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New insights into survival strategies of

Møbjerg, Nadja; Neves, Ricardo

Published in: Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology

DOI: 10.1016/j.cbpa.2020.110890

Publication date: 2021

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Document license: CC BY

Citation for published version (APA): Møbjerg, N., & Neves, R. (2021). New insights into survival strategies of tardigrades. Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology, 254, [110890]. https://doi.org/10.1016/j.cbpa.2020.110890

Download date: 02. okt.. 2021 Comparative Biochemistry and Physiology, Part A 254 (2021) 110890

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Comparative Biochemistry and Physiology, Part A

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Graphical Review New insights into survival strategies of tardigrades

Nadja Møbjerg *, Ricardo Cardoso Neves

Department of Biology, August Krogh Building, University of Copenhagen, Denmark

ARTICLE INFO ABSTRACT

Keywords: Life is set within a narrow frame of physicochemical factors, yet, some species have adapted to conditions far beyond these constraints. Nature appears to have evolved two principal strategies for living to cope with hostile conditions. One way is to remain active, retaining through adaptations that enable the Environmental stress to match the physiological requirements of environmental change. The other is to enter a state of Molecular mechanisms with metabolic suppression. One form of metabolic suppression, known as cryptobiosis, is a wide­ Physiology Tardigrades spread state across life kingdoms, in which metabolism comes to a reversible standstill. Among animals, nem­ Tun state atodes, and tardigrades, comprise species that have the ability to enter cryptobiosis at all stages of their life cycle. Tardigrades are microscopic cosmopolitan metazoans found in permanent and temporal aquatic en­ vironments. They are renowned for their ability to tolerate extreme stress and are particularly resistant after having entered a cryptobiotic state known as a “tun”. As new molecular tools allow for a more detailed inves­ tigation into their enigmatic adaptations, tardigrades are gaining increasing attention. In this graphical review, we provide an outline of survival strategies found among tardigrades and we summarize current knowledge of the adaptive mechanisms that underlie their unique tolerance to extreme or changing environments

Tardigrades are microscopic bilaterians belonging to phylum Tar­ shock, in liquid nitrogen, and even exposure to space vacuum digrada within the protostome superclade Ecdysozoa (Jørgensen et al., and cosmic radiation (Jonsson¨ et al., 2008). When excluding any 2018). These minute animals have an adult body length within the range dormancy periods, tardigrades generally live for a couple of months of 50–1200 μm and are found in permanent and temporary aquatic (Suzuki, 2003). However, cryptobiosis may extend their life span by environments across the globe (Møbjerg et al., 2018). The phylum numerous decades (Tsujimoto et al., 2016). comprises two major evolutionary lineages, i.e. eutardigrades (including With reference to the “Krogh’s principle” (reviewed in Jørgensen, apotardigrades) and the more diverse heterotardigrades. Limno- 2001)—that “for a large number of problems there will be some animal of terrestrial species living in terrestrial habitats (e.g. on mosses or li­ choice or a few such animals on which it can be most conveniently studied” chens) are only active when covered by a thin film of water. During (Krogh, 1929)—and at the same time acknowledging Krogh’s warnings , tardigrades quickly lose extra- and intra-cellular water against generalizations and his profound call to study “vital functions in retaining as little as 2–3% of their body water (Crowe, 1972) and all their aspects throughout the myriads of organisms” (Krogh, 1929), tar­ reducing body volume by as much as 85–90% (Halberg et al., 2013), digrades are obvious organisms for physiological studies. Cryptobiosis while entering the so-called cryptobiotic “tun” state (Fig. 1). The ability challenges our perception of the transition between life and death of an to enter cryptobiosis (latent or hidden life), where metabolism comes to organism. Understanding the mechanisms that underlie the ability to a reversible standstill, is widespread across life kingdoms (Clegg, 2001). stabilize biological structures, from macromolecules across cellular, , rotifers and tardigrades are metazoans with the ability to tissue and organ levels to the whole animal, and subsequently restart life enter cryptobiosis at all stages of their life cycle, as eggs as well as in after years of metabolic suspension has great potential for translational juvenile and adult stages. Cryptobiosis seems to provide animals with a and applied sciences. As new molecular tools allow for increasingly potential to survive conditions that are far beyond any constraints set by detailed investigations, tardigrades are indeed gaining attention by their normal environment. Being particularly resilient in the crypto­ physiologists and biochemists. biotic tun-state, tardigrades are renowned for their ability to tolerate a Survival strategies among extant tardigrades include osmoregula­ variety of extreme conditions, including desiccation, severe osmotic tion, as well as cryptobiosis and diapause in the form of encystment,

