Sea Cucumber-Derived Peptides Alleviate Oxidative Stress in Neuroblastoma Cells and Improve Survival in C. elegans Exposed to Neurotoxic Paraquat.

Oxidative stress results when the production of oxidants outweighs the capacity of the antioxidant defence mechanisms. This can lead to pathological conditions including cancer and neurodegeneration. Consequently, there is considerable interest in compounds with antioxidant activity, including those from natural sources. Here, we characterise the antioxidant activity of three novel peptides identified in protein hydrolysates from the sea cucumber Apostichopus japonicus. Under oxidative stress conditions, synthetic versions of the sea cucumber peptides significantly compensate for glutathione depletion, decrease mitochondrial superoxide levels, and alleviate mitophagy in human neuroblastoma cells. Moreover, orally supplied peptides improve survival of the Caenorhabditis elegans after treatment with paraquat, the latter of which leads to the production of excessive oxidative stress. Thus, the sea cucumber peptides exhibit antioxidant activity at both the cellular and organism levels and might prove attractive as nutritional supplements for healthy ageing.

The structure and global distribution of the endoplasmic reticulum network are actively regulated by lysosomes.

The endoplasmic reticulum (ER) comprises morphologically and functionally distinct domains: sheets and interconnected tubules. These domains undergo dynamic reshaping in response to changes in the cellular environment. However, the mechanisms behind this rapid remodeling are largely unknown. Here, we report that ER remodeling is actively driven by lysosomes, following lysosome repositioning in response to changes in nutritional status: The anchorage of lysosomes to ER growth tips is critical for ER tubule elongation and connection. We validate this causal link via the chemo- and optogenetically driven repositioning of lysosomes, which leads to both a redistribution of the ER tubules and a change of its global morphology. Therefore, lysosomes sense metabolic change in the cell and regulate ER tubule distribution accordingly. Dysfunction in this mechanism during axonal extension may lead to axonal growth defects. Our results demonstrate a critical role of lysosome-regulated ER dynamics and reshaping in nutrient responses and neuronal development.

Preferential adsorption to air-water interfaces: a novel cryoprotective mechanism for LEA proteins.

Late embryogenesis abundant (LEA) proteins comprise a diverse family whose members play a key role in abiotic stress tolerance. As intrinsically disordered proteins, LEA proteins are highly hydrophilic and inherently stress tolerant. They have been shown to stabilise multiple client proteins under a variety of stresses, but current hypotheses do not fully explain how such broad range stabilisation is achieved. Here, using neutron reflection and surface tension experiments, we examine in detail the mechanism by which model LEA proteins, AavLEA1 and ERD10, protect the enzyme citrate synthase (CS) from aggregation during freeze-thaw. We find that a major contributing factor to CS aggregation is the formation of air bubbles during the freeze-thaw process. This greatly increases the air-water interfacial area, which is known to be detrimental to folded protein stability. Both model LEA proteins preferentially adsorb to this interface and compete with CS, thereby reducing surface-induced aggregation. This novel surface activity provides a general mechanism by which diverse members of the LEA protein family might function to provide aggregation protection that is not specific to the client protein.

Structural progression of amyloid-ß Arctic mutant aggregation in cells revealed by multiparametric imaging.

The 42-amino-acid ß-amyloid (Aß42) is a critical causative agent in the pathology of Alzheimer's disease. The hereditary Arctic mutation of Aß42 (E22G) leads to increased intracellular accumulation of ß-amyloid in early-onset Alzheimer's disease. However, it remains largely unknown how the Arctic mutant variant leads to aggressive protein aggregation and increased intracellular toxicity. Here, we constructed stable cell lines expressing fluorescent-tagged wildtype (WT) and E22G Aß42 to study the aggregation kinetics of the Arctic Aß42 mutant peptide and its heterogeneous structural forms. Arctic-mutant peptides assemble and form fibrils at a much faster rate than WT peptides. We identified five categories of intracellular aggregate-oligomers, single fibrils, fibril bundles, clusters, and aggresomes-that underline the heterogeneity of these Aß42 aggregates and represent the progression of Aß42 aggregation within the cell. Fluorescence-lifetime imaging (FLIM) and 3D structural illumination microscopy (SIM) showed that all aggregate species displayed highly compact structures with strong affinity between individual fibrils. We also found that aggregates formed by Arctic mutant Aß42 were more resistant to intracellular degradation than their WT counterparts. Our findings uncover the structural basis of the progression of Arctic mutant Aß42 aggregation in the cell.

