCritical to the hypothesis is that the glass transition tem-peraturemdashTg the temperature which marks the inflectionpoint between the glassy state and the looser although stillhighly viscous lsquorubberyrsquo statemdashshould be high relative toambient temperatures likely to be experienced by theorganism Tg is dependent on water content of the glassincreasing as water is removed (Franks 1985 Sun amp Leo-pold 1997) the drier the glass the more (thermo)stableit is although some degradative reactions continue at aslower rate below Tg (as reviewed by Crowe et al 1998)The glassy state has been observed in various plant seedsArtemia cysts and commercial preparations of bakersrsquoyeast (Sun amp Leopold 1997 Crowe et al 1998 Cerruttiet al 2000 Schebor et al 2000) although clearly glassstability depends on storage temperature and moisturecontent of the anhydrobiote It has been proposed thatvitrification is important for the long-term stability ofanhydrobiotes in the dry state (Sun amp Leopold 1997)However it has been cogently argued (Crowe et al 1998)that vitrification itself is not enoughmdashfor example excel-lent glass formers such as dextran are not effective at stabi-


Figure 3 The Maillard reaction Formation of a Schiff basebetween the carbonyl group of a reducing sugar and theamine group of a protein or nucleic acid The molecule ofwater evolved is removed under conditions of low wateractivity shifting the equilibrium to the right

lizing biomolecules in vitromdashand that native biomoleculeconformation must be maintained during the dehydrationand desiccation stages

Stresses which operate in the dry state include the Mail-lard and Fenton reactions oxidation and irradiation Suchdamaging chemistry will be reduced in the glassy statebut trehalose and sucrose may also have some specific pro-tective activities For example the Maillard reaction(Maillard 1912)mdashalso referred to as non-enzymaticbrowningmdashtakes place between the carbonyl group ofreducing sugars and the amine groups of proteins andnucleic acids leading through a complex cascade ofrearrangements to polymeric pigmented end productsThe initial formation of the Schiff base is an equilibriumreaction which evolves a molecule of water (figure 3)Under conditions of high water activity the equilibriumis driven strongly to the left of the equation shown but inthe dry state there is strong pressure in the forward direc-tion owing to rapid removal of water Maillard activity isa major source of damage in stored dry biologicals (Leaet al 1950) but both trehalose and sucrose are non-reduc-ing disaccharides and therefore should not participateThe glycosidic bond in sucrose is relatively weak howeverand when hydrolysed gives glucose and fructose whichreadily form Maillard products with amine groups at lowwater activity (OrsquoBrien 1996) Trehalose is rather morestable and is less likely to undergo the browning reaction(El-Nockrashy amp Frampton 1967) indeed trehaloseinhibits its progression (Loomis et al 1979) and this isprobably significant for anhydrobiosis as originally notedby Crowe amp Clegg (1973)

Oxidative damage through the generation of free rad-icals is also thought to be incurred during desiccation andthe period in the dry state (Heckley 1978 Senrartna ampMcKersie 1986) Antioxidants which neutralize free rad-icals including enzymes such as superoxide dismutase andcatalase are induced during dehydration in plants(Smirnoff (1993) Farrant (2000) and references therein)and animals (Browne 2001) but their activity will becompromised as water deficit becomes extreme Howeversugars and sugar alcohols such as galactose myo-inositolsorbitol and mannitol are known to scavenge free radicals(Smirnoff amp Cumbes (1989) and references therein) Inprinciple both trehalose and sucrose the main sugarsassociated with anhydrobiosis could also have this pro-perty and indeed trehalose has been indicated in oxidat-ive stress protection of yeast (Benaroudj et al 2001)

In summary trehalose and sucrose are a recurringtheme in anhydrobiosis at least among eukaryotes andmay have multiple protective roles


5 6 7 8 9 10 11 12 13 14 15time (min)

mas

s (a

rbit

rary

uni

ts)

(a)

(b)

Figure 4 Carbohydrate analysis by gas chromatography (a)Carbohydrates extracted from dried nematode Aphelenchusavenae containing glucose and accumulated trehalose (b)Carbohydrates extracted from dried rotifer Philodina roseolacontaining glucose but no trehalose or other disaccharideGlucose is represented by the doublet of peaks emerging at7 min while trehalose gives a single peak at 13 min

(c) Anhydrobiosis without trehalose in bdelloidrotifers

The analysis of sugar accumulation in the rotifers Proseola and Adi vaga as a response to desiccation stressgave surprising results Both rotifer species were collectedfrom a birdbath in Cambridge UK and clone culturesestablished from single individuals these can be expandedto hundreds of thousands or millions of animals asrequired The rotifers were subjected to a drying regimein which animals were placed between two wet filter pap-ers and incubated in a sealed dish for 2ndash3 days The coverwas then removed and the animals were allowed to air dryfor another 3 days This resulted in excellent recovery afterrehydration of between 75 and 80 of the animals inline with Riccirsquos (1998) results for these species By con-trast rotifers dried immediately over silica gel showed amarkedly lower survival of ca 12 as expected from theliterature Analysis of the carbohydrate content of driedrotifers was performed by gas chromatography (GC) Thenematode Ap avenae dried slowly according to estab-lished protocols which elicit extensive accumulation of tre-halose (Madin amp Crowe 1975 Browne 2001) was usedas a reference The GC data showed the expected largeincrease in trehalose content on drying Ap avenae but nosuch change in either rotifer species indeed no trehalosewas observed at all (figure 4) Furthermore it was appar-ent that no other simple sugar was produced in detectablequantities during dehydration as mono- di- and smalloligosaccharides would be detected on the chromato-graph Sucrose when added to the samples as a standardand intracellular glucose are clearly detected Glucose


constitutes ca 1 of dry weight in the nematode Apavenae (Browne 2001) and similar quantities wereobtained for the rotifers P roseola and Adi vaga trehalosecomprises 10 of dry weight in desiccated nematodes Itis therefore reasonable to assume that we would detectchanges in the disaccharide profile at least in this range(ie 1ndash10 of dry weight) which overlaps with trehaloseconcentrations expected in anhydrobiotic animals

Trehalose was also not detected in rotifers subjected toother stresses which might be expected to provoke itsbiosynthesis (ie heat salt cold) If rotifers are able toproduce trehalose they are likely to use the two-step path-way found in all other eukaryotes whose first step is thetransfer of glucose from UDP-glucose to glucose-6-phos-phate to yield trehalose-6-phosphate This step is cata-lysed by trehalose-6-phosphate synthase (EC 24115) anenzyme whose sequence is highly conserved from Gram-negative bacteria to nematodes and higher plants(Blazquez et al 1998 Vogel et al 1998) A polymerasechain reaction (PCR) method with degenerate oligonucle-otide primers based on conserved protein sequence can bedevised to clone the respective gene (tps) from a widerange of species However although tps genes have beenisolated using this technique from species including nema-todes fruitflies yeast and E coli we have been unable toamplify PCR fragments from rotifer genomic DNA indi-cating that tps genes might not be present at all in P roseolaand Adi vaga

Finally we examined the metabolite profile of dried Apavenae and P roseola using proton NMR spectroscopy Inagreement with the GC experiments NMR analysis ofextracts of Ap avenae showed that the general metabolitecontent is raised in the dried compared with the hydratedstate peaks corresponding to trehalose are markedlyincreased in area However no such changes could bedetected in the rotifers suggesting that the overall rep-resentation of metabolites is not significantly altered inpreparation for anhydrobiosis (Lapinski et al 2003)

