Trehalose and anhydrobiosis in tardigrades – evidence for divergence in responses to dehydration Steffen Hengherr 1,2 , Arnd G. Heyer 3 , Heinz-R. Ko ¨ hler 1 and Ralph O. Schill 2 1 Animal Physiological Ecology, Zoological Institute, University of Tu ¨ bingen, Germany 2 Department of Zoology, Biological Institute, Universita ¨ t of Stuttgart, Germany 3 Department of Botany, Biological Institute, Universita ¨ t of Stuttgart, Germany Desiccation in general leads to severe damage of cellu- lar structures, which commonly results in the death of cells and the organism. However, a number of so-called anhydrobiotic organisms have developed remarkable mechanisms, allowing them to minimize or avoid such damage and survive extreme dehydration in a cryptobiotic state [1–6]. Several species of inverte- brate taxa have this ability, including the embryonic cysts of crustaceans, rotifers, insect larvae, nematodes and tardigrades [2,3,7–12]. Additionally, many pro- caryotes, such as bacteria and cyanobacteria [13,14], and even plant seeds [15–19] and adult plants, for example the resurrection lycopode Selaginella lepido- phylla [5,20], demonstrate dehydration tolerance. Although Antonin van Leuwenhoek described anhy- drobiosis over 300 years ago [21], the underlying mech- anisms are still not fully understood. However, over the last three decades, researchers have come to recog- nize the important role of polyhydroxy compounds such as the non-reducing disaccharide trehalose [22– 24]. This sugar is found in high concentrations in a wide variety of anhydrobiotic organisms, including nematodes, embryonic cysts of crustaceans, and yeast. Trehalose concentrations as high as 13–18% of the dry weights have been reported for anhydrobiotic cysts of the crustacean Artemia franciscana [25–27] whereas the nematode Aphelenchus avenae can accumulate 10–15% of its dry weight as trehalose during anhydrobiosis [8,9]. Studies on the anhydrobiotic insect larvae Poly- pedilum vanderplanki report up to 18% trehalose in the dry body mass [11]. Significantly increased trehalose levels also have been found in the Arctic collembolan Onychiurus arcticus during partial desiccation, induced by sub-zero temperatures [28]. The disaccharide sucrose fulfils a similar role in plants and accumulates in desiccation-tolerant plant seeds and resurrection Keywords cryptobiosis; desiccation tolerance; metabolite; nonreducing disaccharide; tardigrade Correspondence R. O. Schill, Department of Zoology, Biological Institute, Universita ¨ t of Stuttgart, Pfaffenwaldring 57, D-70569 Stuttgart, Germany Fax: +49 711 685 6 5096 Tel: +49 711 685 6 9143 E-mail: ralph.schill@bio.uni-stuttgart.de (Received 9 August 2007, revised 31 Octo- ber 2007, accepted 19 November 2007) doi:10.1111/j.1742-4658.2007.06198.x To withstand desiccation, many invertebrates such as rotifers, nematodes and tardigrades enter a state known as anhydrobiosis, which is thought to require accumulation of compatible osmolytes, such as the non-reducing disaccharide trehalose to protect against dehydration damage. The treha- lose levels of eight tardigrade species comprising Heterotardigrada and Eutardigrada were observed in five different states of hydration and dehy- dration. Although many species accumulate trehalose during dehydration, the data revealed significant differences between the species. Although tre- halose accumulation was found in species of the order Parachela (Eutardi- grada), it was not possible to detect any trehalose in the species Milnesium tardigradum and no change in the trehalose level has been observed in any species of Heterotardigrada so far investigated. These results expand our current understanding of anhydrobiosis in tardigrades and, for the first time, demonstrate the accumulation of trehalose in devel- oping tardigrade embryos, which have been shown to have a high level of desiccation tolerance. Abbreviations HPAEC, high-performance liquid anion exchange chromatography; RH, relative humidity. FEBS