Skip to main content
Advertisement

Main menu

  • Home
  • Articles
    • Accepted manuscripts
    • Issue in progress
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Special issues
    • Subject collections
    • Interviews
    • Sign up for alerts
  • About us
    • About JEB
    • Editors and Board
    • Editor biographies
    • Travelling Fellowships
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
    • Outstanding paper prize
    • Biology Open transfer
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contacts
    • Contact JEB
    • Subscriptions
    • Advertising
    • Feedback
    • For library administrators
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

User menu

  • Log in

Search

  • Advanced search
Journal of Experimental Biology
  • COB
    • About The Company of Biologists
    • Development
    • Journal of Cell Science
    • Journal of Experimental Biology
    • Disease Models & Mechanisms
    • Biology Open

supporting biologistsinspiring biology

Journal of Experimental Biology

  • Log in
Advanced search

RSS  Twitter  Facebook  YouTube  

  • Home
  • Articles
    • Accepted manuscripts
    • Issue in progress
    • Latest complete issue
    • Issue archive
    • Archive by article type
    • Special issues
    • Subject collections
    • Interviews
    • Sign up for alerts
  • About us
    • About JEB
    • Editors and Board
    • Editor biographies
    • Travelling Fellowships
    • Grants and funding
    • Journal Meetings
    • Workshops
    • The Company of Biologists
    • Journal news
  • For authors
    • Submit a manuscript
    • Aims and scope
    • Presubmission enquiries
    • Article types
    • Manuscript preparation
    • Cover suggestions
    • Editorial process
    • Promoting your paper
    • Open Access
    • Outstanding paper prize
    • Biology Open transfer
  • Journal info
    • Journal policies
    • Rights and permissions
    • Media policies
    • Reviewer guide
    • Sign up for alerts
  • Contacts
    • Contact JEB
    • Subscriptions
    • Advertising
    • Feedback
    • For library administrators
Research Article
Mechanism allowing an insect to survive complete dehydration and extreme temperatures
M. Watanabe, T. Kikawada, N. Minagawa, F. Yukuhiro, T. Okuda
Journal of Experimental Biology 2002 205: 2799-2802;
M. Watanabe
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
T. Kikawada
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
N. Minagawa
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
F. Yukuhiro
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
T. Okuda
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • Article
  • Figures & tables
  • Info & metrics
  • PDF
Loading

SUMMARY

Cryptobiosis describes the state of an organism whose body water is completely dehydrated and metabolic activity has become undetectable. Our study aimed to elucidate the physiological mechanism of cryptobiosis in the highest cryptobiotic invertebrate, Polypedilum vanderplanki. Larvae of this insect rapidly accumulated a large amount of the carbohydrate, trehalose, (18% of dry body mass) during desiccation for 2 days, suggesting that a high level of trehalose accumulation contributed to the successful induction of cryptobiosis in P. vanderplanki as well as in other lower cryptobiotic organisms. When larvae deprived of the brain, suboesophageal ganglion (SG) and thoracic ganglia (TG) were completely dehydrated and then rehydrated, they were able to recover and move actively. During desiccation, such larvae also accumulated trehalose, although only about half as much as the intact larvae. It is concluded that the brain, SG and TG do not affect the induction and termination of cryptobiosis, and hence in this higher multicellular animal cryptobiosis is independent of brain, SG and TG regulation, just as in plants or in unicellular organisms.

  • Polypedilum vanderplanki
  • Chironomidae
  • cryptobiosis
  • anhydrobiosis
  • trehalose

Introduction

Organisms have evolved various mechanisms for adaptation to adverse environmental conditions such as lack of water (cryptobiosis or anhydrobiosis), freezing temperatures (cryobiosis) and lack of oxygen (anoxia) (Hochachka and Somero, 1984). In plants or unicellular organisms that do not have a clearcut nervous system, individual tissues or cells express stress tolerance in response to a change in the environmental condition (Sakai and Larcher, 1987; Ingram and Bartels, 1996). However, vertebrates and higher invertebrates are believed to regulate stress tolerance directly or indirectly through the brain and become dormant when those stresses are severe and sustaining (Lyman et al., 1982; Denlinger, 1985; Danks, 2000).

A chironomid, Polypedilum vanderplanki Hint., is the largest multicellular animal known to tolerate almost complete dehydration without ill effect (Hinton, 1951, 1960a). Cryptobiotic larvae show extremely high thermal tolerance from -270°C to +106°C and can recover soon after prolonged dehydration of up to 17 years (Hinton, 1960a,b, 1968). However, the underlying molecular and metabolic mechanisms largely remain a mystery. Here we show that rapid accumulation of trehalose plays a key role in the successful induction of cryptobiosis, and that, surprisingly, cerebral regulation is not involved in the process of cryptobiosis.