Live-cell super-resolution microscopy reveals a primary role for diffusion in polyglutamine-driven aggresome assembly.

The mechanisms leading to self-assembly of misfolded proteins into amyloid aggregates have been studied extensively in the test tube under well-controlled conditions. However, to what extent these processes are representative of those in the cellular environment remains unclear. Using super-resolution imaging of live cells, we show here that an amyloidogenic polyglutamine-containing protein first forms small, amorphous aggregate clusters in the cytosol, chiefly by diffusion. Dynamic interactions among these clusters limited their elongation and led to structures with a branched morphology, differing from the predominantly linear fibrils observed in vitro Some of these clusters then assembled via active transport at the microtubule-organizing center and thereby initiated the formation of perinuclear aggresomes. Although it is widely believed that aggresome formation is entirely governed by active transport along microtubules, here we demonstrate, using a combined approach of advanced imaging and mathematical modeling, that diffusion is the principal mechanism driving aggresome expansion. We found that the increasing surface area of the expanding aggresome increases the rate of accretion caused by diffusion of cytosolic aggregates and that this pathway soon dominates aggresome assembly. Our findings lead to a different view of aggresome formation than that proposed previously. We also show that aggresomes mature over time, becoming more compacted as the structure grows. The presence of large perinuclear aggregates profoundly affects the behavior and health of the cell, and our super-resolution imaging results indicate that aggresome formation and development are governed by highly dynamic processes that could be important for the design of potential therapeutic strategies.

Comparative genomics of bdelloid rotifers: Insights from desiccating and nondesiccating species.

Bdelloid rotifers are a class of microscopic invertebrates that have existed for millions of years apparently without sex or meiosis. They inhabit a variety of temporary and permanent freshwater habitats globally, and many species are remarkably tolerant of desiccation. Bdelloids offer an opportunity to better understand the evolution of sex and recombination, but previous work has emphasised desiccation as the cause of several unusual genomic features in this group. Here, we present high-quality whole-genome sequences of 3 bdelloid species: Rotaria macrura and R. magnacalcarata, which are both desiccation intolerant, and Adineta ricciae, which is desiccation tolerant. In combination with the published assembly of A. vaga, which is also desiccation tolerant, we apply a comparative genomics approach to evaluate the potential effects of desiccation tolerance and asexuality on genome evolution in bdelloids. We find that ancestral tetraploidy is conserved among all 4 bdelloid species, but homologous divergence in obligately aquatic Rotaria genomes is unexpectedly low. This finding is contrary to current models regarding the role of desiccation in shaping bdelloid genomes. In addition, we find that homologous regions in A. ricciae are largely collinear and do not form palindromic repeats as observed in the published A. vaga assembly. Consequently, several features interpreted as genomic evidence for long-term ameiotic evolution are not general to all bdelloid species, even within the same genus. Finally, we substantiate previous findings of high levels of horizontally transferred nonmetazoan genes in both desiccating and nondesiccating bdelloid species and show that this unusual feature is not shared by other animal phyla, even those with desiccation-tolerant representatives. These comparisons call into question the proposed role of desiccation in mediating horizontal genetic transfer.

Multiple genes contribute to anhydrobiosis (tolerance to extreme desiccation) in the nematode Panagrolaimus superbus

Abstract The molecular basis of anhydrobiosis, the state of suspended animation entered by some species during extreme desiccation, is still poorly understood despite a number of transcriptome and proteome studies. We therefore conducted functional screening by RNA interference (RNAi) for genes involved in anhydrobiosis in the holo-anhydrobiotic nematode Panagrolaimus superbus. A new method of survival analysis, based on staining, and proof-of-principle RNAi experiments confirmed a role for genes involved in oxidative stress tolerance, while a novel medium-scale RNAi workflow identified a further 40 anhydrobiosis-associated genes, including several involved in proteostasis, DNA repair and signal transduction pathways. This suggests that multiple genes contribute to anhydrobiosis in P. superbus.

Multiple genes contribute to anhydrobiosis (tolerance to extreme desiccation) in the nematode Panagrolaimus superbus.

