Transcriptome survey of the anhydrobiotic tardigrade Milnesium tardigradum in comparison with Hypsibius dujardini and Richtersius coronifer

<p>Abstract</p> <p>Background</p> <p>The phenomenon of desiccation tolerance, also called anhydrobiosis, involves the ability of an organism to survive the loss of almost all cellular water without sustaining irreversible damage. Although there are several physiological, morphological and ecological studies on tardigrades, only limited DNA sequence information is available. Therefore, we explored the transcriptome in the active and anhydrobiotic state of the tardigrade <it>Milnesium tardigradum </it>which has extraordinary tolerance to desiccation and freezing. In this study, we present the first overview of the transcriptome of <it>M. tardigradum </it>and its response to desiccation and discuss potential parallels to stress responses in other organisms.</p> <p>Results</p> <p>We sequenced a total of 9984 expressed sequence tags (ESTs) from two cDNA libraries from the eutardigrade <it>M. tardigradum </it>in its active and inactive, anhydrobiotic (tun) stage. Assembly of these ESTs resulted in 3283 putative unique transcripts, whereof ~50% showed significant sequence similarity to known genes. The resulting unigenes were functionally annotated using the Gene Ontology (GO) vocabulary. A GO term enrichment analysis revealed several GOs that were significantly underrepresented in the inactive stage. Furthermore we compared the putative unigenes of <it>M. tardigradum </it>with ESTs from two other eutardigrade species that are available from public sequence databases, namely <it>Richtersius coronifer </it>and <it>Hypsibius dujardini</it>. The processed sequences of the three tardigrade species revealed similar functional content and the <it>M. tardigradum </it>dataset contained additional sequences from tardigrades not present in the other two.</p> <p>Conclusions</p> <p>This study describes novel sequence data from the tardigrade <it>M. tardigradum</it>, which significantly contributes to the available tardigrade sequence data and will help to establish this extraordinary tardigrade as a model for studying anhydrobiosis. Functional comparison of active and anhydrobiotic tardigrades revealed a differential distribution of Gene Ontology terms associated with chromatin structure and the translation machinery, which are underrepresented in the inactive animals. These findings imply a widespread metabolic response of the animals on dehydration. The collective tardigrade transcriptome data will serve as a reference for further studies and support the identification and characterization of genes involved in the anhydrobiotic response.</p

Transcriptome Analysis in Tardigrade Species Reveals Specific Molecular Pathways for Stress Adaptations

Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum. To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade Milnesium tardigradum were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from Hypsibius dujardini, revealing major pathways involved in resistance against extreme environmental conditions. ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for M. tardigradum are different from typical motifs known from higher animals. M. tardigradum and H. dujardini protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of M. tardigradum. These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and M. tardigradum in particular so highly stress resistant

Filtering duplicate reads from 454 pyrosequencing data

Motivation: Throughout the recent years, 454 pyrosequencing has emerged as an efficient alternative to traditional Sanger sequencing and is widely used in both de novo whole-genome sequencing and metagenomics. Especially the latter application is extremely sensitive to sequencing errors and artificially duplicated reads. Both are common in 454 pyrosequencing and can create a strong bias in the estimation of diversity and composition of a sample. To date, there are several tools that aim to remove both sequencing noise and duplicates. Nevertheless, duplicate removal is often based on nucleotide sequences rather than on the underlying flow values, which contain additional information. Results: With the novel tool JATAC, we present an approach towards a more accurate duplicate removal by analysing flow values directly. Making use of previous findings on 454 flow data characteristics, we combine read clustering with Bayesian distance measures. Finally, we provide a benchmark with an existing algorithm. Availability: JATAC is freely available under the General Public License from http://malde.org/ketil/jatac/. Supplementary information: Supplementary data are available at Bioinformatics onlin