Materials and methods

Insects

Cryptobiotic larvae of Polypedilum vanderplanki Hint. were collected from rock pools in Nigeria in 2000. They were transferred to the laboratory and put into a plastic container (200 mm×300 mm×100 mm) containing water (depth, 20-30 mm) on autoclaved soil (depth, 20-30 mm). The rearing water was aerated continuously. The container was covered with a nylon-mesh cage (200 mm×300 mm×250-300 mm). The larvae were reared for successive generations under controlled light (13 h: 11 h light:dark) and temperature (27°C).

Desiccating procedure

Groups of 3-5 larvae were placed on pieces of filter paper with 0.44 ml of distilled water in a glass Petri dish (diameter 65 mm, height, 20 mm). Two or three of these dishes were immediately transferred to a desiccator (<5% relative humidity) at room temperature (24-26°C) and gradually dried over a period of 48 h (0.22-0.23 ml day-1).

Desiccation and recovery of intact and treated larvae

After ligation (applied behind the head or thorax), the head or head and thorax segments were severed from final instar larvae of a similar body mass (approximately 1 mg) in iced water. The remaining body parts were incubated in distilled water for one day, and then completely dried over 3 days in the desiccator. Intact larvae were transferred directly to the desiccator. Subsequently, after being rehydrated by immersion in distilled water, the larvae were observed closely every 0.5-1 h to check their recovery. Larvae were judged to survive if they could repeatedly contract their abdomen. Because insects have an open circulatory system, radical treatments such as ligation and decapitation are routinely used, particularly in the field of insect endocrinology (Wigglesworth, 1972).

Sugar and polyol measurements

Each group of 3-5 intact or operated larvae was placed in a desiccating Petri dish or in distilled water for 12, 18, 24, 30, 36, 42, 48 or 72 h, and then homogenized individually with 0.1 mg of sorbitol as an internal standard in 0.2 ml of 90% ethanol. After membrane filtration (pore size 0.45 μm), the supernatant was dried under a stream of nitrogen gas at 60°C and the dried residue dissolved in 500 μl of MilliQ water (Millipore). The samples were analysed on a Shimadzu HPLC system (LC-10A system, Shimadzu, Japan) equipped with a guard column (Shim-pack SCR-C, 4.0 mm×50 mm; Shimadzu, Japan) connected to an analytical column (Shim-pack SCR-101C, 7.9 mm×300 mm; Shimadzu, Japan) and a reflective index detector (RID-6A; Shimadzu, Japan). The columns were heated to 80°C, and MilliQ water as the mobile phase was allowed to flow at the rate of 0.8 ml min-1. The injection volume was set at 10 or 20 μl. Standard trehalose and sorbitol solutions were prepared in MilliQ water in the range of 1-5,000 μg ml-1. From the HPLC profile, trehalose and sorbitol could be quantified, at least in the higher range.

Results and discussion

P. vanderplanki lives in the temporary rock pools of tropical Africa. When the pool dries up, the larva in its mud nest becomes dehydrated and remains desiccated until the next rain. Recently, we succeeded in the successive rearing of this species in the laboratory, and established a short-term (2 day) desiccating method for induction of cryptobiosis. Using this method, we examined the process and degree of larval dehydration.

Cryptobiotic larvae of this species were crumpled and sometimes folded in the middle, as is typical for larvae found in mud nests in the field. When given water, all of them recovered within half an hour and moved actively (Fig. 1A, Table 1). During the process of desiccation, the larvae accumulated a large amount of the sugar trehalose (average of approximately 18% of dry body mass: Intact, dry in Fig. 2). Other sugars and polyols were not detected.

Fig. 1.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 1.

P. vanderplanki during recovery from cryptobiosis. (A) Larva recovering from cryptobiosis at 0, 8, 16, 32 and 44 min after rehydration. (B) Decapitated cryptobiotic larva. (C) Decapitated larva on the second day after rehydration.

View this table:
  • View inline
  • View popup
Table 1.

Effect of head and thorax removal on induction and termination of cryptobiosis in larvae of P. vanderplanki

Fig. 2.
  • Download figure
  • Open in new tab
  • Download powerpoint
Fig. 2.

