I. How I didn’t become an immunologist nor a molecular taxonomist

               How did it all begin? Quite differently than I tell the reporters, who come sniffing through the laboratories each year when silly season rolls around. They’re hunting for stories on how scientists battle with nature, day and night, to wrest away her closely guarded secrets. Dear readers, know this: It’s not true that “it all started when I joined the department of parasitology in the 90s, and cast around for a research topic that might encompass both parasitology and evolutionary biology.” Period. Don’t listen to journalists – they’ll believe and publish anything.

            I’d say that the reality was much more interesting. After about four years working in the Department of Immunology of the Institute of Molecular Genetics of Czechoslovak Academy of Sciences, I returned to the Department of Parasitology at Charles University. My primary goal was to form a laboratory of molecular biology, and to dedicate myself to molecular phylogenetics – in other words, to continue with the topics I had researched before leaving the University. Most of all, I was determined to avoid immunology, though Jaroslav Kulda, the department head at the time, had originally invited me to his workplace to study this topic. Since I arrived in the department with my own salary fund, I didn’t feel tied to my bosses’ expectations (however, I always tend to play by my own rules, so the salary fund probably had little to do with it) (Box 1 How to arrive in the department with your own funds).

              Maybe it wasn’t very nice or responsible of me, but I think that I finally gave the head of the department a sufficiently convincing explanation, for why I wanted to form a molecular biology instead of immunology lab (neither of these existed in the department at that time). I told him, “Immunology is an interesting scientific discipline, but under the conditions of the department, which has no history of immunological research, it could not be conducted at a decent level.” Not only did we lack the basic equipment, but we also were missing contacts with top international workaces, as well as sources for literature, chemicals– everything you can think of. Furthermore, we lack

Box 1 How to arrive in the department with your own funds for salaries

This story reminds me of that common scenario in fairytales: kind-hearted Jack, on his way to save an enchanted princess, meets a hungry, weary traveler on the road. Out of the goodness of his heart, Jack shares his last piece of food with the traveler. The weary traveler then turns out to be a powerful sorcerer, who had taken this disguise to test Jack, and now rewards him for his altruistic behavior. About a month after the Velvet Revolution, with the country in disarray, several of us former members of the College of Natural Sciences arranged a meeting. We thought of returning to Charles University and establishing a new workplace, which would study theoretical and evolutionary biology. One of those present was Zdeněk Neubauer, who had been pressured to leave the University in the 80s; with his unorthodox opinions (and this is a very moderate term), he infuriated his more conformist colleagues like a red Soviet flag does an exceptionally irritable Western-oriented bull.  At the end of the meeting, Zdeněk asked each and every one of us, whether we really wanted him on the team. I told him that while I usually disagree with his opinions, I’d be happy for him to be part of our project. At the time, I had no inkling that Zdeněk Neubauer was a good friend of both future president Václav Havel, and of Radim Palouš, the first president of Charles University after the revolution. In the stormy post-revolutionary period, Neubauer had a say in what happened with the salary funds of the former Marxism-Leninism department. Unlike Neubauer, I had no way of knowing that he would be the one to select employees for his Department of Philosophy and Natural history. After ensuring salary funds for most of his conspirators (evidently, not everyone passed the weary traveler test), he told us that we could stay at his new department, or were free to transfer to a department of our choice, along with salary funds. I was among the people who chose the second option (though only transiently, as it turned out).                                


experience: in the Institute of Molecular Genetics, I primarily cloned interleukin genes, an activity closer to molecular engineering than to real immunology. Not even my fourteen months in the Department of Immunology at the University of Tokyo made up for my lack of experience in the field. Rather, they showed me that immunological research is difficult to carry out under the conditions of our University’s Department of Parasitology. I continued, “The most I could do here was assistant work, like preparing antibodies for the experiments of other colleagues, developing a diagnostic system to detect parasites, or look for a way to monitor the immune response of an infected host.” (Saying this, I got the feeling that something flickered in the eyes of the department head. It might have been that proverbial spark of hope, or just a trick of the light.) “But spending my time running a service laboratory was the last thing I wanted to do in a new, or really any, workplace.” (The light in the eyes of the department head dimmed).  If I remember correctly, Jaroslav Kulda never reproached me for moving to molecular biology and then evolutionary parasitology, and made peace with the situation.

Fig. 2 The protozoan Trichomonas. A sexually transmitted parasite that was the subject of my undergraduate and later graduate study. Scanning electron microscope picture by Pavol Demeš.


 In contrast, we at the University had significantly better equipment and practical experience for studying molecular biology. In my undergraduate work, I had looked for DNA in the organelles of the parasitic protozoan trichomonad (Fig. 2). The search was unsuccessful, which might have had something to do with the fact that the particular organelles (hydrogenosomes) in this protozoan have no DNA, as was discovered many years later (notice how carefully the scientist formulates his conclusions). In the ancestors of trichomonads, all the hydrogenosome genes moved into the cell nucleus (see Box 2 Why some cellular organelles have their own DNA, while others, like hydrogenosomes, don’ t

Box 2 Why some cellular organelles have their own DNA, while others, like hydrogenosomes, don’t

Some of the important modern cellular organelles, such as mitochondria and chloroplasts, developed from symbiotic – and in my opinion, probably originally parasitic – bacteria. Chloroplasts and mitochondria kept remnants of their bacterial DNA (most of it gradually moved into the nucleus of their host cell). Hydrogenosomes, on the other hand, peculiar mitochondria found in some protozoans, handed over all their DNA to the cell nucleus. The reason organelles move their DNA into the nucleus is pretty clear. Chloroplasts and mitochondria act as cellular power plants. In the process of converting energy, these organelles produce highly reactive chemical substances that are damaging to DNA and cause mutations. In the cell nucleus, the genes are much better protected. Furthermore, a single cell often has several hundred or even thousand of these organelles, so if each of them had their own DNA, subject to possible mutations, these organelles would begin competing among themselves to reproduce. The victors of the competition will be the organelles with mutations allowing them to reproduce the fastest, even if it detracts from their primary function – producing energy for the cell. So eventually, the predominant organelles will be those that reproduce faster than their competitors, but don’t provide the cell with energy. The fewer genes present in these organelles, the lower the risk that they will mutate and start reproducing faster, while abandoning their power plant function. So transferring organelle genes to the nucleus is a preventative measure against the formation and take-over of “selfish” organelles. But this prevention is not a hundred percent effective. Although the total number of genes in today’s mitochondria and chloroplasts (a couple dozen in mitochondria, a couple hundred in chloroplasts) is negligible when compared to the number of genes in the nucleus, many genetic diseases are caused by mutations in mitochondrion and chloroplast genes. Many of these genetic diseases are probably the result of the cell being overwhelmed by mutated, “selfish” organelles.