Journal 275 (2008) 281–288 ª 2007 The Authors Journal compilation ª 2007 FEBS 281 plants during drying [29–31]. It has been hypothesized that non-reducing disaccharides have a protective role during dehydration. Trehalose has been shown to sta- bilize proteins in their native state and to preserve the integrity of membranes during dehydration in vitro [4,32], Assuming a similar role in vivo, two models for the mechanism of the protective role of trehalose have been proposed that are not mutually exclusive: The water replacement hypothesis [33] states that trehalose forms hydrogen bonds with macromolecules and cellu- lar structures in place of water during dehydration and thus preserves native structures. In addition, the vitrifi- cation hypothesis proposes the formation of amor- phous sugar glasses during desiccation, which protects proteins and membranes [23,32]. Despite evidence for the protective role of trehalose in animals and sucrose in plants, not all organisms that undergo anhydrobiosis contain one of these sug- ars or genes for its synthesis, as seen in bdelloid roti- fers, which appear to lack trehalose synthesis entirely [34,35]. Together with nematodes and rotifers, tardigrades represent one of the three main invertebrate taxa [36], where desiccation tolerance is widespread. Members of these taxa display an exceptional ability to survive high [7,37] and subfreezing temperatures [38–41] while in an anhydrobiotic state. Very few studies have investigated changes of trehalose levels in tardigrades during entry into anhydrobiosis. Crowe [42] compared the trehalose level of hydrated and anhydrobiotic Macrobiotus areo- latus and reported that trehalose accumulated in the dehydrated state. Until now, the only quantitative study to have shown a steady accumulation of treha- lose during anhydrobiosis in tardigrades (up to 2.3% of the dry weight) is that by Westh and Ramløv [43] on Adorybiotus coronifer. To obtain more comprehensive data on whether tre- halose may be important for desiccation tolerance in tardigrades, we investigated eight different species, rep- resenting the orders Parachela and Apochela of the Eutardigrada, and Echiniscoidea of the Heterotardi- grada. The present study represents the first extensive quantitative measure on adult animals of different tar- digrade species and the first observation of trehalose accumulation in tardigrade embryos, using 12 800 indi- viduals and 1440 embryos in total. Results Desiccation and protein level Despite differences between species, none of the tardi- grades investigated showed a significant change in total protein concentration when dehydrated (Table 1). Therefore, we chose total protein as reference parame- ter for quantification of trehalose because of the high sensitivity and accuracy of determination. However, due to the sensitivity of the analysis technique for sam- ple size and the small amount of protein present in tar- digrade eggs, it was not possible to quantify the total protein concentration in tardigrade eggs. Trehalose levels during anhydrobiosis Distinct changes in the level of trehalose during anhy- drobiosis were found only in the species of the Macro- biotidae (Table 2). Although there was a large variation in trehalose concentrations between the Mac- robiotus species, all showed a highly significant accu- mulation of trehalose when passing through state II, in which the animals draw in their legs but still perform distinct movements. Trehalose accumulation reached a maximum in the anhydrobiotic state (III), 48 h after completion of tun formation (P < 0.001). Large dif- ferences occurred in absolute trehalose concentration and accumulation rate between the species. For exam- ple, Macrobiotus tonollii accumulated trehalose by up to sixfold (1.650 ± 0.291 ngÆlg )1 protein; 0.153% of dry weight) whereas Macrobiotus ‘richtersi group’ 2 displayed a 150-fold enrichment of trehalose (up to 7.415 ± 0.580 ngÆlg )1 protein; 0.472% of dry weight) (Fig. 1A and Table 2). Data