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     

Temperature Effect on the Photo-Accumulation and Phobic Response of Volvox aureus*

SYNOPSIS. The effect of temperature on photoaccumulation and photophobic response of Volvox aureus were studied. The algae exhibited positive photoaccumulation at room temperature and negative at low temperature. When stimulated with light of intermediate intensiy (～ 5 × 10³ lux), the phobic response of the algae consisted of a decrease in the frequency or the cessation of flagellar movement in the anterior cells. At room temperature, an increase in light intensity elicited the phobic response, whereas at low temperature a decrease in light intensity was the effective stimulus. The phobic response lasted only a few seconds. The positive and negative photoaccumulations of the algae could be explained by the brief cessation of flagellar movement in the anterior cells, elicited by an increase of stimulus light at room temperature or a decrease of stimulus at low temperature.     

Anion sensitivity of motility and step-down photophobic responses of Euglena gracilis

The motility and step-down photophobic responses of Euglena are influenced by inorganic and organic anions. Persistent motility (with Ca²⁺, Mg²⁺ and K⁺ present) is supported with chloride or sulfate but not with acetate, nitrate or propionate as the only added anions. Cells in media containing acetate displayed a cell aggregation (clumping) behavior that was both red light sensitive and, under some conditions, was accompanied by suppression of the step-down photophobic response. Addition of sodium salts (Cl^-, SO ₄ ^2- , acetate or propionate) to cells in Cl^- or SO ₄ ^2- based media had differential effects on the duration of the step-down photophobic responses induced by blue light removal: anions alter the response. In addition, cells in all Cl^- containing media showed constant photophobic response duration following repeated stimulation. Cells in some SO₄ ^2- containing media, however, showed response summation to repeated stimulation. This latter effect was reversible and was overcome by the addition of chloride anions.     

Role of three basic light reactions in photomovement of desmids

Photoaccumulations in light trap experiments have been studied in the desmids, Cosmarium, Micrasterias and Euastrum. Dependence of accumulation density on exposure time follows saturation curves, while dose response curves show optima. Time-lapse microcinematography and population methods have revealed that all three basic light-induced motor responses known in microorganisms participate in producing photoaccumulations in desmids. During the initial phase the cells are phototactically attracted towards the trap by scattered light. In low light intensity traps photokinetic reactions may play only a minor role, since photokinesis could be evoked only by light intensities100 lx in Cosmarium cucumis. True photophobic reactions have been demonstrated for the first time in desmids. There are two types of phobic responses in desmids: either the cell reverses its movement or it swings sidewise into the new direction. Behaviour of partially shadowed cells suggests that perception of light direction is brough about by simultaneous intensity measurement at two or more sites within the cell.     

Gated ion fluxes involved in photophobic responses of the blue-green alga,Phormidium uncinatum

The sensory transduction chain of photophobic responses in the blue-green alga, Phormidium uncinatum seems to involve a gating cation transport through membrane bound ion channels which provides an effective amplification.The calcium conducting ionophore A23187 inhibits the photophobic response totally and induces frequent reversals which resemble phobic responses but occur without any light stimulation. This indicates that the electrogenic ion conductance may depend on a gradient of divalent cations, esp. calcium. The calcium conductance during a photophobic response is further confirmed by the inhibitory effect of ruthenium red and lanthanum, blockers of the electrogenic calcium transport. In the case of lanthanum this inhibition is found at a concentration at which neither the number of motile filaments nor the average speed of movement is impaired.Incorporation of ionophores for monovalent cations (gramicidin and valinomycin) only partially impairs the response. Similarly, inhibition of the Na⁺/K⁺ pump by ouabain is less effective. Thus, the existence of a countercurrent of monovalent cations during the response, which has been described for e.g. ciliates, is yet obscure in blue-green algae.     