Change of trehalose level in P. vanderplanki larvae during desiccation. Intact, dry: intact larvae (filled circles); -H, dry: decapitated larvae (open triangles); -HT, dry: larvae from which the head and thoraces were removed (open circles); -H, wet: decapitated larvae in water (open squares). There were significant differences in trehalose content 12 h after desiccation in intact larvae, at 48 h and 72 h after desiccation in -H larvae, and at 48 h after dessication in -HT larvae (Mann—Whitney U test, P<0.05). Values are means ± S.D., N=3-9.

Cryptobiotic organisms usually contain a high concentration of disaccharides at the dry state (Crowe, 2002). In general, sucrose is found in seeds and higher plants. Lower plants, lower animals and microorganisms, such as Artemia cysts (Clegg, 1965), nematodes (Madin and Crowe, 1975; Womersley and Smith, 1981), fungus (Sussman and Lingappa, 1959) and bacteria (Payen, 1949; Clegg and Filosa, 1961), all accumulate trehalose at a high concentration of approximately 20% of the dry mass. It has been suggested that trehalose provides the most effective protection against dehydration because of its high ability for water replacement and glass formation (Crowe et al., 1987, 1992; Green and Angell, 1989). Similarly, in P. vanderplanki as a higher invertebrate, a high level of trehalose accumulation would contribute to successful induction of cryptobiosis.

To elucidate the possible role of the brain, suboesophageal ganglion (SG) and thoracic ganglia (TG) in cryptobiosis of P. vanderplanki, we used larvae deprived of the brain, SG and TG by ligation applied behind the head or thorax followed by decapitation or severance of head and thorax. After a 3-day desiccation, such larvae became crumbled, but not folded (Fig. 1B). When completely dehydrated larvae were later submerged in water, most of the decapitated larvae and half the number of those from which the head and thorax were removed were able to recover (Fig. 1C, Table 1). Indeed, some of the decapitated larvae moved actively and survived for more than 2 weeks after recovery.

The rehydration recovery time of the operated larvae was longer than that of intact larvae (Table 1). Larvae of P. vanderplanki are uniformly red due to equal distribution of hemoglobin in their hemolymph. During dehydration, the red color became more intensive in the apical and distal parts of the body (Fig. 1A), suggesting that body water evaporated mainly from the mouth and anus. In the decapitated larvae, both water loss during dehydration and penetration of water during rehydration appeared to occur mainly through the anus. The reduced capacity for water uptake possibly caused the delay in their recovery.

During desiccation, the treated larvae also accumulated a large amount of trehalose, although only about half as much as the intact larvae (-H, dry and -HT, dry in Fig. 2). By contrast, when the decapitated larvae were continuously incubated in water, they did not accumulate trehalose (-H, wet in Fig. 2). It must be stressed that the ligation and subsequent removal of the front body part were carried out in iced water and the larvae had not been exposed to air before transfer to the desiccation dish. Consequently, these larvae received no environmental signal of the forthcoming dehydration before the head and thorax were severed. Paralysis in iced water did not affect the induction of cryptobiosis. Cryptobiosis was successfully induced in decapitated larvae in which ligation and body severing was carried out without ice-water treatment (89%, N=9). We concluded that the brain, SG and TG did not affect the induction and termination of cryptobiosis, i.e. cryptobiosis occurs without the regulation of the brain, SG and TG, just as in plants and unicellular organisms. It would be interesting to examine whether such a regulatory system is operative in other cryptobiotic lower invertebrates, such as nematodes, water flea, rotifers and tardigrades (Young, 1985; Sømme, 1995).

Our findings may be greatly relevant to medical science. Currently, relatively long-term storage of living cells can be done only in cryoprotectant solution at extremely low subzero temperatures (Homma and Morimoto, 1997; Wolkers et al., 2002), and live human organs can be stored for only a short period in vitro (Cooper, 1991; Novick et al., 1992; Kenmochi et al., 1997). From this study, we suggest that individual organs and cells of cryptobiotic larvae, devoid of cerebral regulatory factors, could be stored at room temperature under dehydrated conditions. Further investigations to elucidate the mechanism of the successful induction and recovery of cryptobiosis in the `higher' invertebrate P. vanderplanki might be of enormous consequence for the field of cell and organ storage.

ACKNOWLEDGEMENTS

We thank Drs F. Sehnal, Barbara I. P. Barratt, S. Tanaka and M. Asahina for critical reading of the manuscript. This study was carried out as a part of `Ground-base Research Announcement for Space Utilization' promoted by the Japan Space Forum.

