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Identification of Anhydrobiosis-related Genes from an Expressed Sequence Tag Database in the Cryptobiotic Midge Polypedilum vanderplanki (Diptera; Chironomidae)*

Open AccessPublished:September 10, 2010DOI:https://doi.org/10.1074/jbc.M110.150623
      Some organisms are able to survive the loss of almost all their body water content, entering a latent state known as anhydrobiosis. The sleeping chironomid (Polypedilum vanderplanki) lives in the semi-arid regions of Africa, and its larvae can survive desiccation in an anhydrobiotic form during the dry season. To unveil the molecular mechanisms of this resistance to desiccation, an anhydrobiosis-related Expressed Sequence Tag (EST) database was obtained from the sequences of three cDNA libraries constructed from P. vanderplanki larvae after 0, 12, and 36 h of desiccation. The database contained 15,056 ESTs distributed into 4,807 UniGene clusters. ESTs were classified according to gene ontology categories, and putative expression patterns were deduced for all clusters on the basis of the number of clones in each library; expression patterns were confirmed by real-time PCR for selected genes. Among up-regulated genes, antioxidants, late embryogenesis abundant (LEA) proteins, and heat shock proteins (Hsps) were identified as important groups for anhydrobiosis. Genes related to trehalose metabolism and various transporters were also strongly induced by desiccation. Those results suggest that the oxidative stress response plays a central role in successful anhydrobiosis. Similarly, protein denaturation and aggregation may be prevented by marked up-regulation of Hsps and the anhydrobiosis-specific LEA proteins. A third major feature is the predicted increase in trehalose synthesis and in the expression of various transporter proteins allowing the distribution of trehalose and other solutes to all tissues.

      Introduction

      Water is essential for life, and terrestrial organisms have developed various strategies to avoid water loss, which would ultimately be fatal. Small organisms are particularly sensitive to desiccation due to their relatively large body-surface-to-volume ratio (
      • Danks H.V.
      ,
      • Gibbs A.G.
      ). Under extreme conditions, such as drought or in arid environments, animals may avoid desiccation in a behavioral manner (by migration or burying deeply in the soil) or physiologically (by entering into diapause, or estivation). Some aquatic insects, which can resist moderate desiccation, secrete a thick cuticle, thus limiting the rate of water loss (
      • Suemoto T.
      • Kawai K.
      • Imabayashi H.
      ,
      • Nakahara Y.
      • Watanabe M.
      • Fujita A.
      • Kanamori Y.
      • Tanaka D.
      • Iwata K.
      • Furuki T.
      • Sakurai M.
      • Kikawada T.
      • Okuda T.
      ). Alternatively, various organisms have developed the opposite strategy; they allow water loss relatively easily but are able to survive essentially complete desiccation (
      • Nakahara Y.
      • Watanabe M.
      • Fujita A.
      • Kanamori Y.
      • Tanaka D.
      • Iwata K.
      • Furuki T.
      • Sakurai M.
      • Kikawada T.
      • Okuda T.
      ). This phenomenon, known as anhydrobiosis, is a specific example of the more general cryptobiosis, which is an ametabolic state induced by various adverse environmental conditions, such as desiccation, low temperature, or lack of oxygen. Cryptobiosis was first defined by Keilin (
      • Keilin D.
      ), although the observation of bdelloid rotifers resurrecting from dry mud samples had already been described in the early 18th century. Anhydrobiosis represents an extreme case of dormancy during which the water content falls to less than 3% of the body weight and no metabolic activity can be detected (
      • Crowe J.H.
      • Hoekstra F.A.
      • Crowe L.M.
      ,
      • Watanabe M.
      • Kikawada T.
      • Okuda T.
      ). However, anhydrobiotic individuals can revive and rapidly resume activity once in contact with water (
      • Sømme L.