5 REAPPRAISAL OF THE ROLE OFDISACCHARIDES

The lack of trehalose in rotifers is profoundly disturbingfor our understanding of anhydrobiosis as outlined abovenon-reducing disaccharides are the cornerstones on whichthe various explanatory hypotheses are based Not only isno trehalose present however but there is no apparentchange in the small-molecule profile in dehydrating ani-mals Therefore no analogue of trehalose is producedduring entry into anhydrobiosis despite the requirementfor preconditioning by slow drying which is normallyassociated with adaptive changes So what is going onhere

(a) Re-examination of the link betweendisaccharides and anhydrobiosis

Perhaps we need to look again at the association of tre-halose or sucrose with anhydrobiosis how secure is itCertainly in prokaryotes the link is tenuous Desiccation-tolerant Lactobacillus plantarum apparently contains onlyinsignificant quantities of endogenous sugars (Linders etal 1997) In desiccating cells of the cyanobacterium Nos-toc commune trehalose represents about 1 mg g dry


weight21 and sucrose only 08 mg g dry weight21 ie01 and 008 of dry weight respectively (Hill et al1994) Although it cannot be ruled out that surviving cellsmight contain higher concentrations of sugars at theexpense of non-survivors an equal distribution through-out the population would amount to no more than 1 mMintracellular trehalose prior to desiccation and is probablynot sufficient to stabilize cytoplasmic components duringanhydrobiosis The D radiodurans genome contains genesfor trehalose synthesis (White et al 1999) and Deinococcusspp are known to produce and excrete this sugar in smallamounts (Kizawa et al 1995) but its significance indesiccation tolerance has not been determined althoughthis is currently being examined ( J Battista personalcommunication)

What about tardigrades and nematodes There is a sin-gle report showing that tardigrades accumulate trehaloseup to 2 of dry weight (ca 20 mM) in response to des-iccation (Westh amp Ramloslashv 1991) This is however theapproximate amount of trehalose in the nematode Apavenae before it starts to respond to desiccation stress andbefore acquisition of desiccation tolerance (Madin ampCrowe 1975 Higa amp Womersley 1993 Browne 2001)Trehalose concentrations in tardigrades seem rather lowerthan might be expected and its importance is thereforeunclear (Wright 2001) although interestingly Sano et al(1999) proposed that this is approximately the level atwhich water replacement is complete Nevertheless evenin anhydrobiotic nematodes where trehalose is producedat higher concentrations the correlation between trehaloseaccumulation and acquisition of desiccation tolerance issomewhat loose (Womersley amp Higa 1998) Higa ampWomersley (1993) noted that during dehydration trehal-ose concentrations in Ap avenae reached maximal levelsbefore full desiccation tolerance was attained Browne(2001) has made similar observations on the later stagesof the drying protocol but also noted that in some dehy-dration protocols trehalose content does not increasemarkedly during the first 12 h of desiccation stress despitesurvival levels increasing from 0 to 6 (figure 5a)

A similar situation pertains in yeast Gadd et al (1987)determined that S cerevisiae produces very high concen-trations of intracellular trehalose on entry into stationaryphase increasing from basal levels of ca 35 mM to ca400 mM and that this correlates with improved survivalof desiccation from 0 to 10 Close inspection of thedata points engenders some caution however First tre-halose concentration increased rapidly to 200 mM within4 days in the stationary phase At the same time survivalincreased to ca 1 Maximum trehalose concentration isreached after 8ndash10 days and approaches 400 mM At thispoint survival has still only increased to ca 4 and it isnot until 16 days in the stationary phase that maximumdesiccation tolerance can be measured (figure 5b)

The conventional explanation for these observations innematodes and yeast is that elevated intracellular trehaloselevels are necessary but not sufficient for entry into anhy-drobiosis and that other adaptations are occurring duringthe dehydration stage In yeast where desiccationexperiments are typically performed with stationary-phasecells nutrient and oxygen limitation would stimulatethe general stress response many genes governed bySTRE (stress response element) promoter elements


12

10

8

6

4

2

0

400

350

300

250

200

150100

50

00 4 8 12 16 20 24

0

3

6

9

12

0

6

12

18

24

inte

rnal

treh

alos

e co

nten

t (m

M)

treh

alos

e (

dry

mas

s)

time (d)

time (h)

surv

ival

(

)su

rviv

al (

)

(a)

(b)

0 48 60 72 84 96 10812 24 36

Figure 5 Trehalose content and survival of desiccation ofanhydrobiotic organisms (a) Aphelenchus avenae trehaloseaccumulation (filled squares) and survival (open squares)after different periods of preconditioning at 90 relativehumidity and subsequent drying over silica gel (redrawnfrom data of Browne 2001) (b) Saccharomyces cerevisiaetrehalose accumulation (filled squares) and survival (opensquares) during growth and subsequent drying Stationaryphase is reached after 3ndash4 days in this slow-growth-rateprotocol (redrawn from data of Gadd et al 1987)

would become active (Ruis amp Schuller 1995) includingTPS1 which encodes trehalose-6-phosphate synthase(Winderickx et al 1996) Similarly in heat-shocked yeastcells whose desiccation tolerance is increased (Hottiger etal 1987) a number of genes controlled by heat shockelement (HSE) promoter sequences would be transcribed(Estruch 2000) Some genes including heat shock proteingene HSP104 (Grably et al 2002) and presumably TPS1can be driven by both STRE and HSE sequences

If trehalose is however necessary in these organismsmutation of trehalose synthase genes should lead to a dras-tic reduction in anhydrobiotic capacity Surprisingly noexperiments of this kind have been carried out perhapsowing to technical difficulties The study performed byCoutinho et al (1988) used strain variants of S cerevisiaewith reduced capacity to produce trehalose Strikinglydesiccation tolerance of wild-type and low-trehalose-levelvariants was very similar although another strain overpro-ducing trehalose seemed to show increased survival Thiscould suggest that intracellular trehalose is of limitedimportance in yeast desiccation tolerance However thestrain variants used are uncharacterized and it wouldtherefore be of great interest to repeat these experimentswith defined TPS1 mutants

Panekrsquos group also looked at the effect of extracellulartrehalose on survival Wild-type yeast strains were com-


pared with strains with a non-functional trehalose carrierwhich is apparently responsible for transporting trehaloseacross the membrane into and out of the cell but withunimpaired ability to produce trehalose (Eleutherio et al1993) Strains with the non-functional trehalose trans-porter were found not to survive the desiccation protocolusedmdashdespite producing up to 35 of dry weight (ca35 mM in the wild-type range) trehalose intracellularlymdashwhereas ca 40 survival was observed in the wild-typecontrol When a 10 wv (ie ca 300 mM) trehalose sol-ution was added as a drying excipient survival of the vari-ant rose to more than 40 These results are alsoconsistent with intracellular trehalose playing a minor rolein yeast desiccation tolerance and point to a more criticalrole extracellularly Work with Gram-negative bacteriaalso indicates that extracellular addition of excipients ismore important than elevated intracellular trehalose levels(Garcga de Castro et al 2000a Manzanera et al 2002)

The eukaryotic structure with the most impressiveanhydrobiotic credentials is the orthodox plant seedSeeds are well known to produce very high concentrationsof sucrose in particular and its accumulation coincideswith increased desiccation tolerance (eg Oliver et al2000) In developing soybeans for example conditioningof seeds at high relative humidity increases sucrose raffin-ose and stachyose levels Almost 100 of preconditionedseeds survive desiccation whereas unconditioned seedsare not viable (Blackman et al 1992) Neverthelessthe accumulation of sucrose is not sufficient for anhydro-biosis as shown by abscisic acid (ABA)-insensitivemutants in the abi3 locus (abi3-3 and abi3-4) of Arabidopsisthaliana whose seeds lack desiccation tolerance althoughthe accumulation of sucrose is unhindered and is actuallyhigher than in wild-type (Ooms et al 1993) Interestinglyraffinose and stachyose were not present at all in themutants compared with 2 and 5 of dry weightrespectively in the wild-type which at first sight mightpoint towards a role of those sugars in anhydrobiosisHowever the recalcitrant ie desiccation-sensitive seedsof Avicennia marina (grey mangrove) for example containhigh levels of carbohydrates including stachyose and yetdo not survive drying (Bewley amp Black 1994) Converselycertain plant seeds do not contain significant amounts ofsugars and yet are still desiccation tolerant One exampleis the seeds of Ricinus communis (castor bean) which con-tain only negligible amounts of sugars (Bewley amp Black1994) but desiccate to residual moisture contents below375 with a 100 survival rate on germination Thisdesiccation tolerance is only acquired at days 20ndash25 afterpollination suggesting that specific adaptations take placeduring maturation of castor bean seeds which confer anhy-drobiotic capabilities (Kermode amp Bewley 1985)

