ULTRASTRUCTURE OF NOCTILUCA MILIARIS (PYRROPHYTA) WITH GREEN FLAGELLATE SYMBIONTS1,2

The ultrastructure of the Noctiluca miliaris Suriray from Southest Asian waters which contains the green flagellate, Pedinomonas noctilucae (Subrahmanyan) Sweeney, is in all major respects similar to that of the European strain. New details of the thecal vesicles, pellicle and underlying microtubules are presented. The possibility that the lipid vesicles are identical with the strongly phase-retarding bodies in the surface cytoplasm, some of which are “microsources” of bioluminescence, is suggested.     

Development of the discharge apparatus in the fungus Entophlyctis

Correlative light and electron microscopic observations were used to reconstruct the morphological events involved in the development of the discharge apparatus of Entophlyctis zoosporangia. A discharge plug formed as vesicles containing fibrillar material fused with the plasma membrane and deposited their matrices between the plasma membrane and zoosporangial wall. At the apex of the enlarging plug, the zoosporangial wall lost its microfibrillar appearance, became diffuse, and left an inoperculate discharge pore. The discharge plug exuded through this pore and then expanded into a sphere which rested at the tip of the discharge papilla or tube. After the release of the discharge plug, the number of fibrilla containing vesicles decreased and abundant endoplasmic reticulum appeared in the cytoplasm below the plug. Granular material then accumulated at the interface of the discharge plug and the plasma membrane. This was the endo-operculum. A single layer of endoplasmic reticulum subtended the area of plasma membrane which the endo-operculum covered. Later, dictyosomes appeared in the cytoplasm below the endo-operculum. Fusion of Golgi vesicles with the plasma membrane below the endo-operculum coincided with the initiation of cytoplasmic cleavage. This sequence of events indicates that, unlike the discharge plug, the endo-operculum does not originate by vesicular addition of preformed material.     

Requirement of Indoleamines and a V-type Proton ATPase for the Expression of the Circadian Glow Rhythm in Gonyaulax polyedra

In the dinoflagellate Gonyaulax polyedra , bioluminescence is known to be controlled by proton transfer from an acidic vacuole system to the scintillons. We demonstrate that bafilomycin A 1 , a specific blocker of V-type proton ATPases, inhibits at low concentrations (down to 2 × 10 -8 M) bioluminescence and, in particular, the circadian glow maximum. For many hours bafilomycin A 1 does not interfere with the capacity of the bioluminescent system. Therefore, we conclude on the participation of a V-type ATPase in proton accumulation in the acidic vacuoles. Inhibition of tryptophan hydroxylase by p-chlorophenylalanine, p-fluorophenylalanine, or 5-fluorotryptophan also suppresses the circadian glow maximum. After inhibition of the enzyme by p-chlorophenylalanine, the glow peak can be restored, without any additional unspecific effects on bioluminescence, by supplementation with 5-hydroxytryptophan. Therefore, the availability of indoleamines is required for the expression of the glow maximum. Since 5-methoxytryptamine is the only physiologically occurring indoleamine with substantial effects on bioluminescence at low concentrations (below 10 -7 M), and since this substance accumulates in the second half of the night to stimulatory concentrations, this indolic metabolite may represent the physiologically active substance involved in the expression of the glow maximum.     

Requirement of Indoleamines and a V-type Proton ATPase for the Expression of the Circadian Glow Rhythm in Gonyaulax polyedra

In the dinoflagellate Gonyaulax polyedra, bioluminescence is known to be controlled by proton transfer from an acidic vacuole system to the scintillons. We demonstrate that bafilomycin A 1, a specific blocker of V-type proton ATPases, inhibits at low concentrations (down to 2 × 10 –8 M) bioluminescence and, in particular, the circadian glow maximum. For many hours bafilomycin A 1 does not interfere with the capacity of the bioluminescent system. Therefore, we conclude on the participation of a V-type ATPase in proton accumulation in the acidic vacuoles. Inhibition of tryptophan hydroxylase by p-chlorophenylalanine, p-fluorophenylalanine, or 5-fluorotryptophan also suppresses the circadian glow maximum. After inhibition of the enzyme by p-chlorophenylalanine, the glow peak can be restored, without any additional unspecific effects on bioluminescence, by supplementation with 5-hydroxytryptophan. Therefore, the availability of indoleamines is required for the expression of the glow maximum. Since 5-methoxytryptamine is the only physiologically occurring indoleamine with substantial effects on bioluminescence at low concentrations (below 10 –7 M), and since this substance accumulates in the second half of the night to stimulatory concentrations, this indolic metabolite may represent the physiologically active substance involved in the expression of the glow maximum.     

