Frozen Evolution. Or, that’s not the way it is, Mr. Darwin. A Farewell to Selfish Gene.

Usually the natural scientist cares not just why something happens, but how it happens. He cares not only whether or not Toxoplasma manipulates human behavior, but about the tools which it employs to do so. So of course, from the very start, we pursued this question. Past studies conducted on animals indicate that the manipulation could occur using the neurotransmitter dopamine. Already in the mid-80s, a study showed that latent phase Toxoplasma-infected mice had a 15% higher concentration of dopamine in their brain than did uninfected mice (41). Based on the results of this study, we expected that humans with latent toxoplasmosis might also have higher levels of dopamine. But for a long time we didn’t know how to prove such a thing using the arsenal of methods we had at our disposal. With a human, of course, one cannot simply look into his brain and biochemically measure the concentration of dopamine (our experimental subjects, for example, did not seem enthusiastic about this idea). Fortunately, there are also several possible indirect methods. Of these, we chose the most indirect, but also the cheapest method. It is based on the fact that higher levels of dopamine in some parts of the brain manifest in a lowering of the psychological factor “novelty seeking.” This discovery originally comes from ethopharmacological studies on laboratory rodents. Later, American psychologist Claude R. Cloninger created a five- and then seven-factor questionnaire, called TCI, used to observe these factors in man (see Box 56 What does Cloninger’s TCI measure?).

It is presumed that the levels of some psychological factors measured by this questionnaire reflect the levels of certain neurotransmitters in the human brain. One of these factors is the tendency towards “novelty seeking,” which also occurs in mice. In humans it is expected that “novelty seeking” correlates negatively with the levels of dopamine in the brain. So we gave each of our test subjects, first the soldiers and later the

students and blood donors, Cloninger’s TCI questionnaire, and then looked whether there was a difference in “novelty seeking” between Toxo positives and Toxo negatives⁴³. To our surprise, there was a difference – and it exactly fit our hypothesis that there were heightened levels of dopamine in the brains of humans infected by Toxoplasma (Fig. 28). Toxo positive men and women had lower “novelty seeking” than did Toxo negatives. Up to 2007 we studied this phenomenon in five unrelated subject groups, and in three of them we managed to prove our

Fig. 28 Indirect proof of increased dopamine levels in Toxoplasma positive persons. The levels of this neurotransmitter correlate negatively with the psychological factor novelty seeking, the latter determined using Cloninger’s questionnaire (Temperament and character inventory – TCI). Several of our studies, including this one, have demonstrated that infected individuals have a lower value of this psychological factor. In our study, conducted on soldiers of the mandatory military service, novelty seeking depended on the level of education and type of professions; therefore, we analyzed the relationship between Toxoplasma infection and novelty seeking separately for each sub-group.

hypothesis (42) (34). What is suspicious, however, is that differences in this factor were mostly apparent in test subjects from Prague; Toxo positive people from medium-sized towns (with a population of about 10-100 thousand), even exhibited a nonsignificant greater tendency towards “novelty seeking.” In addition, we repeatedly found that Toxo positive people also have a higher Cloninger’s factor of “self-transcendence.” Unfortunately, this is a factor, the meaning of which probably differs between the Czech and American populations. The values measured in the Czech population reflect completely different psychical traits, than those which Cloninger was trying to measure. It seems that in Czechs it reflects the tendency to be swayed by others, or even (negatively) the level of intelligence. Interpreting changes in this factor as being linked to latent toxoplasmosis is definitely still precocious.

It’s interesting that, unlike a number of previously-studied Cattell’s factors, Cloninger’s factor of “novelty seeking” doesn’t change with time after infection. Furthermore, it always deviates in the same direction for Toxo positive men and women. An explanation for this could be that, after infection by Toxoplasma, change in Cloninger’s personality factors is faster than change in Cattell’s factors. In the case of Cloninger’s factors, infection causes a change in neurotransmitters, which can result in an almost immediate shift in psychological traits, such as a lowered tendency towards “novelty seeking.” In the case of Cattell’s factors, the physiological change must first manifest as a behavioral change. The person must realize his changed behavior, and then adjust his list of personal values accordingly. This recognized shift in values is what shows up as a change in Cattell’s factors, such as a lower tendency to follow social norms (Rule-Consciousness/Superego Strength). In a group of Toxo positives, changes in Cattell’s factors grow gradually more defined, whereas changes in Cloninger’s factors occur immediately after infection – so we cannot measure a correlation with time after infection.

