Michael Levandowsky is a Research Scientist at the Haskins Labs, Pace University, New York, N.Y. 

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Sensory Responses in Euglena 

This representative of an early branch of the eukaryote domain displays a fascinating motility repertoire and multifaceted sensory physiology 

Michael Levandowsky 

Textbook and popular writers once routinely described the green protist Euglena as a kind of mysterious half-plant, half-animal microbe—a missing link. Its photosynthetic capacity appeared plant-like, while its motility and its vitamin B₁₂ requirement is usually animal traits.

We now know from abundant biochemical, ultrastructural, and molecular evidence that it is not very closely related to either animals or plants. Rather, Euglena represents an early branch in the eukaryote domain, and is a distant cousin of the kinetoplastids that became photosynthetic by capturing a chloroplast. Grown heterotrophically in the dark, it stops making chlorophyll and becomes etiolated (colorless) in a few generations, but greens up again when returned to the light. Moreover, it can be permanently cured of its chloroplasts (“bleached”) by exposure to streptomycin and other antibiotics. Streptomycin bleaching of Euglena was an important discovery that contributed to development of the endosymbiotic hypothesis for the origin of eukaryotic cells. Such colorless cells are easily cultured heterotrophically and closely resemble members of the colorless genus Astasia. 

Since its discovery, Euglena's behavior has attracted scientific interest. The organism displays two distinct kinds of movement. During one of them, spiral swimming, its single emergent flagellum points backward at an angle. For the other, which takes place on surfaces, Euglena squirm in a unique way called metaboly or, more simply, euglenoid movement. Though this squirming motion is unusual and intriguing, most research on the sensory biology of this organism has focused on spiral swimming responses to light and other signals. While some of those responses are extensively studied and relatively well-known, researchers are only beginning to examine the underlying sensory transduction chains. 

Responses to Gravity 

Let's start with the response to gravity. Mature Euglena cells respond to gravitational or centrifugal force by turning and swimming away from the source of the field, a negative gravitaxis. Many microorganisms do this, and the response appears to have clear adaptive value, keeping cells near the surface of a water column. 

However, mechanisms whereby different motile microorganisms respond to gravity prove to be quite diverse. For instance, the large ciliate Paramecium apparently orients upward automatically when swimming, responding to a torque generated by the disjunction of the cell's center of gravity (based on distribution of mass) and the center of frictional force (due to its shape). Thus, Paramecium may not need a gravireceptor to move upward in its environment, and so far none has been found. 

By contrast, another ciliate, Loxodes, has a complex gravity-sensing organelle reminiscent of our own: a microscopic crystalline weight (statolith) suspended by a fibril in a vacuole (Mueller's body). The organism uses this apparatus to sense the direction of gravity, according to Tom Fenchel at Copenhagen University's Marine Biological Laboratory, Helsingfors, Denmark, and Bland Finlay at the Institute of Freshwater Ecology, Cumbria, United Kingdom. The cells respond with either negative or positive gravitaxis depending on the levels of dissolved oxygen they encounter while hovering at the interface of oxic and anoxic zones in a water column. 

Figure 1

Euglena seems intermediate between these two examples. Its sensory mechanism is probably based on stretch-sensitive ion channels, affected by mechanical stretching of the entire membrane on the lower side of a swimming cell, according to Donat Häder and his colleagues at the Friedrich-Alexander University in Erlangen, Germany. Experiments in microgravity simulations on earth and experiments in sounding rockets and the space shuttle indicated that Euglena cells have a true gravitaxis, and lose their orientation in the absence of gravity. They then found that in normal gravity, graviorientation ceases when the cells are placed in a medium with density equal to that of the cells and is actually reversed in media with higher densities, where the membrane on the upper side of the cell is stretched, as buoyancy forces push the cell upwards (Fig. 1). In effect, in Euglena the entire cell acts as a statolith! 