Morphological Alterations in Euglena gracilis Induced by Treatment with CTAB (Cetyltrimethylammonium Bromide) and Triton X-100: Correlations with Effects on Photophobic Behavioral Responses*

SYNOPSIS. Treatment of Euglena gracilis with the cationic detergent CTAB at concentrations of 0.05 mM or higher selectively inhibited the ability of the cells to respond with flagellar reorientation to a sudden decrease of light intensity (step-down photophobic response). The ability to respond similarly to an increase in light intensity (step-up photophobic response) was unaffected even at detergent concentrations at which the step-down response was completely inhibited. Electron microscopy of cells treated with 1.0 mM CTAB revealed selective destruction of the membrane of the reservoir and flagellum. No selective effects upon the step-down or step-up photophobic responses were found upon treatment of the cells with Triton X-100.     

Photophobic responses of the cyanobacterium Anabaena variabilis

The photophobic responses in the Cyanobacterium Anabaena variabilis which belongs to the Nostocaceae have been studied with aid of a population method as well as by single trichome observations. In white light experiments both step-up and step-down photophobic responses were observed. The wavelength dependence was examined at a constant fluence rate. The photophobically active light is absorbed by the photosynthetic pigments, mainly by the phycobiliproteins and chlorohyll a. Above 690 nm only negative reactions were observed, i.e. the trichomes left the light trap. In white light experiments DCMU strongly inhibited the photophobic responses, whereas photokinesis was not affected to the same extent indicating that the reaction is coupled with the non cyclic photosynthetic electron transport. DBMIB impaired the photophobic behaviour only slightly. It seems that the photophobic responses of A. variabilis are controlled by a similar mechanism as in Phormidium uncinatum (Oscillatoriaceae) although the two families and, hence, the two species differ in their movement mechanism as well as in their photoactic behaviour.     

Light-adaptation in the photophobic response by Stentor coeruleus

Effects of preillumination on photophobic response (light-adaptation) and recovery of the photophobic sensitivity in the dark (dark-adaptation) in Stentor coeruleus were examined. When the cells were preilluminated with white light of 7.80 W/m² for 2 min, the fluence-rate response curve of photophobic response was shifted toward higher light intensities by half an order of magnitude compared to the one without preillumination. Preillumination with a higher light intensity resulted in a further shift of the fluencerate response curve. An action spectrum for light-adaptation showed a primary peak at 610 nm and secondary peaks at 540 and 480 nm which are almost identical to the peaks observed in the photophobic action spectrum.The light-adapted cells showed a recovery of their photophobic sensing ability following dark treatment. Dark-adaptation resulted in total recovery of photophobic sensing ability in 8 minutes for the most cases examined.     

Phototaxis and photophobic responses in Stentor coeruleus action spectrum and role of Ca2+ fluxes

Negative phototactic orientation, step-up photophobic responses and light-induced action potentials have been studied in the ciliate Stentor coeruleus. A resolved action spectrum, based on fluence rate-response curves, is consistent with stentorin as the photoreceptor. Calcium flux blockers prolong the response time for ciliary stop and reversal and inhibit step-up photophobic responses. Drugs believed to affect the membrane-bound calcium pump likewise inhibit phobic responses. On the other hand, α-phosphatidic acid promotes Ca²⁺-influx and enhances the photophobic sensitivity of the organism, thus providing an unambiguous evidence for the role of Ca²⁺ influx. A change in the response time decreases the degree of phototactic orientation, indicating that negative phototaxis in this organism is brought about by subsequent phobic responses of individual rows of cilia as the associated photoreceptor granules experience an increase in light intensity when the organism rotates during forward locomotion in lateral light.     

Evidence of electrical potential changes in photophobically reacting blue-green algae

The correlation between photophobic responses and light-induced electric potential changes has been studied in the blue-green alga Phormidium uncinatum.