Further contributions to the debate on the role of disac-charides come from attempts to create anhydrobiotic celltypes from desiccation-sensitive cells an approach wehave called lsquoanhydrobiotic engineeringrsquo (Garcga de Castroet al 2000b) Current excitement surrounds work onmammalian cells which are normally sensitive to dehy-dration loss of up to 65 of cellular water from cell lines(eg mouse L cells) can be tolerated but further loss isfatal (eg Mansell amp Clegg 1983) Therefore cell engin-eering to improve desiccation tolerance will be necessaryto achieve a viable dry state and the cytostability it con-


fers If successful this would enhance our understandingof anhydrobiosis as presumably a minimum set of adap-tations would be defined for a particular cell type Butadditionally mammalian cells would become lsquooff-the-shelfrsquo items stored in the dry state instead of deep frozenwhich would greatly reduce storage costs for cell banks orfor products based on cell technology such as monoclonalantibody-producing hybridoma cell libraries or some typesof biosensor This approach may also be applicable totissue engineering which relies on cell-based therapiesincluding the use of cells for tissue repair and as drugdelivery modules and to cell factories producing bioactivemolecules The prize for success is therefore considerable

To date anhydrobiotic engineering of mammalian cellshas been attempted largely by the generation of intracellu-lar trehalose because it is not naturally produced by mam-mals A question central to such projects is how high theintracellular concentration of trehalose should be for areasonable expectation of success Recognized eukaryoticanhydrobiotes define the physiological range as from ca20 mM (in tardigrades) to ca 400 mM (in yeast) Mousecell lines containing bacterial trehalose synthase genes canbe induced to accumulate up to 100 mM trehalose butalthough these cells show increased tolerance of highosmolarity they do not survive desiccation achieved bya number of different methods even in the presence ofextracellular trehalose (Garcga de Castro amp Tunnacliffe2000 Tunnacliffe et al 2001) Similarly when aninducible pore is used to load mouse cells with concen-trations of up to 400 mM trehalose viability is not main-tained after drying although membrane integrity ispreserved with concentrations of 200 mM or above if cellsare stored at sub-zero temperatures (Chen et al 2001)These results indicate that intracellular trehalose concen-trations within the physiological range are insufficient toconfer desiccation tolerance and are consistent with thecurrent understanding of anhydrobiosis By contrast Wol-kers et al (2001) have reported freeze-drying of humanplatelets loaded with only ca 20 mM trehalose withretention of function Although this concentration is 5ndash10-fold lower than that reported by Chen et al (2001) formaintenance of membrane integrity it is possible that thisis sufficient for non-replicating cell fragments This argu-ment does not explain the results of Guo et al (2000)however who claim to have improved desiccation toler-ance in human cells containing no more than 1 mM tre-halose well below the minimum concentration found inanhydrobiotic animals More remarkably still the samegroup has reported similar success without any biochemi-cal modifications to the cell (Gordon et al 2001 Puhlevet al 2001) However there are technical questions relat-ing to this work particularly regarding the degree of celldesiccation which need to be resolved (Garcga de Castroet al 2000b)

It is our opinion that trehalose alone is unlikely todeliver true desiccation tolerance for anhydrobiotic engin-eers and indeed researchers are beginning to investigateother possibilities A preliminary report from Bloom et al(2001) outlines the use of hydrophilic extracellular glycanfrom the cyanobacterium N commune to overlay mam-malian cells prior to drying The authors describe somerecovery of viable cells but an important question withthis approach as with that of Guo and colleagues is


whether treated cells have the low moisture contents typi-cal of dried anhydrobiotes Other adaptations apparentlyrelated to anhydrobiosis including the scavenging ofreactive oxygen species downregulation of metabolismand the accumulation of amphiphilic solutes hydrophilicproteins or peptides heat shock proteins or chaperonesmay also provide protection during dehydration (Oliver etal 2001) but have yet to be assessed for anhydrobioticengineering If these possibilities need to be tested in com-bination however it could be many years before fully des-iccation-tolerant mammalian cells are produced

6 CONCLUSIONS

What does all of this mean for disaccharides as cell-preserving agents In vitro there is very strong evidence forstabilizing properties in the dry state enzymes antibodiesnucleic acids some viruses liposomes and other mem-brane systems can be preserved using non-reducing sugarsas drying excipients (see reviews cited above) Howeverin vivo experiments on recognized anhydrobiotes andattempts at anhydrobiotic engineering are less conclusive

It is apparent that the presence of high concentrationsof trehalose or sucrose is not sufficient for anhydrobiosisEvidence in support of this conclusion includes the pres-ence of disaccharides in recalcitrant seeds the results ofattempts at anhydrobiotic engineering of mammalian cellsand the high disaccharide concentrations in yeast andnematodes which are not fully desiccation tolerant More-over there is little to suggest that trehalose or sucrose isactually necessary for successful anhydrobiosis the litera-ture is sparse and unconvincing we have been unable todetect trehalose or any other disaccharide in two bdelloidrotifer species and there are many bacteria and someplant seeds which survive desiccation without significantdisaccharide accumulation We should stress that this doesnot mean that disaccharides are not critical for anhydro-biosis in some or indeed many species but point out thatthe data to demonstrate this conclusively in living systemsare lacking Because it is apparent that in some casesat least anhydrobiosis is possible without disaccharidessome key in vivo experiments are needed to test therequirement for sugars in anhydrobiosis the most im-portant of which will employ mutants whose disaccharidesynthase genes have been inactivated Some of thesemutants in yeast for example have already been pro-duced for other purposes and should help establishwhether disaccharides confer anhydrobiosis in those org-anisms

If there are questions about the role of disaccharides inanhydrobiosis what else has been learnt in three centuriesof research about which we can be more certain Argu-ably there is not a great deal but two things have beenfirmly established First the intrinsic environment isimportant with many organisms requiring some form ofstress and time to respond to that stress to prepare foranhydrobiosis This need not necessarily be desiccationstress in yeast it could be nutrient depletion and otherstresses associated with the stationary phase or heat stress(Hottiger et al 1987) or osmotic stress (Eleutherio et al1997) in C plantagineum callus it could be ABA treat-ment (Ingram amp Bartels 1996) Most stress responses havea number of adaptations in common ie the general stress


response in E coli this involves 50ndash100 genes (Hengge-Aronis 2002) In yeast and perhaps nematodes and tardi-grades this general stress response may involve theaccumulation of trehalose to varying degrees (Singer ampLindquist 1998 Arguelles 2000) In Arabidopsis inacti-vation of genes which are part of stress response pathwayscan lead to loss of desiccation tolerance in seeds (Oomset al 1993) Although no disaccharide accumulation hasbeen found to occur in rotifers a slow desiccation regimein combination with well-fed animals is stringentlyrequired for high-level survival of desiccation indicatingthat the animals mount some form of energy-expensivestress response Second extrinsic factors are also crucialto successful anhydrobiosis Slow gentle water removalelevated temperatures during drying dry storage con-ditions low storage temperature humidification beforerehydration and elevated water temperature during rehy-dration can influence anhydrobiosis positively Survivalcan be greatly improved or greatly reduced depending onthe conditions chosen However to our knowledge nodesiccation protocol has been devised which enables non-anhydrobiotes to undergo anhydrobiosis successfully

