A little dry history

The history of the study of anhydrobiosis is not a dry topic at all, but an intriguing story involving professional jealousies and matters of life and death. The early history has been told in some detail by David Keilin in his Leeuwenhoek lecture, given to the Royal Society of London in 1958, and much of the following draws on his account. The Leeuwenhoek lecture honours the Dutch scientist Antoni Van Leeuwenhoek, one of the first microscopists. Using his microscopes, Leeuwenhoek made many discoveries, including the first observation of bacteria, and he is regarded as the father of microbiology. His observations on anhydrobiosis were made rather late in his life (in 1702, aged 70) and were recorded in his letter to the Royal Society 'On certain animalcules found in the sediments in gutters of the roofs of houses'. Here is his description of what he found:

I have often placed the Animalcules I have before described out of the water, not leaving the quantity of a grain of sand adjoining to them, in order to see whether, when all the water about them was evaporated and they were exposed to air, their bodies would burst, as I had often seen in other Animalcules. But now I found that when almost all the water was evaporated, so that the creature could no longer be covered with water, nor move itself as usual, it then contracted itself into an oval figure, and in that state remained, nor could I perceive that the moisture evaporated from its body, for it preserved its oval and round shape unhurt.

In order more fully to satisfy myself in this respect, on the third of September, about seven in the morning, I took some of this dry sediment, which I had taken out of the leaden gutter and had stood almost two days in my study, and put a little of it into two separate glass tubes, wherein I poured some rain water which had been boiled and afterwards cooled . . .

As soon as I had poured on the water, I stirred the whole about, that the sediment which, by means of the hairs in it, seemed to adhere like a solid body, might the sooner be mixed with the water: and when it had settled to the bottom of the glass, I examined it, and perceived some of the Animalcules lying closely heaped together. In a short time afterwards they began to extend their bodies, and in half an hour at least a hundred of them were swimming about the glass, though the whole of the sediment which I had put into it did not, in my judgement, exceed the weight of two grains . . .

The preceding kinds of experiment I have many times repeated with the same success, and in particular with some of this sediment, which had been kept in my study above five months, and upon pouring on it rain water, which had been boiled, and afterwards cooled, I saw in a few hours' time many of the Animalcules before described. And if, after being so long in a dry state, these Animalcules, upon water being given to them can unfold their bodies and move about in the usual manner, we may conclude, that in many places, where in summer time the waters stagnate, and at length dry up, there may be many kinds of Animalcules, which, though not originally in those waters, may be carried thither by water fowl, in the water or mud adhering to their feet or feathers.

There are a number of points we might note from this account. The little animals ('Animalcules') were recovered from dry sediment that had been stored for five months, indicating that they could survive desiccation for at least this long. There was a change in the shape of the animals, both during desiccation and during rehydration, and there was a delay in the recovery of activity after rehydration. These are features which have been observed in more recent studies. The 'Animalcules' are believed to have been rotifers, although such sediments are likely to contain other microscopic animals such as tardi-grades and nematodes, as well as protozoa and other microbes.

As Keilin notes, these observations by Leeuwenhoek apparently did not excite the interest of his contemporaries, who may have found the microscopic world which he described a little too outlandish. It was not until 40 years later that the next observation of anhydrobiosis was made, by John Needham, in 1743. Needham was interested in a disease of wheat which results in the formation of galls, known as 'cockles' or 'peppercorns'. Needham describes how, when he opened an infected wheat grain, he found a soft white substance which consisted of fibres. When he added water, the previously dry and lifeless fibres separated and started moving. The fibres were in fact nematodes. Needham's account was not only the first description of anhydrobiosis in nematodes but also the first account of a plant-parasitic nematode. The nematode was probably Anguina tritici which accumulates in infected wheat as an infective larva in the grains, transforming them into galls. Anguina is one of the most desiccation tolerant of plant-parasitic nematodes.

Soon after (in 1753), Needham's observations were confirmed by Henry Baker, the author of several books on microscopy. Baker not only confirmed Needham's and Leeuwenhoek's observations but made a rather extraordinary claim:

We find an Instance here, that Life may be suspended and seemingly destroyed; that by an Exhalation of the Fluids necessary to a living Animal, the Circulations may cease, all the Organs and Vessels of the Body may be shrunk up, dried and hardened; and yet, after a long while, Life may begin anew to actuate the same Body; and all the animal Motions and Faculties may be restored, merely by replenishing the Organs and Vessels with a fresh supply of Fluid.