  • © The Company of Biologists Limited 2002

References

  1. ↵
    Clegg, J. S. (1965). The origin of trehalose and its significance during the formation of encysted dormant embryos of Artemia salina. Comp. Biochem. Physiol. 14,135 -143.
    OpenUrlPubMedWeb of Science
  2. ↵
    Clegg, J. S. and Filosa, M. F. (1961). Trehalose in the cellular slime mould Dictyostelium mucoroides.Nature 192,1077 -1078.
    OpenUrlPubMed
  3. ↵
    Cooper, J. D. (1991). Current status of lung transplantation. Transplant. Proc. 23,2107 -2114.
    OpenUrlPubMed
  4. ↵
    Crowe, L. M. (2002). Lessons from nature: the role of sugars in anhydrobiosis. Comp. Biochem. Physiol. 131A,505 -513.
    OpenUrl
  5. ↵
    Crowe, J. H., Crowe, L. M., Carpenter, J. F. and Wistrom, C. A. (1987). Stabilization of dry phospholipid bilayers and proteins by sugars. Biochem. J. 242, 1-10.
    OpenUrlFREE Full Text
  6. ↵
    Crowe, J. H., Hoekstra, F. A. and Crowe, L. M. (1992). Anhydrobiosis. Annu. Rev. Physiol. 54,579 -599.
    OpenUrlCrossRefPubMedWeb of Science
  7. ↵
    Danks, H. V. (2000). Dehydration in dormant insects. J. Insect Physiol. 46,837 -852.
    OpenUrlCrossRefPubMedWeb of Science
  8. ↵
    Denlinger, D. L. (1985). Hormonal control of diapause. In Comparative Insect Physiology, Biochemistry and Pharmacology (ed. G. A. Kerkut and L. Gilbert), pp.353 -412. Oxford: Pergamon Press.
  9. ↵
    Green, J. L. and Angell, C. A. (1989). Phase relations and vitrification in saccharide-water solutions and the trehalose anomaly. J. Phys. Chem. 93,2880 -2882.
    OpenUrlCrossRef
  10. ↵
    Hinton, H. E. (1951). A new chironomid from Africa, the larva of which can be dehydrated without injury. Proc. Zool. Soc. Lond. 121,371 -380.
    OpenUrl
  11. ↵
    Hinton, H. E. (1960a). Cryptobiosis in the larva of Polypedilum vanderplanki Hint. (Chironomidae). J. Insect Physiol. 5,286 -300.
    OpenUrlCrossRef
  12. ↵
    Hinton, H. E. (1960b). A fly larva that tolerates dehydration and temperatures of -270° to +102°C. Nature 188,336 -337.
    OpenUrlCrossRef
  13. ↵
    Hinton, H. E. (1968). Reversible suspension of metabolism and the origin of life. Proc. R. Soc. B 171, 43-57.
    OpenUrlPubMed
  14. ↵
    Hochachka, P. W. and Somero, G. N. (1984). Biochemical Adaptation. New Jersey: Princeton University Press.
  15. ↵
    Homma, T. and Morimoto, C. (1997). Cryopreservation of human lymphocytes. Low Temp. Med. 23, 146-149 (in Japanese with English summary).
    OpenUrl
  16. ↵
    Ingram, J. and Bartels, D. (1996). The molecular basis of dehydration tolerance in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 47,377 -403.
    OpenUrlCrossRefPubMedWeb of Science
  17. ↵
    Kenmochi, T., Asano, T. and Isono, K. (1997). Preservation of the kidney. Low Temp. Med. 23, 242-248 (in Japanese with English summary).
    OpenUrl
  18. ↵
    Lyman, C. P., Willis, J., Malan, A. and Wang, L. (1982). Hibernation and Torpor in Mammals and Birds. New York, London: Academic Press.
  19. ↵
    Madin, K. A. C. and Crowe, J. H. (1975). Anhydrobiosis in nematodes: carbohydrate and lipid metabolism during dehydration. J. Exp. Zool. 193,335 -342.
    OpenUrlCrossRefWeb of Science
  20. ↵
    Novick, R. J., Menkis, A. H. and McKenzie, F. N. (1992). New trends in lung preservation: a collective review. J. Heart Lung Transplant. 11,377 -392.
    OpenUrlPubMedWeb of Science
  21. ↵
    Payen, R. (1949). Variations des teneurs en glycogène et en tréhalose pendant le séchage de la levurea. Can. J. Res. 27, 749-756 (in French).
    OpenUrl
  22. ↵
    Sakai, A. and Larcher, W. (1987). Frost Survival of Plants: Response and Adaptation to Freezing Stress. Berlin: Springer-Verlag.
  23. ↵
    Sømme, L. (1995). Invertebrates in Hot and Cold Arid Environments. Berlin: Springer-Verlag.
  24. ↵
    Sussman, A. S. and Lingappa, B. T. (1959). Role of trehalose in ascospores of Neurospora tetrasperma.Science 130,1343 .
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Wigglesworth, F. R. S. (1972). The Principles of Insect Physiology. London: Chapman and Hall.
  26. ↵
    Wolkers, W. F., Tablin, F. and Crowe, J. H. (2002). From anhydrobiosis to freeze-drying of eukaryotic cells. Comp. Biochem. Physiol. 131A,535 -543.
    OpenUrl
  27. ↵
    Womersley, C. and Smith, L. (1981). Anhydrobiosis in nematodes — I. the role of glycerol, myo-inositol and trehalose during desiccation. Comp. Biochem. Physiol. 70B,579 -586.
    OpenUrl
  28. ↵
    Young, S. (1985). The dry life. New Sci. 31,40 -44.
Previous ArticleNext Article
Back to top
Previous ArticleNext Article