These reflections point towards a qualitative rather thana quantitative difference between organisms regarded asanhydrobiotic and organisms which are not so Bdelloidrotifers are good examples they are either desiccation tol-erant or they are not (Orstan 1998a Ricci 1998) whichmeans there must be something special about those whichare anhydrobiotic If we are to hope to understand anhyd-robiosis we must continue to search for what is specialabout them and draw on the full power of current andemerging techniques in pursuit of an answer In organismswhere a complete genome sequence is available eg Scerevisiae and Ara thaliana microarrays of all recognizedgenes can be used to determine which transcripts areinduced by desiccation stress With appropriate experi-mental design this should lead to the identification ofthose genes which are specific to this stress responseComplementary information will be derived from experi-ments in proteomics and metabolomics Where genomesequence is unavailable direct analysis of differentiallyexpressed mRNAs and proteins can be attempted It isenvisaged that a key panel of genes and proteinsmdashtheanhydrobiotic gene set and corresponding anhydrobioticprotein setmdashare induced in rotifers in response to waterstress and that these directly govern anhydrobiosis aboveand beyond any general stress response A similarapproach in resurrection plants where Bartelsrsquos group isperhaps furthest advanced has identified many inducedgenes whose function is under investigation (Ingram ampBartels 1996 Bartels amp Salamini 2001) This strategyshould prove especially valuable in rotifers where atten-tion need not be distracted by disaccharide accumulation

7 VAN LEEUWENHOEKrsquoS LEGACY

Antony van Leeuwenhoek (1632ndash1723) was born in theDutch town of Delft where he was educated at a grammarschool until the age of 16 After the death of his fatherVan Leeuwenhoek became an apprentice linen draper andsubsequently established himself as a textile merchantwhere he must have used magnifying glasses routinely toexamine the quality of the cloth He crafted his own


single-lens instruments which at the time outperformedin magnification and resolution all existing compoundmicroscopes He was inspired by Hookersquos Micrographia tolook for subjects other than cloth as shown by an earlystudy of a bee sting which he reported to The RoyalSociety in 1673 He communicated his research in morethan 300 letters of which 190 were sent to The RoyalSociety and is considered the founder of microbiologythrough his observations of bacteria and other micro-scopic organisms

Anhydrobiosis was just one of Van Leeuwenhoekrsquosmany discoveries but it is perhaps one of the most endur-ing puzzles he has bequeathed to us His sense of wonderat the resurrection performed routinely by a simpleaquatic invertebrate the bdelloid rotifer is just as relevanttoday as it was then The rotifer still has much to teachus even 300 years after Van Leeuwenhoek about anintriguing mystery of nature

The authors thank the anonymous reviewers for many com-ments which have helped to improve the manuscript AT isthe AWG Senior Research Fellow and JL is the AWGResearch Student of Pembroke College Cambridge Thiswork was partly funded by The Royal Society research grantno 22084

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Algie J E amp Wyatt I C 1984 Calculation of mass and watercontent between the core cortex and coat of Bacillus stearo-thermophilus spores Curr Microbiol 10 249ndash254

Arakawa T amp Timasheff S N 1982 Stabilization of proteinstructure by sugars Biochemistry 21 6536ndash6544

Arguelles J C 2000 Physiological roles of trehalose in bacteriaand yeasts a comparative analysis Arch Microbiol 174217ndash224

Baker H 1742 Microscope made easy London DodsleyBaker H 1744 Letter to M Folkes President of The Royal

Society Dated 16 Jan 1744Baker H 1753 Employment for the microscope London

DodsleyBartels D amp Salamini F 2001 Desiccation tolerance in the

resurrection plant Craterostigma plantagineum A contri-bution to the study of drought tolerance at the molecularlevel Pl Physiol 127 1346ndash1353

Bartels D Furini A Ingram J amp Salamini F 1996Responses of plants to dehydration stress a molecular analy-sis Pl Growth Regul 20 111ndash118

Battista J R Park M J amp McLemore A E 2001 Inacti-vation of two homologues of proteins presumed to beinvolved in the desiccation tolerance of plants sensitizesDeinococcus radiodurans R1 to desiccation Cryobiology 43133ndash139

Becquerel P 1950 La suspension de la vie au-dessous de120 K absolu par demagnetisation adiabatique de lrsquoalun defer dans le vide le plus eleve Compt Rend Acad Sci 231261ndash263

Benaroudj N Lee D H amp Goldberg A L 2001 Trehaloseaccumulation during cellular stress protects cells and cellularproteins from damage by oxygen radicals J Biol Chem 27624 261ndash24 267

Bewley J D amp Black M 1994 Seeds physiology of developmentand germination New York Plenum


Bianchi G Gamba A Murelli C Salamini F amp BartelsD 1991 Novel carbohydrate metabolism in the resurrectionplant Craterostigma plantagineum Pl J 1 355ndash359

Blackman S A Oberndorf R L amp Leopold A C 1992Maturation proteins and sugars in desiccation tolerance ofdeveloping soybean seeds Pl Physiol 100 225ndash230

Blazquez M A Santos E Flores C-L Martgnez-ZapaterJ M Salinas J amp Gancedo C 1998 Isolation and molecu-lar characterization of the Arabidopsis TPS1 gene encodingtrehalose-6-phosphate synthase Pl J 13 685ndash689

Bloom F R Price P Lao G F Xia J L Crowe J HBattista J R Helm R F Slaughter S amp Potts M 2001Engineering mammalian cells for solid-state sensor appli-cations Biosensors Bioelectron 16 603ndash608

Bolen D W amp Baskakov I V 2001 The osmophobic effectnatural selection of a thermodynamic force in protein fold-ing J Mol Biol 310 955ndash963

Broca P 1860 Reviviscence des animaux sesseches C R SocBiol Paris 3 1ndash139

Browne J A 2001 An investigation into the physiological andgenetic changes associated with anhydrobiosis in the nema-todes Panagrolaimus sp and Aphelenchus avenae PhD thesisNational University of Ireland Maynooth Ireland

Bryce D L 1929 On three cases of encystment among roti-fers J R Microsc Soc 49 217ndash221

Burke M J 1986 The vitreous state and survival of anhydrousbiological systems In Membranes metabolism and dry organ-isms (ed A C Leopold) pp 358ndash364 Ithaca NY CornellUniversity Press

Caprioli M amp Ricci C 2001 Recipes for successful anhydro-biosis in bdelloid rotifers Hydrobiologia 446 71ndash74

Cerrutti P Segovia de Huergo M Galvagno M ScheborC amp Buera M P 2000 Commercial bakerrsquos yeast stabilityas affected by intracellular content of trehalose dehydrationprocedure and the physical properties of external matricesAppl Microbiol Biotechnol 54 575ndash580

Chen T Acker J P Eroglu A Cheley S Bayley HFowler A amp Toner M L 2001 Beneficial effect of intra-cellular trehalose on the membrane integrity of dried mam-malian cells Cryobiology 43 168ndash181

Clegg J S 1962 Free glycerol in dormant cysts of the brineshrimp Artemia salina and its disappearance during develop-ment Biol Bull 123 295ndash301

Clegg J S 1965 The origin of trehalose and its significanceduring the emergence of encysted dormant embryos of Arte-mia salina Comp Biochem Physiol 14 135ndash143