recorded for the other species are provided in Table 2. During the transitional rehydration stage (IV), when the animals had pro- truded their legs and the body again performed move- ments, all Macrobiotidae showed a rapid decline in trehalose concentration, which was further reduced over the next 4 h until the completely re-active state V was reached. Table 1. Total protein content of the hydrated and dehydrated state and dry weight per animal for the different tardigrade species. Data are shown as the mean ± SD. Species Protein (lg) Dry weight (lg) Hydrated Dehydrated Milnesium tardigradum 3.53 ± 0.21 3.80 ± 0.23 6.11 ± 0.20 Macrobiotus tonollii 3.28 ± 0.21 3.41 ± 0.30 3.61 ± 0.15 Macrobiotus richtersi 3.20 ± 0.25 3.65 ± 0.34 5.75 ± 0.19 Macrobiotus sapiens 2.33 ± 0.10 2.15 ± 0.18 2.92 ± 0.14 Macrobiotus ‘richtersi group’ 1 3.69 ± 0.24 3.67 ± 0.13 6.52 ± 0.27 Macrobiotus ‘richtersi group’ 2 3.92 ± 0.34 4.42 ± 0.26 6.55 ± 0.17 Echiniscus granulatus 1.24 ± 0.53 1.60 ± 0.29 2.44 ± 0.11 Echiniscus testudo 0.74 ± 0.18 0.80 ± 0.14 2.14 ± 0.16 Trehalose and anhydrobiosis in tardigrades S. Hengherr et al. 282 FEBS Journal 275 (2008) 281–288 ª 2007 The Authors Journal compilation ª 2007 FEBS Surprisingly, we were unable to detect any trehalose at all in Milnesium tardigradum (Fig. 2 and Table 2). Although we detected low trehalose concentrations in the two Heterotardigrade species (Echiniscus granulatus and Echiniscus testudo), there was no significant change in trehalose level during the anhydrobiotic states (Table 2) as shown by E. granulatus (Fig. 2B) (P = 0.213). Carbohydrate analysis resulted in several other unidentified peaks that differentiated the active state I and the anhydrobiotic state III for M. tardigradum, Macrobiotus richtersi, Macrobiotus ‘richtersi group’ 1 and M. tonollii, respectively, although the increase was low (Table 3). Only one peak (2.233 min retention time) demonstrated significant changes during desicca- tion in more than one species (M. tardigradum and M. ‘richtersi group’ 1). Sugar analysis of tardigrade embryos clearly revealed a low level of trehalose accumulation in Macrobiotus richtersi and M. ‘richtersi group’ 1 (Table 4), although it was not possible to perform statistical tests because of the small number of replicates. No trehalose was detected in eggs or embryos of M. tardigradum or M. tonollii. Discussion Analysis of soluble sugar accumulation in tardigrades during transition to an anhydrobiotic state showed that trehalose accumulates in species of the Macro- biotidae, although the absolute level of trehalose is low compared to other anhydrobitic organisms (e.g. Artemia cysts or the nematode A. avenae), where treha- lose levels can reach 13–18% and 10–15% of the dry weight, respectively [9,25–27]. Westh and Ramløv [43] reported that the Eutardigrade A. coronifer (Parachela) shows a more than 20-fold accumulation of trehalose during transition to anhydrobiosis. This is within the concentration range of trehalose accumulation in the Macrobiotidae investigated in the present study. Table 2. Trehalose concentration (ngÆlg )1 protein). Values in parenthesis represents the amount of trehalose as % of dry weight. Data are shown as the mean ± standard deviation. ND, not detected (beyond detection limit of 0.05 l M). Stages I II III IV V Milnesium tardigradum ND ND ND ND ND Macrobiotus tonollii 0.241 ± 0.202 1.515 ± 0.873 1.650 ± 0.291 0.837 ± 0.272 0.454 ± 0.122 (0.022 ± 0.019) (0.141 ± 0.081) (0.153 ± 0.027) (0.078 ± 0.025) (0.042 ± 0.011) Macrobiotus richtersi 0.055 ± 0.154 1.609 ± 0.586 2.888 ± 0.689 1.291 ± 0.410 0.664 ± 0.190 (0.003 ± 0.009) (0.096 ± 0.035) (0.172 ± 0.041) (0.077 ± 0.025) (0.040 ± 0.011) Macrobiotus sapiens 0.140 ± 0.197 1.193 ± 0.424 1.137 ± 0.263 1.337 ± 0.721 0.316 ± 0.338 (0.011 ± 0.015) (0.092 ± 0.056) (0.087 ± 0.020) (0.103 ± 0.056) (0.024 ± 0.026) Macrobiotus ‘richtersi group’ 1 0.041 ± 0.079 2.950 ± 0.985 4.640 ± 0.677 2.641 ± 1.168 1.033 ± 0.431 (0.002 ± 0.004) (0.166 ± 0.056) (0.262 ± 0.038) (0.149 ± 0.066) (0.058 ± 0.024) Macrobiotus ‘richtersi group’ 2 0.049 ± 0.091 4.228 ± 1.810 7.415 ± 0.580 3.319 ± 1.145 1.394 ± 0.508 (0.003 ± 0.006) (0.269 ± 0.012) (0.472 ± 0.037) (0.211 ± 0.073) (0.089 ± 0.032) Echiniscus granulatus 0.395 ± 0.780 0.030 ± 0.060 0.054 ± 0.153 0.109 ± 0.131 1.394 ± 0.311 (0.023 ± 0.045) (0.002 ± 0.004) (0.003 ± 0.009) (0.006 ± 0.008) (0.014 ± 0.018) Echiniscus testudo 0.158 ± 0.316 0.219 ± 0.437 0.583 ± 0.412 0.889 ± 0.088 1.064 ± 0.096 (0.006 ± 0.010) (0.008 ± 0.014) (0.021 ± 0.013) (0.032 ± 0.003) (0.038 ± 0.003) A B Fig. 1. Alterations of the trehalose level during anhydrobiosis (active stage I; dehydration stage II; anhydrobiotic stage III; rehydra- tion stage IV; active stage, 4 h after rehydration V). (A) Macrobio- tus ‘richtersi group 2’. (B) Echiniscus granulatus. S. Hengherr et al. Trehalose and anhydrobiosis in tardigrades FEBS Journal 275 (2008) 281–288 ª 2007 The Authors Journal compilation ª 2007 FEBS 283 However, the absolute trehalose levels detected in the Macrobiotidae are generally lower than those reported for A. coronifer (2.3% of dry weight) [43]. There is a clear correlation between the accumula- tion of trehalose and the induction of anhydrobiosis, indicating a possible protective role of the disaccharide during desiccation stress at least in the five species of the Macrobiotidae investigated in the present study. The apparent absence of trehalose in M. tardigrad- um, which was the only species of the order Apochela tested here, as well as in bdelloid rotifers [34], which all show desiccation tolerance indistinguishable from that of trehalose accumulating tardigrades, indicates that trehalose accumulation is not essential for desicca- tion tolerance in these animals. Milnesium tardigradum and possibly other Apochela species may have lost the ability to synthesize trehalose and may have replaced its function by some other mechanism that resulted in the unknown peak (2.233 min retention time). It is not uncommon for groups within a taxon to follow different adaptation strategies, as shown in roti- fers by Caprioli et al. [35], who detected only small amounts of trehalose in monogonont and none in bdelloid species. The absence of changes in the level of trehalose in the anhydrobiotic Heterotardigrades E. granulatus and E. testudo and the complete lack of trehalose in M. tardigradum strongly argues against a universal protective role in anhydrobiosis. Using an RNA interference technique, Ratnakunar and Tunnac- liffe [44] confirmed that there is no consistent relation- ship between trehalose accumulation and desiccation tolerance in Saccharomyces cerevisiae. Considering phylogenetic relationships, it would be interesting to investigate whether marine tardigrades accumulate tre- halose as in the Macrobiotidae tested here. Because the selective pressure for desiccation tolerance should be low in a marine habitat, the presence of trehalose in marine tardigrades would allow diagnosis of the direct effects of trehalose on desiccation tolerance in animals Fig. 2. High-performance liquid anion exchange chromatograms of soluble sugars extracted from 40 animals each of seven tardigrade species dried down do anhydrobiotic statge III. Species are: Echiniscus granulatus (EG), E. testudo (ET), Macrobiotus ‘richtersi group’ 1 (CL), M. ‘richtersi group’ 2 (N), M. spaiens (MS), M. richtersi (MR), M. tonollii (MT) and M. tardigradum (Mil). Sugars were eluted isocratically by 150 m M NaOH from an anion exchange column. The retention time for trehalose was 2.65 min. The y-axis shows relative detector units from pulsed chromatography. Table 3. Unidentified peaks of high-performance liquid anion exchange chromatography chromatograms of the state III (dry state), which show a distinct change compared to the state I (active state). Retention time (min) Species P value 2.233 Milnesium tardigradum 0.037* Macrobiotus ‘richtersi group’ 1 0.044* 3.100 Macrobiotus richtersi 0.026* 3.767 Macrobiotus ‘richtersi