Spectral sensitivity in a sponge larva

Cilia at the posterior pole of demosponge larvae are known to cause directional swimming, sometimes in response to light gradients, but so far neither the spectral sensitivity of, nor the molecular basis for, this response has been investigated. We exploited the fact that the larval cilia respond to sudden changes in light intensity, a shadow response, in order to determine the action spectrum of photosensitivity. Our results show that larvae of the haplosclerid sponge Reniera sp. respond most to blue light (440 nm), and have a smaller, secondary response peak to orange-red light (600 nm). These data suggest that the photoreceptive pigment in sponge larvae may be a flavin or carotenoid.     

Phototactic responses of larvae from the marine sponges Neopetrosia proxima and Xestospongia bocatorensis (Haplosclerida: Petrosiidae)

Abstract. Previous studies suggest that phototaxis in sponge larvae is generated by the bending of a tuft of long posterior cilia (LPC). The photoresponsiveness of these cilia is often assayed by examining their reaction to sudden changes in light intensity. Here, we document and describe the larvae of the tropical marine sponges Neopetrosia proxima and Xestospongia bocatorensis and examine the phototactic behavior of their larvae. Both species brood ovoid, tufted parenchymella larvae, clearly countering an earlier hypothesis that all petrosid sponges are oviparous. Larvae of N. proxima were positively phototactic and settled after 2 d, while larvae of X. bocatorensis were negatively phototactic and settled in as little as 4 h. In both species, LPC quickly responded to changes in the light intensity. When the light intensity is reduced, the larvae of N. proxima fold the cilia inwards immediately without beating, then flare them outwards, beating for a few seconds, and then gradually return to the neutral position while continuing to beat. In contrast, the larvae of X. bocatorensis flare the cilia outwards when the light intensity is reduced and fold them inwards when the light intensity is increased. Comparisons with reported ciliary responses to light for other species demonstrate that these responses do not show the hypothesized one-to-one correspondence with phototactic behaviors and are, therefore, of limited use in explaining the mechanisms that coordinate larval swimming.     

Ultrastructure of potential photoreceptor organs in the larva ofScrupocellaria bertholetti (bryozoa)

Summary Pigmented spots have been implicated as potential photoreceptors in many bryozoan larvae which display phototactic behavior. Larvae ofScrupocellaria bertholetti, initially photopositive on release from the brood chambers, have a pair of identical posterolateral pigmented spots and a third morphologically different spot in the anteromedian line. The presumed photoreceptoral organelle in each is composed of numerous unmodified cilia which have the typical 9+2 arrangement of microtubules with electron-dense arms extending from thea-microtubule of each peripheral pair. The posterolateral pigmented spot is composed of two modified coronal cells and a basal sensory cell. Cilia arising from the apical part of this basal cell are aligned vertically. Densely packed pigment vesicles in the three cells form a shield that restricts light entry to one direction. The anteromedian pigmented spot is composed of four cells, two lateral and two posterior. Cilia of the opposing lateral cells are horizontally aligned, whereas cilia of the posterior cells are vertical and curve outward from the oral margin of the pigmented spot. Pigment vesicles are present in all four cells to form a complete shield. Extensions of the larval nervous system are in direct contact with the four cells of the anteromedian spot and the basal cells of the posterolateral spots. The posterolateral pigmented spots share structural and topological similarities with the pigmented spots ofBugula neritina, the only other supposed photoreceptors in lophophorates which have been studied at the ultrastructural level. It is not yet possible to homologize these potential photoreceptors with those of other groups.     

Fine structure of the eyes of Pseudoceros canadensis (Turbellaria,Polycladida)

Summary The cerebral and epidermal ocelli of the Müller's larva and the cerebral and tentacular eyes of the adult turbellarian Pseudoceros canadensis were studied by electron microscopy. The right cerebral ocellus of the larva consists of one cup-shaped pigmented cell and three sensory cells that bear microvilli. The left cerebral eye of the larva has the above named cells plus a sensory cell with many cilia. Evolutionary significance is attributed to the presence of both ciliary and microvillar photoreceptors in an eye of a flatworm. The one epidermal ocellus of the larva is composed of two cells: a cup-shaped pigmented one bearing flattened cilia, the presumed photoreceptors, and a cell above the cup that adds a few nonciliary lamellae to the stack of ciliary ones from the pigmented cell. The adult eyes contain only microvillar receptors; cilia were not observed.     

