Mechanism allowing an insect to survive complete dehydration and extreme temperatures
National Institute of Agrobiological Sciences, Ohwashi 1-2, Tsukuba,
Ibaraki 305-8634, Japan
* Present address: Department of Biological Sciences, State University of New
York, Buffalo, NY 14260, USA
Author for correspondence (e-mail:
oku{at}affrc.go.jp)
Accepted 17 June 2022
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Summary |
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Key words: Polypedilum vanderplanki, Chironomidae, cryptobiosis, anhydrobiosis, trehalose
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Introduction |
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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.
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Materials and methods |
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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 mmx50 mm; Shimadzu,
Japan) connected to an analytical column (Shim-pack SCR-101C, 7.9 mmx300
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.
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Results and discussion |
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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.
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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.
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Acknowledgments |
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