Studies on the ability of multicellular organisms to tolerate specific environmental extremes are relatively rare compared to those of unicellular microorganisms in extreme environments. Tardigrades are extremotolerant animals that can enter an ametabolic dry state called anhydrobiosis and have high tolerance to a variety of extreme environmental conditions, particularly while in anhydrobiosis. Although tardigrades have been expected to be a potential model animal for astrobiological studies due to their excellent anhydrobiotic and extremotolerant abilities, few studies of tolerance with cultured tardigrades have been reported, possibly due to the absence of a model species that can be easily maintained under rearing conditions. We report the successful rearing of the herbivorous tardigrade, Ramazzottius varieornatus, by supplying the green alga Chlorella vulgaris as food. The life span was 35 +/- 16.4 d, deposited eggs required 5.7 +/- 1.1 d to hatch, and animals began to deposit eggs 9 d after hatching. The reared individuals of this species had an anhydrobiotic capacity throughout their life cycle in egg, juvenile, and adult stages. Furthermore, the reared adults in an anhydrobiotic state were tolerant of temperatures of 90 degrees C and -196 degrees C, and exposure to 99.8% acetonitrile or irradiation with 4000 Gy (4)He ions. Based on their life history traits and tolerance to extreme stresses, R. varieornatus may be a suitable model for astrobiological studies of multicellular organisms.

Trehalose is a versatile non-reducing sugar. In some animal groups possessing its intrinsic production machinery, it is used as a potent protectant against environmental stresses, as well as blood sugar. However, the trehalose biosynthesis genes remain unidentified in the large majority of metazoan phyla, including vertebrates. To uncover the evolutionary history of trehalose production machinery in metazoans, we scrutinized the available genome resources and identified bifunctional trehalose-6-phosphate synthase-trehalose-6-phosphate phosphatase (TPS–TPP) genes in various taxa. The scan included our newly sequenced genome assembly of a desiccation-tolerant tardigrade Paramacrobiotus sp. TYO, revealing that this species retains TPS–TPP genes activated upon desiccation. Phylogenetic analyses identified a monophyletic group of the many of the metazoan TPS–TPP genes, namely ‘pan-metazoan’ genes, that were acquired in the early ancestors of metazoans. Furthermore, coordination of our results with the previous horizontal gene transfer studies illuminated that the two tardigrade lineages, nematodes and bdelloid rotifers, all of which include desiccation-tolerant species, independently acquired the TPS–TPP homologues via horizontal transfer accompanied with loss of the ‘pan-metazoan’ genes. Our results indicate that the parallel evolution of trehalose synthesis via recurrent loss and horizontal transfer of the biosynthesis genes resulted in the acquisition and/or augmentation of anhydrobiotic lives in animals.

Tardigrades are aquatic micrometazoans found in various habitats. Many limno-terrestrial species can tolerate desiccation by entering a metabolically inactive dehydrated state called anhydrobiosis, whereas most species in constantly hydrated habitats, such as freshwater or marine species, cannot undergo anhydrobiosis and are susceptible to desiccation. Although comparative analyses between anhydrobiotic species and desiccation-sensitive species will lead to a better understanding of anhydrobiosis, almost all studies on anhydrobiosis have been performed only in anhydrobiotic species. In the present study, we established a rearing system and an isogenic strain of Isohypsibius myrops collected from activated sludge. This strain is highly sensitive to desiccation and cannot tolerate exposure to relative humidity (RH) of 97-98% for 2 days or 94-95% for 1 day. Life history traits revealed that this strain has a short hatching time (3.6 days) and a short lifespan (18.8 days). The strain frequently lays eggs, on average 19 eggs per oviposition at mean interval of 2.5 days, enabling their rapid proliferation. The high fecundity of desiccation-sensitive species implies a possible trade-off between anhydrobiotic ability and fecundity. The established strain provides a good experimental model of desiccationsensitive tardigrades, especially for comparative molecular analyses of anhydrobiosis mechanisms.

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