Clegg J S 1967 Metabolic studies of cryptobiosis in encystedembryos of Artemia salina Comp Biochem Physiol 20801ndash809

Clegg J S 1974 Biochemical adaptations associated with theembryonic dormancy of Artemia salina Trans Am MicroscSoc 93 481ndash490

Clegg J S 1978 Hydration dependent metabolic transitionsand the state of water in Artemia cysts In Dry biological sys-tems (ed J H Crowe amp J S Clegg) pp 117ndash154 NewYork Academic

Clegg J S 1986 The physical properties and metabolic statusof Artemia cysts at low water contents the lsquowater replace-ment hypothesisrsquo In Membranes metabolism and dry organ-isms (ed A C Leopold) pp 169ndash187 Ithaca NY CornellUniversity Press

Clegg J S 2001 Cryptobiosis a peculiar state of biologicalorganization Comp Biochem Physiol 128B 613ndash624

Clegg J S Seitz P Seitz W amp Hazlewood C F 1982Cellular responses to extreme water loss the water replace-ment hypothesis Cryobiology 19 306ndash316

Clement P amp Wurdak E 1991 Rotifera In Microscopic anat-omy of invertebrates vol 4 Aschelminthes (ed F WHarrison) pp 219ndash297 New York Wiley


Colaco C Sen S Thangavelu M Pinder S amp Roser B1992 Extraordinary stability of enzymes dried in trehalosesimplified molecular biology BioTechnology 10 1007ndash1011

Coutinho C Bernades E Felix D amp Panek A D 1988Trehalose as cryoprotectant for preservation of yeast strainsJ Biotechnol 7 23ndash32

Crowe J H 1971 Anhydrobiosis an unsolved problem AmNat 105 563ndash574

Crowe J H 1975 The physiology of cryptobiosis in tardi-grades Mem 1st Ital Idrobiol 32(Suppl) 37ndash59

Crowe J H amp Clegg J S (eds) 1973 Anhydrobiosis Editorrsquoscomments pp 226ndash231 Stroudsburg PA Dowden Hutch-ison amp Ross

Crowe J H amp Madin K A 1974 Anhydrobiosis in tardi-grades and nematodes Trans Am Microsc Soc 93 513ndash524

Crowe J H Hoekstra F A amp Crowe L M 1992 Anhydro-biosis A Rev Physiol 54 579ndash599

Crowe J H Carpenter J F amp Crowe L M 1998 The roleof vitrification in anhydrobiosis A Rev Physiol 60 73ndash103

Crowe J H Crowe L M Oliver A E Tsvetkova N Wol-kers W amp Tablin F 2001 The trehalose myth revisitedintroduction to a symposium on stabilization of cells in thedry state Cryobiology 43 89ndash105

Crowe L M amp Crowe J H 1992 Anhydrobiosis a strategyfor survival Adv Space Res 12 239ndash247

Cubitt C 1872 On the winter habits of Rotatoria MonthlyMicroscopial J 5 168ndash172

Dalyell J G 1803 Tracts on the natural history of animals andvegetables (translated from the original Italian of the AbbeSpallanzani) Edinburgh W Creech and A Constable

Davis H 1873 A new Callidina with the result of experimentson the desiccation of rotifers Monthly Microscopial J 6201ndash209

Dickson M R amp Mercer E H 1967 Fine structural changesaccompanying desiccation in Philodina roseola (Rotifera) JMicrosc 6 331ndash348

Dobers E 1915 Uber die Biologie der Bdelloidea Int RevGes Hydrobiol Biol Suppl 7 1ndash128

Doyere M P L 1842 Memoire sur les Tardigrades AnnalesSoc Nat 18 1ndash35

Ehrenberg G C 1838 Die infusionsthierchen als vollkommeneorganismen ein blick in das tiefere organische Leben der NaturLeipzig Germany Voss

Eleutherio E C A De Araujo P S amp Panek A D 1993Role of the trehalose carrier in dehydration resistance of Sac-charomyces cerevisiae Biochim Biophys Acta 1156 263ndash266

Eleutherio E C A Maia F M Pereira M D Degre RCameron D amp Panek A D 1997 Induction of desiccationtolerance by osmotic treatment in Saccharomyces uvarum varcarlsbergensis Can J Microbiol 43 495ndash498

El-Nockrashy A S amp Frampton V L 1967 Destruction oflysine by nonreducing sugars Biochem Biophys Res Com-mun 28 675ndash681

Estruch F 2000 Stress-controlled transcription factors stress-induced genes and stress tolerance in budding yeast FEMSMicrobiol Rev 24 469ndash486

Farrant J M 2000 A comparison of mechanisms of des-iccation tolerance among three angiosperm resurrectionplant species Pl Ecol 151 29ndash39

Fielding M J 1951 Observations on the length of dormancyin certain plant infecting nematodes Helminthol Soc WashProc 18 110ndash112

Fontana F 1767 Ricerche fisica sopra il veleno della vipereItaly Lucca

Fontana F 1776 Lettre a un de ses amis sur lrsquoergot et la Tre-mella J Physique Par lrsquoAbbe Rozier 7 42

Ford B J 1981 The Van Leeuwenhoek specimens Notes RecR Soc Lond 36 37ndash59


Ford B J 1982 The Rotifera of Antony van LeeuwenhoekMicroscopy 34 362ndash373

Ford B J 1991 The Leeuwenhoek legacy Bristol and LondonBiopressFarrand Press

Franks F 1985 Biophysics and biochemistry at low temperatureCambridge Press

Franks F 2000 Water a matrix of life 2nd edn CambridgeRoyal Society of Chemistry

Gadd G M Chalmers K amp Reed R H 1987 The role oftrehalose in dehydration resistance of Saccharomyces cerevis-iae FEMS Microbiol Lett 48 249ndash254

Gaff D F 1971 Desiccation-tolerant flowering plants insouthern Africa Science 174 1033ndash1034

Gaff D F 1989 Responses of desiccation-tolerant lsquoresurrec-tionrsquo plants to water stress In Structural and functionalresponses to environmental stresses (ed K H Krebb HRichter amp T M Hinkley) pp 1602ndash1606 The Hague TheNetherlands SPB Academic Publishers

Galinski E A Stein M Amendt B amp Kinder M 1997 Thekosmotropic (structure-forming) effect of compensatory sol-utes Comp Biochem Physiol 117A 357ndash365

Garcga de Castro A amp Tunnacliffe A 2000 Intracellular tre-halose improves osmotolerance but not desiccation tolerancein mammalian cells FEBS Lett 487 199ndash202

Garcga de Castro A Bredholt H Stroslashm A R amp Tunna-cliffe A 2000a Anhydrobiotic engineering of gram-negativebacteria Appl Environ Microbiol 66 4142ndash4144

Garcga de Castro A Lapinski J amp Tunnacliffe A 2000bAnhydrobiotic engineering Nature Biotechnol 18 473

Gavarret J 1859 Quelques experiences sur les Rotiferes lesTardigrades et les Anguillules des mousses des toits AnnalesSci Nat Zool 4 315ndash330

Giard A 1894 Lrsquoanhydrobiose ou ralentissement des pheno-menes vitaux C R Soc Biol Paris 46 497ndash500

Gilbert J J 1974 Dormancy in rotifers Trans Am MicroscSoc 93 490ndash513

Gordon S L Oppenheimer S R Mackay A M Brunnab-end J Puhlev I amp Levine F 2001 Recovery of humanmesenchymal stem cells following dehydration and rehy-dration Cryobiology 43 182ndash187

Grably M R Stanhill A Tell O amp Engelberg D 2002HSF and Msn24p can exclusively or cooperatively activatethe yeast HSP104 gene Mol Microbiol 44 21ndash35