It’s a bit harder to explain why mitochondria and chloroplasts, unlike hydrogenosomes, kept some of their genes. Because of their handful of leftover genes, these organelles have to maintain a complicated apparatus to copy the genes, transcribe them into RNA, and translate them into proteins. At the moment, I’m inclined towards the hypothesis that an individual mitochondrion or chloroplast needs these genes in order to directly regulate the activity of its respiratory chain. This respiratory chain produces energy in a mitochondrion or chloroplast. The individual molecules that make up a respiratory chain are anchored in the organelle’s membrane, and during energy production, the molecules pass electrons along the chain while pumping hydrogen ions (protons) from one side of the membrane to the other. The resulting proton gradient is used by the cell to synthesize ATP, a molecule that serves as the cell’s primary source of immediate energy. The activity of the respiratory chain in each organelle must be tuned precisely. The wrong level of molecules in the organelle could quickly lead to the formation of the above-mentioned dangerous chemicals (radicals), which would first damage the proteins and DNA in the organelle, and then spread to the rest of the cell. So each component of the chain must be synthesized in the organelle, because otherwise its rate of synthesis cannot be adjusted to meet the conditions of the specific organelle. If the genes for respiratory synthesis were found in the nucleus, then it would be impossible to transport a protein from the nucleus to only the mitochondria that needed it, without the protein also ending up in hundreds of other mitochondria. This hypothesis additionally explains the lack of DNA in hydrogenosomes, which are organelles present in certain protozoan species that live in low-oxygen environments, and therefore cannot use normal mitochondria to produce energy. These strange mitochondria don’t use a respiratory chain, but synthesize ATP using a different, less dangerous method.


Partly thanks to this, my undergraduate work acquainted me with a variety of interesting techniques from molecular biology, which I used in an effort to find DNA in this trichomonad organelle. I should mention that I selected the topic of my undergraduate work myself, despite emphatic warnings from my mentor Jiří Čerkasov, as well as his remarkable wife and co-worker, Apolena Čerkasová. Instead of organelle DNA, I finally discovered the first dsRNA virus in a protozoan cell, as well as the presence of repetitive DNA in the Trichomonas nucleus. My graduate work included further study of this virus in different strains of trichomonads.

Unfortunately we published the discovery of dsRNA in Trichomonas, along with the subsequent proof of virion particles, later than our American competitors, but at least it was in a significantly worse journal However, in those days that was our workplace tradition. (I sometimes say “we” when discussing the Trichonomas experiments – not as a majestic plural – but rather to express the fact that experiments performed by undergraduate and graduate students are usually financed, planned, or co-planned by the student advisor; and usually several lab members are involved in the study, including other students and lab technicians.) Nevertheless, this time our American competitors didn’t have to try as hard. Unlike me, they didn’t have to drop their research for a year to become a tank platoon commander. Instead of running nucleic acids across an electrophoretic gel in the lab, I found myself, running four tanks and fifteen mischievous scamps across the snow-covered plains on a military base in the Doupov Mountains. I apologize to Captain Šic, the proud head of my battalion – who was famous for saying that hell would freeze over before any of us bean-sprouts (meaning us university types) would get an officer’s star, and yet probably spared me from future military call-backs (I suspect that he scribbled in his report: “Arm Flegr only if all has been lost!”) – again, I hope Captain Šic will forgive me when I say that my American competitors didn’t miss much in military service. The landscape of the Doupov Mountains might look nice from the vantage point of a horse-back rider, but certainly not to the shivering soldier sitting on the steel seat of a T-55 tank. After returning from the mandatory military service, we struggled to catch up to the Americans. Sometimes, in our lab experiments, we even closed the gap, but when it got to the final sprint – getting our work published – we usually came in second place. Even to this day, there are several interesting discoveries about Trichomonas dsRNA viruses that we haven’t yet published (Box 3 Trichonomas double-stranded RNA).


Box 3 Trichonomas double-stranded RNA.

I’ll never forget the day when, after three years of scrutinizing electrophoretic agar gels in the dim red light of the darkroom, I finally shouted “Eureka!” I had discovered something that looked like organelle DNA. I saw a strong, narrow band at the place that normally marks DNA which is three micrometers long (Fig. 3).


Fig. 3 Electrophoretic analysis of Trichomonas nucleic acids. Aside from the nuclear DNA band (on top), other bands are visible. I originally thought that these nucleic acid bands came from the hydrogenesomes, the protozoan’s modified mitochondria. It was later determined they actually come from the double-stranded RNA (dsRNA) of a virus that had infected Trichomonas. This was the first dsRNA virus described for a protozoan. On the left are the results of dsRNA capillary electrophoresis; having no machine ourselves, we resourcefully used a potty chair as the electrophoretic tank, and a car battery as the electric power source. The capillary tubes (from left to right) were filled with increasing concentrations of agarose gel. The picture on the right shows the results of a more modern agarose slab-gel electrophoresis (this time created from a plastic sewing box). The leftmost lane is a sample of total Trichomonas nucleic acids, whereas the last lane represents the same sample treated with DNA digesting enzyme, so it contains only RNA. The second lane is of retroviral dsRNA, and is used as a marker to determine the size of unknown dsRNA segments. Lanes 3 and 4 show retroviral dsRNA loaded together with various amounts of Trichomonas dsRNA.