What do we think is the specific mechanism that changes Cloninger’s factors? A couple years ago I had a clear hypothesis, which agreed with the sources available on the subject, as well as with the results of our studies. Small or large sites of inflammation form and persist in the brain of a Toxoplasma-infected human (or animal). At inflammation sites, active immunocytes release a variety of lymphokines, molecules which immune cells use to communicate. Some of these molecules, such as interleukin 2, stimulate the production of dopamine in surrounding tissue. According to my former hypothesis, higher levels of dopamine, and hence a lowered tendency towards “novelty seeking,” is but one of the side-effects of local inflammation in brain tissue.

The reader may wonder whether it still counts as manipulation, when, according to our hypothesis, the phenomenon seems like just a side-effect of pathogenic processes in the brain. The problem is that there exists a similar dilemma for every useful trait we find in organisms. All such useful traits formed as side-products of random and useless processes. If Toxoplasma today can manipulate the behavior of its host for its own ends, then this is only just the product of a gradual improvement – an evolutionary upgrading of the original abilities which appeared with random mutations. The phenomenon of increased dopamine levels that accompany inflammation in the brain wasn’t originally used by Toxoplasma to manipulate the behavior of its host. Rather, this phenomenon evolved as a means of communication among different types of immune cells (some of which exhibit specific dopamine binding sites). But when it turned out that aspects of the behavior induced by increased levels of dopamine help Toxoplasma spread to another host, then natural selection grasped this side-effect of infection. It took hold of Toxoplasma’s ability to increase the production of dopamine by causing inflammation, and, over the course of several generations, honed it (or at least substantially improved the ability). Strains of Toxoplasma which were able to induce chronic local inflammation in the brain of their host, and which were able to maintain heightened levels of dopamine, had an advantage over strains unable to do so. The strains with the useful ability spread more quickly than the others, so today we encounter only those strains which are able to raise dopamine levels. Today it is unimportant that Toxoplasma’s ability to manipulate the behavior of its host began as a side-effect of pathological processes. What matters is whether or not the ability to raise dopamine levels aids Toxoplasma’s chances of getting from an intermediate host to its definitive felid host. We could determine this using a very complicated experiment, by creating conditions under which manipulation would not be beneficial for Toxoplasma. For example, we could, over a long period of time, artificially transfer Toxoplasma from one host to another, and observe whether its ability to raise dopamine levels might change. If this ability were indeed an active manipulatory activity, one would expect it to gradually weaken. Under such conditions, in which we provide Toxoplasma with a transfer mechanism, the ability to raise dopamine levels wouldn’t benefit the parasite. To the contrary, maintaining the ability would only be a waste of resources which Toxoplasma could invest in something else, such as reproduction. But if raising dopamine levels were truly just a side-effect, then the ability should remain. Of course, such an experiment would be a difficult and long-term study. Moreover, we don’t know how taxing it is, energy-wise, for Toxoplasma to raise dopamine levels, and therefore how quickly this ability would disappear if it were no longer useful for the parasite.

Our hypothesis was that changes in “novelty seeking” occur due to raised dopamine levels, caused by the (possibly evolutionary magnified) inflammatory processes which accompany the formation and maintenance of cysts of the brain. We attempted to prove our hypothesis at least indirectly, using a much simpler method. If a neurophysiologist wished to tackle the question using a classic method, he’d likely try to damp down the inflammatory processes in the body of an infected individual, to observe whether this would manifest in the behavior of that human (or, more likely, the scientist would use a mouse) – to see whether the individual’s tendency towards “novelty seeking” would return to its original level. Such an experiment, however, would also be technically difficult, for damping down inflammatory processes means significantly altering an organism’s immune system. This basically rules out humans as possible test subjects, and we’d have to develop an experimental system which would allow us to observe Toxoplasma-induced changes in the “novelty seeking” of mice. In addition, it would be very difficult to unambiguously interpret the results of such an experiment. For example, the administration of corticoid hormones, which can inhibit inflammatory processes, has drastic effects on the organism; so the lower dopamine levels induced by the decreased inflammation might not even be the cause of most, if any, behavioral changes observed during such an experiment. Interpreting the behavioral changes as a result of the decreased levels of dopamine, would be a ridiculous conclusion, akin to that of a scientist in a famous joke: The scientist concludes that a flea has gone deaf after he removes its last leg – because the flea stops obeying his command to “Jump!”