The molecular basis of anhydrobiosis, the state of suspended animation entered by some species during extreme desiccation, is still poorly understood despite a number of transcriptome and proteome studies. We therefore conducted functional screening by RNA interference (RNAi) for genes involved in anhydrobiosis in the holo-anhydrobiotic nematode Panagrolaimus superbus. A new method of survival analysis, based on staining, and proof-of-principle RNAi experiments confirmed a role for genes involved in oxidative stress tolerance, while a novel medium-scale RNAi workflow identified a further 40 anhydrobiosis-associated genes, including several involved in proteostasis, DNA repair and signal transduction pathways. This suggests that multiple genes contribute to anhydrobiosis in P. superbus.

A functional difference between native and horizontally acquired genes in bdelloid rotifers.

The form of RNA processing known as SL trans-splicing involves the transfer of a short conserved sequence, the spliced leader (SL), from a noncoding SL RNA to the 5' ends of mRNA molecules. SL trans-splicing occurs in several animal taxa, including bdelloid rotifers (Rotifera, Bdelloidea). One striking feature of these aquatic microinvertebrates is the large proportion of foreign genes, i.e. those acquired by horizontal gene transfer from other organisms, in their genomes. However, whether such foreign genes behave similarly to native genes has not been tested in bdelloids or any other animal. We therefore used a combination of experimental and computational methods to examine whether transcripts of foreign genes in bdelloids were SL trans-spliced, like their native counterparts. We found that many foreign transcripts contain SLs, use similar splice acceptor sequences to native genes, and are able to undergo alternative trans-splicing. However, a significantly lower proportion of foreign mRNAs contains SL sequences than native transcripts. This demonstrates a novel functional difference between foreign and native genes in bdelloids and suggests that SL trans-splicing is not essential for the expression of foreign genes, but is acquired during their domestication.

Horizontal gene transfer in bdelloid rotifers is ancient, ongoing and more frequent in species from desiccating habitats.

BACKGROUND: Although prevalent in prokaryotes, horizontal gene transfer (HGT) is rarer in multicellular eukaryotes. Bdelloid rotifers are microscopic animals that contain a higher proportion of horizontally transferred, non-metazoan genes in their genomes than typical of animals. It has been hypothesized that bdelloids incorporate foreign DNA when they repair their chromosomes following double-strand breaks caused by desiccation. HGT might thereby contribute to species divergence and adaptation, as in prokaryotes. If so, we expect that species should differ in their complement of foreign genes, rather than sharing the same set of foreign genes inherited from a common ancestor. Furthermore, there should be more foreign genes in species that desiccate more frequently. We tested these hypotheses by surveying HGT in four congeneric species of bdelloids from different habitats: two from permanent aquatic habitats and two from temporary aquatic habitats that desiccate regularly. RESULTS: Transcriptomes of all four species contain many genes with a closer match to non-metazoan genes than to metazoan genes. Whole genome sequencing of one species confirmed the presence of these foreign genes in the genome. Nearly half of foreign genes are shared between all four species and an outgroup from another family, but many hundreds are unique to particular species, which indicates that HGT is ongoing. Using a dated phylogeny, we estimate an average of 12.8 gains versus 2.0 losses of foreign genes per million years. Consistent with the desiccation hypothesis, the level of HGT is higher in the species that experience regular desiccation events than those that do not. However, HGT still contributed hundreds of foreign genes to the species from permanently aquatic habitats. Foreign genes were mainly enzymes with various annotated functions that include catabolism of complex polysaccharides and stress responses. We found evidence of differential loss of ancestral foreign genes previously associated with desiccation protection in the two non-desiccating species. CONCLUSIONS: Nearly half of foreign genes were acquired before the divergence of bdelloid families over 60 Mya. Nonetheless, HGT is ongoing in bdelloids and has contributed to putative functional differences among species. Variation among our study species is consistent with the hypothesis that desiccating habitats promote HGT.

Determination of Water Content in Dehydrated Mammalian Cells Using Terahertz Pulsed Imaging: A Feasibility Study.

Mammalian cells are involved in a range of biotechnological applications and more recently have been increasingly exploited in regenerative medicine. Critical to successful applications involving mammalian cells are their long-term storage and transport, for which cryopreservation in liquid nitrogen is the most frequently used strategy. However, cryopreservation suffers from high costs, difficulties in transport logistics and the use of undesirable additives (e.g. animal sera or DMSO). An alternative approach, proposed as low cost, low maintenance and process-compatible, is viable desiccation of mammalian cells. Several groups claim to have achieved this, but the extent of desiccation in the cell samples concerned is not always clear, in part because of difficulties in determining very low water content. Although several techniques exist that are frequently used to quantify the amount of water in samples (e.g. FTIR spectroscopy, thermogravimetric analysis (TGA), NMR spectroscopy), the complexity of sample preparation, as well as the costs and time constraints involved are disadvantageous. Here, we assess a novel, rapid and low cost technique, i.e. terahertz (THz) spectroscopy, for the quantification of water content within dehydrated mammalian cell samples.

