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, 5 (11), e14008

Anhydrobiosis-associated Nuclear DNA Damage and Repair in the Sleeping Chironomid: Linkage With Radioresistance


Anhydrobiosis-associated Nuclear DNA Damage and Repair in the Sleeping Chironomid: Linkage With Radioresistance

Oleg Gusev et al. PLoS One.


Anhydrobiotic chironomid larvae can withstand prolonged complete desiccation as well as other external stresses including ionizing radiation. To understand the cross-tolerance mechanism, we have analyzed the structural changes in the nuclear DNA using transmission electron microscopy and DNA comet assays in relation to anhydrobiosis and radiation. We found that dehydration causes alterations in chromatin structure and a severe fragmentation of nuclear DNA in the cells of the larvae despite successful anhydrobiosis. Furthermore, while the larvae had restored physiological activity within an hour following rehydration, nuclear DNA restoration typically took 72 to 96 h. The DNA fragmentation level and the recovery of DNA integrity in the rehydrated larvae after anhydrobiosis were similar to those of hydrated larvae irradiated with 70 Gy of high-linear energy transfer (LET) ions ((4)He). In contrast, low-LET radiation (gamma-rays) of the same dose caused less initial damage to the larvae, and DNA was completely repaired within within 24 h. The expression of genes encoding the DNA repair enzymes occurred upon entering anhydrobiosis and exposure to high- and low-LET radiations, indicative of DNA damage that includes double-strand breaks and their subsequent repair. The expression of antioxidant enzymes-coding genes was also elevated in the anhydrobiotic and the gamma-ray-irradiated larvae that probably functions to reduce the negative effect of reactive oxygen species upon exposure to these stresses. Indeed the mature antioxidant proteins accumulated in the dry larvae and the total activity of antioxidants increased by a 3-4 fold in association with anhydrobiosis. We conclude that one of the factors explaining the relationship between radioresistance and the ability to undergo anhydrobiosis in the sleeping chironomid could be an adaptation to desiccation-inflicted nuclear DNA damage. There were also similarities in the molecular response of the larvae to damage caused by desiccation and ionizing radiation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Time course of DNA repair in the fat body cells of hydrated larvae after 4He+ ion and gamma-ray irradiation and larvae rehydrated after three months of anhydrobiosis.
(A) Typical comet images of nuclear DNA from fat body cells of larvae over a 96 h time course of recovery after irradiation by gamma rays (G-70 Gy) and 4He+ ions (He-70 Gy) to hydrated larvae, respectively and anhydrobiosis (dry: dehydrated larvae). The line marked “0 Gy” corresponds to nuclear DNA from intact hydrated larvae. Bar = 5 µm. (B) Proportion of DNA in the comet tail in the fat body cells of larvae irradiated by gamma rays or 4He+ ions, or following rehydration after anhydrobiosis. Error bars represent mean value ±95% CI.
Figure 2
Figure 2. Ultrastructure of nuclei (n) of two cell types from dry and hydrated larvae.
A: Cells of non-differentiated cell mass in a dry larva. B: Cells of non-differentiated cell mass in a hydrated larva. C: Fat body cells from a dried larva. D: Whole fat body cell from a hydrated larva. In the dry state, chromatin in the nuclei of both cell types showed clear segregation patterns. The chromatin of the cells from hydrated larvae is osmiophilic and widely distributed. n – nuclei. Bar = 1 µm; white arrows indicate location of invaginations in the membranes of the nuclei (A), white arrowheads indicate cell membrane of fat body cells (C, D).
Figure 3
Figure 3. Relative antioxidant activity during dehydration/rehydration cycle, recalculated from ROS-scavenging ability of a P. vanderplanki larva during the course of dehydration and rehydration after anhydrobiosis.
Error bars represent mean value ±95% CI for three replicates. cont. – control hydrated larvae. n –samples with crude from larvae not added.
Figure 4
Figure 4. Accumulation of mature glutathione peroxidase in the desiccated larvae of the sleeping chironomid.
In a fragment of 2D electrophoresis image of total proteins from wet (left image) and dry (right image) larvae the spot corresponding to the glutathione peroxidase is marked and estimated molecular weight, isoelectric point and read sequence of the protein are provided.
Figure 5
Figure 5. Relative mRNA expression profiles for selected genes encoding antioxidants (A, B and C) and DNA repair enzymes (D, E, F) in anhydrobiotic (A, D), heavy ion beam- (B, E) and gamma rays- (C, F) irradiated larvae.
Values for the mRNA level of each gene were corrected for expression level of EF1-alpha, and the relative level of expression changes for each gene was calculated using that of control hydrated larvae as standard (value  = 1). Error bars represent mean value ±95% CI for three replicates. cont. – control hydrated larvae.

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