Grilli Caiola M Ocampo-Friedmann R amp Friedmann E I1993 Cytology of long-term desiccation in the desert cyano-bacterium Chroococcidiopsis (Chroococcales) Phycologia 32315ndash322

Guidetti R amp Jonsson K I 2002 Long-term anhydrobioticsurvival in semi-terrestrial micrometazoans J Zool 257181ndash187

Guo N Puhlev I Brown D R Mansbridge J amp LevineF 2000 Trehalose expression confers desiccation toleranceon human cells Nature Biotechnol 18 168ndash171

Heckley R J 1978 Effects of oxygen in dried organisms InDry biological systems (ed J H Crowe amp J S Clegg) pp257ndash278 New York Academic

Hengge-Aronis R 2002 Recent insights into the general stressresponse regulatory network in Escherichia coli J MolMicrobiol Biotechnol 4 341ndash346

Hershkovitz N Oren A amp Cohen Y 1991 Accumulation oftrehalose and sucrose in cyanobacteria exposed to matricwater stress Appl Environ Microbiol 57 645ndash648

Hickernell L M 1917 A study of desiccation in the rotiferPhilodina roseola with special reference to cytologicalchanges accompanying desiccation Biol Bull 32 343ndash397

Higa L M amp Womersley C Z 1993 New insights into theanhydrobiotic phenomenon the effects of trehalose contentand differential rates of evaporative water-loss on the survivalof Aphelenchus avenae J Exp Zool 267 120ndash129


Hill D R Peat A amp Potts M 1994 Biochemistry and struc-ture of the glycan secreted by desiccation-tolerant Nostoccommune (cyanobacteria) Protoplasma 182 126ndash148

Hill J 1752 The history of animals London OsborneHinton H E 1960 A fly larva that tolerates dehydration and

temperatures of 2270 degC to 1102 degC Nature 188 333ndash337Hoekstra F A Golovina E A amp Buitink J 2001 Mech-

anisms of plant desiccation tolerance Trends Pl Sci 6431ndash438

Hoole S 1807 The select works of Antony van Leeuwenhoek vol2 London G Sidney

Hottiger T Boller T amp Wiemken A 1987 Rapid changesof heat and desiccation tolerance correlated with changes intrehalose content in Saccharomyces cerevisiae cells subjectedto temperature shifts FEBS Lett 220 113ndash115

Ingram J amp Bartels D 1996 The molecular basis of dehy-dration tolerance in plants A Rev Pl Physiol Pl Mol Biol47 377ndash403

Jacobs M H 1909 The effects of desiccation on the rotiferPhilodina roseola J Exp Zool 4 207ndash263

Janson O 1893 Versuch einer Ubersicht uber die rotatorien-familieder philodinaeen Beilage zum 12 Band der Abhandl des nat-urw Bremen Germany Naturwissenschaftlicher Verein zuBremen

Joblot L 1718 Descriptions et usages de plusieurs nouveaux micro-scopes Paris J Collombat

Jonsson K I amp Bertolani R 2001 Facts and fiction aboutlong-term survival in tardigrades J Zool Lond 255 121ndash123

Keilin D 1959 The problem of anabiosis or latent life historyand current concept Proc R Soc Lond B 150 149ndash191

Kermode A R amp Bewley J D 1985 The role of maturationdrying in the transition from seed development to germi-nation J Exp Bot 36 1906ndash1915

Kizawa H Miyazaki J Yokota A Kanagae Y MiyagawaK amp Sugiyama Y 1995 Trehalose production by a strainof Micrococcus varians Biosci Biotechnol Biochem 591522ndash1527

Kleines M Elster R-C Rodrigo M-J Blervacq A-SSalamini F amp Bartels D 1999 Isolation and expressionanalysis of two stress-responsive sucrose-synthase genesfrom the resurrection plant Craterostigma plantagineum(Hochst) Planta 209 13ndash24

Koste W amp Hollowday E D 1993 A short history of westernEuropean rotifer research Hydrobiologia 255256 557ndash572

Lapinski J Clayson E M Brindle K M amp Tunnacliffe A2003 Anhydrobiosis without trehalose in bdelloid rotifers(In preparation)

Lea C H Hannan R S amp Greaves I R N 1950 The reac-tion between proteins and reducing sugars in the lsquodryrsquo statedried human blood plasma Biochem J 47 626ndash629

Leslie S B Israeli E Lighthart B Crowe J H amp CroweL M 1995 Trehalose and sucrose protect both membranesand proteins in intact bacteria during drying Appl EnvironMicrobiol 61 3592ndash3597

Lindau G 1958 Uber die Wiederstandsfahigkeit von Moosro-tatorien aus Wasserkulturen gegen Trocknung und gegenniedrige und hohe Temperaturen Insbesondere uber einigeFaktoren welche die Resistenz begrenzen Z Morph OkolTiere 47 489ndash528

Linders L J M Wolkers W F Hoekstra F A amp VanrsquotRiet K 1997 Effect of added carbohydrates on membranephase behaviour and survival of dried Lactobacillus plan-tarum Cryobiology 35 31ndash40

Loomis S H OrsquoDell S J amp Crowe J H 1979 Anhydro-biosis in nematodes inhibition of the browning reaction ofreducing sugars with dry protein J Exp Zool 208 355ndash360


Louis P Truper H G amp Galinski E A 1994 Survival ofEscherichia coli during drying and storage in the presence ofcompatible solutes Appl Microbiol Biotechnol 41 684ndash688

Madin K A C amp Crowe J H 1975 Anhydrobiosis in nema-todes carbohydrate and lipid metabolism during dehy-dration J Exp Zool 193 335ndash342

Maillard L-C 1912 Actions des acides amines sur les sucresformation des melanoidines par voie methodique C RAcad Sci 154 66ndash68

Mancinelli R L amp Klovstad M 2000 Martian soil and UVradiation microbial viability assessment on spacecraft sur-faces Planet Space Sci 48 1093ndash1097

Mansell J L amp Clegg J S 1983 Cellular and molecularconsequences of reduced cell water content Cryobiology 20591ndash612

Manzanera M Garcga de Castro A Toslashndervik A Rayner-Brandes M Stroslashm A R amp Tunnacliffe A 2002 Hydroxy-ectoine is superior to trehalose for anhydrobiotic engineeringof Pseudomonas putida KT2440 Appl Environ Microbiol 684328ndash4333

Marino C Curto M Bruno R amp Rinaudo M T 1989Trehalose synthase and trehalase behaviour in yeast cells inanhydrobiosis and hydrobiosis Int J Biochem 21 1369ndash1375

Mattimore V amp Battista J R 1996 Radioresistance of Deino-coccus radiodurans functions necessary to survive ionizingradiation are also necessary to survive prolonged desiccationJ Bacteriol 178 633ndash637

May R-M Maria M amp Guimard J 1964 Actions differ-entielles des rayons X et ultraviolets sur le tardigrade Macro-biotus areolatus a lrsquoetat actif et desseche Bull Biol Fr Belg98 349ndash367

Needham J T 1743 A letter concerning chalky tubulous con-cretions with some microscopical observations on the farinaof the red lilly and on worms discovered in smutty cornPhil Trans R Soc Lond 42 634ndash641

Nickle D C Learn G H Rain M W Mullins J I ampMittler J E 2002 Curiously modern DNA for a lsquo250million-year-oldrsquo bacterium J Mol Evol 54 134ndash137

OrsquoBrien J 1996 Stability of trehalose sucrose and glucose tononenzymatic browning in model systems J Food Sci 61679ndash682

Oliver A E Leprince O Wolkers W F Hincha D KHeyer A G amp Crowe J H 2001 Non-disaccharide-basedmechanisms of protection during drying Cryobiology 43151ndash167