Our approach to the question was completely different, and, I’d even say, significantly better (although the methods were less sophisticated, and hence more difficult to publish). We tried to determine whether the same manifestations we saw in Toxo positive humans also occurred in humans infected with cytomegalovirus (CMV). The human CMV is a very widespread virus, belonging to the family Herpesviridae. In the Czech Republic, it has infected about 80% of the population. The virus spreads through close contact between an infected and an uninfected individual (such as kissing; the earlier popular method of transfer on gas masks used in civil defense training in the Czech Republic went out of fashion after we successfully lost the Cold War). This means that the life cycle of CMV is completely different from that of Toxoplasma, since Toxoplasma reaches its definitive host through a so-called alimentary way. In other words, the definitive host catches and devours the intermediate host. Nevertheless, everything else about Toxoplasma and CMV is fairly similar. If you were to take a review article on Toxoplasma, and everywhere replace the word Toxoplasma with CMV, you’d find that the review would still make sense – except for, of course, the part about the parasite’s transmission. Just like Toxoplasma, CMV has a long-term dormant phase in the brain of the infected human. CMV does almost nothing in the infected nervous cells; that is, until the host catches AIDS or is prescribed a strong immunosuppressant, for something like cancer treatment or because of a transplant. Like Toxoplasma, CMV can become active under a weakened immune system, and the resulting encephalitis can be fatal. In addition, CMV also poses a serious risk during pregnancy. When a pregnant woman becomes infected by human CMV or Toxoplasma gondii, there is a chance that the infection could spread to the fetus; both species cause similar types of developmental defects (Box 57 How dangerous is Toxoplasma in pregnant women?).We were primarily interested in that CMV, like Toxoplasma, forms small sites of inflammation in the brain. If the increased “novelty seeking” we observed in Toxo positives were really a side-effect of these small inflammation sites, then we’d expect that the factor would also appear in people infected by CMV. To prove this hypothesis, we tested two large subject groups for the presence of antibodies against CMV (43). One group consisted of soldiers of the mandatory military service, whereas the other was made up of blood donors; each had a couple hundred people. All of these people filled out a Cloninger’s questionnaire so that we could see whether “novelty seeking” differed between those infected and uninfected by CMV. The results of both groups supported our hypothesis that an increase in dopamine levels is a side-effect of long-term local inflammation in brain tissue. Like Toxo positives, people infected by CMV exhibited a decrease in their tendency towards “novelty seeking.” Furthermore, with Toxoplasma we had found no correlation between “novelty seeking” and the concentration antibodies against the parasite – and the same was true for CMV. From this we concluded that the tendency towards “novelty seeking” decreased soon after infection by CMV, just as it did after infection by Toxoplasma. In addition, as was the case with Toxoplasma, we observed that the sharpest effect of CMV on “novelty seeking” was seen in people from big cities. I don’t know why this is so. It’s possible that the set-up of the questionnaire is responsible. Perhaps some of the questions which serve to determine “novelty seeking” may be answered affirmatively by someone from a small village, but negatively by the inhabitant of a larger city (or vice versa). Certain “novelties,” like visiting the zoo, may be commonplace for a big city dweller, whereas others, like riding a goat, may be common for the small towner but not for the city dweller. (Just to be clear, Cloninger’s

questionnaire has no questions about visiting circuses or fooling around with domestic animals – that was just an example.)

An interesting, unintended result of our CMV study had to do with another of Cloninger’s factors, specifically the factor of “harm avoidance.” This factor is supposed to correlate with levels of serotonin, another neurotransmitter. Something I did not mention: while screening our test subjects not only for CMV, we also tested them for Toxoplasma. In the case of “novelty seeking”, we found that the effects of CMV and Toxoplasma were additive. The subjects infected with both parasites had (on average) lower “novelty seeking” than did subjects infected either by CMV or Toxoplasma. But for the factor of “harm avoidance,” the relationship was more complicated. The thing is, Toxo positives and Toxo negatives reacted differently to infection by CMV. In Toxo negatives, CMV infection caused a lower “harm avoidance,” whereas Toxo positives with CMV had a higher “harm avoidance.” Until we confirm this result using other test groups, I’ll refrain from speculating about its meaning.