      ). The most familiar cases of anhydrobiosis are plant seeds or fungal spores, but many microscopic organisms, such as terrestrial tardigrades, bdelloid rotifers, or nematodes, can survive in an anhydrobiotic state (
      • Keilin D.
      ,
      • Sømme L.
      ,
      • Clegg J.S.
      ). For plant seeds or the cysts of some crustacean embryos (Artemia, Triops), anhydrobiosis is only observed in early developmental stages (
      • Hochachka D.W.
      • Guppy M.
      ). In contrast, tardigrades and rotifers enter anhydrobiosis also during postembryonic stages in response to environmental change and can experience anhydrobiosis several times successively (
      • Watanabe M.
      • Kikawada T.
      • Fujita A.
      • Forczek E.
      • Adati T.
      • Okuda T.
      ,
      • Ricci C.
      ). This is also the case for the sleeping chironomid (Polypedilum vanderplanki), the largest anhydrobiotic animal known to date. However, in P. vanderplanki, only the larval stages are able to enter anhydrobiosis; the other stages (eggs, pupae, adults) last only a couple of days, and such a rapid development is more adapted to a strategy of desiccation avoidance.
      P. vanderplanki lives in small temporary rock pools in the semi-arid regions of the African continent (
      • Hinton H.E.
      ). Its larvae are found in small tubular nests in the mud at the bottom of pools, and when the water dries up, they can survive in an almost completely desiccated anhydrobiotic state throughout the dry season, which may last up to 8 months (
      • Hinton H.E.
      ,
      • Kikawada T.
      • Minakawa N.
      • Watanabe M.
      • Okuda T.
      ). To achieve successful anhydrobiosis, P. vanderplanki larvae need an appropriate desiccation rate and, experimentally, at least 2 days are required for the necessary physiological adjustments; appropriate conditions for successful anhydrobiosis have been reproduced in the laboratory (
      • Nakahara Y.
      • Watanabe M.
      • Fujita A.
      • Kanamori Y.
      • Tanaka D.
      • Iwata K.
      • Furuki T.
      • Sakurai M.
      • Kikawada T.
      • Okuda T.
      ,
      • Watanabe M.
      • Kikawada T.
      • Okuda T.
      ,
      • Kikawada T.
      • Minakawa N.
      • Watanabe M.
      • Okuda T.
      ). Viably desiccated larvae will recover normal activity within 1 h of contact with water (see Fig. 1). The mechanism of anhydrobiosis induction in P. vanderplanki was found to be independent of any control by the central nervous system or endocrine influence (
      • Watanabe M.
      • Kikawada T.
      • Minagawa N.
      • Yukuhiro F.
      • Okuda T.
      ), and salt concentration apparently plays an important role in the induction of this phenomenon (
      • Watanabe M.
      • Kikawada T.
      • Okuda T.
      ). Intriguingly, anhydrobiosis can even be successfully induced in isolated fat body tissue (
      • Watanabe M.
      • Kikawada T.
      • Fujita A.
      • Okuda T.
      ). As observed in many other anhydrobionts (
      • Madin K.A.
      • Crowe J.H.
      ,
      • Crowe L.M.
      ,
      • Clegg J.S.
      • Campagna V.
      ) with the exception of bdelloid rotifers and some tardigrades (
      • Hengherr S.
      • Heyer A.G.
      • Köhler H.R.
      • Schill R.O.
      ,
      • Tunnacliffe A.
      • Lapinski J.
      • McGee B.
      ), the accumulation of trehalose in the tissues of P. vanderplanki has a central protective role against desiccation stress, and the induction period of 2 days leading up to successful anhydrobiosis is thus necessary for appropriate levels of trehalose accumulation in the larval body (
      • Watanabe M.
      • Kikawada T.
      • Okuda T.
      ,
      • Kikawada T.
      • Minakawa N.
      • Watanabe M.
      • Okuda T.
      ,
      • Watanabe M.
      • Kikawada T.
      • Minagawa N.
      • Yukuhiro F.
      • Okuda T.