It took me about two months to figure out that it wasn’t actually organelle DNA. The electrophoretic gel had at least six bands, in positions that corresponded to DNA molecules of various lengths. But most of these bands still showed up in the sample after I added a DNA-digesting enzyme, and disappeared when I added an RNA-digesting enzyme, under conditions in which not only single-stranded but also double-stranded RNA (dsRNA) is destroyed (Fig. 4).


Fig. 4 Results of the experiment showing that satellite bands on Trichomonas nucleic acid electrophoretic gel are composed of RNA, not DNA. Samples in each lane (from left to right) were treated with increasing amounts of DNase (an enzyme which digests DNA). In another experiment, the samples were treated with an enzyme which digests double-stranded RNA. This caused all the lower bands to disappear, leaving only the top DNA band intact.

I confirmed the dsRNA nature of the molecules using a combination of fluorescent dyes that glow different colors under UV-light, depending on which nucleic acid they have bonded to.Eventually, I even developed a quick method for isolating dsRNA from a mixture of nucleic acids (1). The Trichomonas virus forms spherical (icosahedral) shells (capsids) about 35 nanometers in diameter. But some of the dsRNA – the short ones – seems to be found in the Trichomonas cell outside these capsids (2); (3); (4). The amount of virus dsRNA in an infected Trichomonas cell is immense – comparable to the amount of DNA in the protozoan’s nucleus. Yet the presence of the dsRNA doesn’t harm the cell; at least under laboratory conditions, it doesn’t slow cellular growth. About 30% of the strains in our lab had the virus, but the amount and lengths of dsRNA in the Trichomonas cells differed from strain to strain (3). Interestingly, it’s almost certain that at least two types of trichomonad dsRNA molecules are covalently bonded to a protein (and the same is probably true for the killer-factor dsRNA found in yeast), which, as far as I know, nobody has published yet. The killer-factor is a mycovirus that is suspiciously similar to the Trichomonas virus, in both the size of its genome and the shape of its capsids. We weren’t able to determine whether the Trichomonas virus, like the killer-factor, also produces a toxin that kills uninfected cells.



But the study of RNA viruses in protozoans wasn’t supposed to be the main research topic in my new laboratory. Already during my post-graduate study, I was well aware that our workplace had two major advantages over labs in other countries. In our department, we had a respectable collection of parasitic protozoans preserved in liquid nitrogen. And outside the liquid nitrogen, we had an equally respectable collection of parasitologists, who, in the quiet sanctuary of the Prague College of Natural Sciences (which, as my colleague Stanislav Komárek like to say, is the last traditional German university in the world), waited out the tumultuous evolution of international biology in the second half of the 20th century. By the start of the 80s, in most Western universities, the departments of parasitology were either dissolved or gradually colonized by molecular biologists and biochemists, who knew little about the parasites they used in their studies. In our university, up to the late 90s, most of the scientists in the department of parasitology, and even most of its graduates, were able to find parasites in nature, determine their species, isolate, and, when technically possible, use them to establish a lab culture. And these abilities can be extremely useful. For example, they served me well in the field of molecular phylogenetics, which I began studying sometime in the mid-80s. In the 90s, scientists had not yet determined the zoological or botanical classification of many unusual groups of protozoa. So it seemed that we had a great chance to use our natural advantages, with the aid of modern molecular biology techniques, to fill in the empty spaces of the taxonomic system. This proved to true, as over the years my molecular phylogenetics research team (Box 4 Research teams) gradually discovered the phylogenetic positions of several interesting groups of parasitic protozoans (5);(6). Other groups of parasites were classified more quickly by our competitors, be it by a couple months or couple years (7) (8), but that’s life.

So how was it that I left my lab of molecular phylogenetics to study Toxoplasma’s effect on host behavior – in other words, to study the manipulation hypothesis? Simply put, it was happenstance. When I arrived in the college, I knew very little about the manipulation hypothesis, and I certainly didn’t expect to study it. In truth, I wasn’t all that interested in it. The manipulation hypothesis states that several parasites alter the behavior of hosts in an effort to spread from the infected host


Box 4 Research teams.

In the college of natural sciences, I always studied whatever seemed most interesting to me at the time. Sometimes, this wasn’t well-received by my surroundings. In the department of parasitology, some of my colleagues were taken aback or even scandalized by the fact that I also taught my students topics that were, let’s say, only loosely related to parasitology. For example, when studying the effect of parasites on human behavior, I also, incidentally (but rather intentionally), collected data related to general evolutionary psychology. If the secondary topic seemed interesting enough, I didn’t hesitate to pursue it. For this reason, I was happy to offer my laboratory as an asylum to students who were unlikely to find support elsewhere, because they had chosen an unusual though interesting topic. So, I might happen to be studying the effect of latent toxoplasmosis on human behavior, the phylogenetics of anaerobic protozoans, the role of scent in choosing a mate, the evolutionary significance of sadomasochistic preferences, and the peptide “dictionary” of parasites and free-living organisms – all at the same time.



into a new, uninfected host. For example, a parasite, in order to complete its life-cycle, might need its current host to get eaten by a certain predator (so that the parasite can spread to the predator). The parasite often is able to alter the behavior of its current host, so it is more likely to get caught and eaten. For example, the infected host may be less careful, more noticeable, spend more time in open space, or react more slowly when in danger (Fig. 5). When I arrived in the department, most of what I knew about the manipulation hypothesis came from my favorite books, The Selfish Gene and The Extended Phenotype by Richard Dawkins (see Box 5 The Selfish Gene).

Among other things, Dawkins discusses the case of the Lancet liver fluke Dicrocoelium dendriticum. During its life-cycle, this fluke needs to get from its intermediate host, an ant, into one of its definitive hosts, such as a sheep. Now, sheep don’t usually eat ants, but the fluke has a very elegant fix for this minor detail. It reprograms the behavior of its host, so that come


Fig. 5 The green-banded broodsac (Urogonimus macrostomus, originally called Leucochloridium macrostomum or Leucochloridium paradoxum), residing in an amber snail (Succinea putris). You can see the large, colorful and swollen broodsacs that this parasitic flatform has created on the snail. During the day, the broodsacs move to the snails’ tentacles and cause them to pulse with light. Insect-eating birds (the definitive hosts of the green-banded broodsac) confuse the tentacles with caterpillars and eat the snail. Inside the broodsacs (sporocysts) are hundreds of microscopic larvae (see the picture on the top left), which infect the finicky bird. The parasite doesn’t harm the bird too much; otherwise the bird would be more careful when hunting for caterpillars.