This was the first claim that life could be a discontinuous process and that an animal could apparently 'die' after desiccation, showing no signs of life, and yet 'come back to life again' when water was added. This claim of death and resurrection had obvious religious implications, with several religions, not only Christianity, including such a process as part of their tradition. The idea that death and resurrection occurs in nature, even among lowly organisms, is thus a controversial one.

Lazzaro Spallanzani, Professor of Natural History at the University of Paris and one of the leading scientists of his time, initially denied the animal nature of the dried fibres observed by Needham in wheat grains (in his book published in 1767). Spallanzani considered them to be dried vegetable fibres, with the movement resulting from water penetrating into them. The reputation of Spallanzani was such that it led Needham to recant his view. Shortly after this, however, two scientists, l'Abbé Roffredi and Felice Fontana, working independently, described the life cycle of the nematode responsible for the formation of galls in wheat and confirmed the animal nature of the fibres. Roffredi and Fontana in their publications attacked Needham for withdrawing his earlier correct view. Needham accepted their findings, but was rather hurt by their criticism. He complained to the editor of the journal which published their papers about instances 'where the critic aims only to wound his adversary'.

Following the work of Roffredi and Fontana, Spallanzani reexamined the phenomenon and was able to confirm the work of Leeuwenhoek, Needham and Baker for himself and to extend their studies greatly. He showed that dried rotifers were more resistant to high temperatures (73 °C) than were hydrated rotifers (which died at 45 °C), but that both dried and hydrated rotifers could survive freezing to — 24 °C. Dried rotifers could also survive exposure to a vacuum. His experiments led him to accept the idea of death and resurrection in these animals, stating in a paper entitled, 'Observations and experiments upon some singular animals which may be killed and revived':

An animal, which revives after death, and which within certain limits, revives as often as we please, is a phenomenon, as incredible as it seems improbable and paradoxical. It confounds the most accepted ideas of animality; it creates new ideas, and becomes an object no less interesting to the researches of the naturalist than to the speculation of the profound metaphysician.

Not everyone accepted Spallanzani's view. One of the strongest opponents of the idea that animals underwent death and resurrection during desiccation and rehydration was Christian Ehrenberg who felt that life processes were greatly slowed down and not stopped altogether during anhydrobiosis and also that not all water was lost. The controversy erupted in 1858 in a debate between two French scientists, PLN Doyere and Felix-Archimede Pouchet, both of whom conducted experiments on tardigrades and rotifers but reached very different conclusions from their work. Doyere considered that these animals could be revived after complete desiccation and the cessation of their life processes, while Pouchet thought that no organism could survive complete desiccation or return to life after all life processes had stopped. The debate became so heated that members of the learned societies and even the newspapers of Paris became divided into two fiercely opposed groups: the resurrectionists and the anti-resurrectionists. In 1859, Doyere and Pouchet approached the Biological Society of France asking them to give an impartial ruling. The Society established a special commission which examined the work of the two scientists and conducted experiments of their own. An extensive report was written by Paul Broca, a distinguished French anatomist, which generally supported the views of Doyere.

the anhydrobiotic state Further defining anhydrobiosis

Broca's report quieted the controversy for a while but it has resurfaced periodically ever since and continues today. The main two points at issue are: do anhydrobiotic organisms lose all their water during desiccation and does metabolism (which we nowadays think of as the primary manifestation of 'life processes') cease? It should be noted that both these questions are essentially unanswerable. If no water is detected in a desiccated anhydrobiotic organism using a particular technique, there might still be some water present which the technique cannot detect and, if no metabolism can be detected, there may be a level of metabolism occurring which the technique used is not sensitive enough to reveal. This latter point was addressed by John Barrett at the University of Wales. He suggested in 1982 that, while we could not prove that metabolism had ceased, we could say what level of metabolism a particular technique should be able to detect. He used three different techniques to look for metabolism in the anhydrobiotic nematode Ditylenchus dipsaci (a parasite of plants): oxygen uptake, heat output and the production of carbon dioxide. No metabolism was detected in the dry nematodes using any of these techniques. Oxygen uptake should have detected a metabolic rate of 0.3 per cent of normal levels, heat output a rate of 0.06 per cent and carbon dioxide a rate of 0.01 per cent. So, if there is metabolism occurring in the desiccated nematodes, it is at a rate which is less than 1/10 000 of that of hydrated nematodes. It is perhaps not unreasonable to accept that metabolism had ceased altogether.