      ). During this induction phase, trehalose is thought first to act as a water replacement molecule and then to form a glassy matrix, which protects cells and their biomolecules against desiccation stress (
      • Sakurai M.
      • Furuki T.
      • Akao K.
      • Tanaka D.
      • Nakahara Y.
      • Kikawada T.
      • Watanabe M.
      • Okuda T.
      ).
      Figure thumbnail gr1
      FIGURE 1Anhydrobiotic cycle of P. vanderplanki larvae. In laboratory conditions, the process of desiccation lasts 48 h, after active larvae have been removed from water. Water loss accelerates after 24 h of desiccation treatment, at which time trehalose accumulates in the body of larvae. After 48 h of desiccation, the larvae reach the anhydrobiotic state and can maintain this state for several months and even years. Once larvae are immersed in water, the process of rehydration takes place rapidly. The anhydrobiotic larvae quickly absorb water, and muscular contractions can be observed after a few minutes. They recover their original active state about 20 min to 1 h after the beginning of rehydration. In the present study, mRNAs used to construct the anhydrobiosis-related EST library were collected from active larvae and from individuals at 12 and 36 h after the beginning of desiccation treatment.
      Apart from the important role of trehalose, the molecular mechanisms of anhydrobiosis are still poorly understood. Molecular studies led to the identification in nematodes and in a rotifer of proteins related to desiccation tolerance, called late embryogenesis abundant (LEA)
      The abbreviations used are: LEA
      late embryogenesis abundant
      EST
      expressed sequence tag
      Hsp
      heat shock protein
      ROS
      reactive oxygen species.
      proteins, which were originally known only in plants (
      • Tunnacliffe A.
      • Lapinski J.
      • McGee B.
      ,
      • Browne J.
      • Tunnacliffe A.
      • Burnell A.
      ,
      • Denekamp N.Y.
      • Reinhardt R.
      • Kube M.
      • Lubzens E.
      ). Anhydrobiosis-related gene expression has been also investigated in a brine shrimp (
      • Qiu Z.
      • Tsoi S.C.
      • MacRae T.H.
      ,
      • Chen W.H.
      • Ge X.
      • Wang W.
      • Yu J.
      • Hu S.
      ) and a rotifer species, allowing the identification of desiccation-responsive genes such as LEA proteins, heat shock proteins, and some antioxidants. However, more information about the molecular mechanisms of desiccation resistance is needed to fully understand the anhydrobiosis phenomenon. The construction of an anhydrobiosis-related EST library in P. vanderplanki (see Fig. 1) has already led to the characterization of genes involved in the desiccation tolerance of this chironomid, such as LEA proteins (
      • Kikawada T.
      • Nakahara Y.
      • Kanamori Y.
      • Iwata K.
      • Watanabe M.
      • McGee B.
      • Tunnacliffe A.
      • Okuda T.
      ), the trehalose transporter Tret1 (
      • Kikawada T.
      • Saito A.
      • Kanamori Y.
      • Nakahara Y.
      • Iwata K.
      • Tanaka D.
      • Watanabe M.
      • Okuda T.
      ), or aquaporins (
      • Kikawada T.
      • Saito A.
      • Kanamori Y.
      • Fujita M.
      • Snigórska K.
      • Watanabe M.
      • Okuda T.
      ). The present work describes the construction and general analysis of this anhydrobiosis-related EST database. Many genes involved in the protection of cellular components and biomolecules, such as chaperone proteins and enzymes governing trehalose metabolism, were found to be up-regulated during the process of desiccation, and the dataset also provides evidence that the response to oxidative stress appears to play a central role in anhydrobiosis.

      Acknowledgments

      We are grateful to Akihiko Fujita and Yoko Saito for the maintenance and rearing of P. vanderplanki. We also thank Takashi Noda for help with statistics.

      REFERENCES

        • Danks H.V.
        J. Insect Physiol. 2000; 46: 837-852
        • Gibbs A.G.
        Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 2002; 133: 781-789
        • Suemoto T.
        • Kawai K.
        • Imabayashi H.
        Hydrobiologia. 2004; 57: 107-114
        • Nakahara Y.
        • Watanabe M.
        • Fujita A.
        • Kanamori Y.
        • Tanaka D.
        • Iwata K.
        • Furuki T.
        • Sakurai M.
        • Kikawada T.
        • Okuda T.
        J. Insect Physiol. 2008; 54: 1220-1225
        • Keilin D.
        Proc. R. Soc. Lond. B Biol. Sci. 1959; 150: 149-191
        • Crowe J.H.
        • Hoekstra F.A.
        • Crowe L.M.
        Annu. Rev. Physiol. 1992; 54: 579-599
        • Watanabe M.
        • Kikawada T.
        • Okuda T.
        J. Exp. Biol. 2003; 206: 2281-2286
        • Sømme L.
        Invertebrates in Hot and Cold Arid Environments. Springer-Verlag, New York1995: 95-114
        • Clegg J.S.
        Comp. Biochem. Physiol. B. 2001; 128: 613-624
        • Hochachka D.W.
        • Guppy M.
        Metabolic Arrest and the Control of Biological Time. Harvard University Press, Cambridge, Massachusetts and London1987: 146-165
        • Watanabe M.
        • Kikawada T.
        • Fujita A.
        • Forczek E.
        • Adati T.
        • Okuda T.
        Eur. J. Entomology. 2004; 101: 439-444
        • Ricci C.
        Hydrobiologia. 2001; 446/447: 1-11
        • Hinton H.E.
        Proc. Zool. Soc. Lond. 1951; 121: 371-380
        • Hinton H.E.
        J. Insect Physiol. 1960; 5: 286-300
        • Kikawada T.
        • Minakawa N.
        • Watanabe M.
        • Okuda T.
        Integ. Comp. Biol. 2005; 45: 710-714
        • Watanabe M.
        • Kikawada T.
        • Minagawa N.
        • Yukuhiro F.
        • Okuda T.
        J. Exp. Biol. 2002; 205: 2799-2802
        • Watanabe M.
        • Kikawada T.
        • Fujita A.
        • Okuda T.
        J. Insect Physiol. 2005; 51: 727-731
        • Madin K.A.
        • Crowe J.H.
        J. Exp. Zool. 1975; 193: 335-342
        • Crowe L.M.
        Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2002; 131: 505-513
        • Clegg J.S.
        • Campagna V.
        Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 2006; 145: 119-125
        • Hengherr S.
        • Heyer A.G.
        • Köhler H.R.
        • Schill R.O.
        FEBS J. 2008; 275: 281-288
        • Tunnacliffe A.
        • Lapinski J.
        • McGee B.
        Hydrobiologia. 2005; 546: 315-321
        • Sakurai M.
        • Furuki T.
        • Akao K.
        • Tanaka D.
        • Nakahara Y.
        • Kikawada T.
        • Watanabe M.
        • Okuda T.
        Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 5093-5098
        • Browne J.
        • Tunnacliffe A.
        • Burnell A.
        Nature. 2002; 416: 38
        • Denekamp N.Y.
        • Reinhardt R.
        • Kube M.
        • Lubzens E.
        Biol. Reprod. 2010; 82: 714-724
        • Qiu Z.
        • Tsoi S.C.
        • MacRae T.H.
        Mech. Dev. 2007; 124: 856-867
        • Chen W.H.
        • Ge X.
        • Wang W.
        • Yu J.
        • Hu S.
        BMC Genomics. 2009; 10: 52
        • Kikawada T.
        • Nakahara Y.
        • Kanamori Y.
        • Iwata K.
        • Watanabe M.
        • McGee B.
        • Tunnacliffe A.
        • Okuda T.
        Biochem. Biophys. Res. Commun. 2006; 348: 56-61
        • Kikawada T.