Ultrastructure and microtubule organization of isolated generative cells ofAllemanda neriifolia at interphase and prophase

Summary The ultrastructure of isolated generative cells ofAllemanda neriifolia at interphase and prophase was studied. The microtubule organization of the isolated cells was also investigated by immunofluorescence microscopy with a monoclonal anti--tubulin. After the generative cells had been isolated from the growing pollen tubes by osmotic shock, most of the cells were at prophase and only a few were at interphase. The interphase cell is spindle shaped and contains an ellipsoidal nucleus. In addition to the usual organelles, the cytoplasm of the interphase cell contains numerous vesicles (each measuring 40–50 nm in diameter) and two sets of longitudinally oriented microtubule bundles — one in the cortical region and the other near the nucleus. Most of the prophase cells are spherical in shape. Based on the ultrastructure and the pattern of microtubule cytoskeleton organization three types of prophase cells can be recognized. (1) Early prophase cell, which contains the usual organelles, numerous vesicles, and a spherical nucleus with condensed chromosomes. Longitudinally oriented microtubule bundles can no longer be seen present in the early prophase cell. A new type of structure resembling a microtubule aggregate appears in the cytoplasm. (2) Mid prophase cell, which has a spherical nucleus containing chromosomes that appear more condensed than those seen in the early prophase cell. In addition to containing the usual organelles, the cytoplasm of this cell contains numerous apparently randomly oriented microtubules. Few vesicles are seen and microtubule aggregates are no longer present. (3) Late prophase cell, typified by the lack of a nuclear envelope. Consequently, the chromosomes become randomly scattered in the cytoplasm. Microtubules are still present and some become closely associated with the chromosomes. The changes in the ultrastructure and in the pattern of microtubule organization in the interphase and prophase cells are discussed in relation to the method of isolation of the generative cells.     

Electron microscopy of infection of nematodes byDactylaria haptotyla

Infection of nematodes byDactylaria haptotyla, a nematode-trapping hyphomycete, was studied by electron microscopy. The cytoplasm of the adhesive knob in the fungus contained a number of electron-dense, membrane-bound vesicles, 0.2–0.5 µm in diam. The vesicles were rarely seen in the stalk cell or vegetative cell cytoplasm. When the adhesive knob came into contact with the nematode's cuticle, it secreted an adhesive which was seen in ultrathin sections between the knob and the cuticle as an amorphous mass. At the same time, electron-dense vesicles in the cytoplasm were reduced in number and many small vacuoles developed. Soon after capture of a nematode, the cell wall of the adhesive knob became obscure at the prospective site of penetration, where a vesicle, 0.7 µm in diam, was found in serial thin sections of the knob's cytoplasm. At the site facing the vesicle, the peripheral part of the nematode's cell exhibited a high electron density. The vesicle, which appeared to be derived from smaller electron-dense vesicles coalesced with each other, released its enzymic contents toward the captured nematodes before penetration by the fungus.     

The mechanical triggering of bioluminescence in marine dinoflagellates: chemical basis

In both photosynthetic (Pyrodinium bahamense, Gonyaulax polyedra, Pyrocystis Iunula, P. noctiluca, P. fusiformis) and nonphotosynthetic (Noctiluca miliaris) bioluminescent dinoflagellates chemical stimulation can by-pass mechanical stimulation. The effective ions are Ca⁺⁺, K⁺, NH₄⁺ and H⁺. Other chemicals found effective are those implicated in Ca⁺⁺ transport or binding. There are interspecies differences in degrees of mechanical and chemical stimulability. Photoinhibition of mechanical stimulability is the result of two effects, the first a reduction in total bioluminescence potential and the second a decrease in mechanical stimulability resulting experimentally in a decreased rate of light emission. This latter effect can be reversed with Ca⁺⁺ ions. Chemicals which bind Ca⁺⁺ or displace Ca⁺⁺ can mimic the effects of photoinhibition. The chemical inhibition of mechanical stimulability is also reversed by Ca⁺⁺ ions. A scheme is proposed which is consistent for all species examined.     