It may not be necessary for an organism to lose all, or nearly all, its water for metabolism to cease and for it to become anhydrobiotic. James Clegg, now at the University of California's Bodega Marine Laboratory, from his studies on the cysts of the brine shrimp Artemia, proposed in the 1970s that, as an organism loses water, it passes through a number of metabolic states. When it has 40-100 per cent of its normal water content, this is sufficient to form a continuous bulk of water within the cells of the organism. This provides a medium for the series of chemical reactions involved in the metabolism of the organism, allowing it to proceed as normal. At 20-40 per cent water, there is insufficient for it to form a continuous medium, only isolated reactions in the remaining pockets of water are possible, most sequences of reactions become impossible and metabolism is depressed. Below 20 per cent water, there is no free water within cells and metabolism may cease (and, if the organism survives, it is anhydrobiotic). The water which remains is thought to be tightly bound to, or in close association with, the proteins and other substances which make up the structure of the cell. This is referred to as 'bound water', 'unfreezable water' (since about 20 per cent of cell water cannot be easily frozen) or 'osmotically inactive water' (since it is not free to move under an osmotic stress, although it will move along gradients of water potential such as evaporation under desiccating conditions). A somewhat smaller quantity of water is actually involved in the structure of biological molecules (0.15-0.4 per cent). The water content of dried cells in anhydrobiosis is too low to provide even a single layer of water molecules to coat their internal surfaces. Under such circumstances, the usual sequence of metabolic reactions is clearly impossible. As well as reducing the availability of water, desiccation will also increase the concentrations of dissolved organic and inorganic substances within the cells. Metabolic enzymes can only tolerate a limited range of concentrations of some of these substances and it is likely that an increase in their levels during desiccation will result in metabolism being inhibited.

Whatever the mechanism, it is clear that normal metabolism ceases when a substantial proportion of water is lost, but long before it is lost altogether. If we define anhydrobiosis as 'the ability to survive the cessation of metabolism due to water loss', it is clear that the organism does not need to lose all its water for this to occur. Since it may be difficult to demonstrate that metabolism has ceased, we can consider an organism to be in a state of anhydrobiosis if it survives the loss of more than 80 per cent of its water (since there is no free water present in cells and normal metabolism ceases at these water contents). The ability to survive the loss of some osmotically inactive water appears to be critical for anhydrobiosis. Water will be lost from an organism whenever the water activity outside its body is lower than that inside its body -that is, when it is drier outside than it is inside. The water will continue to be lost until the water activities inside and outside the body become equal. This equalisation of the water activities may occur by water being lost from the organism or by it producing substances (osmoti-cally active solutes) that may replace some of the water bonded to proteins, and other large molecules, within its cells. Desiccation-sensitive bacterial cells die if their water content is reduced to about 6 per cent and this occurs if they are dried at a relative humidity of 80 per cent or below. At such low water contents, metabolism ceases. Organisms will need the mechanisms which enable them to survive anhydrobioti-cally (if they can) when they are faced with these sorts of relative humidities, unless they can prevent water loss from occurring. The rate of water loss, and hence the time available to the organism to make any necessary adjustments, will depend on how dry the air surrounding it is.

Anhydrobiosis and the nature of life

When metabolism ceases during anhydrobiosis, so do all the processes that we usually consider to be a part of life. I remember that at school I was taught a list of the characteristics of living things which distinguished them from non-living things. The list went something like this: living things move, respire, feed, excrete, grow, reproduce and respond to stimuli. To be considered alive, an object had to exhibit all these characteristics. A car moves and might be considered to feed (on petrol) and excrete (exhaust fumes), but we never see a baby car emerging from its exhaust pipe. What then are we to make of anhydrobiotes which, in the dry state, exhibit none of these characteristics. Should they be considered dead or non-living when in this state? Clearly, they have the capacity for life, since when immersed in water they recover and display the characteristics we associate with life. In anhydrobiosis, life exists in a purely structural state. We can thus recognise three states for an organism: alive, dead and anhydrobiotic (or cryptobiotic, which includes the cessation of metabolism in response to stresses other than desiccation). Perhaps the key feature of living organisms is the potential for reproduction - the presence of a self-replicating molecule, DNA or RNA, which carries the blueprint (genetic code) for their construction and which is the product of a process of evolution.

