250 CHLOROPLAST E.R.: EVOLUTION OF ONE MEMBRANE

Fig. 6.6 Transmission electron micrograph of a section of

the pellicle area of Euglena terricola. (From Leander and

Farmer, 2001a.)

the daughter chromosomes disperse into the two daughter nuclei.

Eyespot, paraflagellar swelling, and phototaxis

The eyespot (stigma) is a collection of orange-red lipid droplets, independent of the chloroplast (Figs. 6.1, 6.2, 6.14). The eyespot is in the anterior part of the cell, curving to ensheath the neck of the reservoir on the dorsal side. In most euglenoids the eyespot consists of a compact group of 20 to 50 droplets, although in Eutreptia and Khawkinea it may consist of just one or two large droplets. The eyespot has been reported to contain -carotene and seven xanthophylls (Sperling Pagni et al., 1981), mainly -carotene (Batra and Tollin, 1964), or a -carotene derivative, echineone (Krinsky and Goldsmith, 1960). The independence of the eyespot is emphasized by the existence of colorless species with an eyespot but no plastids. One flagellum of all green euglenoids bears a lateral swelling near the transition zone from canal to reservoir; in Euglena (Figs. 6.2, 6.14(a)), the swelling is on the longer flagellum. The swelling is composed of a crystalline body next to the axoneme and inside the flagellar membrane.

All euglenoid species with an eyespot and flagellar swelling exhibit phototaxis, usually swimming away from bright light (negative phototaxis) and away from darkness toward subdued light (positive photoaxis) to accumulate in a region of low light intensity. Upon sudden changes in its environment, the cell responds with a transient sideways turn by swinging out its one emergent flagellum. At low light intensities (less than 1.4 W m 2), the alga swims toward the light source, whereas at higher light intensities, it moves away from the light source. According to the shading hypothesis (Häder, 1987), positive phototaxis is brought about by repetitive stepdown photophobic responses. During forward locomotion, the cells rotate helically with a frequency of 1 to 2 hertz (cycles per second). In lateral light, each time the stigma (eyespot) intercepts the light beam impinging on the paraflagellar body, the flagellum swings out temporarily and turns the front end of the cell toward the light source by a fraction until the cell is aligned in the light direction.

Euglena bleached of its chlorophyll but retaining its eyespot and photoreceptor (paraflagellar swelling) is still positively phototactic, eliminating chlorophyll and chloroplasts in the phototaxis directly. A Euglena bleached of all pigments but retaining its photoreceptor is negatively phototactic; this rules out the possibility that the carotenoids of the eyespot are directly stimulatory in phototaxis. A Euglena lacking a photoreceptor and all pigments, like Astasia (Fig. 6.14(b)), is no

EUGLENOPHYTA 251

Fig. 6.7 Euglena gracilis. (a)–(d). Sequence of shape changes photographed at 5-second intervals of a cell undergoing euglenoid movements. The nearly spherical cell in (a) initiates a forward wave of dilation in (b), which reaches the anterior of the cell, and then recovers by an inward flow of cytoplasm to initiate a new wave at (c). The new wave progresses forward, and the cell recovers in (d). Scanning electron microscope micrographs in (e)–(g) illustrate the positions of surface ridges and grooves during selected stages of deformation. Nearly horizontal strips in (e) reorient to longitudinal in cells, initiating (f) or completing (g) the cycle. In (h)–(j), cell ghosts are shown with the changes in cell shape. (From Dubreuil and Bouck, 1985.)

252 CHLOROPLAST E.R.: EVOLUTION OF ONE MEMBRANE

						Fig. 6.8	Semidiagrammatic
						drawings of the anterior portion of
						Bodo (Kinetoplastida) (a), Peranema
						(Euglenophyceae) (b), and Colacium
						(Euglenophyceae) (c), illustrating
						probable evolution of the euglenoids.
						(C) Cytosome; (P) pocket. (After
						Willey and Wibel, 1985b.)
(a)		(b)		(c)

Fig. 6.9 A semidiagrammatic drawing of an anaphase nucleus of Euglena with nuclear envelope intact, the nucleolus pinching in two, and the chromosomes not attached to spindle microtubules.

longer phototactic (Jahn and Bovee, 1968). The flagellar swelling is therefore the photoreceptor or light-sensitive organelle. It has a sensitive maximum at 410 nm, explaining the phototactic peak at that wavelength. Positive phototaxis occurs only if the eyespot, with its absorptive range of 400 to 630 nm, periodically shades the photoreceptor.

There is a circadian rhythm in phototaxis in Euglena, with phototaxis operative during the light period and not operative during the dark

period. Even if light is introduced during the normal dark period, the Euglena cell does not respond phototactically. The phototactic response in the direction of the light source plainly involves the photoreceptor, but why this is incapable of reacting during dark periods when the light is reintroduced is not known. Continuous darkness does not eliminate the rhythm unless continued for so long a period that complete bleaching results, and the cells become photonegative. It is possible that the non-operative nature of photaxis during this time is related to mitotic division occurring during this period. Leedale (1959) found that green euglenoids have almost perfectly circadianly synchronized mitotic cycles, mitosis occurring at the beginning of the dark period, requiring one hour of dark to trigger the division. During mitotic division, Euglena normally rounds up and loses its flagellum; but even if the flagellum is not shed, the cells are still unable to swim ably during this period. Because phototactic insensitivity coincides with the generation time of Euglena during dark hours, it perhaps is of no surprise that motility loss, generation time, and absence of phototaxis should coincide as they do (Jahn and Bovee, 1968).

