The Phenomenon of Cryptobiosis
Introduction
The members of the phylum Tardigrada are tiny (0.1-0.5 mm) animals, most of which live in the film of water on lichens, mosses and other plants, and in soil and forest litter. They have piercing-sucking mouthparts, which most species use to feed on plant cells, though some prey on small animals such as rotifers. Tardigrades are known as "water bears" because of their slow, pawing motion and their resemblance to a miniature eight-legged bear. Water bears are known for their ability to enter cryptobiosis ("hidden life"), or dormant states. This ability has apparently evolved in response to the vulnerability of the water bears' habitat to dessication. As its environment dries, a water bear responds by pulling in its head and legs, and forming a cuticular wall around its shrunken body. A great deal of water is lost from the body, and its metabolic rate becomes almost unmeasurable. The cellular components are tightly packaged in an orderly fashion to prevent damage from dehydration. This condition is known as anhydrobiosis, "life without water," and the barrel-shaped body of the water bear in this condition is called a tun. These anhydrobiotic tuns are extremely resistant to environmental insult: they are able to withstand temperature extremes of -272oC to +151oC, toxic compounds, vacuums, intense radiation, anoxia, and perhaps even the conditions of outer space. More importantly, tuns remain viable for extremely long periods of time, perhaps over one hundred years. When its environment is rehydrated, the tun swells, the head and legs pop out, and the creature resumes activity (King: 1986). Other types of cryptobiosis also occur in waterbears, such as anoxybiosis, a short-term ability to live in water lacking oxygen. During unfavorable periods females may lay thick-walled eggs which develop more slowly than typical thin-walled eggs. This experiment tests the assumption that if Tardigrades in a metabolically almost inactive anhydrobiotic state are placed in water, rehydration will occur in response to osmotic forces, and they will be reanimated.
Experimental Procedure
Each table of students will be provided with a small vial containing a few water bear tuns. Each table will use a NaCl solution in one of the following concentrations: 0.1%, 0.3%, 0.6%, 0.9%, 1.4%, 1.8%. Add to the vial about 1/4-inch of your designated solution. If you use too much, the tuns will be difficult to find later. Cover the vial with its screw-on lid. Swirl the solution briefly to oxygenate it and to free the tuns, which initially will be stuck to the sides. Do not spill it. Use Pasteur pipettes to place individual tuns in concavity slides, so that each member of the group can observe at least one tun. Place your tuns under the microscope for no more than about two minutes at a time, as prolonged exposure to the microscope lamp may heat-stress your animals and kill them. If it's a compound microscope, use low-power magnification. Observe the revival process. Revival is counted only if independent motion of the animal occurs. After ~30 mins. compile results for your group, and report on the board your solution concentration, number reanimated, and number not reanimated. Compile the data for the whole class, as well as that of previous years' classes, if available.
Analysis
Plot the percentage of tardigrades revived against the concentration of the solution. What does this tell you about the osmotic concentration of living water bear tissue? Describe the osmotic gradients and events involved in reanimation.
References
Brusca, R.C. and G.J. Brusca. 1990. Invertebrates. Sinauer Associates. 922 pp.
Kinchin, I.M.. The Biology of Tardigrades. Portland Press. 1994.
Morgan, C.I. and P.E. King. British tardigrades (Tardigrada): keys and notes for the identification of the species. Academic Press, 1976.
King, D.E. 1986. "Water bears." Carolina Tips 49(1):1-3.
Materials required
6 vials of water bears (Carolina L340).
Solutions listed above - easily obtained by dilution.
Pasteur pipettes.
Microscopes, preferably dissecting.
Concavity slides.
Clean-Up
Clean your slides and return them. Put your pipettes in the glass disposal container. Put your microscope away.