Box 5 The Selfish Gene

The book The Selfish Gene was published in 1977 by the British author Richard Dawkins. I believe the first copy to come to Prague was brought by Vladimír Jan Amos Novák, author of a world-renowned book about insect hormones, as well as several fairly nonsensical evolutionary theories; the founder and long-time head of the Laboratory of evolutionary biology, a practically independent research institute of the Czechoslovak Academy of Sciences; a member of the Central Committee of the Communist party of Czechoslovakia but simultaneously a decent sort. Among other things, Novák is famous for his personal letter to Lysenko, in which he tried to convince the latter to stop incarcerating his scientific opponents. Novák himself got locked up by the Russians for half a year, when, toting a backpack, he tried to illegally cross the border into the Soviet Union; he wanted to see for his own eyes, what baffling things were happening during the Lysenkoism in his beloved communist country. (The Lysenkoism was a politically organized witch-hunt of good geneticists and biologists in the name of the obscure, ideologically-backed theories of Trofim Denisovich Lysenko. By the end of the fifties, this persecution spread to the Soviet satellites, including Czechoslovakia, where it fortunately never was as drastic as in the USSR.) Novák apparently got the book from Richard Dawkins in person, and after his return from England, thoughtlessly lent it to my mentor Jiří Čerkasov, who soon lost it to me. I think I was the first Czech to read this book. And if Novák beat me on the trip from London to Prague, it’s quite possible that I win the category of being the Czech to have read it the most times. In a very accessible style, Dawkins shows why Darwin’s theory of the evolution of adaptations by natural selections fails to explain how evolution works in sexually-reproducing organisms. Dawkins proposes the theory of the selfish gene (which is basically an ingenious reformulation of Hamilton’s theory of inclusive fitness). This theory states that in evolution, individuals of a species don’t compete for the best biological fitness (see Box 58How evolution forms useful traits); rather, gene variants (alleles) compete amongst each other to get the greatest number of copies into the next generation of organisms. Definitely read this book (9) – it’s probably the best thing that Richard Dawkins has ever written (and believe me, his writing is not bad at all!). And then, I encourage you to follow up with my book Frozen Evolution in which I, in turn, took on Dawkins’ theory.

morning, the ant rushes out of its nest, goes a meter from the ant-pile, and climbs up a blade of grass. At the top, it chomps into the grass and holds on. There it waits until noon, when it climbs down and finds shade to avoid drying out. In the evening, when the air is cooler, the ant returns to its vigil, waiting until a passing sheep chomps up this blade of grass. I read this beautifully-rendered story so many times as a student, that I’ll never forget the elaborate antics of this clever fluke.

But when I returned to the University in 1991, I had no inkling that I might study something like this. As I mentioned, my primary goal was to form a molecular phylogenetics lab. Above all I tried to get back into normal academic life in the University, but for some reason I could not attain it. I’m not sure, to what extent it was tied to my wandering between the University and the Institute of Academy of Sciences, or to the first symptoms of a mid-life crisis, or to the unexpected arrival of freedom for former Czechoslovakia and the feeling that whatever the future had in store for me, it could not be a wonderful as the fall of Communism. Probably a little bit of each.

In 1987, after finishing my graduate study, I had to leave the University, which couldn’t find a place for me. Other graduates, who, unlike me, were members of the Communist party, were easily accommodated. But I wouldn’t say that reason I “left” was primarily political. Rather, the people in power tried to get rid of independent thinkers, who might someday pose a threat, or at least stand in contrast with the authority’s mediocrity. I have no illusions that this happens only in countries with Real Socialism. Nevertheless, there and then, leaders had an automatic system for chasing out such people – independent thinkers usually weren’t members of the Communist party, so it was easy to get rid of them under this pretext. In my case, it was probably also a matter of common human envy. At the time, I was invited to an excellent laboratory in a California university, where my more publication-successful competitors were studying the Trichomonas virus. And I, still young and naîve, mentioned in front my department head, who “coincidently” was also the leader of the local Communist party cell, what kind of pay I’d be getting at this American laboratory. As a result, I had to find a position outside the University. With a measure of good luck, I finally got a job in the Institute of Molecular Genetics at the Czechoslovak Academy of Sciences, in the laboratory of Vladimír Holáň. But my departure from Charles University lost me the opportunity to go the USA and continue researching Trichomonas viruses. In retrospect, I think it might have been for the better, because otherwise I wouldn’t have begun studying the manipulation hypothesis, nor made the acquaintance of Toxoplasma (however, I say this primarily for the benefit of that department head; if she happens to read this, I hope she stews in her envy).

At my new workplace, I was officially working in biotechnology, developing the already-developed monoclonal antibodies for pregnancy tests (in other words, this is what I got paid for). Unofficially, I did genetic engineering I subcloned sneakily-obtained interleukin genes into expression vectors (the job I was given my boss); semi-illegally, I worked on proteomics, studying the peptide dictionary of parasites; and finally, I worked in immunology, researching DNA synthesis in white blood cells in the presence of nucleotide synthesis inhibitors (a situation under which no DNA synthesis should occur) (see Box 6 DNA synthesis in white blood cells in the presence of a nucleotide synthesis inhibitor).


