Abstract
Tardigrades are microscopic aquatic animals renowned for their tolerance toward extreme environmental conditions. The current study is the first to investigate their tolerance toward heavy metals and we present a novel tardigrade toxicant tolerance assay based on activity assessments as a measure of survival. Specifically, we compare tolerance toward copper in four species representing different evolutionary lineages, habitats and adaptation strategies, i.e., a marine heterotardigrade, Echiniscoides sigismundi, a limno-terrestrial heterotardigrade, Echiniscus testudo, a limno-terrestrial eutardigrade, Ramazzottius oberhaeuseri, and a marine eutardigrade, Halobiotus crispae. The latter was sampled at a time of year, when the population is predominantly represented by aberrant P1 cysts, while the other species were in normal active states prior to exposure. Based on volume measurements and a general relation between body mass and copper tolerance, expected tardigrade EC50 values were estimated at 0.5–2 μg l−1. Following 24 h of exposure, tolerance was high with no apparent link to lineage or habitat. EC50s (95% CI), 24 h after exposure, were estimated at 178 (168–186) and 310 (295–328) μg l−1, respectively, for E. sigismundi and R. oberhaeuseri, whereas E. testudo and H. crispae were less affected. Highest tolerance was observed in H. crispae with a mean ± s.e.m. activity of 77 ± 2% (n = 3) 24 h after removal from ~3 mg l−1 copper, suggesting that tardigrade cysts have increased tolerance toward toxicants. In order to identify putative tolerance related genes, an E. sigismundi transcriptome was searched for key enzymes involved in osmoregulation, antioxidant defense and copper metabolism. We found high expression of Na/K ATPase and carbonic anhydrase, known targets for copper. Our transcriptome, furthermore, revealed high expression of antioxidant enzymes, copper transporters, ATOX1, and a Cu-ATPase. In summary, our results indicate that tardigrades express well-known key osmoregulatory enzymes, supporting the hypothesis that copper inhibits sodium turnover as demonstrated for other aquatic organisms. Tardigrades, nevertheless, have high tolerance toward the toxicant, which is likely linked to high expression of antioxidant enzymes and an ability to enter dormant states. Tardigrades, furthermore, seem to have a well-developed battery of cuproproteins involved in copper homeostasis, providing basis for active copper sequestering and excretion.
Highlights
Tardigrades are microscopic metazoans that require a film of water to be in an active and reproducing state
E. sigismundi, E. testudo, H. crispae, and R. oberhaeuseri, representing different evolutionary lineages, habitats, and adaptation strategies were chosen for the current investigation into copper tolerance within phylum Tardigrada
We exposed groups of ∼20 tardigrades for 24 h of measured copper concentrations of up to 2991 μg l−1 in E. sigismundi, 3897 μg l−1 in E. testudo, 3065 μg l−1 in H. crispae and 3643 μg l−1 in R. oberhaeuseri (Figure 1)
Summary
Tardigrades are microscopic metazoans that require a film of water to be in an active and reproducing state These minute aquatic animals have adapted to terrestrial environments by evolving dormant states, cryptobiosis, and cysts, and they are famous for their ability to survive a range of environmental extremes, including complete desiccation, high levels of radiation, extreme hydrostatic, and osmotic pressures, very low sub-zero temperatures as well as space conditions (e.g., Rebecchi et al, 2007; Jönsson et al, 2008; Hengherr et al, 2009; Guidetti et al, 2011; Møbjerg et al, 2011; Persson et al, 2011; Welnicz et al, 2011; Clausen et al, 2014; Zawierucha et al, 2015; Hashimoto et al, 2016; Heidemann et al, 2016; Hygum et al, 2016). Copper, due to its variable valencies, promotes the formation of free radicals leading to oxidative stress (e.g., Balamurugan and Schaffner, 2006)
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