Rhodopsin-mediated photosensing in green flagellated algae

Green flagellated algae possess a primitive visual system that regulates the activity of their motor apparatus. Photoexcitation of a rhodopsin-type photoreceptor protein gives rise to the photoreceptor current, which, above a certain threshold of stimulus intensity, induces the flagellar current. It is probable that the photoinduced alteration in flagellar beating is governed by changes in intracellular Ca2+ concentration. This rhodopsin-mediated sensory system serves to align the swimming path with the direction of the light stimulus, whereas processes of energy metabolism determine whether the oriented movement is directed towards or away from the light source.     

Photomovement of the red alga Porphyridium cruentum (Ag.) Naegeli

Photophobic reactions of the red alga Porphyridium cruentum have been studied by single cell observations and by population experiments with the light trap method. In white light traps photoaccumulation is saturated at about 6000 lx. Experiments with monochromatic light demonstrate the necessity of carefully separating the three basic light reactions, viz. phototaxis, photokinesis and photophobic response by an appropriate experimental set-up: In single-beam experiments trap wavelengths >695 nm cause photodispersal which is not due to photophobic entrance reactions, but is exclusively due to the positive photokinetic effect of the trap light. This photodispersal can be cancelled by a photokinetically active background light. In the short wavelength range not only photokinesis, but also phototaxis interferes with photophobic reactions thus affecting the density of photoaccumulations in the light trap. Phototactic and photokinetic interference can be avoided by a blue background light. The action spectrum measured this way indicates activity of photosystem I and photosystem II pigments in the perception of the step-down photophobic stimulus. Varying the wavelength of the background light at constant trap light absorbed mainly by photosystem I or photosystem II respectively, efficient spill-over of light energy from photosystem II to the light reaction of photosystem I could be demonstrated. From the results it is concluded that phobic reactions are induced by a decrease of the electron flow rate in the linear electron transport chain.     

Two photophobic responses in Volvox carteri

Phototaxis of Volvox carteri is the result of two photophobicresponses: stop and accelerationof flagellar activity in theanterior region of the colony. These responses of a colony fixedunder a microscope were analyzed quantitatively by cine-micrography. The phototactic sign of Volvox is temperature-dependent: itis positive at room temperature and negative at low temperature.When the temperature was lowered, the stop response to the on-stimuluswas reduced and changed to the acceleration response, whilethe acceleration response to the off-stimulus changed to thestop response. Decrease in light inteasity resulted in reduction in both stopresponses, i.e., the response to the on-stimulus at 24?C andthat to the off-stimulus at 16?C, but scarcely affected theacceleration responses. The action spectra of the photophobic responses at 24? and 14?Care similar, with a peak at 520 nm. (Received January 24, 1979; )     

Mechanism of Photoaccumulation in Paramecium bursaria

Responses of Paramecium bursaria to light intensity changes were investigated. The resting paramecia show a direction changing response (photophobic response) to a sudden decrease of light intensity, whereas no response was shown to an increase in intensity. The critical intensity decrease dI_c necessary to show the response was measured at various values of initial light intensity, and the ratio dI_c/I was found to be equal to ～0.15. The swimming paramecia show different behavior depending on their swimming direction in the spatial gradient of light intensity. Paramecia show direction change more frequently when they are swimming down the gradient than in the opposite direction. This difference in the rate of direction changing is 13–17%. These results may offer an explanation for the mechanism of photoaccumulation.     

Photoreception inEuglena gracilis: Fluence-rate and time dependence of photoaccumulation and photodispersal

The fluence-rate and time dependence for photoaccumulation and photodispersal ofEuglena gracilis was measured for the wild-type strain and three white mutants. For wavelengths of 453 or 463 nm the threshold for photoaccumulation was close to 6×10⁻²Wm⁻². Photoaccumulation increased steadily with time and reached a maximum after about 4 hr. Red light elicited substantial photoaccumulation in the wild type and photodispersal in the white, non-photosynthetic mutant 1224-5/9f. The chromophore mediating the red-light response needs to be a non-photosynthetic pigment which remains presently unidentified. A whiteEuglena mutant, FB, which had retained a reduced stigma and a paraflagellar body, showed weak photoaccumulation. Two white mutants, 1224-5/1f and 1224-5/9f, both of which lacked the stigma and positive phototaxis, displayed during the first 90 min of irradiation photodispersal; after longer irradiations they showed instead photoaccumulation. These results contradict a widely held belief that the presence of a stigma represents a stringent requirement for photoaccumulation. Our results imply that phototaxis is not a prerequisite for photoaccumulation. Exogenous flavins and 5,10-methenyl-tetrahydrofolate (MTHF) influenced in a wavelength-dependent manner photoaccumulation and photodispersal. In the wild type FAD and riboflavin (RB) caused at 453 nm an increase of the responsiveness for photoaccumulation. The photoaccumulation of the white mutant FB, was sensitized by FMN and FAD. In the white mutant 1224-5/9f exogenous flavins lowered the threshold for photodispersal. FMN, which absorbs only blue light, altered also the responsiveness to red light: in the wild type FMN reduced photoaccumulation and in the white mutant 1224-5/9f it reduced photodispersal.     

