BIOlogical SignaTures of AnhydrobioSIS via comparative transcriptomics on different evolutionary lineages within tardigrades

Project Information

BIOSTASIS

Grant agreement ID: 747087

DOI

10.3030/747087

Project closed

EC signature date 17 March 2017

Start date 1 February 2018

End date 31 January 2020

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 200 194,80

EU contribution

€ 200 194,80

Coordinated by

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 1 - BIOSTASIS (BIOlogical SignaTures of AnhydrobioSIS via comparative transcriptomics on different evolutionary lineages within tardigrades)

Reporting period: 2018-02-01 to 2020-01-31

Summary of the context and overall objectives of the project

The BIOSTASIS project revolved around the study of anhydrobiosis in a tardigrade species ie. Ramazzottius varieornatus. Anhydrobiosis (life without water) is a survival strategy that confers extreme desiccation tolerance. This remarkable phenomenon is present in a limited number of species across the tree of life (animals from four phyla, i.e. Tardigrades, Rotifers, Nematods and Arthropods), selected land plants, certain fungi and bacteria. This strategy is a form of cryptobiosis (latent life), defined by the reversible shut-down of metabolism (biostasis). While desiccated, organisms show no visible signs of life, but they can resume activity in a few minutes to a few hours when placed in a drop of water.

Of all the abiotic environmental stressors, desiccation is considered the most detrimental to organisms. Water availability plays a crucial role in defining individual species activity and their ecological interactions, and organisms are frequently confronted with water shortage in both aquatic and terrestrial ecosystems. The most common drivers of dehydration are the evaporative water loss, freezing during winter (and the subsequent reduction of liquid water in extracellular fluids), and hypersalinity. Global environmental change is expected to greatly affect the magnitude of all of these drivers, impacting life at all hierarchical levels, from genes, to species, to ecosystems.

Genomic studies on anhydrobionts have led to rapid progress on multiple fronts, yet to date, little is known on the relevance of molecules, genes and mechanisms during the entry and the exit of organisms from anhydrobiosis. Within the BIOSTASIS project, using an experimental set up with several time points, I set the ambitious aim to try and reveal key-player genes involved in the mechanism of anhydrobiosis in a strong cryptobiont tardigrade species ie. Ramazzottius varieornatus. My hypothesis: there could be a potential gene signature for anhydrobiosis, establishing a transcription profile that is characteristic of a given physiological state (active, desiccated or rehydrated) and that could possibly reflect the phylogenetic position of a taxon (ie. organisms that are close relatives could have the same transcription profiles).

The project results revealed that the duration of the desiccation stress impacts both the total number of genes that are regulated at the “tun state” and also the recovery time needed for the individuals to return to their normal gene expression levels (as compared to the control samples). The analysed data resulted to new information regarding genes and mechanisms implicated in the mechanisms of anhydrobiosis.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Ramazzottius varieornatus individuals were collected from a roof gutter in Copenhagen area and kept in environmental chambers at 10oC with moderately hard water containing roof-gutter sediment. The experimentation included 11 time points throughout different conditions, ie. control (normal/active animals), desiccated animals (1hour and 72 hours of desiccation) and a series of rehydrated time points (active animals after 10min, 1 hour, 12 hours and 24 hours (Fig1). Prior to the experiments for anhydrobiosis the animals were cleaned from debris and starved for one day. Using BGI500SEQ (BGI-Shenzhen) 37 transcriptomes were sequenced in total. After the de novo assembly, annotation and differential gene expression between the different conditions/time points, the results reveal that multiple molecular components seem to contribute to the intracellular mechanisms for coping with desiccation in Ramazzottius varieornatus. My data indicate a significant contribution of protective stress proteins such as heat shock proteins (HSPs) and antioxidative enzymes. Focus has also been rising towards the Intrinsically Disordered Proteins (IDPs) such as Late Embryogenesis Abundant proteins (LEA) that can undergo functional conformational changes in response to desiccation. These proteins were initially described in land plants and later detected also in bacteria, cyanobacteria, fungi and metazoans. Solutes, such as trehalose, that can stabilize biological structures during severe desiccation have been identified as crucial for survival in some organisms. The data on R. varieornatus suggested that this solute is potentially involved into the recovery of the tardigrades during the post-anhydrobiosis stages. Finally, key-player mechanisms that have been identified are: a) stabilization of proteins, b) prevention of oxidative stress, c) elimination of damage from structural stress and d) alternative regulation of signalling pathways responsible for the epagoge of these protective mechanisms. Interestingly, my results reveal a large number of differentially expressed transcripts that are hypothetical proteins; their open reading frames have been predicted through genome sequencing projects and functions cannot be readily assigned. These results indicate that there is a need to experimentally verify the expression of those transcripts and proceed with their functional characterization.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The work carried out within the BIOSTASIS project has resulted to deepening the knowledge and understanding of the intriguing phenomenon of anhydrobiosis. The comparative transcriptomics on the comprehensive multi-time experimental set up has verified previous studies and provided new information regarding a) important genes and pathways involved in desiccation tolerance and b) the gene regulation during the transition between the entry and exit (ie. recovery) of R. varieornatus from desiccation. Among the novel results of the BIOSTASIS project is the identification of a number of transcription factors that regulate their expression levels in the different stages of anhydrobiosis. The data also implicated pathways such as polyamine utilization, glyoxalase-dependent detoxification, and lipid desaturation. Furthermore, the comparison of the gene expression among different tardigrade species and also other model organisms indicated unique molecular adaptations within tardigrade lineages.
The overall results present a lot of potential of high impact for natural and biomedical sciences. Understanding the mechanisms and identifying the molecules implicated in desiccation tolerance is fundamental for improving methods for example in storing biological materials. The “dry” alternative will significantly reduce energy use while enhancing sample availability and management and space utilization with the potential to generate great revenue. Stress biology research is highly relevant to the ongoing EU 2018-2020 focus areas (i) Building a low-carbon, climate resilient future2, (ii) Connecting economic and environmental gains – the Circular Economy. Moreover, unveiling the mechanisms of desiccation tolerance will greatly enhance our understanding of species and ecosystems persistence to ongoing and future environmental change, in line with the commission’s priorities 2019-2024 for the European Green Deal under the Clean Energy and Biodiversity policies.

BIOSTASIS experimental set up