Fine structure of monoamine-containing basal cells in the taste buds on the barbels of three species of teleosts

Summary The taste buds on the barbels in three species of teleosts (Cyprinus carpio, Misgurnus anguillicaudatus, Parasilurus asotus) were studied by means of fluorescence and electron microscopy. Intensely yellow-fluorescent cells, which are disk-shaped and located exclusively in a basal position, are observed in the barbel-buds of all fishes examined. The basal cells contain a large number of small clear vesicles approximately 40–60 nm in diameter, which show a tendency to aggregate in the cytoplasm facing the junction of the nerve terminals; chemically transmitting synapses are seen in the latter region. It is suggested from the present observations that the basal cells in the barbel-bud may originate from Schwann cells and have a dual function both as mechanoreceptors and paracrine elements. Since the administration of 5,6-DHT results in an appearance of small dense vesicles among the small clear vesicles, the possibility exists that the basal cell may be capable of taking up monoamines and storing them in the small clear vesicles.     

The Subcellular Origin of Bioluminescence in Noctiluca miliaris

The light emitted by Noctiluca has its origin in 1 to 5 x 10⁴ organelles ("microsources") which are scattered throughout the perivacuolar cytoplasm, and which appear to be the elementary functional units of bioluminescence. Microscopical techniques, image intensification, and microphotometry were employed in their investigation. Microsources are fluorescent, strongly phase-retarding, and range widely in diameter below 1.5 microns. The number of quanta emitted in a flash from a microsource ("microflash") is of the order of 10⁵ photons. However, microflashes show a wide range of intensities, which are correlated with the size of the organelles from which they arise. Each organelle responds repetitively and with reproducible time course to a succession of invading triggering potentials. Reversible changes in the intensity of the flash emitted by the whole cell ("macroflash") occur because of graduations in intensity of microflashes rather than as a result of changes in the number of responsive organelles. The shape of the flash emitted by individual microsources resembles that of the macroflash except for slightly shorter rise and decay times. It is concluded that the macroflash results from somewhat asynchronous, but otherwise parallel summation of microflashes.     

SPONTANEOUS AND STIMULATED BIOLUMINESCENCE OF THE DINOFLAGELLATE CERATOCORYS HORRZDA (PERIDINIALES)1

This is the first report of spontaneous bioluminescence in the autotrophic dinoflagellate Ceratocorys horrida von Stein. Bioluminescence was measured, using an automated data acquisition system, in a strain of cultured cells isolated from the Sargasso Sea. Ceratocorys horrida is only the second dinoflagellate species to exhibit rhythmicity in the rate of spontaneous flashing, flash quantum flux (intensity), and level of spontaneous glowing. The rate of spontaneous flashing was maximal during hours 2–4 of the dark phase [i.e. circadian time (CT)16–18 for a 14:10 h LD cycle (LD14:10)], with approximately 2% of the population flashing-min^?1, a rate approximately one order of magnitude greater than that of the dinoflagellate Gonyaulax polyedra. Flash quantum flux was also maximal during this period. Spontaneous flashes were 134 ms in duration with a maximum flux (intensity) of 3.1×10⁹ quanta-s^?1. Light emission presumably originated from blue fluorescent microsources distributed in the cell periphery and not from the spines. Values of both spontaneous flash rate and maximum flux were independent of cell concentration. Isolated cells also produced spontaneous flashes. Spontaneous glowing was dim except for a peak of 6.4× 10⁴quanta-s^?1 cell^?1, which occurred at CT22.9 for LD14:10 and at CT22.8 for LD12:12. The total integrated emission of spontaneous flashing and glowing during the dark phase was 4×10⁹ quantacell^?1, equivalent to the total stimulable luminescence. The rhythms for C. horrida flash and glow behavior were similar to those of Gonyaulax polyedra, although flash rate and quantum flux were greater. Spontaneous bioluminescence in C. horrida may be a circadian rhythm because it persisted for at least three cycles in constant dark conditions. This is also the first detailed study of the stimulated bioluminescence of C. horrida, which also displayed a diurnal rhythm. Cultures exhibited >200 times more mechanically stimulated bioluminescence during the dark phase than during the light phase. Mechanical stimulation during the dark phase resulted in 6.7 flashes. cell^?1; flashes were brighter and longer in duration than spontaneous flashes. Cruise-collected cells exhibited variability in quantum flux with few differences in flash kinetics. The role of dinoflagellate spontaneous bioluminescence in the dynamics of near-surface oceanic communities is unknown, but it may be an important source of natural in situ bioluminescence.     