Light-Induced Body Movement of Euglena gracilis Coupled to Flagellar Photophobic Responses by Mechanical Stimulation*†

SYNOPSIS. In low viscosity media, Euglena gracilis strain Z responds to a sudden change in light intensity by a cessation of forward movement, followed by a reorientation of the locomotor flagellum which results in turning of the cell around the lateral axis (photophobic response). At a viscosity interface between low [～ 1 cP (centipoise)] and high (4000 cP) media, the cells exhibit avoidance responses or become immobilized in the higher viscosity medium. Upon changing the light intensity, free swimming cells have photophobic responses, while immobilized ones undergo body contractions. For cells immersed in media of varying viscosity, the delay between light stimulation and body contraction (transduction time) is shortest at high viscosities. From 500 to 2000 cP, where the cells are capable of both movement and light-induced body contractions, there is a logarithmic dependence of the transduction time on the viscosity. The transduction time does not vary appreciably with the intensity of the primary light stimulus within a range of 0.14-1.13 kW/m².     

The Relationship between Stimulus Intensity and Oriented Phototactic Response (Topotaxis) in Chlamydomonas

Two techniques have been used to study the quantitative relationship between stimulus intensity and oriented phototactic response (topotaxis) in Chlamydomonas. The net response of a cell population was monitored photometrically and was recorded continuously against time. The responses of individual cells were observed through a microscope and their swimming tracks were recorded on film. The net response of the population is positive at low stimulus intensity and negative at high intensity. The direction of response can be reversed within two seconds by raising or lowering the intensity. The intensity-response curve for phototaxis is similar to the dose-response curve for phototropism. The net response has no distinct threshold; it increases linearly with log intensity; then it decreases and finally becomes negative. The individual-cell studies reveal that the intensity-dependent increase in net topotactic response is due primarily to an increase in the number of cells responding and in the directness of their swimming path. As stimulus intensity is raised, the swimming path becomes increasingly well-aligned with the stimulus beam, whether net response is positive throughout the intensity range tested, negative throughout that range, or changing from positive to negative. Changes in swimming rate do not contribute significantly to the intensity-dependent changes in net response. Swimming rate shows virtually no change throughout the intensity range of positive topotaxis and shows only a small increase in the negative range. However, a transient decrease in swimming rate (stop response) is often observed at the onset of stimulation. The implications of these results for the orientation mechanism are discussed.     

Photosensory transduction in the flagellated alga, Euglena gracilis. II. Evidence that blue light effects alteration in Na+/K+ permeability of the photoreceptor membrane

1. The blue light-induced cell tumbling behavior (the step-down photophobic response) and the accumulation of cells into a blue light trap (photoaccumulation) were investigated in Euglena. Dose response plots for these phenomena which we collectively term 'photobehavior' show both threshold and saturation characteristics. 2. NaCl effects apparent elevation in the photosensitivity of the cell as evidenced by alteration of the dose response plot character and lowering of the light intensity saturation level. 3. NaCl and ouabain enhance the duration of the photophobic responses and the rate of photoaccumulation. KCl and NH4Cl have lesser or inhibitory effects. 4. Choline chloride reduces the duration of the photophobic responses and the rate of photoaccumulation. 5. KCl reduces the enhancement of photobehavior induced by NaCl and at constant chloride concentration, photobehavior is unaffected by the relative KCl and NaCl concentrations. 6. Antagonists of voltage-dependent, monovalent cation fluxes in membranes (tetrodotoxin, procaine, tetraethylammonium, 4-aminopyridine) do not alter photobehavior. 7. The results suggest a role for a photoreceptor membrane-located transport system for Na+/K+ as a key step in control of the intraflagellar free Ca/+ levels that determine the photobehavior mediated by flagellar reorientation.     