* Corresponding author. E-mail address: [email protected] (N. Møbjerg). https://doi.org/10.1016/j.cbpa.2020.110890 Received 14 December 2020; Received in revised form 21 December 2020; Accepted 21 December 2020 Available online 26 December 2020 1095-6433/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). N. Møbjerg and R.C. Neves Comparative Biochemistry and Physiology, Part A 254 (2021) 110890

Fig. 1. Light micrographs of the limno- terrestrial eutardigrade Ramazzottius varieornatus in the active state (dorsal view) and tun state (ventral view); anterior faces right in both aspects. With strong cryptobiotic abilities, this extremotolerant endures a range of extreme conditions, including desiccation and high osmolalities (> 2000 mOsm kg 1) of non-electrolytes (Heidemann et al., 2016). During desic­ cation and exposure to high osmolality the tardigrade will contract its body and withdraw its legs to form a tun. Upon return to favourable condi­ tions (i.e., high water potential) the tun extends its body, draws its legs out and, eventually, resumes activity. The cur­ rent tun was induced by a high osmo­ lality sucrose .

cyclomorphosis and resting eggs (Fig. 2). Extremotolerant tardigrades external changes in factors such as and tension rapidly enter cryptobiosis in response to a sudden environmental (Guidetti and Møbjerg, 2018). During encystment, tardigrades produce change, and quickly revive from their ametabolic state upon removal of several protective cuticular coats, while undergoing profound morpho­ the stress factor. Cryptobiosis may be induced by a range of extreme logical changes in the buccal-pharyngeal apparatus, claws and gonads conditions leading to different cryptobiotic sub-states, namely anhy­ (Guidetti and Møbjerg, 2018). Cyst formation in moss-dwelling tardi­ drobiosis, osmobiosis, cryobiosis and possibly anoxybiosis and chemo­ grades may occur as part of a seasonal cyclic event, and in the marine biosis. Most notably are stress conditions that force the tardigrade to eutardigrade Halobiotus crispae cyclomorphosis includes the production deal with water loss, i.e. desiccation (anhydrobiosis), rise in external of a cyst-like stage (Guidetti and Møbjerg, 2018). In addition, resting osmotic (osmobiosis) or freezing (cryobiosis). Lack of oxygen eggs have been reported. These diapausing eggs, which are laid in (anoxybiosis) and environmental toxicants (chemobiosis) may also response to an environmental deterioration, require a sequence of induce quiescence; however, conclusive empirical evidence on these two stimuli (e.g. dehydration followed by rehydration) in order to hatch forms of cryptobiosis is currently lacking. Anhydrobiosis, osmobioisis (reviewed in Guidetti et al., 2011). and possibly chemobiosis are cryptobiotic states characterized by tun Tardigrades can be extremely tolerant to environmental stress even formation. The ability to form tuns is present among all extant tardi­ in their active states (Møbjerg et al., 2011). The latter is exemplifiedby grade lineages, indicating that this is an ancient and homologous trait marine tidal species that osmoregulate over a range of external salinities ◦ (Hygum et al., 2016). Tun formation likely evolved within an ancient (2–40 ppt) and avoid freezing by supercooling to around 20 C (Hal­ marine environment as a response to fluctuationsin abiotic factors, such berg et al., 2009). Furthermore, limno-terrestrial species generally as external osmotic pressure. During desiccation, limno-terrestrial tar­ handle extreme levels of radiation with LD50 values for gamma and digrades generally enter the tun state within half an hour, and marine heavy ion radiation in adults of up 5 and 10 kGy, respectively (Jonsson,¨ tidal species may enter the state in the course of seconds (Sørensen- 2019). Interestingly, however, active tardigrades are sensitive to high ◦ Hygum et al., 2018). This is possibly due to the highly permeable nature with estimated LD50 values in the range 29–37 C, of tardigrade epithelia and cell membranes as well as a strategy among depending on species and experimental approach (reviewed in Neves extremotolerant species to constitutively express bioprotectants et al., 2020a). Indeed, tardigrades seem to have a restricted tolerance to required for cryptobiotic survival. high temperatures even in the tun state, with estimated LD50 values for ◦ ◦ ◦ Whereas cryptobiosis evolved as a quick response to sudden changes desiccated Ramazzottius varieornatus of 83 C, 63 C and 56 C following in the environment, encystment represents a slower emerging form of 1 h, 24 h and 1 week exposures, respectively (Neves et al., 2020a, dormancy that likely is induced by endogenous stimuli as well as 2020b). The latter may suggest that high temperatures destabilize and