The Aquaporin Channel Repertoire of the Tardigrade

Limno-terrestrial tardigrades are small invertebrates that are subjected to periodic drought of their micro-environment. They have evolved to cope with these unfavorable conditions by anhydrobiosis, an ametabolic state of low cellular water. During drying and rehydration, tardigrades go through drastic changes in cellular water content. By our transcriptome sequencing effort of the limnoterrestrial tardigrade Milnesium tardigradum and by a combination of cloning and targeted sequence assembly, we identified transcripts encoding eleven putative aquaporins. Analysis of these sequences proposed 2 classical aquaporins, 8 aquaglyceroporins and a single potentially intracellular unorthodox aquaporin. Using quantitative real-time PCR we analyzed aquaporin transcript expression in the anhydrobiotic context. We have identified additional unorthodox aquaporins in various insect genomes and have identified a novel common conserved structural feature in these proteins. Analysis of the genomic organization of insect aquaporin genes revealed several conserved gene clusters

Towards Decrypting Cryptobiosis—Analyzing Anhydrobiosis in the Tardigrade <i>Milnesium tardigradum</i> Using Transcriptome Sequencing

<div><p>Background</p><p>Many tardigrade species are capable of anhydrobiosis; however, mechanisms underlying their extreme desiccation resistance remain elusive. This study attempts to quantify the anhydrobiotic transcriptome of the limno-terrestrial tardigrade <i>Milnesium tardigradum</i>.</p><p>Results</p><p>A prerequisite for differential gene expression analysis was the generation of a reference hybrid transcriptome atlas by assembly of Sanger, 454 and Illumina sequence data. The final assembly yielded 79,064 contigs (>100 bp) after removal of ribosomal RNAs. Around 50% of them could be annotated by SwissProt and NCBI non-redundant protein sequences. Analysis using CEGMA predicted 232 (93.5%) out of the 248 highly conserved eukaryotic genes in the assembly. We used this reference transcriptome for mapping and quantifying the expression of transcripts regulated under anhdydrobiosis in a time-series during dehydration and rehydration. 834 of the transcripts were found to be differentially expressed in a single stage (dehydration/inactive tun/rehydration) and 184 were overlapping in two stages while 74 were differentially expressed in all three stages. We have found interesting patterns of differentially expressed transcripts that are in concordance with a common hypothesis of metabolic shutdown during anhydrobiosis. This included down-regulation of several proteins of the DNA replication and translational machinery and protein degradation. Among others, heat shock proteins Hsp27 and Hsp30c were up-regulated in response to dehydration and rehydration. In addition, we observed up-regulation of ployubiquitin-B upon rehydration together with a higher expression level of several DNA repair proteins during rehydration than in the dehydration stage.</p><p>Conclusions</p><p>Most of the transcripts identified to be differentially expressed had distinct cellular function. Our data suggest a concerted molecular adaptation in <i>M</i>. <i>tardigradum</i> that permits extreme forms of ametabolic states such as anhydrobiosis. It is temping to surmise that the desiccation tolerance of tradigrades can be achieved by a constitutive cellular protection system, probably in conjunction with other mechanisms such as rehydration-induced cellular repair.</p></div

Mapping qualities of Illumina datasets mapped onto transcriptome assembly.

<p>MapQ 0: several equally best hits, MapQ 37: maximum MapQ for single end reads in BWA.</p

Transcriptome assembly contig distributions.

<p>(A) Final assembly contig length distribution. (B) Illumina data assembly contig length distribution before and after error correction using Reptile (red: decrease, green: increase).</p

DESeq differential transcript expression analysis.

<p>(A–C) Log ratio versus abundance plots (MA-plot) for each of the anhydrobiotic stages versus active stage (A: active vs. dehydration; B: active vs. inactive tun; C: active vs rehydration; red dots: significantly regulated at FDR of 0.1, red triangle regions pointed by arrow: significantly regulated without counts in one of the stages and outliers). (D) Venn diagram of differentially expressed transcripts compared to active stage shared between stages (generated using <a href="http://bioinformatics.psb.ugent.be/webtools/Venn/" target="_blank">http://bioinformatics.psb.ugent.be/webtools/Venn/</a>).</p