CALCIUM RELEASE FROM INTRACELLULAR STORES IS NECESSARY FOR THE PHOTOPHOBIC RESPONSE IN THE BENTHIC DIATOM NAVICULA PERMINUTA (BACILLARIOPHYCEAE)1

Complex photoreceptor pathways exist in algae to exploit light as a sensory stimulus. Previous studies have implicated calcium in blue‐light signaling in plants and algae. A photophobic response to high‐intensity blue light was characterized in the marine benthic diatom Navicula perminuta (Grunow) in van Heurck. Calcium modulators were used to determine the involvement of calcium in the signaling of this response, and the fluorescent calcium indicator Calcium Crimson was used to image changes in intracellular [Ca²⁺] during a response. A localized, transient elevation of Calcium Crimson fluorescence was seen at the cell tip at the time of cell reversal. Intracellular calcium release inhibitors produced a significant decrease in the population photophobic response. Treatments known to decrease influx of extracellular calcium had no effect on the population photophobic response but did cause a significant decrease in average cell speed. As the increase in intracellular [Ca²⁺] at the cell tip corresponded to the time of direction change rather than the onset of the light stimulus, it would appear that Ca²⁺ constitutes a component of the switching mechanism that leads to reversal of the locomotion machinery. Our current evidence suggests that the source of this Ca²⁺ is intracellular.     

The role of endosymbiotic algae in photoaccumulation of green Paramecium bursaria

The endosymbiotic unit of Paramecium bursaria with Chlorella sp. photoaccumulates in white, blue-green, and red light (<700 nm), whereas alga-free Paramecia never do. The intensity of photoaccumulation depends on both the light fluence rate and the size of the symbiotic algal population. Photoaccumulation can be stopped completely with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosynthetic electron transport. Hence the photosynthetic pigments of the algae act as receptors of the light stimulus for photomovement and a close connection must exist between photosynthesis of the algae and ciliary beating of the Paramecium.     

Vision in microalgae

Flagellate green algae such as Chlamydomonas and related genera are guided by their eyes to places where light conditions are optimal for photosynthetic growth. These eyes constitute the simplest and most common visual system found in nature. The eyes contain optics, photoreceptors and the elementary components of a signal-transduction chain. Rhodopsin serves as the photoreceptor, as it does in animal vision. Upon light stimulation, its all-trans-retinal chromophore isomerizes into 13-cis and activates a photoreceptor channel which leads to a rapid Ca²⁺ influx into the eyespot region. At low light levels, the depolarization activates small flagellar currents which induce in both flagella small but slightly different beating changes resulting in distinct directional changes. In continuous light, Ca²⁺ fluxes serve as the molecular basis for phototaxis. In response to flashes of higher energy the larger photoreceptor currents trigger a massive Ca²⁺ influx into the flagella which causes the well-known phobic response. The identification of proteins contributing to this signalling system has just begun with the isolation and cloning of the opsins from Chlamydomonas and Volvox. These plant opsins are highly charged, are not typical seven-helix receptors, and are believed to form a protein complex with the photoreceptor channel. In Spermatozopsis, a G-protein has been found which interacts either directly with the rhodopsin or with the rhodopsin-ion channel complex. By using insertional mutagenesis, genes coding for proteins that are involved in signalling have been tagged. One of them is connected to the flagellar channel and crucial for the flagellar action potential. Elucidation of photoreception in flagellated algae will provide deeper insight into the development of visual systems, starting from single-celled organisms and moving up through higher animals. Received: 10 March 1997 / Accepted: 18 April 1997     

THE FLAGELLAR PHOTORESPONSE IN VOLVOX SPECIES (VOLVOCACEAE,CHLOROPHYCEAE)1

Steering their swimming direction toward the light is crucial for the viability of Volvox colonies, the larger members of the volvocine algae. While it is known that this phototactic steering is achieved by a difference in behavior of the flagella on the illuminated and shaded sides, conflicting reports suggest that this asymmetry arises either from a change in beating direction or a change in beating frequency. Here, we report direct observations of the flagellar behavior of various Volvox species with different phyletic origin in response to light intensity changes and thereby resolve this controversy: Volvox barberi W. Shaw from the section Volvox sensu Nozaki (2003) changes the direction of the flagellar beating plane, while species encompassed in the group Eudorina (Volvox carteri F. Stein, Volvox aureus Ehrenb., and Volvox tertius Art. Mey.) decrease the flagellar beating frequency, sometimes down to flagellar arrest.     

