title: Comparative Investigation of Copper Tolerance and Identification of Putative Tolerance Related Genes in Tardigrades.
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, forE. sigismundiandR. oberhaeuseri, whereasE. testudoandH. crispaewere less affected. Highest tolerance was observed inH. crispaewith amean±s.e.m. activity of 77 ± 2% (n= 3) 24 h after removal from ~3 mg l-1copper, suggesting that tardigrade cysts have increased tolerance toward toxicants. In order to identify putative tolerance related genes, anE. sigismunditranscriptome 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.
authors.affiliation: Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen Copenhagen, Denmark.
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Hygum
authors.affiliation: Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen Copenhagen, Denmark.
authors.forename: Dannie
authors.initials: D
authors.lastname: Fobian
authors.affiliation: Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen Copenhagen, Denmark.
authors.forename: Maria
authors.initials: M
authors.lastname: Kamilari
authors.affiliation: Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen Copenhagen, Denmark.
authors.forename: Aslak
authors.initials: A
authors.lastname: Jørgensen
authors.affiliation: Centre for Social Evolution, Department of Biology, University of Copenhagen Copenhagen, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Schiøtt
authors.affiliation: Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami Miami, FL, USA.
authors.forename: Martin
authors.initials: M
authors.lastname: Grosell
authors.affiliation: Section for Cell Biology and Physiology, Department of Biology, University of Copenhagen Copenhagen, Denmark.
authors.forename: Nadja
authors.initials: N
authors.lastname: Møbjerg
keywords: chemobiosis; copper tolerance; cyst; osmoregulation; stress tolerance; tardigrades; toxicants; transcriptome