Oliver M J 1996 Desiccation tolerance in vegetative plantcells Physiol Plant 97 779ndash787

Oliver M J 1997 How some plants recover from vegetativedesiccation a repair based strategy Acta Physiol Plant 19419ndash425

Oliver M J amp Bewley J D 1997 Desiccation-tolerance ofplant tissues a mechanistic overview Hort Rev 18 171ndash213

Oliver M J Tuba Z amp Mishler B D 2000 The evolutionof vegetative desiccation tolerance in land plants Pl Ecol15 85ndash100

Ooms J J J Leon-Kloosterziel K M Bartels DKoornneef M amp Karssen C M 1993 Acquisition of des-iccation tolerance and longevity in seeds of Arabidopsis thali-ana Pl Physiol 102 1185ndash1191

Ophir T amp Gutnick D L 1994 A role for exopolysaccharidesin the protection of microorganisms from desiccation ApplEnviron Microbiol 60 740ndash745

Orstan A 1995 Desiccation survival of the eggs of the rotiferAdineta vaga (Davis 1873) Hydrobiologia 313314 373ndash375

Orstan A 1998a Factors affecting long-term survival of drybdelloid rotifers a preliminary study Hydrobiologia 387388327ndash331


Orstan A 1998b Microhabitats and dispersal routes of bdel-loid rotifers Sci Nat 1 27ndash36

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Perry R N 1999 Desiccation survival of parasitic nematodesParasitology 119 S19ndashS30

Pierce S K Maugel T K amp Reid L 1987 Illustrated invert-ebrate anatomy a laboratory guide New York Oxford Univer-sity Press

Potts M 1994 Desiccation tolerance of prokaryotes MicrobiolRev 58 755ndash805

Potts M 1999 Mechanisms of desiccation tolerance in cyano-bacteria Eur J Phycol 34 319ndash328

Potts M 2001 Desiccation tolerance a simple process TrendsMicrobiol 9 553ndash559

Pouchet F A 1859a Nouvelles experiences sur les animauxpseudo-resuscitans Comp Rend Acad Sci 49 492ndash494

Pouchet F A 1859b Experiences sur la resistance vitale desanimalcules pseudoresuscitans Comp Rend Acad Sci 49886ndash888

Pouchet F A 1859c Generation spontanee et resurrection desRotiferes Cosmos 14 380ndash382

Puhlev I Guo N Brown D R amp Levine F 2001 Des-iccation tolerance in human cells Cryobiology 42 207ndash217

Quillet M amp Soulet M 1964 Sur lrsquoaccumulation concomit-ante du saccharose et du trehalose chez plusieurs especes deSelaginelles indigenes et exotiques C R Acad Sci Paris 259635ndash637

Rahm P G 1923 Biologische und physiologische Beitrage zurKenntnis der Moosfauna Z Allg Physiol 20 1ndash34

Rahm P G 1926 Die trockenstarre (Anabiose) der moosti-erwelt Biol CentBl 46 452ndash477

Rahm P G 1937 A new ordo of tardigrades from the hotsprings of Japan (Furu-yu section Unzen) Annot Zool Japn16 345ndash352

Ricci C 1987 Ecology of bdelloids how to be successful Hyd-robiologia 147 117ndash127

Ricci C 1998 Anhydrobiotic capabilities of bdelloid rotifersHydrobiologia 387388 321ndash326

Ricci C 2001 Dormancy patterns of rotifers Hydrobiologia446 1ndash11

Ricci C amp Melone G 1984 Macrotrachela quadricornifera(Rotifera Bdelloidea) a SEM study on active and cryptobi-otic animals Zool Scrt 13 195ndash200

Ricci C Vaghi L amp Manzini M L 1987 Desiccation ofrotifers (Macrotrachela quadricornifera) survival and repro-duction Ecology 68 1488ndash1494

Roffredi lrsquoAbbe 1775a Sur lrsquoorigine des petits vers ou Angu-illes du Bled rachitique J Physique Par lrsquoAbbe Rozier 5 1ndash19

Roffredi lrsquoAbbe 1775b Seconde lettre ou suite drsquoobservationssur le rachitism du Bled sur les Anguilles de la colle de far-ine et sur le grain charbonne J Physique Par lrsquoAbbe Rozier5 197ndash225

Roffredi lrsquoAbbe 1776 Memoir pour servir de sopplement etdrsquoeclaircissement aux memoires sur les anguilles du Bled etde la colle de farine J Physique Par lrsquoAbbe Rozier 7 369ndash385

Ruis H amp Schuller C 1995 Stress signalling in yeast Bio-Essays 17 959ndash965

Sano F Asakawa N Inoues Y amp Sakurai M 1999 A dualrole for intracellular trehalose in the resistance of yeast cellsto water stress Cryobiology 39 80ndash87

Schebor C Galvagno M Buera M P amp Chirife J 2000Glass transition temperatures and fermentative activity of


heat-treated commercial active dry yeasts Biotechnol Prog16 163ndash168

Schramm U amp Becker W 1987 Anhydrobiosis of the bdel-loid rotifer Habrotrocha rosa (Aschelminthes) Z Mikrosk-Anat Forsch 101 1ndash17

Seki K amp Toyoshima M 1998 Preserving tardigrades underpressure Nature 395 853ndash854

Senrartna T amp McKersie B D 1986 Loss of desiccation tol-erance during seed germination a free radical mechanism ofinjury In Membranes metabolism and dry organisms (ed A CLeopold) pp 85ndash101 Ithaca NY Cornell University Press

Shen M J Mudgett M B Schopf J W Clarke S ampBerger R 1995 Exceptional seed longevity and robustgrowth ancient sacred lotus from China Am J Bot 821367ndash1380

Singer M A amp Lindquist S 1998 Multiple effects of trehal-ose on protein folding in vivo and in vitro Mol Cell 1639ndash648

Smirnoff N 1993 The role of active oxygen in the responseof plants to water deficit and desiccation New Phytol 12527ndash58

Smirnoff N amp Cumbes Q J 1989 Hydroxyl radical scaveng-ing activity of compatible solutes Phytochemistry 28 1057ndash1060

Spallanzani L 1765 Saggio di osservazioni microscopiche concer-nenti il sistema della generaziones dei Signori de Needham e Buf-fon Italy Modena

Spallanzani L 1776 Opuscoli fisica animale et vegetabileItaly Modena

Stroslashm A R 1998 Osmoregulation in the model organismEscherichia coli genes governing the synthesis of glycine beta-ine and trehalose and their use in metabolic engineering ofstress tolerance J Biosci 23 437ndash445

Stryer L 1999 Biochemistry New York FreemanSun W Q amp Leopold A C 1997 Cytoplasmic vitrification

and survival of anhydrobiotic organisms Comp BiochemPhysiol 117B 327ndash333

Szent-Gyorgyi A 1979 Welcoming address In Cell-associatedwater (ed W Drost-Hansen amp J S Clegg) pp 1ndash2 NewYork Academic

Timasheff S N 1992 Water as ligand preferential bindingand exclusion of denaturants in protein unfolding Biochem-istry 31 9857ndash9864

Trembley A 1747 Observations upon several species of waterinsects of the Polypus kind Phil Trans R Soc Lond 44627ndash655

Tunnacliffe A Garcga de Castro A amp Manzanera M 2001Anhydrobiotic engineering of bacterial and mammalian cellsis intracellular trehalose sufficient Cryobiology 43 124ndash132


Umezawa S 1958 On the desiccation death of the rotiferPhilodina roseola Res Rep Kochi Univ 7 1ndash15

Van Leeuwenhoek A 1702 Letter to Hendrik van Bleyswijkdated 9 February 1702

Van Rijnberk G amp Palm L C (eds) 1999 The collected lettersof Antoni van Leeuwenhoek vol 14 1701ndash1704 pp 55ndash73Lisse The Netherlands Swets and Zeitlinger

