MICROSCOPIC LOCH NESS MONSTER
Editor's note: a popular article 'Tear of the swan' on Lacrymaria olor was presented in Micscape 1998. This little known organism is so fascinating, the author has kindly offered a more extensive essay on his observations. It includes a successful protocol developed by the author for the cultivation of Lacrymaria, in the hope that it will encourage other enthusiasts to study this remarkable protozoan.
In this essay, I simply want to give you some sense of the excitement of investigating a largely unknown and fascinating organism and secondly, to show how starting by with a few simple questions I got involved in asking a lot of complicated questions, many of which are still unanswered. For me much of the fun of this research was a consequence of the process of discovering just how much there is to find out about an apparently simple organism and I was a bit overwhelmed by the number of problems, questions, and experiments that arose out of the investigation of a single microscopic organism.
The title of my essay is "A Microscopic Loch Ness Monster" and I have been negotiating with the Public Broadcasting System to see if they will present this as a series on Monsterpiece Theatre. An alternative title for this lecture is "Sex, Dyes, and Videotapes." In order to study micro-organisms, one needs to culture them and in order to do that one often needs to know something about their sex lives. In order to study their structural detail, it is often necessary to use biological stains or dyes in order to get a better idea of the morphology. As for the video tapes, I have used those to document the antics of this extraordinary creature.
Lacrymaria olor is a protozoan cell with the colorful name of "tear of a swan". For a very long time, protozoa have been described as single-celled animals. However, some recent biological taxonomists have tried to move away from that description by creating an entirely new kingdom for a large set of groups of organisms that have never quite comfortably fit into the traditional plant/animal distinction. The more I studied Lacrymaria, the more sympathy I developed for the view that denies saying that protozoa can be accurately described as "one-celled animals." I have described this organism in the title of my lecture as a "microscopic Loch Ness monster" for reasons which will become apparent when you observe Lacrymaria. Let me begin by briefly describing the organism before I tell you about the research which I undertook and what I learned about this remarkable creature.
General Morphology and Behavior
1. Tear-shaped body
2. Shows strong contraction and extensility
3. Head with "nose cone" and ciliary bands
4. Macro- and micronuclei
5. Contractile vacuoles
6. Diagonal ciliary bands
7. Posterior mucocysts
8. Trichites in nose cone and fibrils that control the mouth (cystostome)
9. Toxicysts in nose cone
10. Birefringent crystals especially in the anterior end
11. Extensility to a remarkable degree
Lacrymaria olor is an incredible creature on a number of counts. To begin with the cell is both quite small and yet can achieve a length rivaling some of the largest so-called single-celled animals. The body of the entire organism when contracted is often not more than 100mm in length. A micron (mm) is one thousandth of a millimeter, thus an inch is about 2,540 microns. Ciliated protozoa range in size from just a few microns to a few that reach a size of nearly 2000mm. However, Lacrymaria olor has a highly extensile "neck" which ends in a "head-like" structure where the mouth is located. This neck is capable of astounding extension and when the organism is feeding, the total organism can achieve a length of nearly 1200mm! In other words, if my neck had such extensility, I could virtually instantaneously achieve a height of about 70 feet. But, in addition this remarkable creature's neck is so flexible that it can angle back upon itself in such a way that it produces gymnastic feats that are quite astonishing.
The general structure or morphology of this animal is not well understood. It is usually described as having two macronuclei and a single micronuclei but is sometimes depicted as having just one large lobed macronucleus. My own observations revealed specimens with one, two, three, four, five, and seven macronuclei, although special reasons obtain for those having more than two. A Lacrymaria olor has two contractile vacuoles to regulate the osmotic pressure in the cell. One of these is typically located at the posterior end of the cell and the other at the anterior end of the body near the beginning of the neck.