Ciliary sieving and active ciliary response in capture of particles by suspension-feeding brachiopod larvae

Larvae of a brachiopod, Glottidia pyramidata, used at least two ciliary mechanisms to capture algal cells upstream from the lateral band of cilia that produces a feeding/swimming current. (1) Filtration: the larvae retained algal cells on the upstream (frontal) side of a sieve composed of a row of stationary laterofrontal cilia. Movement of the laterofrontal cilia could not be observed during capture or rejection of particles, but the laterofrontal cilia can bend toward the beating lateral cilia, a possible mechanism for releasing rejected particles from the ciliary sieve. (2) Localized changes of ciliary beat: the larvae may also concentrate particles by a local change in beat of lateral cilia in response to particles. The evidence is that the beat of lateral cilia changed coincident with captures of algal cells and that captured particles moved on paths consistent with a current redirected toward the frontal side of the tentacle by an induced local reversal of the lateral cilia. The change of beat of lateral cilia could have been an arrest rather than a reversal of ciliary beat, however. The similar ciliary bands in adult and larval lophophorates (brachiopods, phoronids, and bryozoans) suggest that these animals share a range of ciliary behaviours. The divergent accounts of ciliary feeding of lophophorates could be mostly the result of different authors observing different aspects of ciliary feeding.     

Versatile ciliary behaviour in capture of particles by the bryozoan cyphonautes larva

Cyphonautes larvae of a bryozoan, Membranipora membranacea, used several ciliary mechanisms to capture algal cells upstream from the lateral band of cilia that produces a feeding current. (1) Lateral cilia changed beat and a backcurrent occurred at the time and place that particles were retained. (2) Algal cells were sieved and held stationary at the upstream (frontal) side of a row of laterofrontal cilia that were not beating. (3) Localized extension of cilia toward the inhalant chamber, coincident with particle captures, indicated that laterofrontal cilia flick toward the inhalant chamber. These flicks may aid transport of captured particles toward the mouth. Thus my earlier report that larvae only sieve, in contrast to the adults (which have an active ciliary response) was in error. The similar ciliary bands in adult and larval bryozoans and in other lophophorates (brachiopods, and phoronids) suggest that these animals share a core repertoire of ciliary behaviours in the capture and concentration of suspended food particles.     

Proximate control of diel vertical migration in Phyllosoma larvae of the Caribbean spiny lobster Panulirus argus

Phyllosoma larvae of the spiny lobster Panulirus argus undergo diel vertical migration (DVM), in which they are at depth during the day and nearer the surface at night. This study determined the visual spectral sensitivity of Stage I larvae and investigated whether light plays a proximate role in DVM as an exogenous cue and as an entrainment cue for an endogenous rhythm in vertical migration. Under constant conditions, larvae have a circadian rhythm (24.5-h period) in vertical swimming that resulted in a twilight DVM pattern. The behavioral response spectrum and electroretinogram recording indicated two photoreceptor spectral classes with maxima at 360 and 486 nm. When stimulated in an apparatus that simulated the underwater angular light distribution, dark-adapted larvae showed only positive phototaxis, with a threshold intensity of 1.8 × 10(13) photons m(-2) s(-1) (3.0 × 10(-5) μmoles photons m(-2) s(-1)). They have an avoidance response to predator shadows in which they descend upon sudden decreases in light intensity of more than 69%. When stimulated with relative rates of decrease in light intensity as occur at sunset they ascended, whereas they descended upon relative rates of light intensity increase as occur at sunrise. Thus, the DVM pattern is controlled by both an endogenous circadian rhythm in swimming and behavioral responses to light at sunrise and sunset.     