1. The photophobic reaction time depends on both length of preillumination and presentation time of stimulus. Under optimal conditions a reaction time of about 10 s has been determined.
2. Light-induced potential changes can be measured by means of external electrodes with a small gap between them bridged by a population of perpendicularly oriented trichomes. These potential changes follow a light-dark cycle with a lag phase of about 10 s.
3. The amplitude of these light-induced potential changes increases with light intensity until it reaches a saturation value of about 12 mV at 10000 lx. The action spectrum resembles the photophobic action spectrum with peaks in the absorption region of C-phycoerythrin and chlorophyll a.

The significance of light-induced potential changes as a means of sensory transduction for photophobic responses in blue-green algae is being discussed.     

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     

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.     

Thin-blooded Antarctic fishes: a rheological comparison of the haemoglobin-free icefishes Chionodraco kathleenae and Cryodraco antarcticus with a red-blooded nototheniid, Pagothenia bernacchii

The icefishes (family Channichthyidae) comprise a unique group of teleost fishes endemic to Antarctic and sub-antarctic seas. All members of the family totally lack haemoglobin. Haematological parameters and viscosity were determined for blood from 11 specimens of two channichthyid species (Chionodraco kathleenae Regan, 1914; Cryodraco antarcticus Dollo, 1900), and 14 specimens of a red-blood Antarctic nototheniid species (Pagothenia bernacchii (Boulenger, 1902)), captured near the Italian research station at Terra Nova Bay, Ross Sea, Antarctica. Channichthyid blood contained only a small number of non-pigmented cells (10 000-40 000 cells μI^?1, depending on species) in contrast to nototheniid blood (360 000-450 000 cells μI^?1 in unstressed specimens). Blood viscosity was measured by cone plate viscometry over a range of shear rates (11.3-450s ^?1), at six temperatures between – 1.8°C and + 15°C. At the ambient Antarctic seawater temperature of – 1.8° C, and at low shear rate (22.5 s^?1), the viscosity of channichthyid blood was relatively low (3.99 ± 0.40 cP) compared with blood taken from unstressed P. bernacchii, which was about 25% more viscous (4.91 ± 0.59 cP). The viscosity of channichthyid blood was almost independent of shear rate, approximating an ideal Newtonian fluid, while the viscosity of nototheniid blood was much more dependent upon both shear rate and temperature, increasing sharply at low shear rates and low temperatures. Viscosity of nototheniid blood varied with haematocrit, which was in turn strongly influenced by stress. Blood samples taken from P. bernacchii under moderate stress induced by handling during acute caudal venepuncture had haematocrit values in the range 15–20% and viscosities of 8-l0cP, while undisturbed specimens sampled through a venous cannula yielded haematocrits of 8–10%. The viscosity of nototheniid plasma did not differ significantly from that of channichthyid whole blood or channichthyid plasma. The higher viscosity of nototheniid blood is attributable to cell content, and in stressed specimens possibly also to adrenergic swelling of erythrocytes. The absence of erythrocytes in channichthyid blood avoids the great increases in viscosity which are induced in corpusculate blood by sub-zero seawater temperatures.     

Photoisomerization of retinal at 13-ene is important for phototaxis of Chlamydomonas reinhardtii: simultaneous measurements of phototactic and photophobic responses

A real-time automated method was developed for simultaneous measurements of phototactic orientation (phototaxis) and step-up photophobic response of flagellated microorganisms. Addition of all-trans retinal restored both photoresponses in a carotenoid-deficient mutant strain of Chlamydomonas reinhardtii in a dose-dependent manner. The phototactic orientation was biphasic with respect to both the light intensity and the concentration of retinal. All-trans retinal was more effective than 11-cis retinal to regenerate both photobehavioral responses. Analogs having locked 11-cis configurations and a phenyl ring in the side chain also induced photoresponses, although at concentrations more than two orders of magnitude higher than all-trans retinal. According to the present assay method, the responses were hardly detectable in cells incubated with retinal analogs in which the 13-ene was locked in either its trans or cis configuration. The results strongly suggest that the isomerization of the 13-14 double bond is important for photobehavioral signal transduction and that a single retinal-dependent photoreceptor controls both phototactic and photophobic responses.     