The ageing process is suspended while an organism is dry. Many nematodes complete their life cycle and die within a matter of weeks of hatching from the egg. They will, however, survive in a state of anhy-drobiosis for many years. We thus need to distinguish between the chronological and physiological age of an organism. There are many records of plant parasitic nematodes surviving anhydrobiotically for decades. In tardigrades, the record is 120 years, in animals recovered from a dried plant stored in an herbarium. The proportion of organisms which recovers when stored dry, however, slowly declines. What causes this slow death rate among organisms in an anhydrobiotic state? Many survive better if stored in an atmosphere of nitrogen gas, rather than air. Some biological molecules react with the oxygen in the air (oxidise) and this is often destructive. Storing the material in the dark and at low temperature also improves survival.

A living organism continually repairs any damage which occurs to its cells. During anhydrobiosis, normal metabolism ceases and this repair cannot occur. Any damage to the cells of the organism thus accumulates and may reach a point where death occurs when it is reim-mersed in water. Just as life processes are suspended during anhydrobiosis, so are some of the processes which lead to death. Howard Hinton, formerly Professor of Entomology at the University of Bristol, working on anhydrobiotic larvae of the midge P. vanderplanki, reported that a larva which had suffered damage to its body wall while dry had a portion of its gut forced through the wound when it was immersed in water; it recovered activity only to die some four hours later.

Anhydrobiosis and extreme survival

In the dry state, anhydrobiotes are resistant to physical insults which would be fatal to them if they were hydrated. Since there is no or little water present to freeze, they are resistant to low temperatures. Survival to temperatures within a fraction of a degree of absolute zero (—273 °C) has been reported. At the other extreme, anhydrobiotic nematodes have survived two minutes at 105°C, tardigrades and rotifers several minutes at 151°C, Artemia cysts an hour and a half at 103.5 °C and P. vanderplanki larvae a minute at 102-104°C. Anhydrobiotic animals have been reported to survive high doses of X-ray radiation and immersion in alcohol, and anhydrobiotic nematodes can tolerate the nematocides designed to kill them. Anhydrobiotic nematodes have survived exposure to vacuums and tardigrades have survived high pressures of 6000 atmospheres. Some of these reports must be treated with caution, however, because what may have been observed was delayed death rather than survival. Tardigrades have revived after exposure to the vacuum and high doses of radiation inside the column of a scanning electron microscope, only to die after a few minutes of movement.

The remarkable survival abilities of anhydrobiotic animals have led some to suggest that they may be able to survive conditions in space. Reinhardt Kristensen of the Zoological Museum in Copenhagen is quoted as saying with regards to tardigrades: 'They can tolerate outer space, no doubt about it'. The survival abilities of Ditylenchus dipsaci, a plant-parasitic nematode, led John Barrett of the University of Wales and myself to wonder whether they might survive exposure to space. In what we called (among ourselves) the 'Worms in Space Project' in the mid-1980s, we booked them on the European Retrievable Carrier (EURECA) due to be launched by the European Space Agency on the Space Shuttle. However, although Ditylenchus can survive exposure to a vacuum, it failed to survive in the ultra-high vacuum facility operated by the German Aerospace Centre in Cologne which simulates the vacuum of space. Perhaps they can not survive losing that last drop of water or some other critical material is lost in extreme vacuums. I am afraid that our worms lost their seat on the Space Shuttle and our experiments have remained earth bound.