Photoresponse and Learning Behavior of Ascidian Larvae, a Primitive Chordate, to Repeated Stimuli of Step-Up and Step-Down of Light

Ascidians are lower chordates and their simple tadpole-like larvae share a basic body plan with vertebrates. Newly hatched larvae show no response to a stimulus of light. 4 h after hatching, the larvae were induced to swim upon a step-down of light and stop swimming upon a step-up of light. At weaker intensity of light, the larvae show the same response to a stimulus after presentation of repeated stimuli. When intensity of actinic light was increased, the larvae show sensitization and habituation of the swimming response to a stimulus after repeated stimuli of step-down and step-up of the light. Between 2 h 20 min and 3 h 40 min after hatching the larvae did not show any response to the first stimulus, but after several repeatedstimuli they show swimming response to a step-down of light. A repeated series of stimulus cause sensitization. Between 4 h and 7 h after hatching, the larvae show photoresponse to the first stimulus, but after several repetition of the stimuli, the larvae could not stop swimming to a stimulus of a step-up of the actinic light. A repeated series of stimulus cause greaterhabituation. Both sensitization and habituation depend upon intensity ofactinic light.     

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.     

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.     

An electro-optic monitor of the behavior of Chlamydomonas reinhardtii cilia

The unicellular green alga Chlamydomonas reinhardtii steers through water with a pair of cilia (eukaryotic flagella). Long-term observation of the beating of its cilia with controlled stimulation is improving our understanding of how a cell responds to sensory inputs. Here we describe how to record ciliary motion continuously for long periods. We also report experiments on the network of intracellular signaling that connects the environment inputs with response outputs. Local spatial changes in ciliary response on the time scale of the underlying biochemical dynamics are observed. Near-infrared light monitors the cells held by a micropipette. This condition is tolerated well for hours, not interfering with ciliary beating or sensory transduction. A computer integrates the light stimulation of the eye of Chlamydomonas with the ciliary motion making possible long-term correlations. Measures of ciliary responses include the beating frequency, stroke velocity, and stroke duration of each cilium, and the relative phase of the cis and trans cilia. The stationarity and dependence of the system on light intensity was investigated. About 150,000,000 total beat cycles and up to 8 h on one cell have been recorded. Each beat cycle is resolved so that each asynchronous beat is detected. Responses extend only a few hundred milliseconds, but there is a persistence of momentary changes that last much longer. Interestingly, we see a response that is linear with absolute light intensity as well as different kinds of response that are clearly nonlinear, implying two signaling pathways from the cell body to the cilia.     

Light induced larval release of a colonial ascidian

Larval release and photobehavior were studied in the colonial ascidian Polyandrocarpa zorritensis. The test hypothesis was that if larval release is induced by light, then larvae should be attracted to settlement areas where light is sufficient for larval release. Light induced larval release but the time course varied with light intensity. As the intensity of either sunlight or blue-green light decreased (1) the time until the beginning of larval release (latency) became longer, (2) the mean time of larval release increased, and (3) the time interval over which larvae were released increased. The threshold light intensity to induce larval release in blue-green light (8.75x10(12) photons cm(-2) s(-1)) was lower than that in sunlight (3.6x10(13) photons cm(-2) s(-1)). Light induced larval release was not affected by currents up to 15 cm s(-1). Larvae aggregate in light when given a choice between light and dark. This response did not vary with larval age. The lowest light intensity, at which larvae could distinguish between light and dark was 5.0x10(12) photons cm(-2) s(-1) in blue-green light and 2.9x10(14) photons cm(-2) s(-1) in sunlight. Thus, the hypothesis is supported because larvae are attracted to areas where light intensity is sufficient for larval release.     