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Fig. 2. Schematic illustration of survival strategies in tardigrades, including cryptobiosis, diapause and regulation in the active state. Cryptobiosis can be induced by a range of extreme environmental conditions. Anhydrobiosis, osmobioisis and possibly chemobiosis are cryptobiotic sub-states characterized by formation of a tun (compare to Fig. 1). In striking contrast, anoxybiosis (prompted by oxygen depletion) and cryobiosis (induced by extremely low temperatures) do not lead to tun formation. A turgid and elongated body characterizes oxygen depleted tardigrades, while tardigrades in cryobiosis may have a partly contracted body. Importantly, true cryobionts seem to lack a lower lethal temperature. Diapause in the form of encystment and cyclomorphosis implicate cyclic changes in the morphology and physiology of the tardigrades and, noteworthy, are directly related to the molting process. The hatching of resting eggs requires an environmental cue such as rehydration after a period of desiccation. In addition, tardigrade eggs may also enter any of the cryptobiosis forms endured by adults. Active state tardigrades can be extremely tolerant to environmental stress, osmoregulating when facing large fluctuation in external salinity, avoiding freezing by supercooling and handling extreme levels of ionizing radiation. Interestingly, however, tardigrades are sensitive to high temperatures and even the otherwise highly resistant tun, seems to have a restricted timeframe for high temperature tolerance. denature proteins and other essential biological macromolecules. dimensional structure of the desiccated tun state, with filaments lock­ Tardigrades lack discrete respiratory and circulatory systems. In­ ing in a rigor mortis-like state. In addition, actin and other cytoskeletal ternal organs of active, hydrated tardigrades are surrounded by a fluid filaments within epithelia may stabilize anhydrobiotic cell structure. filled body cavity that may assist in movement of various solutes Physiological and molecular studies on tardigrades have been chal­ (Møbjerg et al., 2018). Somatic muscles attach to the cuticle (Fig. 3) and lenged by their small size and the small (picogram) quantities of DNA locomotion relies on regulation of body volume—tardigrades hyper- and RNA available from single individuals, but a number of recent regulate ensuring a constant influx of water with excess water likely studies involve genomics, transcriptomics and proteomics. The compact being expelled by osmoregulatory epithelia of the epidermis, midgut genomes of the extremotolerant, Ramazzottius cf. varieornatus (~55 Mb), and, in eutardigrades, Malpighian tubules (Møbjerg et al., 2018). So­ and the much less tolerant, but commercially available Hypsibius exem­ matic as well as visceral muscles participate in tun formation with the plaris (~100 Mb), were recently released (Hashimoto et al., 2016; latter directing internal organ rearrangement, while dorsal, ventral, and Koutsovoulos et al., 2016) and candidate genes with proposed signifi­ lateral muscle groups provide body contraction and the leg musculature cance for tardigrade stress tolerance are currently being identified.The retracts the four pair of legs (Halberg et al., 2013). Importantly, muscle latter, for example, involve several groups of intrinsically disordered protein filaments seem to play a vital role for stabilizing the three- proteins found within the eutardigrade lineage (Hashimoto et al., 2016).

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Fig. 3. Schematic representation of somatic muscles (ventral view) involved in tun formation of a eutardi­ grade, Ricthersius cf. coronifer (modified from Halberg et al., 2013); anterior faces upwards in both aspects. The somatic musculature attaches to the cuticle at distinct attachment sites, not all visible in the current represen­ tation. Muscle fibers involved in locomotion of the hy­ drated, active tardigrade (A) participate in body contraction during tun formation (B). Indeed, the contraction of dorsal, lateral and ventral muscles as well as leg muscles mediate a structural reorganization of the tardigrade during formation of the desiccated tun state. Muscle protein filaments likely play a vital role for stabi­ lizing the three-dimensional structure of the desiccated tun state, locking filamentsin a rigor mortis-like state. The body musculature is also essential for maintaining the structural integrity when the tardigrade resumes activity following rehydration. Note the relative position of the pharyngeal bulb (pb) and the midgut (mg) both in the extended and contracted body. Visceral muscles (not shown) likely participate in tun formation with the latter directing internal organ rearrangement.