A phototactic circadian rhythm also exists in the movement of mud flat Euglena. The mouth of the River Avon in Bristol, England, is an estuary with very large tidal differences in water level. The mud flats which are exposed at low tide become green in spots owing to an accumulation of Euglena obtusa on the surface of the mud (Bracher, 1937; Palmer and Round, 1965). Before the incoming tide floods the mud flats, the Euglena cells move back down into the mud, a pattern of behavior that avoids the washing away of the Euglena by the tide. Euglena proxima exhibits a similar movement and it has been shown that the

EUGLENOPHYTA 253

Fig. 6.10 The vertical migration rhythm in Euglena obtusa on a mud flat (top curve), and in constant light in the laboratory (bottom curve). The dark bars on the upper abscissa represent night, whereas the wavy lines indicate when the tide covered the mud flats. (After Palmer and Round, 1965.)

cells absorb a significant amount of nutrients in the mud during this part of the migration cycle (Kingston, 2002). Euglena cells accumulate at the surface of the mud only when the low tides occur during daylight, no cells being found on the surface during low tides (Fig. 6.10). The above observation indicate that phototaxis is the main mechanism involved, with the Euglena cells swimming toward the light during the day to reach the surface of the mud. If the Euglena cells are brought into the laboratory and placed in constant light (980 lux), the cells display a circadian rhythm in their vertical migration. The phase of this circadian rhythm is determined by the last dark period in nature, be it night or a dark period resulting from the covering of the mud flat by murky water at high tide. Thus the rhythm is actually circadian, entrained by the tidally caused light–dark cycle, rather than truly tidal.

Muciferous bodies and extracellular structures

Muciferous bodies (mucocysts), containing water-soluble polysaccharides, occur in helical

rows under the pellicle in all species of euglenoid flagellates (Fig. 6.2). The muciferous bodies open to the outside of the cell through pores that course between the strips of the pellicle. Euglenoid cells are permanently coated with a thin slime layer from the muciferous bodies (Rosowski, 1977). It sometimes accumulates at the posterior end of the cell as a trailer of slime, and some species have the habit of sticking to a substratum by their posterior ends. Species with large muciferous bodies eject the contents on irritation and produce a copious slime layer around the cell (Hilenski and Walne, 1983). In Euglena gracilis (Figs. 6.7, 6.14(a)) the slime is composed of glycoproteins and polysaccharides (Cogburn and Schiff, 1984). The envelopes and stalks of Colacium (Figs. 6.15, 6.16) are formed of carbohydrate extruded by mucocysts in the anterior portion of the cell (Willey, 1984). The cylindrical stalks are composed of an inner and outer core of mildly acidic carbohydrate. The stalk is continuous with the canal and anterior part of the cell (Willey et al., 1977). The envelopes of such species as Trachelomonas (Figs. 6.12, 6.14(d)) are built up by inorganic deposition on a foundation of mucilaginous threads.

254 CHLOROPLAST E.R.: EVOLUTION OF ONE MEMBRANE

Fig. 6.11 Scanning electron micrographs of the encystment of Eutreptiella gymnastica. The vegetative cell (a) loses its flagella, forms a large number of paramylon grains, and begins to round up (b). The cell swells and produces a mucilaginous covering (c). (From Olli, 1996.)

Fig. 6.12 Scanning electron micrograph of the mineralized envelope of Trachelomonas lefevrei. (From Dunlap et al., 1983.)

Cysts are formed by euglenoids as a means of surviving unfavorable periods. The cell rounds off and secretes a thick sheath of mucilage (Fig. 6.11) that survives for months until the cell emerges by cracking the cyst. In conditions of partial desiccation or excessive light, the slime sheath sometimes acts as a temporary cyst, cells emerging from the sheath as soon as conditions improve. In certain genera (Euglena and Eutreptia), cell divi-

sion within the slime layer leads to the formation of a palmelloid colony, which may form extensive sheets of cells covering many square feet of mud surface (Leedale, 1967).

Trachelomonas is a large genus of freeswimming green euglenoids, characterized by encasement of the cell in a patterned mineralized envelope with a rimmed apical pore through which the flagellum emerges (Figs. 6.12, 6.14(d)). Most of the species are defined by the form and ornamentation of the envelopes, characteristics that can be changed by varying conditions of growth, especially iron and manganese supply. This has resulted in some described species being growth forms of other species (Pringsheim, 1953). The process of envelope formation involves mineralization of an initial fibrillar envelope which is probably derived as a secretion of the mucilaginous bodies (Pringsheim, 1953; Leedale, 1975). It begins with longitudinal division of the parent protoplast, the two daughter cells rotating within the envelope, and one or both squeezing out through the pore. Each naked daughter cell then secretes a new envelope externally, at first delicate and colorless but already the size and shape of the old one. Under good growth conditions, the envelope slowly becomes thicker and ornamented, first yellow, then brown. Under conditions of manganese deficiency, the envelope usually remains thin and unornamented. Envelope substructure is species specific, with a given species producing one of two general types of substructure: (1) weft or fibrillar deposits composed primarily of manganese, or (2) fine