Step-up photophobic response in the ciliate,Stentor coeruleus

The avoidance by Stentor coeruleus of a light trap is caused by a step-up photophobic response. The phobic response invariably consists of a delay of about 200 ms, a stop response, a turn to one side, and resumption of swimming in the new direction. After this the cells enter a refractory period of 1–3 s following a phobic response, during which they will not give a second response. Phobic responses can be elicited by spatial and temporal increases in light intensity. The action spectrum for the step-up photophobic response resembles the absorption spectrum of stentorin, the proposed photoreceptor pigment, and of its chromophore, hypericin.The phobic response is specifically inhibited by the protonophorous uncouplers TPMP⁺ and FCCP but not by the ionophores gramicidin and A23187. Since the uncouplers block light-induced membrane potential changes at the same concentrations, it has been proposed that the primary photoreception causes a light-induced potential change, which in turn, induces a motor response.Abbreviations TPMP⁺ triphenyl methyl phosphonium bromide - FCCP carbonylcyanide p-trifluoromethoxy-phenylhydrazone     

Dependence of the photophobic response of the blue-green alga,Phormidium uncinatum,on cations

It is suggested that photophobic responses caused by a sudden step-down in light intensity require the presence of cations in the blue-green alga, Phormidium uncinatum.Drastic removal of cations abolishes the phobic response, which recovers after addition of Ca²⁺ ions. Calcium can be substituted for partially by other cations with an effectivity following the sequence Ca>Mg>Na>Ba>Co=0. During the photophobic response there is a 25% increase in ⁴⁵Ca binding by the cells related to a step-down in light intensity. Three seconds after a light-dark transition there is a sharp increase in the binding of labelled calcium, followed by a subsequent release.Flushing the filaments with high cation concentrations, esp. calcium causes a reversal of movement in the absence of a light stimulus similar to a photophobic reversal. This stimulus could trigger the same sequence of events in the transduction chain bypassing the primary photoresponse.Abbreviations EDTA Ethylene diaminetetraacetic acid - EGTA ethylene glycol-bis (2-aminoethylether) N,N tetraacetic acid     

Chemotaxis in Laminaria digitata (Phaeophyceae): I. ANALYSIS OF SPERMATOZOID MOVEMENT

The chemotactic behaviour of Laminaria digitata spermatozoidsupon stimulation with sex pheromone was studied using videomicroscopyand high speed cinematography. The cells show phobic (‘shock-’)reorientation reactions when perceiving decreasing stimulusconcentrations in a pheromone gradient and are able to adaptto constant concentrations. The phobic responses are executedby regulation of flagellar activity: drastic changes in thedirection of cell movement are induced by rapid deflexions ofthe posterior flagellum. Key words: Laminaria, chemotaxis, pheromone, flagellar movement.     

A TEST OF TWO POSSIBLE MECHANISMS FOR PHOTOTACTIC STEERING IN VOLVOX CARTERI (CHLOROPHYCEAE)

We tested two competing models that could explain how differential flagellar activity leads to phototactic turning in spheroids of Volvox carteri f. weismannia (Powers) Iyengar. In one model, turning results from the flagella of anterior cells in the lighted and shadowed hemispheres beating at different frequencies. In a competing model, turning results from a change in beat direction in these flagella. Both models successfully explain phototactic steering under constant illumination, but they make different predictions when colonies are exposed to abrupt changes in light intensity. If turning is due to control of flagellar beat frequency, both progression and rotation rates will change in the same direction and with similar magnitudes. If spheroid turning is due to a change in flagellar beat direction, a decreased rate of progression will accompany an increased rate of rotation and vice versa. We used video-microscopy to observe the behavior of positively phototactic V. carteri spheroids exposed to 10× step-up and step-down stimuli. After a step-up stimulus, spheroids slow their progression and rotation by equal amounts. No significant changes are reported in these parameters after the reciprocal step-down response. These observations are consistent with the variable flagellar frequency model and inconsistent with the variable flagellar direction model for phototactic turning. Switching the direction of light stimulus by 180° results in reorientation of positively phototactic spheroids. The kinetics of this reorientation did not precisely match the predictions of either model.     