In the year 2009 our hypothesis that raised dopamine levels are a side-effect (or an evolutionarily improved side-effect) of inflammatory processes in the brain suffered quite a blow from an unexpected direction. Molecular neurophysiologists at Leeds and Manchester discovered that Toxoplasma is the only protozoan known to possess genes for enzymes which catalyze dopamine synthesis in the brain (44). It’s not definite why it has these genes, but of course there is the simple explanation that Toxoplasma can use the enzymes to increase synthesis of dopamine, which has already been proven (45). Toxoplasma may be increasing dopamine synthesis in order to manipulate the behavior of the intermediate host, to raise that host’s chances of being eaten by a felid – this, however, remains to be definitively proven. Naturally, the discovery that Toxoplasma possesses genes for dopamine synthesis does not rule out our inflammation hypothesis. Inflammation in the brain, among other things, causes increased levels of interleukin, which in turn signal the brain to produce higher levels of dopamine. The cysts of Toxoplasma, by causing inflammation, could have at first increased dopamine as a side-effect; the parasite may have later improved on this ability by providing the brain with additional molecules of enzymes for dopamine synthesis. (Box 58 How evolution forms useful traits).

By the way, we have completely neglected the basic question of how Toxoplasma could benefit from lowering the “novelty seeking” of its intermediate host. But it’s not difficult to formulate a hypothesis to explain it. For example, we might say that a mouse with a lower tendency towards “novelty seeking” won’t become familiar with the area around its burrow; it won’t know of the possible hiding places and dangers of the area, and will be more liable to become the prey of a cat. The problem is that we could just as easily formulate a hypothesis if our results were to show that Toxoplasma increases, rather than decreases, the tendency towards “novelty seeking.” We’d probably reason that a “novelty seeking” mouse, instead of safely remaining in its burrow, chooses to venture out at every possible moment, to roam its surrounding and investigate every unfamiliar object – such a mouse would be more likely to wind up in the belly of a cat. Certainly, we could test both of these hypotheses. There undoubtedly exists a strain of lab mice with more and less curious individuals; in any case, we could probably find a way to lower or rise the “novelty seeking” of mice through pharmaceutical means. The difficulty of the experiment lies in that we cannot determine which species was Toxoplasma’s natural host, before the spread of humans caused enormous growth in the cat (and mouse) populations. As was discussed earlier, it’s unclear whether the original, and thus more natural Toxoplasma host may have been a monkey (or an ape) rather than a mouse. Which host is Toxoplasma actually best adapted to – which host should we study?

There is one more problem I should mention. Does dopamine always lower the tendency towards “novelty seeking?” Based on our experiences with Toxo’s effects on human and rodent behavior, I doubt it (46) (47). Humans and mice certainly have one thing in common – they are irrepressibly curious creatures. If we give a mouse a new and old toy, then it will definitely play longer with the new toy (48). And when a mouse is infected with toxoplasmosis, its interest in “novelty seeking” decreases (49). On the other hand, Norwegian rats (including both laboratory Norwegian rats and wild-laboratory hybrids, which Joanne P. Webster used in her experiments on the effect of toxoplasmosis on the behavior of its host) almost anxiously avoid new things (50). After becoming infected by Toxoplasma, such rats have a greater tendency towards “novelty seeking.” If I had to guess, I’d say that dopamine (and Toxoplasma) primarily lowers the individual’s ability to recognize a novelty, to distinguish, for example, the novelty of a new toy. In species such as Norwegian rats, which normally avoid unfamiliar things, this leads to an increased factor of “novelty seeking;” whereas in species like mice and humans, which are naturally curious, it leads to a lowered tendency towards “novelty seeking.” And so, my dear ethopharmocologists, I have a question for you to resolve: is the effect of dopamine on “novelty seeking” really the same in Norwegian rats, as it is in mice?