group’ 1 0.003** 8.300 Macrobiotus tonollii 0.043* 10.333 Macrobiotus richtersi 0.003** *P £ 0.05 (weakly significant); **P £ 0.01 (significant). Table 4. Trehalose concentration (ng) per embryo. Data are shown as the mean ± standard deviation. ND, not detected (beyond detec- tion limit of 0.05 l M). Species Hydrated Dehydrated Milnesium tardigradum ND ND Macrobiotus tonollii ND ND Macrobiotus richtersi 1.16 ± 0.0003 1.32 ± 0.002 Macrobiotus sapiens 1.10 ± 0.003 1.08 ± 0.001 Macrobiotus ‘richtersi group’ 1 1.92 ± 0,021 4.32 ± 0.002 Macrobiotus ‘richtersi group’ 2 1.25 ± 0.002 1.30 ± 0.002 Trehalose and anhydrobiosis in tardigrades S. Hengherr et al. 284 FEBS Journal 275 (2008) 281–288 ª 2007 The Authors Journal compilation ª 2007 FEBS that probably have not evolved alternative adaptation strategies for desiccation tolerance. However, to date, no studies have been conducted on the anhydrobiotic abilities of tardigrades from the tidal zone. Besides the animals themselves, eggs of the tardi- grades also show a good desiccation tolerance [45, unpublished data] and, in a first non-quantitative car- bohydrate analysis of eggs of the Macrobiotidae, we found that dry embryos also show low levels of tre- halose accumulation. However, embryos of M. tar- digradum and M. tonollii contained no trehalose. There appears to be no correlation between trehalose content and morphology, colour or type of oviposi- tion because there are no differences between species that deposit eggs in the exuvium (Milnesium) and free ovipositing species (M. tonolli). However, it is possible that eggs of M. tardigradum are more protected due to a slower dehydration inside the exuviae compared to eggs of the free ovipositing species. No additional studies concerning this issue are available. Although more quantitative studies on eggs of different species are required to prove a correlation between the accu- mulation of trehalose and desiccation tolerance in tar- digrade embryos, it appears that the ability to synthesise trehalose during desiccation is not just a feature of fully developed animals. However, relation- ships between the developmental stage and the ability to accumulate trehalose cannot be established based on the data available in the present study because of the unknown age of the eggs and developmental stages of the embryos used. In conclusion, our results support the hypothesis that non-reducing disaccha- rides such as trehalose may be involved in the desic- cation tolerance of anhydrobiotic invertebrates [4,12,32]. However, trehalose does not appear to be essential. The fact that some anhydrobiotic organisms do not require trehalose does not argue against an important function of this non-reducing disaccharide in organ- isms where it does occur. Trehalose may play an important primary role as an energy source, as demon- strated for yeast [46]. Additionally, in the organisms where it accumulates, trehalose could constitute a general stress defensive molecule and minimize in- activation of proteins by functioning as a ‘chemical chaperone’. In this case, it may not have an immediate effect on protein folding on its own, as is the case for other protein chaperones [47,48], but could act as a stabilizing agent. A small a-crystalline stress protein, p26, which was discovered in desiccation tolerant Art- emia cysts [49–52], was demonstrated to protect native protein conformation in vitro synergistically with treha- lose [53]. The disaccharide in turn acts synergistically with heat shock proteins during protein folding [49]. Because of the synergistic mode of action, neither the protein nor the sugar alone are sufficient for improved survival. This leads to the question as to what protective mechanisms are employed by anhydrobiotic organisms such as M. tardigradum or bdelloid rotifers, which do not accumulate trehalose. Multiple strategies may exist and complex mechanisms may be required where changes in protein composition may be of greater importance than changes in