Visualization of calcium transients controlling orientation of ciliary beat

To image changes in intraciliary Ca controlling ciliary motility, we microinjected Ca Green dextran, a visible wavelength fluorescent Ca indicator, into eggs or two cell stages of the ctenophore Mnemiopsis leidyi. The embryos developed normally into free-swimming, approximately 0.5 mm cydippid larvae with cells and ciliary comb plates (approximately 100 microns long) loaded with the dye. Comb plates of larvae, like those of adult ctenophores, undergo spontaneous or electrically stimulated reversal of beat direction, triggered by Ca influx through voltage-sensitive Ca channels. Comb plates of larvae loaded with Ca Green dextran emit spontaneous or electrically stimulated fluorescent flashes along the entire length of their cilia, correlated with ciliary reversal. Fluorescence intensity peaks rapidly (34-50 ms), then slowly falls to resting level in approximately 1 s. Electrically stimulated Ca Green emissions often increase in steps to a maximum value near the end of the stimulus pulse train, and slowly decline in 1-2 s. In both spontaneous and electrically stimulated flashes, measurements at multiple sites along a single comb plate show that Ca Green fluorescence rises within 17 ms (1 video field) and to a similar relative extent above resting level from base to tip of the cilia. The decline of fluorescence intensity also begins simultaneously and proceeds at similar rates along the ciliary length. Ca-free sea water reversibly abolishes spontaneous and electrically stimulated Ca Green ciliary emissions as well as reversed beating. Calculations of Ca diffusion from the ciliary base show that Ca must enter the comb plate along the entire length of the ciliary membranes. The voltage-dependent Ca channels mediating changes in beat direction are therefore distributed over the length of the comb plate cilia. The observed rapid and virtually instantaneous Ca signal throughout the intraciliary space may be necessary for reprogramming the pattern of dynein activity responsible for reorientation of the ciliary beat cycle.     

Changes of the lingual epithelium in Ambystoma mexicanum

Changes in the lingual epithelium during ontogenesis and after induced metamorphosis in Ambystoma mexicanum are described as observed by light microscopy and scanning electron microscopy. The epithelium of the tongue is always multilayered in the larva as well as in the adult. It consists of a stratum germinativum with little differentiated basal cells and a stratum superficiale (superficial layer) with specialized superficial cells and goblet cells. Usually, there are more than two layers because of a stratum intermedium consisting of replacement cells. The apical cell membrane of the superficial cells is perforated by fine pores. Its most typical feature are microridges. Maturing superficial cells possess microvilli. Goblet cells occur in early larvae primarily in the centre of the tongue. They spread throughout the dorsal face of the tongue as their numbers increase during ontogenesis. The small apices of the goblet cells are intercalated in the wedges between the superficial cells. Leydig cells are not found on the larval tongue but on that of adults. Due to metamorphosis, the epithelium of the tongue changes. It is furrowed in its anterior part. The furrows house the openings of the lingual glands. The surface is further modulated by ridges which are densely coated by microvilli and which bear the taste buds. The villi of the tongue which lack extrusion pores show cilia and microvilli but lack microridges. The Leydig cells disappear during metamorphosis. In addition to the two types of goblet cells found in different regions of the glandular tubules, goblet cells occur in the caudal part. They secrete directly into the cavity of the mouth. The posterior part is characterised by a dense coat of cilia.     

Life History Changes in Coral Fluorescence and the Effects of Light Intensity on Larval Physiology and Settlement in Seriatopora hystrix

Fluorescence is common in both coral adult and larval stages, and is produced by fluorescent proteins that absorb higher energy light and emit lower energy light. This study investigated the changes of coral fluorescence in different life history stages and the effects of parental light environment on larval fluorescence, larval endosymbiotic dinoflagellate abundance, larval size and settlement in the brooding coral Seriatopora hystrix. Data showed that coral fluorescence changed during development from green in larvae to cyan in adult colonies. In larvae, two green fluorescent proteins (GFPs) co-occur where the peak emission of one GFP overlaps with the peak excitation of the second GFP allowing the potential for energy transfer. Coral larvae showed great variation in GFP fluorescence, dinoflagellate abundance, and size. There was no obvious relationship between green fluorescence intensity and dinoflagellate abundance, green fluorescence intensity and larval size, or dinoflagellate abundance and larval size. Larvae of parents from high and low light treatments showed similar green fluorescence intensity, yet small but significant differences in size, dinoflagellate abundance, and settlement. The large variation in larval physiology combined with subtle effects of parental environment on larval characteristics seem to indicate that even though adult corals produce larvae with a wide range of physiological capacities, these larvae can still show small preferences for settling in similar habitats as their parents. These data highlight the importance of environmental conditions at the onset of life history and parent colony effects on coral larvae.     

