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Under the yoke of Chronos

Team verifies the variety of rhythms that organize life in animals

MIRIAN MARQUES / USPThe Folsomia collembolan: regular oviposition and change of skinMIRIAN MARQUES / USP

There was a time when, throughout four consecutive months – without so much as a thought for resting at weekends and holidays -, a group of biologists from the Zoology Museum of the University of São Paulo (USP) accompanied, day by day, the growth and reproduction of collembolans, primitive insects without eyes and pigmentation, which measure less than one millimeter in length and live deep down in caves. Taking turns in shift work, the researchers lost count of the nights they spent awake, or with just a nap on a mat stretched out in the corner of the room; every eight hours, they had to note down what was happening in each one of the 486 flasks, each one with a single insect. There were also moments of discomfort when they followed the daily life of solitary bees of the Tetraglossula anthracina species, in the interior of upstate São Paulo, or the development of the anopheles mosquito, which transmits malaria, this time in the Atlantic Rain Forest in Paraná.

The hundreds of pages of notes bear witness that the existence of a complex temporal organization must be considered, to understand how the daily activities of living beings works, like feeding, sleep, rest and work generally. With reinforcement from extra data, obtained with leaf-cutting ants (Ata sexdens ) and stingless social bees (Frieseomelitta doederleini ), the species being studied today, the group coordinated by Mirian David Marques discovered that this temporal organization involves not only a biological clock, as it was expected, but at least two other types. Of all, the best known is the circadian one – with a duration of roughly 24 hours, it is governed by the alternation between light and dark, and it coordinates, for example, sleep in human beings. But there are also the ultradian one, which last less than 20 hours, like heartbeats and respiratory rhythms, and the infradian ones, which exceed 28 hours, such as happens with the shedding of the skin of some insects, which takes place in periods that vary from two days to one year.

“The tendency used to be to regard the circadian rhythm as a kind of sole clock”, Mirian comments. In 2000, her work with biological rhythms, started 18 years ago, was joined by a group of physicists and geologists, who helped to interpret the animals’ behavior – one of the results of this joint work is a new mathematical model that explains the infradian rhythms, published in November 2000 in the Journal of Theoretical Biology. “The consistency and beauty of the projects resides precisely in the constant dialog with other groups”, says the researcher.

If, on the one hand, the team broke the monopoly of the circadian rhythm, on the other, itreasserted the importance of this kind of temporal organization in organisms. The circadian rhythm caused a surprise by showing that it is part of the life of theStrinatia brevipennis cave cricket – always in the dark, it seemed that they would not need this kind of clock – and how early it comes in human life. A study published in February in Biological Rhythm Research , which counted on collaboration from the team from USP, proves that the circadian rhythm is manifested in premature babies: contrary to what used to be imagined, as babies still do not relate to light and dark, research showed a variation in the temperature of the newly-born which is repeated at each cycle of roughly 24 hours – a clear indication of the circadian record.

How can this be explained?According to Mirian, there is a sort of memory or primitive circadian record in all living beings, like a legacy from the most remote ancestors of each species. It was already known that the control of the biological rhythm is associated with theper (from period) genes, identified in the 70s, and others like thetim (timeless) and cry (cryptochrome) genes, discovered just a few years ago – making up together what is called the temporal genome.

Mirian widens the debate: besides the biological clocks of living beings, it is nature that shows a rhythm of work. “All the cycles, working in a harmonious manner, guarantee the interaction between organisms and the environment they live in”, she says. The light and dark cycle is an important point of reference for fostering this harmony, in order to indicate the moment for eating, waking up and sleeping. The works by USP are helping to expand the horizons of chronobiology, the area that studies biological cycles, by showing that other cyclical elements – such as the availability of food, the alternations between hot and cold and wet and dry, reproduction and social relations – may help to fine tune life.

“I wanted to open up new horizons”, Mirian confesses. It was in 1987, having concluded her post-doctoral studies in the United States, that she inaugurated the Chronobiology Laboratory in USP’s Zoology Museum. Right at the start she decided to study biological systems that diverged from the hallowed classical models – rats, mice, seaweeds, fruit flies and moths. Time has shown that the challenge was greater than she had thought.

In 1993, the thesis for a doctorate by Miriam Gimenes, one of Mirian Marques’ pupils, inaugurated a new stage in chronobiology: very probably, it was the first study in this area carried out in a natural environment, far from the laboratories. Mirian Gimenes worked with solitary bees of the Tetraglossula anthracina genus, accompanied in their own habitat, the marshes, close to the cities of Campos do Jordão, in the Mantiqueira mountain range, and to Mairinque, also in the interior of São Paulo. The places were chosen because they have the same latitude – it was a way of guaranteeing similarity between the annual variations of the light and dark parameter.

These bees seek exclusively a yellow flower,Ludwigia elegans , found in the marshes. Very early, at daybreak, before even the Ludwigia flowers have opened, the insect appears, helps the flower to open, collects the pollen, and then the nectar. When the job is done, it disappears. It only reappears the following day, at exactly the same time. A biological cycle is formed, set into action by the variation between dark and light – daybreak – , but with the important participation of food being available, which until then had not been given much importance. The Tetraglossula realizes that at every beginning of a morning it finds food in abundance – a thick nectar, rich in sugars and amino acids. In the afternoon, there is still light, but the insect disappears, because it has collected all it needed, and, furthermore, it would be likely to find more suitable food for its needs on that day.

The assessment of the behavior of the mosquito of the anopheles genus, an insect of nocturnal habits and responsible for the transmission of malaria, reserved a greater challenge. After all, different biological rhythms were in play: the parasite’s, the host’s, and that of the transmitter of the disease itself. It was not easy to separate these variables. The field of work involved four months of observations, from October 1995 to January 1996, on the foothills of the Marumbi mountain range, 6 kilometers to the north of Morretes, in Paraná.