The body is covered with small hair-like processes called cilia used for locomotion. The cilia on the body are arranged in spirals down the neck and along the length of the body and spiral to the right. The cilia around the head are longer and often are moved in bundles. The body and the neck usually contain crystals which show up strikingly under polarized light. These crystals are especially concentrated at the posterior end of the cell. The structure of the neck is particularly problematic and a better understanding of its construction waits upon electron microscopic studies. It is almost certain that the neck consists of complex arrangements of microtubules and perhaps myonemes and a study by Tatchell in England has partially mapped out the ultrastructure, but more remains to be done. Another particularly mysterious aspect of this organism is the extraordinary extensility of the neck in relation to the cellular membrane. Briefly, it can simply be said that membranes aren't supposed to behave in the manner observed.
Now, let me turn to a discussion of how this particular project came about. From time to time in examining pond samples, I happened across Lacrymaria olor. I watched it with fascination and enjoyed it for its elegance, but thought little more about it. Then about fourteen years ago, I found five specimens in a culture sample which I collected from Hutton Lake in the game preserve located about 15 miles southwest of Laramie. On earlier occasions I had found a few specimens and both Bob Jenkins, a cell biologist at the University of Wyoming, and I attempted to culture the organism with no success. However, this time, I was determined to establish cultures. Beginning with those five cells, I coaxed them along, making hundreds of cultures and trying dozens of different types of culture media.
This process was both tedious and aromatic as my long-suffering wife will attest. I found a reference that gave a very simple medium of malted milk in distilled water, but this was successful only in a limited way. I tried making extracts of hay, wheat, cow dung, malted milk, alfalfa, lettuce, sheep dung, rice, and all kinds of combinations. A very fragrant process and the worst of the extracts were malted milk media which after a few days smelled like regurgitated baby formula. Finally after a long period of trial and error combined with a consultation of literature on various types of media used for culturing protozoa, I found a medium that has provided moderate success. This was a frustrating period, because there are many protozoa that culture very readily and can be maintained with little difficulty. But I was soon to discover that nothing about Lacrymaria was going to be easy. I also learned that making generalizations about this wee beastie was risky.
Culturing Lacrymaria was the first step, since it is essential to have a large number of specimens to carry out even relatively simple observations and experiments. Now, I have a quite reliable method that provides several hundred specimens in stable cultures which I have been able to keep going for over ten years. I should perhaps qualify that remark a bit. The method is reliable for me. Four other people have tried it and one has had good success, but the other three have had difficulty with long term maintenance and I think that I have finally figured out why. And here's where we get to the sex part. I can already see the headline in the "National Inquirer" "University of Wyoming Professor Conducts Bizarre Sex Experiments in Laboratory" and admittedly Lacrymaria's sex life is, by our standards, a bit strange. It not only does the old anti-arithmetic trick of multiplying by dividing, it also, if it can find a different mating type, will conjugate and exchange genetic material. But this business of mating types is a very peculiar business. As Margulis reports:
"Mating types can change or be lost according to the time of day (Barnett, 1966). During the day one clone will be of mating type 1 and will mate only with mating type 2. At dusk members of the clone will not mate at all. By nightfall the mating type 1s will have changed into 2s and thus will refuse to mate with other mating types 2s. By changing the lighting conditions, making "dawn" and "dusk" come at different times, the formation of different sexes or mating types in these paramecia can be controlled. Mating type is determined by the presence of proteins on the surface of the cilia."
(Margulis, A Handbook of Protoctista, p.200).
And we think human sexual relationships are complicated! Now, if there is no other mating type available, then Lacrymaria may simply start dividing up its own genetic material into six, eight, or more pieces and begin a process of repair and recombination. This mechanism is called autogamy. After this reorganization, the organism can then begin to reproduce again by division. Some scientists contend that the evolution of sex is primarily a repair system for damaged DNA. Richard Michod, University of Arizona, states this view in a highly unromantic fashion. "Mates," he says "are essentially spare-parts shops."
Certain conditions obtain for autogamy to take place and the cells must still be healthy enough to undergo this radical repair process. If the conditions are not right, then the organism will reach a limit in terms of the number of times it can divide and the culture will die out. What is intriguing is that there is apparently a genetic switch operative here. It functions rather like a counter and keeps track of the number of divisions. If there has been no new genetic material introduced by conjugation nor any reorganization of the genetic material by means of autogamy then when the counter reaches its preprogrammed limit, the cells die off. Joan Smith-Sonneborn, another cell biologist at the University of Wyoming, has experimentally determined the number of generations for certain subspecies of Paramecium aurelia.