Characterization of the Eyespot Regions of "Blind"Chlamydomonas Mutants after Restoration of Photophobic Responses

Chlamydomonas reinhardtii exhibits photophobic and positive and negative phototactic responses that can be defined for cell populations using computerized cell tracking and motion analysis. Mutants CC-2359 and FN68 are pigment deficient mutants that are blocked in carotenoid synthesis and lack these photo responses. In particular, neither mutant exhibits flash-induced photophobic responses to visible light stimuli to which wild-type gametic cells exhibit a strong response, with several behavioral stages. Upon addition of all-trans retinal to these mutants, the photophobic responses are restored with minor quantitative differences from wild-type populations. Using both light and electron microscopy, we have compared the ultrastructural characteristics of wild-type C. reinhardtii to those of both mutants. As previously described, wild-type cells contain an eyespot consisting of 2–4 layers of pigmented granules encased within thylakoid membranes, located between the distal extremities of the flagellar root. This structure is also visible as an orange-red spot in light microscopy. The photoreceptor is thought to be concentrated in the plasma membrane above the eyespot. The mutant, CC-2359, lacks this eyespot as seen by both light and electron microscopy, even when the photophobic response has been restored. FN68-like mutants studied earlier by Morel-Laurens and Feinlieb and others contain an eyespot which can be seen only by electron microscopy. In FN-68, the eyespot generally has the same dimensions as in wt cells, differing mainly in pigment granule appearance. Consistent with these findings, several laboratories have shown that the full range of phototactic responses can be reconstituted in FN68 and CC-2359, but that negative phototaxis requires a significantly stronger light stimulus in the latter strain. We confirm the suggestion that the eyespot is not necessary for the photophobic response, and is not critical for the appropriate assembly and function of the photophobic response receptor in the membrane. Furthermore, the locus of reconstitution of the functional receptor is not the eyespot. Because of the definitive demonstration of the absence of the eyespot in CC-2359, however, the eyespot may play a role in negative phototaxis.     

Photoactivated adenylyl cyclase controls phototaxis in the flagellate Euglena gracilis

Euglena gracilis, a unicellular freshwater protist exhibits different photomovement responses, such as phototaxis (oriented movement toward or away from the light source) and photophobic (abrupt turn in response to a rapid increase [step-up] or decrease [step-down] in the light fluence rate) responses. Photoactivated adenylyl cyclase (PAC) has been isolated from whole-cell preparations and identified by RNA interference (RNAi) to be the photoreceptor for step-up photophobic responses but not for step-down photophobic responses (M. Iseki, S. Matsunaga, A. Murakami, K. Ohno, K. Shiga, C. Yoshida, M. Sugai, T. Takahashi, T. Hori, M. Watanabe [2002] Nature 415: 1047-1051). The present study shows that knockdown of PAC by RNAi also effectively suppresses both positive and negative phototaxis, indicating for the first time that PAC or a PAC homolog is also the photoreceptor for photoorientation of wild-type E. gracilis. Recovery from RNAi occurred earlier for step-up photophobic responses than for positive and negative phototaxis. In addition, we investigated several phototaxis mutant strains of E. gracilis with different cytological features regarding the stigma and paraxonemal body (PAB; believed to be the location for the phototaxis photoreceptor) as well as Astasia longa, a close relative of E. gracilis. All of the E. gracilis mutant strains had PAC mRNAs, whereas in A. longa, a different but similar mRNA was found and designated AlPAC. Consistently, all of these strains showed no phototaxis but performed step-up photophobic responses, which were suppressed by RNAi of the PAC mRNA. The fact that some of these strains possess a cytologically altered or no PAB demonstrates that at least in these strains, the PAC photoreceptor responsible for the step-up photophobic responses is not located in the PAB.     