Box 6 DNA synthesis in white blood cells in the presence of a nucleotide synthesis inhibitor

A lipopolysaccharide, extracted from the cell walls of bacteria, is thought to stimulate the division of B cells (white blood cells (WBCs), or leukocytes, that create antibodies). Scientists have determined this by adding a radioactively-marked DNA building block - thymidine (one of four types of nucleotides) into the culture medium given to the leukocytes. If the B cells are dividing, they should incorporate the thymidine, along with the radioactive marker, into the DNA of the new cells. We conducted this experiment using the mouse WBCs. Vladimír Holáň noticed that when we added the nucleotide synthesis inhibitor aminopterin (more specifically, purine nucleotide synthesis inhibitor) into the medium, then intensity of radioactive thymidine incorporation significantly increased. Actually, one would expect the opposite: in the presence of aminopterin, cells shouldn’t be able to synthesize purine nucleotides, and therefore should not be able to synthesize the new DNA they need for cellular division. We never figured out why our B cells had no regard for theories and cheerfully incorporated radioactive thymidine into their DNA, even in the presence of the inhibitor. But it’s possible – and, I think, quite probable – that thymidine incorporation into WBC DNA, which is generally considered to be a measure of how quickly these cells are dividing, is actually tied to a completely different process, which, unlike DNA synthesis, doesn’t require a supply of new nucleotides and therefore is not inhibited by the presence of aminopterin. My guess is that in certain areas, WBCs continuously degrade and re-synthesize their own DNA, which might be related to the antibody affinity maturation (see Box 22 Antibodies and myths about them. If I hadn’t left the Institute of Academy of Sciences for the University, I probably would have continued solving the mystery of what lipopolysaccharide-stimulated B cells are actually doing, when immunologists think they are synthesizing new DNA. If one of the readers wants to take a crack at this problem, then I must warn him – only some lipopolysaccharides have this effect on lymphocytes (of causing thymidine incorporation even in the presence of a nucleotide synthesis inhibitor).



I wasn’t too happy in the academic institute, probably because I had been spoiled by the atmosphere in the University. To say it more positively, in my time at the College of natural sciences, I was most grateful for the fact that people on different research teams helped each other, borrowed each other materials and equipment, gave advice and most of all, had a true interest in their work – an interest which often even bordered on enthusiasm. They did science for the sake of science, not for money or their career. In the Institute, I got to study something I was interested in (by that I mean the semi-illegal part of my work), and I had incomparably better equipment than at the University, but I felt out of place. There was great rivalry among the research teams, and when I needed to borrow a certain chemical from a friend, I had to creep into his lab after working hours, so that his boss wouldn’t see. And so it’s not surprising, that immediately after the fall of Communism, I happily returned to my former position at the University. But to my dismay, soon after I got the unshakable feeling that I no longer belonged there. You can never step into the same river twice. Fortunately, it all straightened out after a couple years, but the first few years back in the College were strange and certainly not pleasant.

Maybe for this reason, for the first time in my life, I began paying attention to my own psyche. I began considering past events in which my reactions had surprised me, but which I’d had neither the time nor the inclination to dwell on. As long as I can remember, maybe since I was six years old, my opinions and way of thinking hasn’t really changed. In any case, the mistakes I make are the same. But suddenly it seemed that in certain situations, I behaved differently than I would’ve expected. I’ve always considered myself to be someone who doesn’t give up easily, and who carries out important things to the end. Usually, I am very reluctant to submit to authority, and even less to “authority;” and if I must do so, then it is only for strategical or tactical reasons, decided after serious consideration and borderline self-denial. Life taught me not to insist upon my opinions at any cost. Nevertheless, I am sometimes unsuccessful, which has gotten me into several tight spots. But to tell the truth, I didn’t mind the occasional “tight spot.” I was quite happy to tussle, time to time, with an unwelcoming environment. Of course, if only due to my physical disposition, I rarely fought physically; but that’s probably because in today’s cushy times, conflicts between adults are rarely addressed with a stick or a fist. Furthermore, several times in my childhood (and also a few times later on), I was able to avert a physical conflict at the last minute with an unexpected psychological maneuver: You’d be surprised at how quickly an enthusiastic aggressor on the ridge of the Krkonos mountains will be thrown off balance when you politely inquire after the soonest bus to Neratovice. In any case, I must agree with a friend who would always say: “Whenever you see a hornet’s nest, then you pick up a stick and start poking at it, to see what the stupid insects will do. And then you have to find a way to get out of the mess.”

And certain recent behavior patterns did not fit my personality. A simple example: buying salami sold by weight. In my childhood, butcher and delicatessen shops – and actually, pretty much any shop – that sold goods by weight, cheated their customers. Today, of course, they still do, but impersonally and on a professional basis – the cashier scans your vacuum-sealed bag of ham with added water, wishes you a good meal and hopes you’ll come again (or you can go to another store, if you think their water is better). During my childhood, salespeople apparently considered it a matter of personal pride, to cheat their customer of a couple ounces on each portion they sold. So when a person bought ten ounces of ham, he could count on either receiving less of the meat, or at least getting the wrong amount of change – or possibly, both. Of course, this also happened to me. And instead of protesting and causing a nice ruckus (and losing the possibility of returning to the store), I kept quiet. And what’s more, keeping quiet for some reason seemed more beneficial. As if staying mum and accepting the scam would somehow gain me a future advantage over the salesman. “I sure fooled him, letting him cheat me for the salami.” All in all, completely irrational behavior, which in retrospect, try as I might, I cannot see as beneficial. And after leaving the store, I could not understand the motive behind my actions, nor the feelings that accompanied them. Could the salesperson have hypnotized me?

Now for a different example. The house I live in had bad electrical wiring, so time to time I blew a fuse. One day I was particularly successful, and blew not only the secondary circuit breaker, but also the main circuit breaker, and finally the power inlet box on the outside of my house. I knew that there are high currents running through this area, and that it’s safer not to approach it, but rather to call the professionals. I also happened to know that replacing this fuse positioned in front of the electricity meter is the business of Electrical Company (or whatever its name is) and that it’s free of charge. And I told myself – few people know that it‘s free of charge, so when the repairmen arrive, they’ll probably ask to be paid. No doubt they’ll say that I caused the damage, therefore I should pay for their trip and the broken fuse.