Although the survival abilities of anhydrobiotic animals in space have yet to be tested, some microorganisms have made the journey into the void. In 1984, the Long Duration Exposure Facility (LDEF) was launched by the National Aeronautics and Space Administration (NASA) on board the Space Shuttle Challenger and placed in low Earth orbit at an altitude of 476 kilometres above the Earth. The original intention was that the LDEF be retrieved after a year, but the 1986 Challenger disaster, in which the launch rocket exploded during takeoff killing all seven crew members, and other problems with the Space Shuttle programme meant that it was not recovered until 1990, after 69 months in orbit. Of the biological samples on board the LDEF, only bacterial spores were exposed to the environment of space. Other material included Artemia (brine shrimp) cysts and plant seeds, but these were shielded from the vacuum of space within sealed containers. Bacterial spores (Bacillus subtilis), however, were exposed to space for nearly six years. Even some unprotected spores survived after their return to Earth (about 2 per cent), but survival was greater (up to 70 per cent) if they were protected by buffer salts, glucose or by an aluminium cover which shielded them from ultraviolet (UV) radiation. Since then, bacterial spores have flown on other missions which have exposed them, along with other microbes, to the space environment. Fungal spores, viruses and salt-tolerant bacteria and cyanobacteria, as well as bacterial spores, have all survived exposure to space.

Damage to the organisms' DNA, caused by exposure to the vacuum and radiation of space, appears to be important in inducing mutations and in determining their survival. UV radiation from the sun causes the most damage and is directly absorbed by the DNA molecule. It reacts with the DNA to produce products which are highly lethal and mutagenic. Microorganisms can, however, be protected from UV radiation by dust particles or other substances which shield them from it. Even a multiple layer of bacterial cells will protect those at the bottom of the layer from the harmful effects of UV radiation. Organisms have so far been exposed to space only in low Earth orbit. Temperatures during these exposures varied from — 30 °C to +45 °C due to direct solar heating or to heat reflected from the Earth. Temperatures in deep space are much lower (—269 °C), within a few degrees of absolute zero. At such low temperatures, the harmful effects of UV radiation are greatly reduced.

Among the cosmic radiation, heavy high-energy (HZE) particles cause the most damage to biological materials. These particles are of galactic rather than solar origin and are atomic nuclei which are stripped of their electrons and accelerated to high speeds. These particles are fairly rare (about 1 per cent of the particulate radiation in space), but a single hit by a HZE particle can kill a spore and the chances of such a hit will set the ultimate time limit on their survival in space.

the mechanisms of anhydrobiosis in animals How do anhydrobiotic animals survive in their remarkable way? Most of the information we have is from studies on nematodes and tardi-grades, so I will focus on these groups but add results of studies on other organisms where appropriate.

Controlling water loss

A slow rate of water loss appears to be important to prevent damage to the structure of the animal and to allow it to make biochemical changes in preparation for the desiccated state. Anhydrobiotic nema-todes are divided into two broad groups: slow-dehydration strategists and fast-dehydration strategists. Slow-dehydration strategists rely on the characteristics of their environment to produce the slow rate of water loss needed. For a nematode living in soil or moss, the material in which it lives will lose water slowly when it is exposed to desiccation. This will produce the slow rate of water loss necessary for the nema-tode's survival. Some nematodes, however, live in more exposed sites such as the aerial parts of plants and drying plant tissue which will lose water quickly when exposed to desiccation (Figure 3.2). In these habitats, the nematode itself needs to control the rate at which water is lost from its body.

Nematodes are covered by a cuticle which, in many cases, has a very low permeability to water, slowing the rate at which they lose water. Some appear to decrease the permeability of their cuticles when they are exposed to desiccation. This also happens in tardigrades, where the secretion of lipids through pores in the cuticle and changes in the properties of the cuticle itself reduce water loss. A similar job is performed

Through Air Dried Tissue

plant/air interface high or rapidly drying plant tissue slow-drying plant tissue soil/air upper soil lower soil

HABITAT DESICCATION RATE OF STRESS WATER LOSS

plant/air interface high or rapidly drying plant tissue slow-drying plant tissue soil/air upper soil lower soil high low fast slow high fast intermediate slow very slow or zero figure 3.2 The desiccation stress and rates of water loss likely to be experienced by plant-parasitic and free-living nematodes in various plant and soil habitats. Based on a figure in Womersley (1987). Drawing of plant by Jo Ogier.

by the cuticles of other animals and of plants, by eggshells, the cases of seeds and the walls of cysts and spores. Some algae, and other microorganisms, secrete a gelatinous material which slows the rate of water loss. Changes in behaviour may also act to reduce the rate of water loss. Many nematodes coil up when desiccated (Figure 3.3). This reduces the surface area exposed to the air and consequently reduces the rate of water loss. Tardigrades withdraw their legs into their bodies forming a tun (so-called because it is barrel shaped). This reduces the area of exposed surface but may also slow water loss by removing the more permeable areas of the tardigrade's cuticle from exposure to the air (Figure 3.4). Some plant-parasitic nematodes form dense aggregations known as 'eelworm wool'. The formation of these aggregations may

Tardigrade Tun

figure 3.3 The anhydrobiotic plant-parasitic nematode Ditylenchus dipsaci, coiled in response to desiccation. Each worm is about 1 millimetre long.