Long Term Aggresome Accumulation Leads to DNA Damage, p53-dependent Cell Cycle Arrest, and Steric Interference in Mitosis.

Juxtanuclear aggresomes form in cells when levels of aggregation-prone proteins exceed the capacity of the proteasome to degrade them. It is widely believed that aggresomes have a protective function, sequestering potentially damaging aggregates until these can be removed by autophagy. However, most in-cell studies have been carried out over a few days at most, and there is little information on the long term effects of aggresomes. To examine these long term effects, we created inducible, single-copy cell lines that expressed aggregation-prone polyglutamine proteins over several months. We present evidence that, as perinuclear aggresomes accumulate, they are associated with abnormal nuclear morphology and DNA double-strand breaks, resulting in cell cycle arrest via the phosphorylated p53 (Ser-15)-dependent pathway. Further analysis reveals that aggresomes can have a detrimental effect on mitosis by steric interference with chromosome alignment, centrosome positioning, and spindle formation. The incidence of apoptosis also increased in aggresome-containing cells. These severe defects developed gradually after juxtanuclear aggresome formation and were not associated with small cytoplasmic aggregates alone. Thus, our findings demonstrate that, in dividing cells, aggresomes are detrimental over the long term, rather than protective. This suggests a novel mechanism for polyglutamine-associated developmental and cell biological abnormalities, particularly those with early onset and non-neuronal pathologies.

A plant cell model of polyglutamine aggregation: Identification and characterisation of macromolecular and small-molecule anti-protein aggregation activity in vivo.

In vitro studies have shown that LEA proteins from plants and invertebrates protect and stabilise other proteins under conditions of water stress, suggesting a role in stress tolerance. However, there is little information on LEA protein function in whole plants or plant cells, particularly with respect to their anti-aggregation activity. To address this, we expressed in tobacco BY-2 suspension cells an aggregation-prone protein based on that responsible for Huntington's disease (HD). In HD, abnormally long stretches of polyglutamine (polyQ) in huntingtin (Htt) protein cause aggregation of Htt fragments within cells. We constructed stably transformed BY-2 cell lines expressing enhanced green fluorescent protein (EGFP)-HttQ23 or EGFP-HttQ52 fusion proteins (encoding 23 or 52 glutamine residues, pertaining to the normal and disease states, respectively), as well as an EGFP control. EGFP-HttQ52 protein aggregated in the cytoplasm of transformed tobacco cells, which showed slow growth kinetics; in contrast, EGFP-HttQ23 or EGFP did not form aggregates and cells expressing these constructs grew normally. To test the effect of LEA proteins on protein aggregation in plant cells, we constructed cell lines expressing both EGFP-HttQ52 and LEA proteins (PM1, PM18, ZLDE-2 or AavLEA1) or a sHSP (PM31). Of these, AavLEA1 and PM31 reduced intracellular EGFP-HttQ52 aggregation and alleviated the associated growth inhibition, while PM18 and ZLDE-2 partially restored growth rates. Treatment of EGFP-HttQ52-expressing BY2 cells with the polyphenol epigallocatechin-3-gallate (EGCG) also reduced EGFP-HttQ52 aggregation and improved cell growth rate. The EGFP-HttQ52 cell line therefore has potential for characterising both macromolecular and small molecule inhibitors of protein aggregation in plant cells.

Expression-level dependent perturbation of cell proteostasis and nuclear morphology by aggregation-prone polyglutamine proteins.

We describe a gene expression system for use in mammalian cells that yields reproducible, inducible gene expression that can be modulated within the physiological range. A synthetic promoter library was generated from which representatives were selected that gave weak, intermediate-strength or strong promoter activity. Each promoter resulted in a tight expression range when used to drive single-copy reporter genes integrated at the same genome location in stable cell lines, in contrast to the broad range of expression typical of transiently transfected cells. To test this new expression system in neurodegenerative disease models, we used each promoter type to generate cell lines carrying single-copy genes encoding polyglutamine-containing proteins. Expression over a period of up to three months resulted in a proportion of cells developing juxtanuclear aggresomes whose rate of formation, penetrance, and morphology were expression-level dependent. At the highest expression levels, fibrillar aggregates deposit close to the nuclear envelope, indicating that cell proteostasis is overwhelmed by misfolded protein species. We also observed expression-level dependent, abnormal nuclear morphology in cells containing aggresomes, with up to ∼80% of cells affected. This system constitutes a valuable tool in gene regulation at different levels and allows the quantitative assessment of gene expression effects when developing disease models or investigating cell function through the introduction of gene constructs.