And I was right. The contractors arrived, changed the fuse, and told me to pay them ten bucks. At the time, five bucks was something like fifty bucks today. I knew that they had no right to the money; and what’s more, I had anticipated their maneuver. But precisely because I knew it was a scam, and they thought that I didn’t know, then, with a feeling of superiority, I gave them those ten dollars. All the while, I told myself: there, that’ll show them. Of course, I didn’t show them anything – there was nothing to show. The contractors cheerfully stuck the ten dollars into their pocket and drove off. And I stood there in disbelief, waiting for the punch line. How is it possible, that whenever I let somebody cheat me, I have a strong feeling that I’m actually winning? At that moment, I understood that that feeling of superiority is only illusion, and incompatible with reality. The person who cheats me leaves with a feeling of smug satisfaction (he’d probably be even more pleased to know that I let him cheat me) and I am left only with the strange feeling, that I cannot grasp how I let it happen, and why on earth I thought it was a good idea.

Like I said, after my return to the College of natural sciences, I started to see this pattern of strange behavior as an interesting psychological problem. I asked around, whether others had experienced something similar, and discussed the problem with my friends. I inquired whether anyone ever got the feeling, that in a particular situation they behave according to a foreign will and foreign interests, even though it seems like they are acting voluntarily. We probably discussed hypnosis and suggestion, since I thought they might be related. We tossed around the idea that certain people could be able to impose their will upon other people, so that victim behaves (with a feeling of superiority) in a way that benefits the suggester. I don’t recall what we concluded, but it was clear that none of my acquaintances had experienced this situation.

About a month after one such debate, played out in a cozy, poorly lit corner of a pub close to the college building, Jaroslav Kulda stopped in my lab, in dire need of volunteers to test a new Toxoplasma antigen. For a long time, the department of parasitology conducted a study of toxoplasmosis. When I arrived in the College of natural sciences, this study was nearing its end, but a small part of original activities still existed, namely the testing Toxoplasma antigens.

The method for producing this antigen for toxoplasmosis diagnosis was probably developed by the former researchers in the department of parasitology. By the time I arrived, the antigens had long been commercially produced by the Institute of Sera and Vaccines; but whenever they came out with a new batch of antigens, it brought them to our department for testing. In addition, several clinics sent us pregnant women suspected of having toxoplasmosis, and we were to screen them using a method known as the skin test. In this test, a Toxoplasma antigen (a mixture of macromolecules obtained by solubilization of Toxoplasma cells) is injected into the skin. Within two days, people infected with the parasite – who are Toxo positive – get a red spot surrounding the injection site. Based on the size of the spot (scientifically-speaking, based on the intensity of the delayed hypersensitivity reaction), we could determine how long ago a person had been infected, as well as the risk of health complications for a pregnant woman (see Box 57 How dangerous is Toxoplasma in pregnant women?).

Testing antigens and screening pregnant women went well together; we had a regular supply of fresh antigen to test, and simultaneously a regular stream of patients who we could screen using the antigen. But before screening the women, we had to quickly determine how the antigen worked, and in what concentration we’d use it. And that was when Jaroslav Kulda ran from lab to lab, looking for volunteers for antigen testing.

Today, of course, it would probably be impossible for a parasitologist who was not a physician to inject people with an antigen, but back then, they weren’t so strict about it. The times were a bit wilder, and such things weren’t as tied down by various regulations. For example, in one of the labs for the parasitology class, students injected each other with antigens to test for toxoplasmosis. In 1993, we even had an undergrad student in her second or third year of study conducting the skin test. I get goose bumps when I imagine the mess we’d be in if someone had complained about it, or even reported us to the authorities. For this reason, we quickly abandoned this method of testing, and switched over to the classic serological method. Serological tests, which determine the level of antibodies in a blood sample, are neither as sensitive nor as specific as a skin test, which means that one risks a false positive or false negative result (see Box 7 The specificity and sensitivity of diagnostic tests).


Box 7 The specificity and sensitivity of a diagnostic test.

The quality of a diagnostic test is primarily based on its specificity and sensitivity. A test with low sensitivity is one unable to detect low concentrations of a particular substance; therefore, it sometimes gives us a false negative result. So a person with low levels of Toxoplasma antibodies may be diagnosed as Toxo negative by an immunological test, even though he has actually been infected. On the other hand, a test with low specificity is one unable to distinguish Toxoplasma antibodies from antibodies against a different parasite. Such a test might diagnose someone who never had the parasite as Toxo positive – in other words, a false positive reading. Unfortunately, the specificity and sensitivity of a test often work like two communicating vessels. A test with high specificity has low sensitivity, and vice versa. Therefore, physicians select a test based on whether a false negative result or false positive result would do less harm to the patient. (In which case, they would select a high sensitivity low specificity, or high specificity low sensitivity test, respectively.) But in basic research, low specificity or sensitivity usually doesn’t pose such a serious problem, and the test is chosen according to different criteria. If a cheaper and faster, but less specific or sensitive method allows us to test ten times as many individuals than a more expensive and time-consuming, albeit more specific or sensitive method, then it’s usually better to go with the former, “inferior” method. The possible greater error will be made up for by the opportunity to test substantially more people.


On the other hand, the researcher doesn’t risk get in getting trouble. One of our doctor friends, who came to the college once or twice a year, would draw (and still does) the blood for testing. The student volunteers would enter the “office” in four-minute – in later years, even three-minute – intervals, sign the informed consent form, get about 4 mL of blood drawn by the doctor, and pick up a psychological questionnaire to fill in. And we were lucky to have the doctor we did, because just his name guaranteed that the students would come in droves to have their blood drawn. Whenever I lured the students to this testing, I always emphasized that the doctor’s name was Zdeněk Hodný – something like “Joe Gentle” – and therefore that the blood draw wouldn’t hurt a bit. The students probably didn’t believe me, so they came to see for themselves.

