about Sleeping chironomid
About the Sleeping Chironomid
Cryptobiosis
The surprising resistance of the Sleeping Chironomid
Why does the sleeping chironomid present such a resistance to drought?
The role of trehalose
How to acquire resistance?
Whereas chironomids are taxonomically related to mosquitoes and other flies, they are not blood-sucking insects. Those midges are common in Japan and other parts of the world, and the characteristic swarming of the males can be observed from spring to summer.
  The sleeping chironomid (Polypedilum vanderplanki) is a species living in rocky areas of the semiarid region of central Africa, in countries such as Nigeria, Uganda and Malawi. This area is situated near to the equator and is subjected to high temperatures throughout the year, with the clear succession of a rainy season and a dry season. The dry season may last for eight months without even a single drop of rain.
  アフリカの地図と岩山と水たまり
The larvae of the sleeping chironomid live in small water pools, which appear in depressions on granititic rocks. They live in the mud at the bottom of those water pools and feed on organic matter and bacteria. Since water temperature exceeds 40 degrees Celsius during the daytime, if no rain falls for one week, the small water pools quickly dry up in no time. Therefore, other midges and natural predators (dragonfly nymphs, for example) cannot survive in such an instable environment.

Then how can the sleeping chironomid survive? The sleeping chironomid builds up mud with its saliva, making a pipe shape called tubular nest, and stands inside this nest. When water evaporates, the larva desiccates little by little, protected in the tubular nest and its body water content finally drops to as low as 3% of body weigth. In this state, the sleeping chironomid waits until next rainfall.
ネムリユスリカ幼虫と干上がった水たまり
When the long-awaited rain falls, as soon as its body is in contact with water, the larva absorbs water rapidly and recovers its original living state. The larva can revive in a time as short as 1 hour, and swim again in the small water pool. Then, it begins to grow again. If no other rain falls in the following days, the larva of the sleeping chironomid will desiccate again and wait for the next rain, just as it did before. This kind of resistance ability is observed throughout the larval life, except just after eclosion. Eggs, pupae and adults of the sleeping chironomid do not present any drought resistance.

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ネムリユスリカの生活史
Human body contains about 80% of water, and which water plays important roles in the metabolism (chemical reactions that occur into the body and the cell in order to maintain life) or as a structural element constituting the body. That is why a water loss corresponding to only 10-12% of the body weight is critical for the maintenance of life. However, the sleeping chironomid can survive to a loss of about 97% of its body water. Of course, in such a dehydrated state, the normal metabolism is stopped.

This ability to survive through a state without any metabolic activity to is called cryptobiosis (meaning hidden life). This is different from the phenomenon of dormancy, a state of low metabolism that is observed in other insects. This phenomenon of cryptobiosis was discovered at the beginning of the 18th century in microorganisms such as rotifers and was considered later as a proof supporting the theory of autogenesis. Organisms undergoing cryptobiosis may be classified into two categories; in fungi spores, plant seeds or Artemia eggs, the parental generation foresees drought and prepares the next generation for cryptobiosis, whereas in rotifers, nematode worms, water bears and insects, the same individual engages cryptobiosis in response to a dry environment. A characteristic of the latter category is that individuals can repeat the cycle of desiccation and revival many times. The sleeping chironomid is the only insect known to possess this ability and may be considered as the largest and most highly derived animal performing cryptobiosis.
Categories Parental generation foresees drough Individual engages cryptobiosis in response to a dry environment
Organisms

fugai spores

Plant seeds

Cysts of Artemia

P. vanderplanki

Tardigrades

Rotifers

Nematodes

Reversibly switching between active(development) and anhydrobiotic phases. ×
In addition, the term of cryptobiosis includes four phenomena. The loss of body water content in response to desiccation, as observed in the sleeping chironomid leads to anhydrobiosis. Similarly, resistance to water loss caused by diffusion in a hypertonic solution (with high salt concentration) is referred as osmobiosis. The capacity to survive at low oxygen concentrations under the level required for the maintenance of metabolic activity is known as anoxybiosis. Finally, cryobiosis is the resistance of an organism to freezing and extremely low temperatures.
The surprising resistance of the Sleeping Chironomid
The sleeping chironomid is not only able to survive in a completely dry state to the African dry season, that lasts for several months, but also it has the capacity to resist to various extreme conditions. Concerning the viability, desiccated larvae, which were maintained into a desiccator (a recipient filled with chemical compounds that absorb relative humidity) did revive after 17 years of desiccation, when placed into water again. On the contrary, dried larvae kept in conditions with high relative humidity absorb moisture and their revival rate drops little by little. The absence of water is thus a crucial factor for the maintenance of cryptobiosis.

A much more surprising tolerance is observed toward temperature. Larvae normally swimming in the water do not survive for a long time at 5ºC and die within 1 hour at 43ºC. By contrast, desiccated larvae boiled at 103ºC for 1 min and then placed into water at room temperature recovered movement rapidly and some of them resumed their development until the final molt to the adult form. Even after boiling for 3 hours at 106ºC or for 5 min at 200ºC some individuals did revive. safely. Generally, if we consider that the proteins constituting an organism are completely denatured at 70ºC and loose their biologic activity, this resistance of dried sleeping chironomid larvae to high temperatures is really astonishing. Desiccated larvae are not only resistant to high temperatures, but also to lower temperatures. Even treatments for 5 min at -270ºC or 77 hours at -190ºC did not affect development and normal metamorphosis of larvae after revival.

