Comparative proteome analysis of Milnesium tardigradum in early embryonic state versus adults in active and anhydrobiotic state.

Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state.

Integrated pathway modules using time-course metabolic profiles and EST data from Milnesium tardigradum.

BACKGROUND: Tardigrades are multicellular organisms, resistant to extreme environmental changes such as heat, drought, radiation and freezing. They outlast these conditions in an inactive form (tun) to escape damage to cellular structures and cell death. Tardigrades are apparently able to prevent or repair such damage and are therefore a crucial model organism for stress tolerance. Cultures of the tardigrade Milnesium tardigradum were dehydrated by removing the surrounding water to induce tun formation. During this process and the subsequent rehydration, metabolites were measured in a time series by GC-MS. Additionally expressed sequence tags are available, especially libraries generated from the active and inactive state. The aim of this integrated analysis is to trace changes in tardigrade metabolism and identify pathways responsible for their extreme resistance against physical stress. RESULTS: In this study we propose a novel integrative approach for the analysis of metabolic networks to identify modules of joint shifts on the transcriptomic and metabolic levels. We derive a tardigrade-specific metabolic network represented as an undirected graph with 3,658 nodes (metabolites) and 4,378 edges (reactions). Time course metabolite profiles are used to score the network nodes showing a significant change over time. The edges are scored according to information on enzymes from the EST data. Using this combined information, we identify a key subnetwork (functional module) of concerted changes in metabolic pathways, specific for de- and rehydration. The module is enriched in reactions showing significant changes in metabolite levels and enzyme abundance during the transition. It resembles the cessation of a measurable metabolism (e.g. glycolysis and amino acid anabolism) during the tun formation, the production of storage metabolites and bioprotectants, such as DNA stabilizers, and the generation of amino acids and cellular components from monosaccharides as carbon and energy source during rehydration. CONCLUSIONS: The functional module identifies relationships among changed metabolites (e.g. spermidine) and reactions and provides first insights into important altered metabolic pathways. With sparse and diverse data available, the presented integrated metabolite network approach is suitable to integrate all existing data and analyse it in a combined manner.

Mechanisms associated with cellular desiccation tolerance of Artemia encysted embryos from locations around the world.

Using differential scanning calorimetry we demonstrated the presence of biological glasses and measured the glass transition temperatures (Tg) in dry encysted gastrula embryos (cysts) of the brine shrimp, Artemia, from eleven different locations, two of which provided cysts from parthenogenetic animals. Values for Tg were highest, by far, in Artemia franciscana cysts from the Mekong Delta, Vietnam (VN), these cysts having been produced from previous sequential inoculations into growth ponds of cysts from the San Francisco Bay, California, USA. Tg values for three groups of A. franciscana cysts were significantly higher than those of other cysts (except those of Artemia persimilis) studied here, as well as all other desiccation-tolerant animal systems studied to date. We also measured three stress proteins (hsc70, artemin and p26) in all these cysts as well as the total alcohol soluble carbohydrates (ASC), about 90% of which is the disaccharide trehalose, a known component of biological glasses. We interpret the results in terms of mechanisms involved with desiccation tolerance and, to some extent, with thermal conditions at the sites of cyst collection.

Mechanisms associated with cellular desiccation tolerance in the animal extremophile artemia.

Using differential scanning calorimetry, we demonstrated the presence of biological glasses and measured the transition temperatures in dry encysted embryos (cysts) of the brine shrimp, Artemia franciscana. Cysts from the following three geographic locations were studied: San Francisco Bay (SFB); the Great Salt Lake, Utah (GSL); and the Mekong Delta, Vietnam (VN; these cysts were produced from previous sequential inoculations of SFB cysts into growth ponds). Values for the glass transition temperature, T(g), were highest in VN cysts. This study indicates that the composition and properties of these biological glasses can be altered by natural selection and thermal adaptation. To our knowledge, T(g) values for all three kinds of cysts were significantly higher than those for any other desiccation-tolerant animal system. To gain insight into the significance of T(g), we examined the thermal stability of these dry cysts at 80 °C. GSL cysts were the least tolerant, by far, with VN cysts being extremely tolerant and SFB cysts not far behind. Those results correlated with the thermal transition values. Also measured were alcohol-soluble carbohydrates, ~90% of which is the disaccharide trehalose, a known component of biological glasses. Amounts in the GSL cysts were significantly less than those in the other two kinds of cysts. Several stress proteins were measured in the three groups of cysts, with all of them being in lesser amounts in GSL cysts compared with the SFB and VN cysts. We interpret the data in terms of mechanisms involved with desiccation tolerance and thermal conditions at the sites of cyst collection.

