cDNA representational difference analysis for identifying transcripts regulated under anhydrobiosis in the tardigrade Milnesium tardigradum

Abstract

Although water is a prerequisite for life, some organisms are capable of surviving complete cellular dehydration by entering a state called anhydrobiosis. In this state, no metabolic activity is measurable and these organisms can survive several years in dry form until water becomes available again. Anhydrobiosis has been described in bacteria, fungi, plants and many invertebrates. Among those invertebrates, tardigrades are also known to be capable to undergo anhydrobiosis by forming a so-called ÔtunÕ stage. Therefore, they represent an interesting model system for studying the cellular response to desiccation. We have applied cDNA representational difference analysis to investigate the transcriptional response of the tardigrade Milnesium tardigradum to desiccation. This resulted in the identification of two transcripts that showed significant regulation between active and anhydrobiotic animals as validated by quantitative real-time PCR. One of these putative transcripts was downregulated fivefold. It did not show any significant similarity to known transcripts, but had previously been cloned in M. tardigradum. The second transcript could be identified as the M. tardigradum homologue of a subunit of replication factor C and was downregulated more than sixfold in the anhydrobiotic state. Replication factor C plays an important role in DNA replication during cell division. Both candidates showed an even lower expression in the transition stage between active and the tun stage (27-and 25-fold reduction, respectively), implying active inhibition of transcription of these two transcripts. Our results suggest that the potential modulation of DNA replication and its role in protecting genome stability should attract more attention when studying anhydrobiotic animals.

Towards Decrypting Cryptobiosis—Analyzing Anhydrobiosis in the Tardigrade Milnesium tardigradum Using Transcriptome Sequencing

“…Previously identified differential transcripts fit well into the picture of inhibited cell division. The cloning of replication factor C subunit 2 by means of representational difference analysis has been previously reported [73] . The replication factor C is a direct binding partner of proliferating cell nuclear antigen (PCNA).…”

Mechanisms of Desiccation Tolerance: Themes and Variations in Brine Shrimp, Roundworms, and Tardigrades

“…Recently, there has been an effort to develop tardigrades as a model for the study of the molecular biology of extremes like anhydrobiosis (see Hashimoto and Kunieda, 2017;Goldstein, 2018 for review). As tardigrades have gained in popularity as a research organism, many studies have focused on transcriptional responses to desiccation in various species (Schill et al, 2004;Reuner et al, 2010;Grohme et al, 2011;Wang et al, 2014;Hashimoto et al, 2016;Yoshida et al, 2017). In contrast, few studies test for endogenous functions of proteins in tardigrades (Tenlen et al, 2013;.…”

Life-history traits in the tardigrade species Paramacrobiotus kenianus and Paramacrobiotus palaui

“…Tardigrades show extraordinary tolerance to physical extremes including high pressure (Seki and Toyoshima, 1998;Horikawa et al, 2009), UV and high energy radiation (Jönsson et al, 2005;Horikawa et al, 2006;Jönsson et al, 2008;Rebecchi et al, 2009Rebecchi et al, , 2011Persson et al, 2011), exposure to high temperatures (Doyère, 1842;Pouchet, 1859;Rahm, 1921a;Ramløv and Westh, 2001;Hengherr et al, 2009c) and exposure to low temperatures (Rahm, 1921b;Ramløv and Westh, 1992;Hengherr et al, 2009a, b;Guidetti et al, 2011a). Over the past few years, more and more emphasis has been placed on the genome (Schill et al, 2004;Förster et al, 2009Förster et al, , 2011aFörster et al, , 2011bGrohme et al, 2011), proteome (Schokraie et al, 2010(Schokraie et al, , 2011(Schokraie et al, , 2012Ya-maguchi et al, 2012) and metabolome (Kunieda and Kubo, 2006;Hengherr et al, 2008b;Beisser et al, 2012;Cesari et al, 2012) of tardigrades for a better understanding of the survival mechanisms. However, not much is known about their life-history traits, because only few species are in culture and life-cycle observations in nature are quite rare (Altiero and Rebecchi, 2001;Suzuki, 2003;Horikawa et al, 2008;Altiero et al, 2010;Lemloh et al, 2011).…”