With immense care lest they themselves should catch malaria, the researchers concluded that, in the adult phase, the mosquito’s biological clock is directly linked to the cycles of the moon: during the fourth quarter of the moon, the population of this insect becomes five times larger. On the other hand, in the larva stage, the anopheles may follow different rhythms of development, since it is an opportunistic species, which lives in the water held in bromeliads. When the insect finds an attractive and propitious environment, its growth speeds up, without this bringing the species any metabolic or physiological problems. In adverse moments, when the anopheles finds itself in dry places or under low temperatures, for example, its development is delayed, also without any damage to the mosquito.

Secrets of the caves
The Folsomia candida collembolans, insects that live in the depths of caves – and the main culprits for the biologists’ lost nights -, brought other doubts. It used to be believed that that this animal, regarded as primitive, with neither eyes nor pigmentation, would lead a completely ungoverned life, in a sort of temporal chaos, as it had no way of relying on references to light or dark. But it is not like that. The biologists from USP did not identify any indications of an active circadian rhythm, but they did detect two very strict cycles: oviposition at every seven days, and changing its cuticle (skin) every three and a half days.

But the results had to be confirmed, substantiated by observation of the animals in darkrooms in the laboratory that simulate their natural environment. Since the 60s, chronobiology has had at its disposal a method of work that results in a graph called a phase response curve, which maps the workings of circadian rhythm. The method works on the basis of stimuli that speed up or slow down the biological clock. For example, if a hen sleeps at 5 p.m. and is then woken up with the light from a spotlight, it gets up and starts to scratch for food as it always does. One hour later, it is back asleep again. Its brain records the alteration and makes the hen only go to sleep again at 6 p.m. on the following day. When these interferences are repeated, they indicate on a graph what has happened during the 24 hours or so.

The researchers applied this method to collembolans, stimulated by means of variations in temperature, Firstly, they confirmed the absence of the circadian rhythm. They also discovered that this methodology was not efficient in this kind of situation, since the response to the stimuli, assessed by means of a change of cuticle or by oviposition, were not simultaneous. Conclusion: another mathematical approach had to be built, that could also handle infradian rhythms, already evident at that time, though still little explained.

It was then that Mirian asked the physicists for help. Through a doctorate student, Gisele Akemi Oda, she arrived at Iberê Caldas, from USP’s Institute of Physics. The coordinator of a thematic project about chaotic systems (see Pesquisa FAPESP nº. 65) and Gisele’s co-supervisor, Caldas observed carefully the numerical series from the team from the museum – notes and graphs about the rhythm of the daily activities of the animals collected in the course of ten years – and the scientific interests immediately added up, on the basis of the physical principle of oscillation. “Between a wave caused by a stone thrown into the water and another wave, from a second stone, there is an interaction that is not random”, Mirian explains. “The same line of thought is valid for biological clocks”.

Together, the researchers from the Zoology Museum and the Institute of Physics created a graphic model that detects the responses offered to the same stimulus, organizes them mathematically, and produces a portrait of a set of phenomena, in a wide-ranging and coherent manner. Gisele concluded her doctorate and then signed an article about the new method of assessing infradian rhythms with Caldas and Mirian, published in 2000 in the Journal of Theoretical Biology .

But the model did not prove capable of explaining the biological rhythm of the Strinatia brevipennis cricket, the subject of a thesis by another of Mirian’s pupils, Sonia Hoenen. Regarded as a species that makes the transition between the dark zone and the light one, as it inhabits the open spaces in the caves of the Ribeira valley region, between the states of São Paulo and Paraná, Strinatia is intensely pigmented and does not emit any sound. In the laboratory, it was kept in cages with walls made of transparent acetate, which would vibrate in accordance with the movement of the crickets. Put into contact with each one of the four walls of the cage, a needle would indicate whether the animal was standing still or active. The problem is that the quantity of data was so large – dozens of pages just for the crickets – that it was no longer known for sure which information was important and how it could be interpreted. The answer this time came from seismology.

German geologist Martin Schimmel, currently with the Jaume Almera Institute of Sciences of the Earth, in Spain, was doing post-doctoral studies at USP’s Institute of Astronomy, Geophysics and Atmospheric Sciences (IAG) when he heard about the Strinatia for the first time. The researcher had developed a way of differentiating two kinds of seismic waves: those generated in the inside of the Earth, which may cause earthquakes, and those that do not offer any danger. Invited by the biologist, Schimmel decided to employ the same method to interpret, in a suitable manner, the records made by the biologists. One day, he telephoned her and asked what the cave crickets did every 24 hours. She was exultant when she saw that the geologist had managed to separate the important movements from the irrelevant ones and detected the circadian cycle in these animals, which every 24 hours show sleep, hunger, in short, a more intense biological activity.

“It is ancient information, that biologists call a relictual characteristic, probably a remnant of the animal’s ancestors”, the researcher, who can also call on collaborators from universities in England, Germany, Canada and Argentina. The next challenge that has been posed is to discover the plasticity of the biological clocks – to what point they can be compressed or stretched, without losing their properties. Mirian now knows that nature itself works with organisms like the conductor of an orchestra. “Challenging the conductor”, says she, “always ends up in playing out of tune, sometimes fatally”.

The project
Rhythms of Insect Activities: Implementation of a Unit with Constant Environmental Conditions (nº 92/04445-9); Modality Regular research benefit line; Coordinator Mirian David Marques – USP; Investment R$ 20,034.65