This Issue

 Download PDF

Email

Thank you for your interest in spreading the word on Journal of Experimental Biology.

NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. We do not capture any email address.

Enter multiple addresses on separate lines or separate them with commas.
Mechanism allowing an insect to survive complete dehydration and extreme temperatures
(Your Name) has sent you a message from Journal of Experimental Biology
(Your Name) thought you would like to see the Journal of Experimental Biology web site.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
Research Article
Mechanism allowing an insect to survive complete dehydration and extreme temperatures
M. Watanabe, T. Kikawada, N. Minagawa, F. Yukuhiro, T. Okuda
Journal of Experimental Biology 2002 205: 2799-2802;
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
Research Article
Mechanism allowing an insect to survive complete dehydration and extreme temperatures
M. Watanabe, T. Kikawada, N. Minagawa, F. Yukuhiro, T. Okuda
Journal of Experimental Biology 2002 205: 2799-2802;

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Alerts

Please log in to add an alert for this article.

Sign in to email alerts with your email address

Article navigation

  • Top
  • Article
    • SUMMARY
    • Introduction
    • Materials and methods
    • Results and discussion
    • ACKNOWLEDGEMENTS
    • References
  • Figures & tables
  • Info & metrics
  • PDF

Related articles

Cited by...

More in this TOC section

  • Unexpected lack of specialisation in the flow properties of spitting cobra venom
  • Responses of Manduca sexta larvae to heat waves
  • Heat hardening in a pair of Anolis lizards: constraints, dynamics and ecological consequences
Show more RESEARCH ARTICLE

Similar articles

Other journals from The Company of Biologists

Development

Journal of Cell Science

Disease Models & Mechanisms

Biology Open

Advertisement

Predicting the Future: Species Survival in a Changing World

Read our new special issue exploring the significant role of experimental biology in assessing and predicting the susceptibility or resilience of species to future, human-induced environmental change.


Big Biology Podcast - Hollie Putnam and coral bleaching

Catch the next JEB-sponsored episode of the Big Biology Podcast where Art and Marty talk to Hollie Putnam about the causes of coral bleaching and the basic biology of corals in the hope of selectively breeding corals that can better tolerate future ocean conditions.

Read Hollie's Review on the subject, which is featured in our current special issue. 


Stark trade-offs and elegant solutions in arthropod visual systems

Many elegant eye specializations that evolved in response to visual challenges continue to be discovered. A new Review by Meece et al. summarises exciting solutions evolved by insects and other arthropods in response to specific visual challenges.


Head bobbing gives pigeons a sense of perspective

Pigeons might look goofy with their head-bobbing walk, but it turns out that the ungainly head manoeuvre allows the birds to judge distance.

Articles

  • Accepted manuscripts
  • Issue in progress
  • Latest complete issue
  • Issue archive
  • Archive by article type
  • Special issues
  • Subject collections
  • Interviews
  • Sign up for alerts

About us

  • About JEB
  • Editors and Board
  • Editor biographies
  • Travelling Fellowships
  • Grants and funding
  • Journal Meetings
  • Workshops
  • The Company of Biologists
  • Journal news

For Authors

  • Submit a manuscript
  • Aims and scope
  • Presubmission enquiries
  • Article types
  • Manuscript preparation
  • Cover suggestions
  • Editorial process
  • Promoting your paper
  • Open Access
  • Outstanding paper prize
  • Biology Open transfer

Journal Info

  • Journal policies
  • Rights and permissions
  • Media policies
  • Reviewer guide
  • Sign up for alerts

Contact

  • Contact JEB
  • Subscriptions
  • Advertising
  • Feedback

 Twitter   YouTube   LinkedIn

© 2021   The Company of Biologists Ltd   Registered Charity 277992