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.     

A chamber for applying pressure to roots of intact plants

A chamber was designed to apply up to 20 bars pressure to roots of intact plants. The unique features of this chamber are a split top arrangement to permit enclosing roots of intact plants within the chamber, a circulation coil to control temperature of rooting media, and a valve arrangement to permit changing solution without disturbing the plant. Changes in transpiration in response to changes in the pressure applied to roots of intact pepper plants illustrate one use of the equipment. Well watered plants at low light (0.05 langley/min) were observed to exude water from the leaf margins when 5 bars pressure was applied to the roots. When roots were cut off, a 1 bar pressure caused exudation. Plants with cooled roots or plants in dry soil did not exude water when as much as 6 bars pressure was applied. Transient response of transpiration rates to sudden application and release of pressure was observed in pepper and bean plants but not in rhododendron. The magnitude of this transient response was highly dependent upon light intensity and CO₂ concentration in the aerial environment.     

Planktonic sea urchin larvae change their swimming direction in response to strong photoirradiation

To survive, organisms need to precisely respond to various environmental factors, such as light and gravity. Among these, light is so important for most life on Earth that light-response systems have become extraordinarily developed during evolution, especially in multicellular animals. A combination of photoreceptors, nervous system components, and effectors allows these animals to respond to light stimuli. In most macroscopic animals, muscles function as effectors responding to light, and in some microscopic aquatic animals, cilia play a role. It is likely that the cilia-based response was the first to develop and that it has been substituted by the muscle-based response along with increases in body size. However, although the function of muscle appears prominent, it is poorly understood whether ciliary responses to light are present and/or functional, especially in deuterostomes, because it is possible that these responses are too subtle to be observed, unlike muscle responses. Here, we show that planktonic sea urchin larvae reverse their swimming direction due to the inhibitory effect of light on the cholinergic neuron signaling>forward swimming pathway. We found that strong photoirradiation of larvae that stay on the surface of seawater immediately drives the larvae away from the surface due to backward swimming. When Opsin2, which is expressed in mesenchymal cells in larval arms, is knocked down, the larvae do not show backward swimming under photoirradiation. Although Opsin2-expressing cells are not neuronal cells, immunohistochemical analysis revealed that they directly attach to cholinergic neurons, which are thought to regulate forward swimming. These data indicate that light, through Opsin2, inhibits the activity of cholinergic signaling, which normally promotes larval forward swimming, and that the light-dependent ciliary response is present in deuterostomes. These findings shed light on how light-responsive tissues/organelles have been conserved and diversified during evolution.     

Ciliary behavior of a negatively phototactic Chlamydomonas reinhardtii

With an instrument that can record the motion of both cilia of the unicellular alga Chlamydomonas reinhardtii for many hours, the behavioral differences of its two cilia have been studied to determine their specific role in phototaxis. The organism was held on a fixed micropipette with the plane of ciliary beating rotated into the imaging plane of a quadrant photodetector. The responses to square-wave light patterns of a wide range of temporal frequencies were used to characterize the responses of each cilium. Eighty-one cells were examined showing an unexpectedly diverse range of responses. Plausible common signals for the linear and nonlinear signals from the cell body are suggested. Three independent ciliary measures--the beat frequency, stroke velocity, and phasing of the two cilia--have been identified. The cell body communicates to the cilia the direction of phototaxis the cell desires to go, the absolute light intensity, and the appropriate graded transient response for tracking the light source. The complexity revealed by each measure of the ciliary response indicates many independent variables are involved in the net phototactic response. In spite of their morphological similarity, the two cilia of Chlamydomonas respond uniquely. Probably the signals from the cell body fan out to independent pathways in the cilia. Each cilium modifies the input in its own way. The change in the pattern of the effective and recovery strokes of each cilium associated with negative phototaxis has been demonstrated and its involvement in phototactic turning is described.