What goes down must come up: symmetry in light-induced migration behaviour of Daphnia

During a short period of the year, Daphnia may perform a phenotypically induced diel vertical migration. For this to happen, light-induced swimming reactions must be enhanced both at dawn and at dusk. Enhanced swimming in response to light intensity increase can be elicited by fish-associated kairomone in the laboratory, if food is sufficiently available. However, during the light change at dusk the Daphnia are still in the hypolimnion, where no fish kairomone is present and both temperature and food availability is low. Still, what goes down must come up. This raises questions about how Daphnia tunes its light-induced swimming behaviour to prevailing conditions such that a normal diel vertical migration can be performed. We investigated the symmetry in behavioural mechanism underlying these diel vertical migrations in the hybrid Daphnia galeata × hyalina (Cladocera; Crustacea), with special interest for the environmental cues that are known to affect swimming in response to light increase. That is, we tested whether fish- associated kairomone, food availability, and temperature affected both swimming in response to light intensity increase and decrease similarly. We quantified swimming behaviour during a sequentially increased rate of light change. Vertical displacement velocity was measured and proved to be linearly related to the rate of the light change. The slope (PC) of the function depends on the value of the factors kairomone concentration, food availability, and temperature. The changes of the PC with kairomone concentration and with temperature were similar both at light intensity increases and decreases. The PC also increased with food concentration, although during light increases in a different way from during light intensity decreases. Low food availability inhibited swimming in response to light intensity increase, but enhanced swimming in response to light intensity decrease. Hence, ascent from the deep water layers with low food concentration at dusk is facilitated. These causal relations are part of a proximate decision-making mechanism which may help the individual Daphnia to tune migration to predation intensity and food availability.     

Migration of hatchery reared juvenile Atlantic salmon, Salmo salar L., smolts down a release ladder. 1. Environmental effects on migratory activity

The effects of light intensity, water temperature and river spate conditions on the rate of migration of hatchery-reared Atlantic salmon smolts down a release ladder were examined. Low light intensity and high day time water temperatures raised smolt migration rates: water temperature had littleor no effect at night. Thediel patternofdown-ladder movement wasdetermined by these two environmental stimuli. Smolts demonstrated a threshold response to both light and water temperature: a fall in light intensity, or increase in water temperature, below or above their respective thresholds elicited no further response. As fish progressed through the ladder their movement became increasingly nocturnal, and most entry into the estuary occurred at night. Heavy rainhll caused rapid migration of smolts through the ladder. As ladder discharge remained constant, smolts must have been responding to some other change in the water conditions. Smolts responded to spate conditions irrespective of water temperature and light intensity, and they responded to light intensity irrespective of water temperature. A hierarchy of environmental cues, responsible for triggering migratory behaviour on a day to day basis during the smolt run, of spate> light intensity > water temperature is therefore suggested.     

Sensitization and habituation of the swimming behavior in ascidian larvae to light

Ascidian larvae of Ciona intestinalis change their photic behavior during the course of development. Newly hatched larvae show no response to a light stimulus at any intensity. At 4 hr after hatching, larvae were induced to start to swimming upon the cessation of illumination, and to stop swimming upon the onset of illumination. At a weaker light intensity (5.0 x 10(-3) J/m (2).s), the larvae showed similar responses to either a single stimulus or repeated stimuli of onset and cessation of light until 10 hr after hatching. At a stronger light intensity (3.2 x 10(-1) J/m(2).s), when the stimulus was repeated, they showed sensitization and habituation of the swimming response. At 3 hr after hatching the larvae failed to show any response to an initial stimulus at any intensity of light, but after several repeated stimuli (sensitization) they showed a swimming response at light intensities above 4.0 x 10(-2) J/m (2).s. At 5 hr and with intensity above 1.0 x 10 (-2) J/m(2).s, the larvae showed photoresponses to the first stimulus, but after several repetitions the larvae failed to stop swimming upon the onset of light (habituation). A repeated series of stimuli at stronger intensities of light caused greater habituation; this habituation was retained for about 1 min. Since the larval central nervous system in Ciona is comprised of only about 100 neurons, learning behavior in ascidian larvae should provide insights for a minimal mechanism of memory in vertebrates.     

Bacterial motility and chemotaxis: Light-induced tumbling response and visualization of individual flagella

High intensity visible light induces tumbling in Salmonella typhimurium, the effect being reversible provided short pulses are used. Fully aerated cultures are more sensitive to light than cultures with a lower oxygen tension. Bacteria which have been subjected to a sufficient positive chemotactic stimulus (concentration increase of attractant) are immune to the light stimulus. Light with a wavelength of 510 nm or shorter is effective, suggesting that absorption by a flavin may be involved. The light source used, with a yellow filter inserted to prevent light-induced tumbling, is bright enough to allow visualization of individual flagella as well as flagellar bundles. This has provided information on flagellar aggregation, rigidity, and behavior during tumbling. Since the occurrence of tumbles is modified as a result of chemical gradient sensing, these techniques are valuable in the study of bacterial chemotaxis.