carbohydrate levels. We could not identify other carbohydrate compounds that specifically accumulated in non-trehalose tardigrade species during drying. Molecular chaperones may be particularly important in protecting proteins against stress-induced denaturation [54]. In nematodes, as well as in plants, the induction of late embryogenesis abun- dant proteins [55] has been associated with dehydra- tion tolerance [5,18,20,56]. Studies on anhydrobiotic tardigrades also indicate that these peptides might be involved in protective mechanisms (McGee B, Schill R & Tunnacliffe A, unpublished data). Besides the induc- tion of putative late embryogenesis abundant proteins, higher expression of stress genes of the hsp70 family have been detected in M. tardigradum undergoing an- hydrobiosis [57], indicating a function within the multi- ple strategies to withstand desiccation stress. Apart from the function of protein chaperones in assisting nascent and misfolded proteins to gain their correct conformation, the exact role of heat shock proteins in the protection of cells during dehydration has yet to be addressed. A continued search for additional mechanisms allow- ing organisms such as tardigrades to survive almost complete desiccation, as well as the further study of such adaptations, should lead to a better understanding of the remarkable phenomenon of anhydrobiosis. Experimental procedures Tardigrade culture Individuals of six eutardigrade species and two heterotardi- grade species were used to investigate changes of trehalose concentrations during anhydrobiosis. For the investigation, M. tardigradum Doye ` re 1849 (order Apochela), M. tonollii Ramazotti 1956, M. richtersi Murray 1911, Macrobio- tus sapiens Binda & Pilato, 1984, M. ‘richtersi group’ 1 and M. ‘richtersi group’ 2 (all order Parachela) were maintained in laboratory cultures. The cultures were scaled up for growth on agar plates (3%) covered with a thin layer of water. Tardigrades were fed bdelloid rotifers, Philodina citrina, which had been raised on the green algae, S. Hengherr et al. Trehalose and anhydrobiosis in tardigrades FEBS Journal 275 (2008) 281–288 ª 2007 The Authors Journal compilation ª 2007 FEBS 285 Chlorogonium elongatum. The colourless eggs of the Macorbiotus species are laid free and show the characteris- tic truncate conical shape with projections. Animals of Milnesium deposit the colourless or rosy, oval and smooth eggs in exuvium. Because it was not possible to maintain hatcheries of the heterotardigrades E. granulatus (Doye ` re 1840) and E. testudo (Doye ` re 1840), the animals were manually isolated directly from mosses using a pipette and a stereomicroscope. Tardigrade sampling Tardigrades were starved over 2 days before harvesting to avoid contamination with food-organisms. After repeated washing with clean water, animals were transferred into microliter tubes (40 individuals per tube). Fertilized eggs of the cultured species were isolated 4 days after fertilization (only known for M. tardigradum) cleaned with water and subsequently transferred into microliter tubes (60 eggs per tube). The age of fertilized eggs of the other species was not known. The remaining fluid in the tubes was reduced to approximately 1–2 lL. Stages of anhydrobiosis Five different states according to Schill et al. [57] were investigated. Microliter tubes containing animals repre- senting the active state (I) were frozen directly in liquid nitrogen. To produce different anhydrobiotic states, the animals were dried in open microliter tubes at room tem- perature, exposed to 85% relative humidity (RH) in a chamber containing a saturated solution of KCL (Roth, Karlsruhe, Germany). State II was defined as the state at which the legs were drawn in but the body still per- formed distinct movements. Animals in the anhydrobiotic state (III) were sampled after completed tun formation and further drying at 35% RH over 48 h in a chamber containing a saturated solution of MgCl 2 (Roth), The remaining