Ditylenchus

figure 3.4 An active tardigrade (left) and one which has withdrawn its appendages into its body to form a tun during desiccation and entry into anhydrobiosis (right). The animal is about 1 millimetre long. Drawing by Jo Ogier, based on photographs in Crowe & Cooper (1971).

aid desiccation survival since the nematodes on the outside will dry first and slow the rate of water loss of those in the centre of the aggregation (the so-called 'eggshell effect').

In the dry state

A slow rate of water loss allows the animal to shrink and to pack its internal structures together in an orderly fashion so that disruption during desiccation is prevented or at least minimised. The structure of anhydrobiotic organisms in the dry state can be revealed using techniques for preparing specimens for electron microscopy which do not involve exposure to water. Studies on a wide range of anhydrobiotic organisms from cyanobacteria to nematodes reveal a similar picture. Most anhydrobiotes keep their internal structures intact in the dry state. The main change during desiccation is that the cytoplasm of the cell is reduced in volume and becomes condensed around the mitochondria and other cell structures. Organisms in a state of anhydrobio-sis have much the same appearance as they do in the active hydrated state. The main difference is that the cells shrink and the cytoplasm and organelles pack closely together. Some more complex changes have been observed in anhydrobiotic plant tissues. Multilayered structures and dense spheres, which are absent in wet tissue, have been seen in some plant pollen. The origin and function of these structures is unknown.

You might think that desiccation would harm the enzymes, DNA and other macromolecules which make up the bodies of organisms. However, enzymes show no loss of activity in anhydrobiotic nema-todes and there is also no evidence of the break up of DNA molecules.

The role of trehalose

Trehalose is a sugar which is closely related to the sugar (sucrose) which you put in your tea. A number of anhydrobiotes, including nem-atodes, tardigrades, Artemia and yeast, produce trehalose as they dry out. Anhydrobiosis is thus the sweet life as well as the dry life. John Crowe of the University of California at Davis has been the main proponent of the theory that trehalose plays a crucial role in anhydrobi-osis. He found that Aphelenchus avenae, a fungus-eating nematode which could be cultured in large quantities for biochemical analysis, converted its carbohydrate (sugar) stores from glycogen (the main store of carbohydrates in animals) into trehalose as they dried out. James Clegg found a similar phenomenon in Artemia cysts. This increase in trehalose levels led these researchers to suggest that trehalose plays an important role in anhydrobiosis. They also found that anhydrobiotic organisms converted lipid into glycerol (a polyol or sugar alcohol) and suggested that the glycerol replaced the bound water which makes up 20 per cent of the water in the body. It is now thought that the glycerol was produced in response to conditions within their samples becoming low in oxygen. Glycerol is produced in response to a variety of stresses such as low oxygen concentrations, osmotic stress and low temperatures. It thus appears to be a general response to stress and is now not thought to be specifically involved in anhydrobiosis. There is, however, strong evidence that trehalose plays an important role.

A biological membrane consists of two layers of phospholipids with proteins immersed within the bilayer and carbohydrates associated with its surface. The structure of the membrane is maintained by its interaction with water. The phospholipid molecules have two ends, one end of which is attracted to water (the hydrophilic heads) and the other is repelled by water (the hydrophobic tails). Water molecules attach to the hydrophilic heads. If you remove the water, the structure of the membrane is changed. The normal condition of a membrane is a liquid crystalline state. In this state, the membrane is fluid, with the same mobility as salad oil, and its molecules can move around. This fluidity is important for the biological functions of membranes. The condition of the membrane will, however, change as conditions within the organism change. If the temperature falls, for example, the membrane becomes less fluid and eventually solidifies, like melted butter when it cools. Desiccation also affects the condition of the membrane. Removal of water allows the phospholipid molecules to pack more closely together and the membrane undergoes a change in state from a

HYDRATED

DESICCATED

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