To get back to the start of our research: when Jaroslav Kulda came looking for volunteers, I naturally stepped forth. In the first place, I was interested in the testing process; and furthermore, in whether or not I was infected. To my surprise – and by that, I don’t mean pleasant – I found, two or three days after the testing, that I am Toxo positive. A red spot had formed around the injection site on my arm, and based on its size, I’d been infected relatively recently. At the time, I suspected this had happened in Japan, where I’d sampled raw or undercooked meat, and where the Toxoplasma prevalence is about the same as in the Czech Republic – that is, around 30%. But today I think my infection probably happened before my Japan trip, maybe when I was handling hay from a rabbit run (see Toxoplasma risk factors, discussed in chapter XXI).

At the time of my testing, I knew little about Toxoplasma and toxoplasmosis (the disease caused by this parasite). Although I worked in the department of parasitology, and even in the room where Toxoplasma research was conducted; and my undergraduate work was carried out on protozoans in a lab shared by the department of parasitology and the department of animal physiology, my research focused on a different protozoan, the sexually-transmitted parasite Trichomonas. In any case, by education I was more of a physiologist or cellular physiologist; at heart an evolutionary biologist; and in terms of professional experience, primarily a molecular and cellular biologist. And as I mentioned, I’d spent four years working in


Fig. 6 The life cycle of Toxoplasma gondii. The definitive felid host releases oocysts in his feces (a). Oocysts reside in the soil and enter intermediate (c) and definitive hosts (e). In their bodies the oocysts quickly become tachyzoites (d), which divide rapidly. Tachyzoites spread the infection through the entire body (f), often using the host’s free-moving cells, such as white blood cells, as vectors. In host tissues, particularly in muscular, nervous, and connective tissue, tachyzoites change into slowly dividing bradyzoites (d), which permanently remain in the host in the form of tissue cysts. If the infected individual is eaten (b), then the tissue cysts release bradyzoites, which change into tachyzoites in the new host (h). If the consumer is felid, then the parasite differentiates in its intestinal cells. The tachyzoites first become merozoites and finally gametocytes, which fuse in pairs and form resistant oocysts. In a pregnant woman (or gravid female of any host species), tachyzoites can infect the developing fetus (g). Humans can be infected with oocysts from things like unwashed vegetable; or with bradyzoites from sources like raw or improperly cooked meat. Other sources of infection include contaminated water, and blood transfusion or organ transplantation from infected donors.

immunological labs. The most I knew about Toxoplasma was that it’s some protozoan; but I sometimes mixed it up with Toxocara, which is a parasitic helminth (or, as an old-timer would say, a parasitic worm).

When I discovered that I was Toxoplasma-infected, I quickly got to know my enemy. The first thing that caught my attention was the interesting life cycle of this parasite (Fig. 6). Toxoplasma is a coccidium, and thus related to the protozoan which causes malaria. Both Toxoplasma and malaria belong to the group known as the Apicomplexa. Toxoplasma reproduces sexually in the intestinal epithelium of felids. Cats, therefore, are Toxoplasma’s definitive hosts (see Box 8 Intermediate and definitive hosts).


Box 8 Intermediate and definitive hosts

In their life-cycle, many parasites go through several host species, each of which plays a specific role. The most important role is that of the trophic host,who the parasite pumps for maximum nourishment –resources used to form the biomass of its offspring. Then the vector, or transport host, ensures a transfer into a new host, and, if possible, an uninfected population. Finally, the definitive hostis the organism in which the parasite reproduces sexually – in the other, intermediate host(s), it reproduced only asexually. Whereas in the trophic host, the parasite‘s primary concern was the number of offspring, in the definitive host it’s also concerned about variety. Therefore, the definitive host serves as the place for the parasites’ “rendez-vous.” The parasite usually doesn’t harm the definitive host, because it needs this host to survive as long as possible and accumulate as many distinct strains of the parasite as possible. Quite often, the definitive host also serves as a vector, and the intermediate host also serves as a trophic host, although it’s not a rule – there are plenty of exceptions. Beyond that, parasitologists define a paratenic host as a host in which the parasite cannot reproduce, but can sometimes accumulate. Through such a host, a parasite can get into an intermediate or definitive host – usually when the paratenic host is eaten, intentionally or unintentionally, by the new host. Sometimes the existence of a paratenic host increases a parasite’s chances of getting into another host; but other times, it’s to the parasite’s disadvantage. The parasite might get into the paratenic host by accident, and only get to the definitive host if it’s lucky.



Toxoplasma’s intermediate host, on the other hand, can be any warm-blooded animal – so under normal circumstances, a mammal or bird. It’s rumored that under “abnormal” circumstances, a fish can also become a Toxoplasma host, if kept at warm enough temperatures. It probably wouldn’t work with a carp – I doubt he’d appreciate water at 30 or more degree Celsius (86°F) – but with a tropic fish, it might be feasible. Toxoplasma’s life-cycle usually begins when an infected cat, the definitive host, excretes cysts in its scat. A cat is usually infected as a kitten, and releases cysts for only two or three weeks, although in large amounts. Then it stops releasing cysts, and thereafter is not contagious. Through the feces, the cysts make their way into the soil. From there they can infect any organism, be it mouse or man, that digs in the dirt, or eats something with dirt still on it. (For example, a child playing in the sandbox, or an adult munching on a carelessly-washed carrot). When a mouse becomes infected, it usually suffers only a mild illness (although this depends on how “bad,” or virulent, the strain of Toxoplasma is, and how many parasites got into the mouse); something similar happens with a human. Certain mammals get sicker than others. Upon reaching the intestines of an intermediate host (mouse or man), the protozoan exits the cysts and form tachyzoites, a motile form of Toxoplasma. Tachyzoites attack various cells in the body, and inside, they quickly reproduce. A healthy immune system deals with these tachyzoites pretty quickly. It develops a strong immune response, and the tachyzoites have to “retreat into illegality.” The quickly-reproducing tachyzoites become slowly-reproducing bradyzoites, which remain in the body as tissue cysts until the host’s death. If the infected intermediate host is eaten by a felid, the protozoan begins sexually reproducing in the cells on the internal surface of the intestines, and releasing hardy cysts into the feces. And so it comes full circle. If the intermediate host (mouse or man) is eaten by an organism other than a felid, it behaves just as it would in any intermediate host, reproducing only asexually and eventually forming tissue cysts. Immediately after infection, a person undergoes a phase known as acute toxoplasmosis, during which the tachyzoites are rapidly reproducing. In someone with a healthy immune system, this