Expression of multiple horizontally acquired genes is a hallmark of both vertebrate and invertebrate genomes.

BACKGROUND: A fundamental concept in biology is that heritable material, DNA, is passed from parent to offspring, a process called vertical gene transfer. An alternative mechanism of gene acquisition is through horizontal gene transfer (HGT), which involves movement of genetic material between different species. HGT is well-known in single-celled organisms such as bacteria, but its existence in higher organisms, including animals, is less well established, and is controversial in humans. RESULTS: We have taken advantage of the recent availability of a sufficient number of high-quality genomes and associated transcriptomes to carry out a detailed examination of HGT in 26 animal species (10 primates, 12 flies and four nematodes) and a simplified analysis in a further 14 vertebrates. Genome-wide comparative and phylogenetic analyses show that HGT in animals typically gives rise to tens or hundreds of active 'foreign' genes, largely concerned with metabolism. Our analyses suggest that while fruit flies and nematodes have continued to acquire foreign genes throughout their evolution, humans and other primates have gained relatively few since their common ancestor. We also resolve the controversy surrounding previous evidence of HGT in humans and provide at least 33 new examples of horizontally acquired genes. CONCLUSIONS: We argue that HGT has occurred, and continues to occur, on a previously unsuspected scale in metazoans and is likely to have contributed to biochemical diversification during animal evolution.

Effects of secondary metabolite extract from Phomopsis occulta on ß-amyloid aggregation.

Inhibition of ß-amyloid (Aß) aggregation is an attractive therapeutic and preventive strategy for the discovery of disease-modifying agents in Alzheimer's disease (AD). Phomopsis occulta is a new, salt-tolerant fungus isolated from mangrove Pongamia pinnata (L.) Pierre. We report here the inhibitory effects of secondary metabolites from Ph. occulta on the aggregation of Aß42. It was found that mycelia extracts (MEs) from Ph. occulta cultured with 0, 2, and 3 M NaCl exhibited inhibitory activity in an E. coli model of Aß aggregation. A water-soluble fraction, ME0-W-F1, composed of mainly small peptides, was able to reduce aggregation of an Aß42-EGFP fusion protein and an early onset familial mutation Aß42E22G-mCherry fusion protein in transfected HEK293 cells. ME0-W-F1 also antagonized the cytotoxicity of Aß42 in the neural cell line SH-SY5Y in dose-dependent manner. Moreover, SDS-PAGE and FT-IR analysis confirmed an inhibitory effect of ME0-W-F1 on the aggregation of Aß42 in vitro. ME0-W-F1 blocked the conformational transition of Aß42 from α-helix/random coil to ß-sheet, and thereby inhibited formation of Aß42 tetramers and high molecular weight oligomers. ME0-W-F1 and other water-soluble secondary metabolites from Ph. occulta therefore represent new candidate natural products against aggregation of Aß42, and illustrate the potential of salt tolerant fungi from mangrove as resources for the treatment of AD and other diseases.

Extracellular monomeric tau protein is sufficient to initiate the spread of tau protein pathology.

Understanding the formation and propagation of aggregates of the Alzheimer disease-associated Tau protein in vivo is vital for the development of therapeutics for this devastating disorder. Using our recently developed live-cell aggregation sensor in neuron-like cells, we demonstrate that different variants of exogenous monomeric Tau, namely full-length Tau (hTau40) and the Tau-derived construct K18 comprising the repeat domain, initially accumulate in endosomal compartments, where they form fibrillar seeds that subsequently induce the aggregation of endogenous Tau. Using superresolution imaging, we confirm that fibrils consisting of endogenous and exogenous Tau are released from cells and demonstrate their potential to spread Tau pathology. Our data indicate a greater pathological risk and potential toxicity than hitherto suspected for extracellular soluble Tau.

Critique of the use of fluorescence-based reporters in Escherichia coli as a screening tool for the identification of peptide inhibitors of Aß42 aggregation.