High Speed Cinemicrography of the Direct Photophobic Response of Euglena and the Mechanism of Negative Phototaxis*

SYNOPSIS. The shock reaction of Euglena gracilis strain Z to a sudden increase in light intensity (the “direct photophobic response”) was examined by high speed cinemicrography. The response is expressed as a turning reaction toward the dorsal side of the cell, after a transduction time of 0.1–0.5 sec after the onset of stimulation. Transduction times, turning rates, and flagellar beat frequencies were measured by analyzing the filmed sequences. The experimental data are consistent with a mechanism of directional homeostasis in negative phototaxis that is based upon shading of the photoreceptor by the cell's posterior end.     

Comparative study of phototactic and photophobic receptor chromophore properties in Chlamydomonas reinhardtii.

The motile, unicellular, eukaryotic alga Chlamydomonas reinhardtii exhibits two distinct behavioral reactions to light stimuli, phototaxis and the photophobic response. Both are mediated by retinal-containing receptors. This paper focuses on a direct comparison of the two photoresponses and the chromophore requirements for their photoreceptor(s). Using computerized motion analysis assays for phototaxis and photophobic responses by the same populations of cells, we measured the ability of various isomers and analogues of retinal to reconstitute photobehavior in the pigment-deficient mutant FN68. The results indicate that photophobic and phototaxis responses each require chromophores with an all-trans polyene chain configuration, planar ionone ring/polyene chain conformation, and the ability to isomerize around the retinal C13-C14 double bond. One difference between the two behaviors is that the photophobic response becomes highly desensitized after light stimuli to which the phototaxis response does not become desensitized, indicating the existence of at least one distinct step in the photophobic response pathway. A second difference is that the retinal regeneration of the photophobic response but not of phototaxis is inhibited by a 5-membered ring 13-trans-locked analogue. While showing close similarity in the chromophore structural requirements of the two behaviors, the results indicate that differences exist between the two responses at the level of their photoreceptor proteins and/or in their transduction processes.     

Phototaxis in the flagellates,Euglena gracilis and Ochromonas danica

Oriented movement with respect to laterally impinging white light of the flagellates Euglena gracilis and Ochromonas danica has been analyzed in an individual cell study with a microvideographic technique. Using the deviation of track segments (in given time intervals of 1 s) from the light direction as raw data allowed a computer based analysis of the direction distribution. A number of statistical methods employed to test the significance of the obtained results demonstrated an obvious phototactic orientation in Ochromonas which was positive (toward the light source) in low illuminance (1.25 lx=5.3×10^-3 Wm^-2) and negative in higher illuminance (>12.5 lx=5.3×10^-2 Wm^-2). Since in this flagellate the threshold for negative phototaxis is much lower than that for the step-up photophobic response, the hypothesis that negative phototaxis may be brought about by repetitive step-up phobic responses can be rejected for at least this organism. In Euglena positive phototaxis was observed in 50 lx (=0.21 Wm^-2), while an illuminance of 500 lx (=2.1 Wm^-2) caused a negative phototaxis.The experiments were carried out in this laboratory     

光谱和光强度对棕榈蓟马雌成虫行为反应的影响

室内研究了光谱、光强度对棕榈蓟马雌成虫的趋、避光行为的影响。结果显示:在340—605 nm波谱内棕榈蓟马雌成虫对14个单色光刺激的趋光行为反应为多峰型。其中蓝光483 nm处峰最高,趋光反应率达34.96%,其次为绿光498—524 nm、562—582 nm、紫外光340 nm处;其避光行为反应共有3个峰,其中紫外光380 nm处最高,避光率18.08%,另外2个峰分别在橙光605 nm、紫光420 nm处。在趋光率较高的单色光(340、483、524、582 nm)和避光率较高的单色光(380、605 nm)以及白光刺激下,棕榈蓟马雌成虫的趋光率随光强增强的增强而提高,而避光率则随着光强的增强而降低;光强最弱时仍均有一定趋光率,最强时均未出现高端平台。因此:棕榈蓟马雌成虫对不同单色光具有明显的选择性,光谱和光强度对其趋光行为和避光行为有较大影响,光强度的影响作用与波长因素有关。