Fig. 7 A Toxoplasma cyst in a murine brain. The small particles inside the cyst are individual bradyzoites, “silently” awaiting the moment when the host is eaten by a predator (hopefully by a felid, the definitive host of Toxoplasma). The bradyzoites will be released by the action of digestive enzymes and penetrate the host from the intestinal lumen through the endothelium. Photo by Mirka Pečálková-Berenreiterová

resembles mild virosis, and is often accompanied by swelling of the lymph nodes. The following phase, in which the parasites withdraw in tissue cysts, is known as latent toxoplasmosis. From a clinical standpoint, it is symptomless. Only in a small fraction of cases does a person ever realize that he was infected, and that he will carry Toxoplasma tissue cysts in his body for the rest of his life (Fig. 7).

When reading up on Toxoplasma, one thing in particular caught my notice. This parasite can serve as a model organism for studying the manipulation hypothesis, because it seems that Toxoplasma alters the behavior of its intermediate host in order to up its chances of getting into a definitive host. In the 80s, an English team led by notable parasitologist William M. Hutchison (who discovered the life-cycle of Toxoplasma) tested this hypothesis. They published a number of studies showing that, in certain situations, Toxoplasma-infected mice behave differently than healthy mice. And in a natural setting, some of the behavioral differences of uninfected mice could have made them more likely to get eaten by the definitive host, a cat. It wasn’t


Box 9 Manipulative parasites: body snatchers

A manipulating parasite, figuratively speaking, battles over the body with its host. The host was evolutionarily programmed to behave in a manner that would get as many copies of its own genes into the next generation; whereas the parasite was programmed to make the host behave in a way that would pass on the parasite’s genes. At first glance, it might seem that the host will win the tug-of-war; after all, he’s fighting on his own turf. But in reality, the parasite is usually the victor. The main reason is because, evolutionarily, the parasite is much more experienced than his host in battling over body and behavior. The parasite battles with a host in every generation; and only the parasites who win will pass their genes to the next generation. In contrast, only a part of the host population battles the parasite in a particular generation. Moreover, it’s still possible for hosts who would lose the battle to pass on their genes – for example, by reproducing before getting infected or manipulated. Because of these two reasons, the parasite is exposed to more systematic selection than the host; so he usually wins the evolutionary battle. Probably the most sophisticated “body snatcher” is a type of barnacle, which belongs among marine crustaceans. These parasites don’t waste time re-programming the behavior of their host, but instead take-over the whole body. The miniature larva of the barnacle genus Sacculina penetrates the body of a crab and then grows into network of threads, which most closely resembles the mycelium of wood-decaying fungi (and I’ll have you know that barnacles are actually the distant cousins of crawfish and crabs!). They hormonally castrate their host and divest him of the ability to shed his cuticle, so that he won’t waste energy on such trivialities like his own growth and reproduction. After some time, the reproductive organ of the barnacle bursts out of the crab’s abdomen, and from then on the crab produces the larvae of his parasite. The great marine robbery is complete. The crab lives as he would normally, but instead of producing his own offspring, he makes that of the parasite. So the next time you’re in Hawaii, you can easily find one of these crabs. In some areas, the majority of the population has been infected, and already from a distance you can distinguish them by the fact that their shells are overgrown with barnacles and algae – the crabs, you see, aren’t allowed to molt.


long before this new knowledge about Toxoplasma’s “life-style” clicked with what I’d had observed regarding my own behavior. In my muscle and nervous tissues, lies a protozoan whose interests are undoubtedly different from my own. Whereas evolution programmed me to survive and reproduce, it programmed Toxoplasma to try to manipulate me into getting eaten by a felid. Well, at least in the case of mice it can do so by altering their behavior (see Box 9 Manipulative parasites: body snatchers). So why couldn’t it also manipulate a human? Hidden in its tissue cysts, Toxoplasma has no way of knowing that it’s in a human instead of a mouse brain; and even less so, of knowing that, for several millennia now, the chances of humans becoming cat chow are very slim. (I wanted to write that the chances are non-existent; but not long after I started my studies, one of our students was killed by a tiger in the Prague Zoo. I don’t know whether the student was Toxo positive, seeing as he climbed into the enclosure, I wouldn’t be surprised.) So Toxoplasma continues doing what it learned from its evolution, from those tens of millions of years that it’s lived on this planet, attacking warm-blooded animals. It lies in its cysts, awaiting its opportunity, and manipulating its intermediate host so that he’ll quickly wind up in a felid’s stomach.

Suddenly I had an explanation for my strange behavior, for those situations in which I sought to be the victim of an attack and voluntarily cooperated with the person doing me harm. Even this irrational behavior is, from an evolutionary standpoint, an advantageous adaptation – of course, it’s not good for me, the unwilling host of Toxoplasma, but it’s very useful for the parasite trying to get into a felid’s body. All the pieces fit. And I realized that, if my hypothesis were correct, then its implications applied not only to myself, but to an enormous amount of people. A third of the world’s population was Toxo positive in the early 90s. In the Czech Republic, it was also a third; in France, 60 to 70%; in Germany and Hungary, 50 to 60%; and in USA, about 20%. Although in China, or at least in those areas covered by the studies, the prevalence of toxoplasmosis is significantly lower – usually between 5 and 10% – in South America more than 60% of the population is infected, and in parts of Africa, over 90%. Just the number of potentially afflicted people is very disturbing; without taking into consideration the possible health and economic impacts. At this point, my primary concern wasn’t just my own behavior, but rather the possible effects of Toxoplasma on a third of the world’s population. And I immediately began planning, how to attack my hypothesis.






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The classical Darwinian theory of evolution can explain the evolution of adaptive traits only in asexual organisms. The frozen plasticity theory is much more general: It can also explain the origin and evolution of adaptive traits in both asexual and sexual organisms Read more