Gravisensory transduction apparently involves modulation of the membrane potential. For example, ion channel blockers, ionophores, and ATPase inhibitors impair graviperception. Inhibitors include the calcium ionophore calcimycin (A23187); gadolinium ions, which affect calcium-selective stretch-sensitive ion channels in other systems; and vanadate, which inhibits cytoplasmic ATPase, thought to be responsible for maintenance of the ionic gradient. Details of these systems remain to be studied, but both potassium and calcium evidently play important roles in gravitaxis in this organism. 

Complex Responses to Light

Euglena responds in various ways to light. More than a century ago, observers noted that Euglena cells are attracted to moderate light, but are repelled by strong light, such as direct sunlight. Careful cinematographic observations show that a quick increase in light above a certain threshold leads to a halt in swimming, reorientation of the flagellum to point away from the cell body, and consequent turning away from the light (a step-up photophobic response). Similarly, rapidly dimming light below a threshold (a step-down photophobic response) leads Euglena cells to turn toward the light source. Constant illumination leads to continuous swimming toward moderate light or away from bright light, due mainly to a taxis (oriented response) but also involving a weak kinesis (nondirected response—e.g., a biased random walk leading to accumulation).

In nature, the combination of negative gravitaxis, positive phototactic response to moderate light levels, and negative phototactic response to high light levels causes the cells to accumulate in the water column in a band in suitable light conditions. This is important: the bright light at the water's surface can bleach cellular pigments, and the UV-B component of solar radiation damages both motility and photoorientation. 

Euglena's photoreceptor is an organelle called the paraxonemal body (PAB, also termed paraflagellar body, PFB), situated at the base of its single emergent flagellum. The PAB contains a flavoprotein arranged in a paracrystalline array, as well as pterin pigments. The flavoproteins appear to be the primary chromophore, while the pterins serve as auxiliary light antennas. Curiously, though Euglena also contains retinal, its function is not known and appears not to be involved in the phototactic response. 

The Euglena stigma or “eyespot,” an orange-red spot containing carotenoids at the cell's anterior, apparently shades the PAB photoreceptor as the cell turns during spiral swimming. This swimming can serve to orient the cell during the phobic responses. When illuminated from the side, a swimming cell sticks its flagellum out laterally and turns toward moderate light and away from strong light. 

It was once thought that a variation of the phobic response modulated cellular orientation during continuous tactic swimming. In particular, researchers believed that the eyespot shaded the PAB photoreceptor when the cell wandered off course in its spiral upward swimming. However, it was found that calcium ionophores affect the phobic response, but not taxic swimming, and it now appears that taxic swimming orientation is based on a dichroic orientation of the photoreceptor pigments in the paracrystalline array in the PAB, heightening the cells' sensitivity to light in certain orientations.

Recent experiments indicate that applied DC electric fields do not affect positive or negative light responses, suggesting that electric potential changes are not involved in sensory transduction of these responses, and earlier reports of a galvanotactic response to electric fields need to be reexamined. On the other hand, experiments with impaled cells exposed to various ionic and electric field environments suggest that electrode voltage can affect flagellar beating frequency and waveform by modulating the flow of Ca²⁺ and Mg²⁺ ions across the Euglena cell membrane. The flagellar motion responds to changes in intracellular concentrations of intracellular bivalent ions rather than to transmembrane voltage per se.

Response to Chemical Signals 

Over a century ago, W. Pfeffer, H. S. Jennings, and others observed chemosensory responses in a number of bacterial and protistan microbes. In the first decades of the 20th century, Ernst Pringsheim and his colleagues at Göttingen University in Germany conducted detailed studies of several flagellate species, including the colorless euglenid Astasia, which responds positively to an array of short-chain fatty acids and other potential carbon sources. Such behavioral studies then became rare, as biochemistry and then molecular biology took center stage in microbiology. In the 1960s, however, Julius Adler at the University of Wisconsin and his collaborators began studying the genetics of bacterial chemotaxis, and others identified cAMP as the “acrasin” signal in aggregation by the slime mold Dictyostelium, stimulating renewed interest in microbial chernoreception.