animals were rehydrated. Animals were sam- pled in this transitional state IV, that was defined as the state at which the legs protruded and the body again per- formed movements. This state was achieved after approxi- mately 15–20 min. State V was an active one in which the animals moved around 4 h after state IV. This method of producing distinct anhydrobiotic states has been tested for survival and no animals died during the procedure. For all investigated states, eight batches containing 40 animals each were used for each species (n = 8). Eggs of the hydrated state were also frozen directly in liquid nitrogen. Eggs of the dehydrated state were treated like animals of stage III. Due to the small size of the objects for investigating the embryos, we could use only two batches for each hydrated and dehydrated state per species (n = 2). Carbohydrate analysis Samples were homogenised with an ultrasonicator (SONO- PULS, HD3100; Bandelin Electronic, Berlin, Germany) in 100 lL of distilled water. After incubating at 95 °C for 60 min the homogenate was centrifuged (20 000 g at 4 °C for 15 min) and the supernatant was used for carbohydrate analysis. Water-soluble carbohydrates were determined by high-performance liquid anion exchange chromatography (HPAEC) using a CarboPac PA-1 column on a Dionex DX-500 gradient chromatography system coupled with pulsed amperometric detection by a gold electrode (Dionex, Sunnyvale, CA, USA). The detector settings for the deter- minations were: T 1 = 0.4 s, T 2 = 0.02 s, T 3 = 0.01 s, T 4 = 0.07 s, E 1 = 0.1 V, E 2 = )2.0 V, E 3 = 0.6 V, E 4 = )0.1 V, sensitivity range = 0.1 lC and integration range = 0.2–0.4 s. Eluents were A = 0.15 m NaOH, B = 0.85 m sodium acetate in 0.15 m NaOH. Following 10 min of isocratic elution with A, a linear gradient to 100% B within 2 min and an isocratic step with B for 2 min was used for column cleaning, before the column was again equilibrated with A for 5 min. Trehalose eluted at a retention time of 2.6 min. Pure commercially available trehalose (Sigma-Aldrich Chemie GmBH, Munich, Ger- many) in the concentration range 1–100 lm was used for calibration. Protein quantification Samples were homogenised in two batches of 40 animals and 60 eggs for each active and cryptobiotic state by ultra- sonication (SONOPULS, HD3100, Bandelin Electronic) in 30 lL of a buffered extraction solution [80 mm KCH 3 CO 2 , 5mm Mg(CH 3 CO 2 ) 2 ,20mm Hepes, 2% protease inhibitor cocktail (Sigma-Aldrich Chemie GmBH), pH 7.4]. The homogenate was subsequently centrifuged (20 000 g at 4 °C for 5 min). The total protein concentration in each superna- tant was determined by a protein quantification mini-assay based on the method of Bradford using Coomassie Protein Assay Reagent (Pierce, IL, USA). Dry weight determination For each tardigrade species, four samples of 20 individuals each were dried (35% RH, 20 °C) over 4 days and weighed on a Supermicro S4 2405 (Sartorius, Go ¨ ttingen, Germany). Statistical analysis The statistical significance of differences in the trehalose and protein level between the samples was tested using one-way analysis of variance and the Bonferroni t-test. Significance levels were P £ 0.001 (highly significant), Trehalose and anhydrobiosis in tardigrades S. Hengherr et al. 286 FEBS Journal 275 (2008) 281–288 ª 2007 The Authors Journal compilation ª 2007 FEBS 0.001 < P £ 0.01 (significant) and 0.01 < P £ 0.05 (weakly significant). Acknowledgements Many thanks to Roger Worland and James S. Clegg for critical discussion and manuscript pre-review. This study was supported by the German Research Foundation (DFG), SCHI865 ⁄ 1-1, and enabled using the equipments made available by the project FUNCRYPTA (0313838A), funded by the German Federal Ministry of Education and Research, BMBF. References 1 Keilin D (1959) The problem of anabiosis or latend life: history and current concept. Proc R Soc Lond B 150, 149–191. 2 Crowe JH & Clegg JS (eds.) 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