Vogel G Aeschbacher R A Muller J Boller T ampWiemken A 1998 Trehalose-6-phosphate phosphatasesfrom Arabidopsis thaliana identification by functional comp-lementation of the yeast tps2 mutant Pl J 13 673ndash683

Vreeland R H 2000 Isolation of a 250 million-year-old bac-terium from a primary salt crystal Nature 407 897ndash900

Warner D T 1962 Some possible relations of carbohydratesand other biological components with the water structure at37deg Nature 196 1055ndash1058

Webb S J 1965 Bound water in biological integrity SpringfieldIL Thomas

Westh P amp Ramloslashv H 1991 Trehalose accumulation in thetardigrade Adorybiotus coronifer during anhydrobiosis J ExpZool 258 303ndash311

White O (and 31 others) 1999 Genome sequence of the radi-oresistant bacterium Deinococcus radiodurans R1 Science 2861571ndash1577

Winderickx J de Winde J H Crauwels M Hino AHohmann S Van Dijck P amp Thevelein J M 1996 Regu-lation of genes encoding subunits of the trehalose synthasecomplex in Saccharomyces cerevisiae novel variations ofSTRE-mediated transcription control Mol Gen Genet 252470ndash482

Wolkers W F Walker N J Tablin F amp Crowe J H 2001Human platelets loaded with trehalose survive freeze-dryingCryobiology 42 79ndash87

Womersley C 1981 Biochemical and physiological aspects ofanhydrobiosis Comp Biochem Physiol 70B 669ndash678

Womersley C Z amp Higa L M 1998 Trehalose its role inthe anhydrobiotic survival of Ditylenchus myceliophagus Nem-atologica 44 269ndash291

Wright J C 2001 Cryptobiosis 300 years on from VanLeeuwenhoek what have we learned about tardigradesZool Anz 240 563ndash582

Wright K A 1991 Nematoda In Microscopic anatomy ofinvertebrates vol 4 Aschelminthes (ed F W Harrison) pp111ndash195 New York Wiley

Yancey P H Clark M E Hand S C Bowlus R D ampSomero G N 1982 Living with water stress evolution ofosmolyte systems Science 217 1214ndash1222

Zacharias O 1886 Konnen die Rotatorien und Tardigradennach vollstandiger Austrocknung wieder aufleben odernicht Biol CentBl 6 230ndash235


Figure 1 Antony van Leeuwenhoek at the age of 54 from amezzotint reproduction of an oil painting by JohannesVerkolje 1686 (The Royal Society collection) Reproducedwith permission

unsegmented pseudocoelomates ranging in size from 100to 2000 mm the body (figure 2c) is divided into threeregions the foot with toes the trunk and the head bearingthe characteristic cilia whose action induces a vortex inwater drawing small food particles towards the mouthBdelloids inhabit freshwater ponds lakes temporarypools brackish waters sewage and the interstices of soilmosses and lichens (Ricci 1987) Some of these habitatsdry out frequently and the ability of most bdelloid speciesto withstand desiccation and the dry state (Ricci 1998)must represent a clear selective advantage This abilitymdashsurvival of extreme desiccation and entry into a state ofsuspended animation pending recovery by rehydrationmdashis known as anhydrobiosis a term derived from the Greekfor lsquolife without waterrsquo

Van Leeuwenhoek had described rotifers in earlier cor-respondence with The Royal Society most clearly in hisletter of 17 October 1687 (Van Rijnberk amp Palm 1999)and had sent specimen packages with some letters whichwere rediscovered by Ford (1981) When Ford rehydratedthese specimens some contained animals identifiable asrotifers even allowing a proposed species designationalthough none had apparently survived its three centuriesof dryness (Ford 1982 1991) Nevertheless viability canbe maintained for extended periods in the dry state thecurrent record for (well-documented) survival of a driedanimal being of the order of decades for nematodes andbrine shrimp cysts (Fielding 1951 Clegg 1967 Jonsson ampBertolani 2001) The longest authenticated period sur-vived by a rotifer after desiccation appears to be 9 years(Guidetti amp Jonsson 2002) although there is potential fora much longer phase of suspended animation 1000-year-


Figure 2 The bdelloid rotifer Philodina roseola (a) Drawingsof the rotifers that Van Leeuwenhoek (1702) observed (b) Aphotograph of P roseola obtained from a bird bath (c) Thestructure of a bdelloid rotifer reproduced from Pierce et al(1987) with permission

old lotus seeds have germinated successfully (Shen et al1995) and there are regular claims of the recovery ofancient bacteria up to 250 million years old (eg Vreeland2000) although these claims are equally regularlydebated (eg Nickle et al 2002)

In the past the tolerance of extreme dehydration andthe dry state that anhydrobiosis represents has challengedsome fundamental biological precepts particularly regard-ing the necessity of water for life and the requirement formetabolic continuity and has contributed to the debateon spontaneous generation (Keilin 1959) Modernresearch places emphasis on unravelling the molecular andcellular mechanisms underlying anhydrobiosis as we arestill a long way from a full understanding of these pro-cesses This review will summarize what is known aboutanhydrobiosis in rotifers with reference to other speciesand include some surprising recent results which maynecessitate modification of prevailing models


2 ANHYDROBIOSIS














































































5 6 7 8 9 10 11 12 13 14 15time (min)

mas

s (a

rbit

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uni

ts)

(a)

(b)


















12

10

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6

4

2

0

400

350

300

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200

150100

50

00 4 8 12 16 20 24

0

3

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0

6

12

18

24

inte

rnal

treh

alos

e co

nten

t (m

M)

treh

alos

e (

dry

mas

s)

time (d)

time (h)

surv

ival

(

)su

rviv

al (

)

(a)

(b)

0 48 60 72 84 96 10812 24 36















6 CONCLUSIONS













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2 ANHYDROBIOSIS














































































5 6 7 8 9 10 11 12 13 14 15time (min)

mas

s (a

rbit

rary

uni

ts)

(a)

(b)


















12

10

8

6

4

2

0

400

350

300

250

200

150100

50

00 4 8 12 16 20 24

0

3

6

9

12

0

6

12

18

24

inte

rnal

treh

alos

e co

nten

t (m

M)

treh

alos

e (

dry

mas

s)

time (d)

time (h)

surv

ival

(

)su

rviv

al (

)

(a)

(b)

0 48 60 72 84 96 10812 24 36















6 CONCLUSIONS













REFERENCES









































































































































































































































































5 6 7 8 9 10 11 12 13 14 15time (min)

mas

s (a

rbit

rary

uni

ts)

(a)

(b)


















12

10

8

6

4

2

0

400

350

300

250

200

150100

50

00 4 8 12 16 20 24

0

3

6

9

12

0

6

12

18

24

inte

rnal

treh

alos

e co

nten

t (m

M)

treh

alos

e (

dry

mas

s)

time (d)

time (h)

surv

ival

(

)su

rviv

al (

)

(a)

(b)

0 48 60 72 84 96 10812 24 36















6 CONCLUSIONS













REFERENCES



















































































































































































































12

10

8

6

4

2

0

400

350

300

250

200

150100

50

00 4 8 12 16 20 24

0

3

6

9

12

0

6

12

18

24

inte

rnal

treh

alos

e co

nten

t (m

M)

treh

alos

e (

dry

mas

s)

time (d)

time (h)

surv

ival

(

)su

rviv

al (

)

(a)

(b)

0 48 60 72 84 96 10812 24 36















6 CONCLUSIONS













REFERENCES











































































































































































































6 CONCLUSIONS













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resurrecting van leeuwenhoek's rotifers: a reappraisal of the role

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