Figure 2

Early workers noted that Euglena is attracted to low pH, but, oddly, this response has been little studied. In the 1970s Bodo Diehn and his colleagues at Toledo University studied the aerotaxis response by Euglena to sharp oxygen gradients. Dark-grown etiolated cells in capillaries or under cover slips accumulate in dynamic bands or rings (Fig. 2). These patterns appear to be due to a response to oxygen gradients that develop in the medium. Cells swimming out of the aggregates toward regions of greater or lesser oxygen concentration undergo a “shock” (chemophobic) reaction, abruptly stopping and turning back to rejoin the aggregates. Because individual cells orient themselves in the gradient, this behavior probably reflects a true taxis. Moreover, low levels of cyanide and carbon monoxide but not other respiratory poisons abolish these patterns, leading Diehn and coworkers to suggest that cytochrome a3 oxidase may be the receptor. However, these agents do not effect photosensory responses of Euglena.

Since Euglena grows well heterotrophically in the dark on peptone media, we are testing whether it displays behavioral responses to amino acids and other potential nutritional signals. To do so, we use a flat capillary assay, originally developed for studying chemosensory responses in the ciliate Tetrahymena, to measure responses to amino acids. Euglena cells respond positively to 10 mM levels of a number of amino acids: lysine, ornithine, arginine, cysteine, histidine, leucine, isoleucine, valine and alanine. Cells also respond to histamine, ethanol, and acetate.

Figure 3

Thus, Euglena can detect and aggregate in response to certain chemical signals. Because sensory transduction in other protists, such as Paramecium and Chlamydomonas, involves calcium channels, we examined the effects of putative Ca²⁺ blockers. For instance, adding 0.1 mM lanthanum, barium, or cobalt ions to cell suspensions causes cells to elongate and also to swim faster, in straighter paths (Fig. 3). However, adding these ions does not diminish the chemosensory response to oxygen gradients--on the contrary, this response is mildly enhanced, perhaps due to increased motility. These observations suggest that, while motility and shape are influenced by calcium channels, some chemosensory responses are not dependent on them. 

What's Next? 

In the last 5 years or so, several reports have suggested the presence in Euglena of putative elements of signal transduction chains, some of which may be involved in these sensory responses. Thus, for example, adding inositol 1,4,5-trisphosphate (InsP3) or cyclic ADP-ribose (cADPR) releases Ca²⁺ from a microsome fraction from Euglena. Caffeine also induces Ca²⁺ release, desensitizing the response to cADPR but not to InsP3. Ruthenium red inhibits the cADPR but not the InsP3 response. Activity of ADP-ribosyl cyclase, which produces the cADPribose, oscillates in synchronized cell populations.

The significance of these observations for the cell's physiology, in particular the significance if any for sensory transduction, is not known. Although a number of putative elements of signal transduction are present in Euglena, their functions in its biology remain to be determined.

Euglena is an amazingly versatile organism. It can grow in the light or in the dark with a wide variety of carbon sources. It is grown in the lab over a wide pH range-from pH 3 to 9—and is often found in unsavory but nutrient-rich habitats such as sewage treatment lagoons. It displays an interesting repertoire of sensory responses to light, chemical signals, and gravity. Since it causes no human diseases, it has tended to be understudied, and there is ample opportunity for research on its fascinating and surprisingly multifaceted sensory physiology. 

SUGGESTED READING 

Hader, D.-P., M. Lebert, and P. Richter. 1998. Gravitaxis and graviperception in Euglena gracilis. Adv. Space Res. 21:1277-1283.

Jennings, H. S. 1906. Behavior of the lower organisms. (Reprinted 1976 by Indiana University Press).

Lebert, M., and D. Hader. 1996. How Euglena tells up from down. Nature 379:590.

Miller, S., and B. Diehn. 1978. Cytochrome c oxidase as the receptor molecule for chemoaccumulation (chemotaxis) of Euglena toward oxygen. Science 200:548-549.