Properties and cellular localization of a luciferin binding protein in the bioluminescence reaction of Gonyaulax polyedra

A luciferin binding protein LBP involved in the bioluminescence reaction of Gonyaulax polyedra was purified and used for antibody production. Luciferin bound to LBP is fluorescent and can be used as a marker in living cells, allowing the localization of LBP in cortical organelles to be visualized. In cell sections, the same peripheral localization was observed using anti-LBP and immunofluorescence microscopy. The amount of LBP is ten-fold greater from cells from in night phase compared to those from in day phase, as determined both by immunoblots of cell extracts, and in vivo fluorescence. These changes correlate with the circadian changes in bioluminescence of living cells.     

Compartmentalization of algal bioluminescence: autofluorescence of bioluminescent particles in the dinoflagellate Gonyaulax as studied with image-intensified video microscopy and flow cytometry

Compartmentalization of specialized functions to discrete locales is a fundamental theme of eucaryotic organization in cells. We report here that bioluminescence of the dinoflagellate alga Gonyaulax originates in vivo from discrete subcellular loci that are intrinsically fluorescent. We demonstrate this localization by comparing the loci of fluorescence and bioluminescence as visualized by image-intensified video microscopy. These fluorescent particles appeared to be the same as the previously described in vitro "scintillons." We attribute the endogenous fluorescence to that of the bioluminescence substrate, luciferin, because (a) the fluorescence excitation and emission characteristics are comparable, (b) the autofluorescence is lost after exhaustive stimulation of bioluminescence, and (c) the fluorescence of discharged particles in vitro can be restored by adding luciferin. The fluorescence in vivo exhibits a standard property of circadian (daily) rhythmicity: under constant environmental conditions, the intensity of the particle fluorescence fluctuates cyclically (it is maximal during the night phase and is low during the day). Thus, luciferin is localized within the cell at discrete loci from which the bioluminescence emanates; the cellular quantity of luciferin is rhythmically modulated by the circadian clock.     

Bioluminescence of Marine Dinoflagellates : I. An underwater photometer for day and night measurements

Portable light-baffled underwater photometers have been designed for the measurement of dinoflagellate bioluminescence by day and night. Maximal light emission is obtained by mechanical stimulation in a defined volume. The pump which stimulates the dinoflagellates also constantly replenishes the sample volume so that continuous measurements are possible. Evidence for both diurnal variation and vertical migration is presented. Using luminous bacteria for calibration a single dinoflagellate has been found to emit of the order of 10¹⁰ light quanta per flash. The technique suggests that large scale mapping of bioluminescence is feasible.     

Sensitivity to stimulation, a component of the circadian rhythm in luminescence in gonyaulax

A new method for the stimulation of bioluminescence in the dinoflagellate Gonyaulax polyedra is described. With this technique, in which cells flow through a capillary coil, it is possible to graduate the intensity of the stimulus by varying the flow rate. In continuous darkness, the threshold stimulus for cells in the middle of the day phase is greater than that for cells in the middle of the night phase. Some evidence suggests heterogeneity of sensitivity to stimulation among either cells or individual luminescent sources within a cell. At stimulus intensities much above threshold, the luminescence of both day- and night-phase cells is proportional to the number of cells within the capillary coil. Night-phase cells emit about 14 times as much light as do day-phase cells in continuous darkness.     

