Differential expression profiling of heat stressed tardigrades reveals major shift in the transcriptome

Diversity in Expression Biases of Lineage-Specific Genes During Development and Anhydrobiosis Among Tardigrade Species

“…The expansion of gene families coding for proteins with chitin binding properties in tardigrades might have evolved to help regulate physiological responses to environmental stresses. 41 , 42 Numerous other GO terms were found enriched among TSGs and TSG expansions including functions related to DNA binding, peptidase activity, and G protein-coupled receptor activity ( Figure 4 ), which have been previously reported in Yoshida et al 17 based on analyses of only He and Rv (before data from Echiniscoides sigismundi and Richtersius coronifer were available). Lineage-specific genes and expansions (ie, genes specific to either He or Rv) showed little overlap in enriched GO terms between species, suggesting species-specific emergence and duplications of unrelated gene families that code for proteins with distinct functions.…”

Single-animal, single-tube RNA extraction for comparison of relative transcript levels via qRT-PCR in the tardigradeHypsibius exemplaris

“…We replicated the previous experiment with H. exemplaris by treating tardigrades with prolonged heat (30 °C) or cold stress (4°C) (Figure 4b ). Tardigrades in their active state are highly sensitive to increases in temperature 18 ; thus, we selected 30°C as the high temperature for H. exemplaris , as this resulted in minimal lethality ( Figure S7 ). We then extracted RNA from single tardigrades, generated cDNA, and analyzed the expression levels of HSP70 β 2 and HSP90 α normalized to actin by qRT-PCR.…”

“…Stable genetic manipulation of these remarkable animals, including transgenesis and germline gene modification, has remained elusive until very recently 15, 16 . As such, most experimentation to reveal molecular mechanisms of extremotolerance is performed through transcriptional profiling 4, 5, 17, 18 . To test the functionality of identified genes, such studies are often followed by RNAi-mediated knockdown of molecular targets 6 and quantification of extremotolerant capacity after knockdown 5 .…”

“…Importantly, these studies often analyze heterogeneous populations of animals that include both animals that survive environmental stressors and those that do not. As such, the studies are confounded by averaging expression data from multiple and potentially dramatically different states of response 4, 5, 18 . Although previous methods have been developed to extract RNA from single tardigrades, these protocols as reported in the literature do not yield concentrations of RNA that are quantifiable by Real-Time PCR; rather they rely on optical density-based methods to monitor transcriptional changes 6, 19 .…”

The major inducible small heat shock protein HSP20-3 in the tardigrade Ramazzottius varieornatus forms filament-like structures and is an active chaperone

“…Comparison of the molecular adaptions across Eutardigrada ( Hypsibius exemplaris, R. varieornatus , Richtersius coronifer ) and Heterotardigrada 21 with two other related invertebrates, the arthropod, Drosophila melanogaster and the nematode, Caenorhabditis elegans and various vertebrates and yeast 7 established that proteins in addition to the tardigrade specific proteins (CAHS, MAHS, SAHS) are necessary for the unique molecular adaptions that supports cryptogenic responses to extreme stresses 7 , 21 . With this in mind, we investigated the small heat shock protein (sHSP) family present in R. varieornatus because sHSPs have properties in common with CAHS and SAHS proteins, such as their ability to prevent protein aggregation and precipitation 22 , 23 , 24 , 25 , form biomolecular condensates 26 , 27 , 28 and to be upregulated in response to cold and heat shock of R. varieornatus 29 , 30 .…”