High-throughput screens that dispense with the need for expensive synthetic Aß peptide would be invaluable for identifying novel anti-aggregants as potential treatments for Alzheimer's disease. A biosynthetic in vivo approach, using a recombinant fluorescent green fluorescent protein (GFP) reporter for the aggregation state of Aß in Escherichia coli, has been reported by other workers. Here, inducible Aß-GFP expression in E. coli was coupled to the concurrent constitutive production of a quasi-random peptide library to screen for anti-aggregant activity. To attempt to introduce greater robustness, mCherry was also co-expressed as an internal fluorescence standard to allow ratiometric comparison between samples. However, fluctuations in mCherry expression levels, as well as a low dynamic range of GFP output between positive and negative anti-aggregant peptides, highlighted limitations with the approach. Despite this, two novel peptides were identified that showed an equivalent in vitro anti-aggregant activity to that of epigallocatechin-3-gallate. Thus, although biosynthetic in vivo strategies show promise as screens for novel activities, unforeseen problems can arise because of the variability inherent in any biological system.

Biochemical diversification through foreign gene expression in bdelloid rotifers.

Bdelloid rotifers are microinvertebrates with unique characteristics: they have survived tens of millions of years without sexual reproduction; they withstand extreme desiccation by undergoing anhydrobiosis; and they tolerate very high levels of ionizing radiation. Recent evidence suggests that subtelomeric regions of the bdelloid genome contain sequences originating from other organisms by horizontal gene transfer (HGT), of which some are known to be transcribed. However, the extent to which foreign gene expression plays a role in bdelloid physiology is unknown. We address this in the first large scale analysis of the transcriptome of the bdelloid Adineta ricciae: cDNA libraries from hydrated and desiccated bdelloids were subjected to massively parallel sequencing and assembled transcripts compared against the UniProtKB database by blastx to identify their putative products. Of ~29,000 matched transcripts, ~10% were inferred from blastx matches to be horizontally acquired, mainly from eubacteria but also from fungi, protists, and algae. After allowing for possible sources of error, the rate of HGT is at least 8%-9%, a level significantly higher than other invertebrates. We verified their foreign nature by phylogenetic analysis and by demonstrating linkage of foreign genes with metazoan genes in the bdelloid genome. Approximately 80% of horizontally acquired genes expressed in bdelloids code for enzymes, and these represent 39% of enzymes in identified pathways. Many enzymes encoded by foreign genes enhance biochemistry in bdelloids compared to other metazoans, for example, by potentiating toxin degradation or generation of antioxidants and key metabolites. They also supplement, and occasionally potentially replace, existing metazoan functions. Bdelloid rotifers therefore express horizontally acquired genes on a scale unprecedented in animals, and foreign genes make a profound contribution to their metabolism. This represents a potential mechanism for ancient asexuals to adapt rapidly to changing environments and thereby persist over long evolutionary time periods in the absence of sex.

MAPK phosphorylation is implicated in the adaptation to desiccation stress in nematodes.

Some nematodes can survive almost complete desiccation by entering an ametabolic state called anhydrobiosis requiring the accumulation of protective molecules such as trehalose and LEA proteins. However, it is not known how anhydrobiotic organisms sense and regulate the response to water loss. Mitogen-activated protein kinases (MAPKs) are highly conserved signalling proteins that regulate adaptation to various stresses. Here, we first compared the anhydrobiotic potential of three nematode species, Caenorhabditis elegans, Aphelenchus avenae and Panagrolaimus superbus, and then determined the phosphorylation status of the MAPKs p38, JNK and ERK during desiccation and rehydration. Caenorhabditis elegans was unable to undergo anhydrobiosis even after an initial phase of slow drying (preconditioning), while A. avenae did survive desiccation after preconditioning. In contrast, P. superbus withstood desiccation under rapid drying conditions, although survival rates improved with preconditioning. These results characterise C. elegans as desiccation sensitive, A. avenae as a slow desiccation strategist anhydrobiote and P. superbus as a fast desiccation strategist anhydrobiote. Both C. elegans and A. avenae showed increased MAPK phosphorylation during drying, consistent with an attempt to mount protection systems against desiccation stress. In P. superbus, however, MAPK phosphorylation was apparent prior to water loss and then decreased on dehydration, suggesting that signal transduction pathways are constitutively active in this nematode. Inhibition of p38 and JNK in P. superbus decreased its desiccation tolerance. This is consistent with the designation of P. superbus as a fast desiccation strategist and its high level of preparedness for anhydrobiosis in the hydrated state. These findings show that MAPKs play an important role in the survival of organisms during anhydrobiosis.