        • Saito A.
        • Kanamori Y.
        • Nakahara Y.
        • Iwata K.
        • Tanaka D.
        • Watanabe M.
        • Okuda T.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 11585-11590
        • Kikawada T.
        • Saito A.
        • Kanamori Y.
        • Fujita M.
        • Snigórska K.
        • Watanabe M.
        • Okuda T.
        Biochim. Biophys. Acta. 2008; 1778: 514-520
        • Suetsugu Y.
        • Minami H.
        • Shimomura M.
        • Sasanuma S.
        • Narukawa J.
        • Mita K.
        • Yamamoto K.
        BMC Genomics. 2007; 8: 314
        • Eigenheer A.L.
        • Keeling C.I.
        • Young S.
        • Tittiger C.
        Gene. 2003; 316: 127-136
        • Tagu D.
        • Prunier-Leterme N.
        • Legeai F.
        • Gauthier J.P.
        • Duclert A.
        • Sabater-Muñoz B.
        • Bonhomme J.
        • Simon J.C.
        Insect Biochem. Mol. Biol. 2004; 34: 809-822
        • Wu-Scharf D.
        • Scharf M.E.
        • Pittendrigh B.R.
        • Bennett G.W.
        Sociobiology. 2003; 41: 479-490
        • Hankeln T.
        • Amid C.
        • Weich B.
        • Niessing J.
        • Schmidt E.R.
        J. Mol. Evol. 1998; 46: 589-601
        • Hoback W.W.
        • Stanley D.W.
        J. Insect Physiol. 2001; 47: 533-542
        • Pereira Ede J.
        • Panek A.D.
        • Eleutherio E.C.
        Cell Stress Chaperones. 2003; 8: 120-124
        • França M.B.
        • Panek A.D.
        • Eleutherio E.C.
        Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 2007; 146: 621-631
        • Corona M.
        • Robinson G.E.
        Insect Mol. Biol. 2006; 15: 687-701
        • Maulik N.
        • Das D.K.
        Biochim. Biophys. Acta. 2008; 1780: 1368-1382
        • Franco R.
        • Sánchez-Olea R.
        • Reyes-Reyes E.M.
        • Panayiotidis M.I.
        Mutat. Res. 2009; 674: 3-22
        • Limón-Pacheco J.
        • Gonsebatt M.E.
        Mutat. Res. 2009; 674: 137-147
        • Denekamp N.Y.
        • Thorne M.A.
        • Clark M.S.
        • Kube M.
        • Reinhardt R.
        • Lubzens E.
        BMC Genomics. 2009; 10: 108
        • Freitas D.R.
        • Rosa R.M.
        • Moraes J.
        • Campos E.
        • Logullo C.
        • Da Silva Vaz Jr., I.
        • Masuda A.
        Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 2007; 146: 688-694
        • Rizzo A.M.
        • Negroni M.
        • Altiero T.
        • Montorfano G.
        • Corsetto P.
        • Berselli P.
        • Berra B.
        • Guidetti R.
        • Rebecchi L.
        Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 2010; 156: 115-121
        • Haegeman A.
        • Jacob J.
        • Vanholme B.
        • Kyndt T.
        • Mitreva M.
        • Gheysen G.
        Mol. Biochem. Parasitol. 2009; 167: 32-40
        • Schokraie E.
        • Hotz-Wagenblatt A.
        • Warnken U.
        • Mali B.
        • Frohme M.
        • Förster F.
        • Dandekar T.
        • Hengherr S.
        • Schill R.O.
        • Schnölzer M.
        PLoS One. 2010; 5: e9502
        • Cuming A.C.
        Shewry P.R. Casey R. Seed Proteins. Kluwer Academic Publishers, The Netherlands1999: 753-780
        • Chakrabortee S.
        • Boschetti C.
        • Walton L.J.
        • Sarkar S.
        • Rubinsztein D.C.
        • Tunnacliffe A.
        Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 18073-18078
        • Shimizu T.