Dormant stages in freshwater bryozoans--an adaptation to transcend environmental constraints.

Freshwater invertebrates often disperse between discrete habitat patches via the production of dormant propagules. Being dispersed passively by animal vectors or wind, certain adaptations for exposures to terrestrial and aerial conditions like desiccation and freezing are required. In the present study, we investigate the mechanisms of survival and physiological adaptations due to desiccation and low temperatures in the statoblasts of two populations of the freshwater bryozoan Cristatella mucedo. Our results show that both sessoblasts and floatoblasts tolerate almost complete desiccation and subzero temperatures. Trehalose, a non-reducing disaccharide which has been related to desiccation tolerance, was detected by amperometric chromatography. However, due to the low concentrations found, it is unlikely that trehalose is playing a major part in desiccation tolerance of bryozoan statoblasts. Vitrification is assumed to be important in the survival of desiccation tolerant organisms. Differential scanning calorimetry revealed thermal transitions (T(g) onset around 70°C) in desiccated statoblasts, indicating that a vitreous matrix is present. During the exposure to subzero temperatures, freeze tolerance of statoblasts was confirmed by the detection of internal ice formation, which took place at a crystallisation temperature of between -6°C and -12°C.

Proteomic analysis of tardigrades: towards a better understanding of molecular mechanisms by anhydrobiotic organisms.

BACKGROUND: Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered. PRINCIPAL FINDINGS: Here we present to the best of our knowledge the first comprehensive study of Milnesium tardigradum on the protein level. To establish a proteome reference map we developed optimized protocols for protein extraction from tardigrades in the active state and for separation of proteins by high resolution two-dimensional gel electrophoresis. Since only limited sequence information of M. tardigradum on the genome and gene expression level is available to date in public databases we initiated in parallel a tardigrade EST sequencing project to allow for protein identification by electrospray ionization tandem mass spectrometry. 271 out of 606 analyzed protein spots could be identified by searching against the publicly available NCBInr database as well as our newly established tardigrade protein database corresponding to 144 unique proteins. Another 150 spots could be identified in the tardigrade clustered EST database corresponding to 36 unique contigs and ESTs. Proteins with annotated function were further categorized in more detail by their molecular function, biological process and cellular component. For the proteins of unknown function more information could be obtained by performing a protein domain annotation analysis. Our results include proteins like protein member of different heat shock protein families and LEA group 3, which might play important roles in surviving extreme conditions. CONCLUSIONS: The proteome reference map of Milnesium tardigradum provides the basis for further studies in order to identify and characterize the biochemical mechanisms of tolerance to extreme desiccation. The optimized proteomics workflow will enable application of sensitive quantification techniques to detect differences in protein expression, which are characteristic of the active and anhydrobiotic states of tardigrades.

Stress response in tardigrades: differential gene expression of molecular chaperones.

Semi-terrestrial tardigrades exhibit a remarkable tolerance to desiccation by entering a state called anhydrobiosis. In this state, they show a strong resistance against several kinds of physical extremes. Because of the probable importance of stress proteins during the phases of dehydration and rehydration, the relative abundance of transcripts coding for two alpha-crystallin heat-shock proteins (Mt-sHsp17.2 and Mt-sHsp19.5), as well for the heat-shock proteins Mt-sHsp10, Mt-Hsp60, Mt-Hsp70 and Mt-Hsp90, were analysed in active and anhydrobiotic tardigrades of the species Milnesium tardigradum. They were also analysed in the transitional stage (I) of dehydration, the transitional stage (II) of rehydration and in heat-shocked specimens. A variable pattern of expression was detected, with most candidates being downregulated. Gene transcripts of one Mt-hsp70 isoform in the transitional stage I and Mt-hsp90 in the anhydrobiotic stage were significantly upregulated. A high gene expression (778.6-fold) was found for the small alpha-crystallin heat-shock protein gene Mt-sHsp17.2 after heat shock. We discuss the limited role of the stress-gene expression in the transitional stages between the active and anhydrobiotic tardigrades and other mechanisms which allow tardigrades to survive desiccation.

Trehalose and anhydrobiosis in tardigrades--evidence for divergence in responses to dehydration.

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 trehalose levels of eight tardigrade species comprising Heterotardigrada and Eutardigrada were observed in five different states of hydration and dehydration. Although many species accumulate trehalose during dehydration, the data revealed significant differences between the species. Although trehalose accumulation was found in species of the order Parachela (Eutardigrada), 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 developing tardigrade embryos, which have been shown to have a high level of desiccation tolerance.