Very little is known of cell physiology in tardigrades. The available contribute to protection of cell structure in a synergistic effect with other transcriptomic data indicate that tardigrades express a full repertoire of bioprotectants, such as heat shock proteins (HSP) and late embryogen­ membrane transporters, including membrane pumps, numerous solute esis abundant (LEA) proteins. Intrinsic disordered LEA proteins appear carriers, a variety of ion channels and aquaporins. For example, exper­ to be present in all investigated tardigrade species. In addition, a full imental evidence of organic anion secretion by the tardigrade midgut repertoire of heat shock proteins seems conserved in tardigrades, suggests a potential pathway for secretion of nitrogenous waste with including Hsp70, Hsp40 and various small heat shock proteins. More + + + apical H and basolateral Na /K pumps possibly delivering the driving recently, it has been suggested that tardigrade-specific intrinsically force for transepithelial transport (Fig. 4; Halberg and Møbjerg, 2012). disordered proteins can provide structural stabilization during desicca­ Importantly, transcriptomics as well as experimental data indicate that tion through vitrification (Boothby et al., 2017). In addition, a highly strong cryptobionts, such as R. cf. varieornatus, constitutively express effective antioxidant defense apparatus as well as high fidelity DNA bioprotectants necessary for cryptobiotic survival, whereas less tolerant repair systems are likely crucial for cryptobiotic survival (Kamilari et al., species, such as H. exemplaris need a period of preconditioning in order 2019). Accordingly, tardigrades seem to have a comprehensive number to upregulate expression of essential protectants and survive extreme of genes encoding proteins involved in antioxidant defense. It was conditions. Generally, comparative genomics and transcriptomics reveal recently shown that the tardigrade damage suppressor protein (Dsup) a high degree of divergence between tardigrade species, indicating that binds to nucleosomes protecting chromosomal DNA from reactive hy­ different evolutionary lineages exhibit unique physiological and mo­ droxyl radicals (Chavez et al., 2019). Interestingly, Dsup gene expres­ lecular adaptations (Kamilari et al., 2019). Among the bioprotectants sion enhances tolerance of tobacco plants to genomutagenic stress with relevance for cryptobiotic survival are disaccharides such as (Kirke et al., 2020). Thus, while the secrets of latent life in many aspects and sucrose, in addition to the polyol glycerol. Trehalose has still remain a mystery, recent research is beginning to unravel some of traditionally been identified as a molecular stabilizer replacing water the underlying mechanisms. during desiccation in Artemia (brine shrimp) cysts, thereby protecting cellular structures such as plasma membranes, and at the same time Declaration of competing interest stabilizing the cell through vitrification (reviewed in Hibshman et al., 2020). Some, but not all, tardigrades accumulate trehalose during The authors declare that they have no known competing financial desiccation, though in much lower concentration as compared with interests or personal relationships that could have appeared to influence Artemia, and several tardigrade species appear to lack enzymes required the work reported in this paper. for trehalose synthesis. Trehalose accumulation may, nevertheless,

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Fig. 4. Schematic illustration of cellular and molecular mechanisms potentially involved in epithelial transport and anhydrobiotic survival of tardigrades as exemplifiedby a midgut epithelial cell (based on several sources, including Boothby et al., 2017, Chavez et al., 2019, Halberg and Møbjerg, 2012, Hashimoto et al., + + + 2016, Hibshman et al., 2020; Kamilari et al., 2019). The Na /K -ATPase and the V-type H -ATPase are likely candidates providing electrochemical driving force for transepithelial movement of ions and organic molecules over cell membranes of hydrated active tardigrades. Experimental evidence in the eutardigrade Halobiotus crispae indicates that solute carriers may transport organic anions (AO ), providing a potential pathway for secretion of nitrogenous waste. An assortment of bio­ protectants may play important roles in tardigrade , protecting cell membranes, proteins and DNA against damage during desiccation. Among the proposed protectants are disaccharides such as trehalose, heat-shock proteins, LEA (Late Embryogenesis Abundant) proteins and tardigrade-specificintrinsically disordered proteins. Trehalose may help stabilize proteins, nucleic acids and membrane lipids by replacing water during desiccation, but also by immobilizing macromolecules following formation of a glassy matrix through vitrification. Heat-shock and LEA proteins are suggested to have a role preventing protein aggre­ gation during desiccation, acting as molecular shields and chaperones, and/or by being involved in the repair process of damaged proteins during rehydration. Recently identified, heat soluble tardigrade-specific intrinsically disordered proteins (CAHS, MAHS, SAHS) appear to be unique to the eutardigrade lineage. These proteins have been associated with anhydrobiotic survival and are constitutively expressed during dehydration and rehydration in Ramazzottius cf. varieornatus. In this species, RvLEAM (a group 3 LEA protein) and MAHS have been identifiedas potent mitochondrial protectants, while the unstructured-nuclear protein Dsup binds to nucleosomes and protects DNA from reactive hydroxyl radicals. Potential cell damage due to is furthermore prevented by superoxide dismutases, glutathione peroxidases and catalases (catalases are not present in all tardigrades).

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