        • Kanamori Y.
        • Furuki T.
        • Kikawada T.
        • Okuda T.
        • Takahashi T.
        • Mihara H.
        • Sakurai M.
        Biochemistry. 2010; 49: 1093-1104
        • Parsell D.A.
        • Lindquist S.
        Annu. Rev. Genet. 1993; 27: 437-496
        • Berjak P.
        • Farrant J.M.
        • Pammenter N.W.
        Jenks M.A. Wood A.J. Plant Desiccation Tolerance. Blackwell Publishing, Oxford2007: 151-192
        • Hinault M.P.
        • Goloubinoff P.
        Adv. Exp. Med. Biol. 2007; 594: 47-54
        • Bukau B.
        • Horwich A.L.
        Cell. 1998; 92: 351-366
        • Narberhaus F.
        Microbiol. Mol. Biol. Rev. 2002; 66 (table of contents): 64-93
        • Lopez-Martinez G.
        • Benoit J.B.
        • Rinehart J.P.
        • Elnitsky M.A.
        • Lee Jr., R.E.
        • Denlinger D.L.
        J. Comp. Physiol. B. 2009; 179: 481-491
        • Viner R.I.
        • Clegg J.S.
        Cell Stress Chaperones. 2001; 6: 126-135
        • Arrigo A.P.
        Adv. Exp. Med. Biol. 2007; 594: 14-26
        • Lopez-Martinez G.
        • Elnitsky M.A.
        • Benoit J.B.
        • Lee Jr., R.E.
        • Denlinger D.L.
        Insect Biochem. Mol. Biol. 2008; 38: 796-804
        • Clegg J.S.
        Comp. Biochem. Physiol. 1965; 14: 135-143
        • Mitsumasu K.
        • Kanamori Y.
        • Fujita M.
        • Iwata K.
        • Tanaka D.
        • Kikuta S.
        • Watanabe M.
        • Cornette R.
        • Okuda T.
        • Kikawada T.
        FEBS J. 2010; 277: 4215-4228
        • Hediger M.A.
        • Romero M.F.
        • Peng J.B.
        • Rolfs A.
        • Takanaga H.
        • Bruford E.A.
        Pflugers Arch. 2004; 447: 465-468
        • Tarr P.T.
        • Tarling E.J.
        • Bojanic D.D.
        • Edwards P.A.
        • Baldán A.
        Biochim. Biophys. Acta. 2009; 1791: 584-593
        • Narayanaswami V.
        • Ryan R.O.
        Biochim. Biophys. Acta. 2000; 1483: 15-36
        • Wharton D.A.
        J. Comp. Physiol. B. 2003; 173: 621-628
        • Adhikari B.N.
        • Wall D.H.
        • Adams B.J.
        BMC Genomics. 2009; 10: 69
        • Meier R.
        • Tomizaki T.
        • Schulze-Briese C.
        • Baumann U.
        • Stocker A.
        J. Mol. Biol. 2003; 331: 725-734
        • Burmester T.
        • Hankeln T.
        J. Insect Physiol. 2007; 53: 285-294
        • Wajcman H.
        • Kiger L.
        • Marden M.C.
        C. R. Biol. 2009; 332: 273-282
        • Fan G.H.
        • Lapierre L.A.
        • Goldenring J.R.
        • Richmond A.
        Blood. 2003; 101: 2115-2124
        • Sugasawa K.
        • Shimizu Y.
        • Iwai S.
        • Hanaoka F.
        DNA Repair. 2002; 1: 95-107
        • Hand S.C.
        • Jones D.
        • Menze M.A.
        • Witt T.L.
        J. Exp. Zool. A Ecol. Genet. Physiol. 2007; 307: 62-66
        • Sharon M.A.
        • Kozarova A.
        • Clegg J.S.
        • Vacratsis P.O.
        • Warner A.H.
        Biochem. Cell Biol. 2009; 87: 415-430