Now, I wasn't looking for any such phenomenon as this when I started trying to culture Lacrymaria. However, on three occasions, I had all of my cultures die off within a period of about two weeks and on each occasion that involved over 50 cultures. This meant starting completely over again each time and this was extremely frustrating, since even with my culture method, Lacrymaria is exceptionally difficult to get started from a few wild specimens. In the old cultures, there was no indication of viral infection, chemical contamination, or predation. This led me to the suspicion that Lacrymaria might have a "genetic switch" such as the one which has been described for Paramecium. As a consequence, I spent a summer doing intensive collection from lakes, rivers, ponds, streams, springs, and puddles. If you were driving out in the plains or in the mountains near water and saw a tall overweight middle-aged figure in hip-waders with nets and bottles instead of a fly rod, that was probably me.
What I was after was specimens of Lacrymaria from a variety of habitats which would I hoped have some slight genetic variation and provide me with different mating types. As I mentioned I obtained the first organisms from Lake Hutton which is a highly alkaline lake with an extremely large sulfate peak. In practical terms what this means is wading through stinky, black, sulfurous mud. Most accounts describe the habitat of Lacrymaria as low or only moderate in oxygen with little water movement allowing the organisms to attach to the substrate in the detritus and feed. However, that summer I found specimens in six distinctly different environments ranging from the one I described at Hutton to a moss sample from a cold, clear, shallow gravel-bottomed stream fed by a nearby spring. I managed to culture these six different strains or eco-types and all of them flourished at first. However, after some months two strains began to get smaller and smaller, a clear sign of weakening. Four strains still survive along with several mixes. The crossing of the different strains sometimes produces a new hardier strain, but not always. In any case, all of this is a long-winded way of saying that those other three people who did not have success with my culture method may have run into the problem of a "genetic switch" triggered as a result of insufficient genetic diversity. Or another possible explanation is that they don't know how to follow culture directions.
I now want to turn briefly to three other aspects of the behavior of this organism, feeding, encystment, and regeneration.
Searching for food is completely random and prey capture is apparently governed only by direct contact with the "nose cone." There is undoubtedly some incidental ingestion of bacteria but it is extremely unlikely that this could be a food source sufficient to sustain Lacrymaria for any period of time. Experiment shows that there is little ingestion of random particles. Adding powdered carmine, indigo, or India ink to cultures does not result in the accumulation of these particles in food vacuoles. Lacrymaria feed primarily on small ciliates, flagellates, and amoeba, although on occasion they will, if they come in contact with a large ciliate such as Blepharisma or Stentor, tear chunks out of these cells as food. The flagellate Chilomonas is swallowed whole by Lacrymaria and even though there is then considerable contraction of the neck, the Lacrymaria looks rather like an ostrich that has just swallowed a grapefruit. The feeding process is so frustratingly random that one can wait for a very long time to observe it and during the wait is strongly inclined to regard this organism as very stupid, if not retarded and wonder how it ever survived at all.
2) ENCYSTMENT or THE RESURRECTION CAPER
Some protozoa, bacteria, and algae are able to go through a remarkable process wherein they radically reorganize their cell structure, produce one or more protective "casings" or "walls" and go dormant. The cell differentiates becoming essentially a blob of protoplasm containing a smaller blob of genetic material and all of this surrounded by an extraordinary membrane or two or three or in some cases four, which allow the organism to survive dehydration, heat, cold, oxygen loss and some kinds of contamination. Then later, sometimes years later, when conditions get back to normal, a complex biochemical and/or biophysical signal activates the organism through the membrane and the reverse process excystment begins. The cell begins to reform its own special organelles and then finally emerges from the cyst and takes up where it left off. Fortunately there are a few protozoa in which the cysts are both large and relatively distinctive allowing observation of both processes. Unfortunately, this is not so with Lacrymaria which does go into a "resting stage" and then sometimes into a cyst which is extraordinarily small and not at all distinctive and soon becomes virtually impossible to trace. However, I was able to get experimental evidence for encystment and excystment in Lacrymaria by letting culture dry completely and sit for weeks or months then adding new culture medium and occasionally Voila! Resurrection.
