Comparative transcriptomics suggest unique molecular adaptations within tardigrade lineages

Abstract

Background: Tardigrades are renowned for their ability to enter cryptobiosis (latent life) and endure extreme stress, including desiccation and freezing. Increased focus is on revealing molecular mechanisms underlying this tolerance. Here, we provide the first transcriptomes from the heterotardigrade Echiniscoides cf. sigismundi and the eutardigrade Richtersius cf. coronifer, and compare these with data from other tardigrades and six eukaryote models. Investigating 107 genes/gene families, our study provides a thorough analysis of tardigrade gene content with focus on stress tolerance.

Results: E. cf. sigismundi, a strong cryptobiont, apparently lacks expression of a number of stress related genes. Most conspicuous is the lack of transcripts from genes involved in classical Non-Homologous End Joining. Our analyses suggest that post-cryptobiotic survival in tardigrades could rely on high fidelity transcription-coupled DNA repair. Tardigrades seem to lack many peroxins, but they all have a comprehensive number of genes encoding proteins involved in antioxidant defense. The "tardigrade unique proteins" (CAHS, SAHS, MAHS, RvLEAM), seem to be missing in the heterotardigrade lineage, revealing that cryptobiosis in general cannot be attributed solely to these proteins. Our investigation further reveals a unique and highly expressed cold shock domain. We hypothesize that the cold shock protein acts as a RNA-chaperone involved in regulation of translation following freezing.

Conclusions: Our results show common gene family contractions and expansions within stress related gene pathways in tardigrades, but also indicate that evolutionary lineages have a high degree of divergence. Different taxa and lineages may exhibit unique physiological adaptations towards stress conditions involving possible unknown functional homologues and/or novel physiological and biochemical mechanisms. To further substantiate the current results genome assemblies coupled with transcriptome data and experimental investigations are needed from tardigrades belonging to different evolutionary lineages.

Keywords: Cold shock domain; Functional gene categories; Model organisms; Stress genes; Tardigrada; Transcriptomics.

Fig. 1

Global comparisons of transcripts and predicted protein sequences. Transcript and protein sequences of E. cf. sigismundi and R. cf. coronifer were compared to sequences from other tardigrades and model organisms. Global comparisons were conducted using BLASTX for the transcripts alignments (left) and BLASTP (right) for the predicted protein sequences. Heatmaps represent the percentage of transcripts (left) or predicted protein (right) with detectable sequence similarity at an e-value threshold of 10e⁻⁵

Fig. 2

Comparison of shared and species-specific orthologous protein groups as revealed by OrthoMCL analyses. a Shared and species-specific orthologous protein groups within tardigrades; b Shared and species-specific protein groups between tardigrades and other ecdysozoans as revealed by a comparison between E. cf. sigismundi, R. cf. coronifer, D. melanogaster and C. elegans; c Comparison of shared and species-specific orthologous protein groups between E. cf. sigismundi and four model eukaryote organisms; d Comparison of shared and species-specific orthologous protein groups between R. cf. coronifer and four model eukaryote organisms

Fig. 3

Comparative investigation of gene expression in tardigrades. Comparative data on the 10 most highly expressed protein coding genes within tardigrade transcriptomes and cumulative expression of the stress related gene categories under study (for the complete list of genes refer to Additional file 1). The predicted protein coding genes were obtained from the transcriptomes of three tardigrade species (Echiniscoides cf. sigismundi, Richtersius cf. coronifer and Ramazzottius cf. varieornatus). Values are depicted as TPM. Gradient purple columns: dark = 1st transcript, light = 10th transcript. Rv1,2,3,5,6,7,9 = hypothetical proteins; Rv4 = SAHS1 (Secretory Abundant Heat Soluble 1); Rv10 = SAHS2 (Secretory Abundant Heat Soluble 2); Rv8 = cuticular protein. Rc1,Rc6 = hypothetical proteins; Rc2,5 = uncharacterized proteins; Rc3 = PE-1(Peritrophin-1); Rc4 = rhogef domain containing protein; Rc7 = NOTCH1-like (Neurogenic locus notch-like protein 1); Rc8 = SAHS1 (Secretory Abundant Heat Soluble 1); Rc9 = COI (Cytochrome Oxidase subunit I); Rc10 = periostin. Es1,6,7,8,=hypothetical proteins; Es2,3,9 = uncharacterized proteins; Es4 = CSRP3 (cysteine and glycine rich protein 3 precursor); Es5 = proactivator polypeptide-like; Es10 = HSP20 (Heat Shock Protein 20)

Fig. 4

Alignment of amino acid sequences containing a Cold Shock Domain (CSD). Data obtained from representative bacteria and animals including tardigrades. Tardigrade sequences are indicated by bold in the left margin of the figure. RNP1 and RNP2 (shaded in grey) represent consensus RNA binding domains. DNA binding sites are highlighted in yellow. Note the RGG (green) and RG (orange) repeats. In the graphical representation below the CSD, the overall height of the stack indicates the sequence conservation at the specific position, while the height of symbols within the stack indicates the relative frequency of each amino acid at the position

Fig. 5

Phylogenetic analysis of Cold Shock Domain proteins. Protein sequences of Cold Shock Domain containing proteins from various species of bacteria and animals aligned using Muscle. The Maximum Likelihood phylogenetic tree was constructed using RAxML. Bootstrap values (1000 trials) are shown on branches. Clades with bootstrap values < 50 have been collapsed into polytomies using TreeGraph2 [92]. All animal taxa are clustered together, and are separated into a clade containing the YB proteins and a clade containing the Lin28 proteins with Zn finger motifs. The Es_CSP form a separate clade as sister-group to the YB cluster of the rest of the animal taxa analyzed