Moreover, active larvae immersed into 100% ethanol, which is commonly used for fixation and dehydration of tissues in histological experiments, die within 1 min. In contrast, desiccated larvae can survive even after 168h into 100% ethanol, they revive when transferred into water and some of them may complete their development to the adult stage. Interestingly, just a little water added to ethanol is enough to prevent the revival of larvae once transferred back into water. This phenomenon may be explained by the absorption of additive water into the cells and tissues of the larval body, resulting in a destabilization of the cryptobiotic state. Ethanol was thus allowed to exert its fixative effect. Consequently, desiccated larvae need almost complete dehydration to undergo successful cryptobiosis.
ネムリユスリカの能力
What about the resistance to radiations? In general, a stronger resistance to radiations is observed in insects, compared to vertebrates. It depends on the species, but a dose of about 200 to 2000 Gy (Gray: unit of absorption of radioactive energy) is lethal. Sleeping chironomid larvae that are not desiccated also die in majority after exposure to radiations over 2000 Gy. However, desiccated larvae in cryptobiosis were found to resist to an irradiation of 7000 Gy and some of them survived 48 hours after recovery into water. Those larvae did not succeed to develop into pupae, but our results show that cryptobiosis confers a surprising resistance to radiations.
Why does the sleeping chironomid present such a resistance to drought?
There are several reasons why the sleeping chironomid can tolerate drought and presents a resistance to various kinds of environmental stress.

When exposed to environmental stress, replacement of water by low weight molecules occurs in the body, in order to protect biological components in the cells. Such molecules are known as compatible solutes. Molecules such as sugars or proline are well-known compatible solutes and are involved in the improvement of resistance to various environmental conditions. In the sleeping chironomid, we found that the role of compatible solute was played by a sugar called trehalose. Since trehalose is the hemolymphatic sugar of insects, it represents 0.5 to 1% of the body lymph in larvae. However, during the process of desiccation, trehalose is rapidly synthesized to reach finally a content of 20%. In the Japanese chironomid Chironomus yoshimatsui, which is not resistant to drought, the same process of desiccation does not induce the accumulation of trehalose and those insects finally die. Consequently, the accumulation of trehalose is a key factor for the drought resistance of the sleeping chironomid.

Nevertheless, the sleeping chironomid is not able to synthesize trehalose and to revive under any condition of desiccation. If dried too rapidly, larvae fail to accumulate enough trehalose and do not recover when placed in water again. On the contrary, when desiccated slowly by lowering the relative humidity little by little, larvae of the sleeping chironomid accumulate a large quantity of trehalose and thus revival becomes possible. The induction of cryptobiosis needs a minimum of time for physiological preparation and we demonstrated that the speed of desiccation is very important for this phenomenon.
The role of trehalose
What kind of compound is trehalose, which is so important for cryptobiosis?
トレハロースの構造
Trehalose is a disaccharide (two sugar molecules linked together) and this sugar is naturally present in animals, plants, bacteria and even in seawater. Trehalose is found in many types of food that we regularly eat, such as mushrooms, algae or shrimps. The fact that dried mushroom can recover their original shape after being immersed into water, is due to the presence of trehalose in the tissues of mushrooms. Trehalose is also the main sugar in the hemolymph (blood) of insects.
トレハロースを含むもの
Trehalose is known to play the role of compatible solute in various organisms, which undergo cryptobiosis: bacteria, spores from molds, yeast, nematod worms, eggs of the brine shrimp (Artemia) and also the resurrection plant (Selaginella). The fact, that trehalose can be accumulated in high quantity in the body without toxicity and that its synthesis or degradation are relatively easy, may explain why trehalose has been selected as a compatible solute in such a wide range of taxa, from plants to animals. Moreover, among other sugars, trehalose presents chemico-physical properties that enable particularly high protection against desiccation. Since trehalose contains several hydroxyl groups that resemble water molecules, it can easily replace the water, which lays around membranes and inside the 3D structure of proteins. Furthermore, at high concentration, trehalose can easily undergo vitrification (the transition to an amorphous state different from crystal, which generates extremely low molecular activity and high stability) and by lowering the activity of biological subtances, it protects them against denaturation. Each of these particular properties of trehalose taken together are thought to make possible the phenomenon of Cryptobiosis.
How to acquire resistance?
What are the environmental cues that induce exponential synthesis of trehalose in the larvae of the sleeping chironomid? Investigation of water and trehalose contents in larvae body during the course of desiccation showed that trehalose titer was stable until water content falls under 75%. There should be some mechanism allowing the perception of chemicophysical changes that result from the loss of water in the body of the sleeping chironomid.

Many insects survive to seasons that are not favorable for reproduction and development in a state of dormancy. Concerning the mechanism of dormancy, insects integrate cues such as photoperiod or temperature in their brain, and dormancy is induced through hormonal secretion. What about the cryptobiosis in the sleeping chironomid? We did the following experiment: with a thin thread, larvae were ligated underwater between the head and the body, or between the thorax and the abdomen. Abdomens from both groups isolated in this way were desiccated and accumulated an amount of trehalose equivalent to about the half of the trehalose accumulated by unwounded individuals. Furthermore, treated head-cut individual did succeed to revive after placing them into water again. This experiment demonstrates that the brain is not directly involved in the physiological mechanism leading to the induction of cryptobiosis and to the revival of desiccated larvae. We are now discovering that the process of cryptobiosis depends on a mechanism, which is completely different from what is known in classical dormancy systems.
幼虫の断頭実験