3) A REGENERATION or HOW TO GROW A NEW HEAD
We all know how starfish can grow new "arms" or certain kinds of lizards can grow new tails, but Lacrymaria has them all beat. It can grow a new head. I was able to demonstrate this by making a micro-scalpel out of a very fine glass needle and then as the organism extended its neck I would very quickly draw the needle across the neck. This requires some practice, but eventually one gets good enough at it that the head is severed and if this produces ethical qualms, one can always pretend that it was John the Baptist or Medusa. Nonetheless, I have a vision of the headline in the "National Inquirer": "Professor Brutally Decapitates Research Animals."
This bit of micro-surgery produces two remarkable results. 1) The head goes zipping off quite like a small and powerful motor. This strongly suggests that the head pulls the neck out from the body thus in part accounting for the remarkable extensility. This is also evidenced by the fact that when the organism is anchored the extensility is much greater than when the organism is free-swimming. 2) The main body and neck of the organism continue to swim and the wound is closed off virtually immediately. In a matter of minutes the process of regeneration has begun and in a remarkably short time, the body has produced a new head and resumes feeding. The severed head swims rapidly for several minutes, but will not regenerate, and gradually ceases motion and disintegrates.
Before I conclude, I want to mention one other intriguing aspect of Lacrymaria, and that is its ability to mutate in a bizarre manner. Twice I have found naturally occurring mutations and this is not too unusual with protozoa. However, I also accidentally induced mutations when I was studying the autogamy process. I used a dye known as Acridine Orange. This dye is a carcinogen, a well-known mutagen, and is photoactive. In my initial experiments, I had no guidelines at all to determine the desirable concentration of the dye and so I simply added a few drops of a 1% solution to one culture a few more drops to another one and a few fewer to yet another. The moral of this story is always keep precise records! And here we get another "National Inquirer" headline: "Researcher Creates 3-Headed Mutant Monsters." There were some other kinds of mutations as well, but the common pattern was three heads extending from three necks and all of them naturally trying to swim in different directions. I did manage to video-tape some mutant forms, but not these wonderfully tricephalic forms, since the video camera had begun picking up interference that made the picture unusable. It took five weeks to sort that problem and by that time no more mutants. I did get one rather blurry Polaroid picture which shows two of the heads clearly, but the third is hidden behind the other two.
My attempts to recreate these lovely monsters have not yet been successful. But by using different concentrations of Acridine Orange, I have discovered several things. One is that the organisms absorb the dye quickly and unless kept in dim light or in the dark the dye becomes photoactive and toxic. Another is that in very dilute solutions Acridine Orange seems to accelerate reproduction, activity, and perhaps even vitality.
I have conducted a number of other experiments including attempts to anesthetize the organism and then preserve it with the neck in an extended state. The substances used must often be diluted to concentrations of one ten-thousandth of one percent. These ventures have been maddeningly frustrating. Nonetheless I am proceeding with these experiments as time permits. Clearly there are many issues which I mentioned above which require further investigation; extensility, mutagenic responses, regeneration, and encystment. However, my most pressing project is the attempt to produce 80 foot specimens that I can breed in lakes. Needless to say, Scotland's Office of Tourism is very interested in these experiments.
Comments to the author Richard Howey welcomed.
Click here for a protocol to cultivate Lacrymaria olor written by Richard Howey.
Read the Micscape 1998 article 'Tear of a Swan - Lacrymaria olor' compiled by Maurice Smith and which presents the work of Richard Howey and features video clips / stills by Ken Jones.
The author's other articles on-line can be found by typing 'Howey' in the search engine of the Article Library, link below.
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