Same Temporal Niche,Opposite Rhythmicity: Two Closely Related Bioluminescent Insects With Opposite Bioluminesce Propensity Rhythms

Arachnocampa species, commonly called glowworms, are flies whose larvae use light to attract prey. Here we compare rhythmicity in two of the nine described species: the Tasmanian species, Arachnocampa tasmaniensis, which inhabits caves and wet forest, and the eastern Australian mainland species, A. flava, primarily found in subtropical rainforest. Both species show the same nocturnal glowing pattern in external (epigean) environments and the same inhibition of bioluminescence by light and both species show circadian regulation of bioluminescence. We find that the underlying circadian bioluminescence propensity rhythm (BPR) of the two species peaks at opposite phases of the day:night cycle. Larvae of A. flava, placed in constant darkness in the laboratory, bioluminesce during the subjective scotophase, typical of nocturnal animals, whereas A. tasmaniensis shows the opposite tendency, bioluminescing most intensely during the subjective photophase. In A. tasmaniensis, which are exposed to natural day:night cycles, light exposure during the day overrides the high bioluminescence propensity through negative masking and leads to a release of bioluminescence after dusk when the BPR is on the wane. A consequence is that A. tasmaniensis is able to start glowing at any phase of the light:dark cycle as soon as masking by light is released, whereas A. flava is locked into nocturnal bioluminescence. We suggest that the paradoxical BPR of A. tasmaniensis is an adaptation for living in the cave environment. Observations of bioluminescence in colonies of A. tasmaniensis located in the transition from a cave mouth to the dark zone show that glowing is inhibited by light exposure but a peak bioluminescence follows immediately after “dusk” at their location. The substantial difference in the circadian regulation of bioluminescence between the two species probably reflects adaptation to the cave (hypogean) habitat in A. tasmaniensis and the forest (epigean) habitat in A. flava. (Author correspondence: d.merritt@uq.edu.au)     

Oogenesls in the terrestrial hermit crab,coenobita clypeatus (decapoda,anomura): II. Vitellogenesis

Oocytes from the land hermit crab, Coenobita clypeatus, in various stages of vitellogenesis were examined by light and electron microscopy. Early vitellogenic oocytes are characterized by accumulations of discrete vesicles of endoplasmic reticulum in the perinuclear cytoplasm. As oocytes develop, the endoplasmic reticulum becomes abundant, and numerous Golgi complexes are seen. There is a well developed Golgi-endoplasmic reticulum interaction. Within the confines of the reticulum are discrete intracisternal granules, which can be seen coalescing into electron-dense yolk bodies. Lipid accumulation is seen throughout the cytoplasm. Coincident with the burst of intra-oocytic metabolism are oolemma modifications and micropinocytosis, which provide ultrastructural evidence for extra-oocytic yolk production. The mature oocyte contains numerous yolk and lipid vesicles of varying electron density that comprise both intra- and extra-oocytic substrates.     

A three-dimensional study of organelle interrelationships in regenerating rat liver. 3. Organelles related to bile.

A number of cell structures are described which show a morphological relationship to the bile canaliculi. Two types of peribiliary vesicles are identified: osmication positive ones occurring between the bile canaliculi and the osmicated immature Golgi cisternae and probably deriving from the latter, and osmication negative ones related to MVB, on which they appear as buds. Small coated vesicles are seen attached to this second type. Large lacunae may originate from MVB, as suggested by the MVB-like internal vesicles they may contain. Some stay in luminal continuity with the bile canaliculi. Canalicular coated vesicles are seen as parts of the canalicular plasma membrane and free in the cytoplasm.     

On the formation of the pattern of crystal idioblasts — in Canavalia ensiformis DC

Summary Light and electron-microscope observations were made of the crystal idioblasts in the leaves of Canavalia. The crystal-containing cells occur as pairs in which the crystals, nuclei, and the majority of the chloroplasts are symmetrically arranged with regard to the common wall. The chloroplasts are found in the cytoplasm along this wall.The crystals originate in a vacuole. The space in which the young crystal develops is delimited by a membrane. One to several additional membranes surround the crystal inside the vacuole. Numerous vesicles are distributed between these vacuolar membranes. Dense groups of tubules or fibrils are oriented toward a portion of the crystal surface, suggesting that the material forming the crystal might be transported to the surface by these structures.The cytoplasm of the young idioblasts contains many mitochondria and dictyosomes with associated vesicles. Concentrations of what is assumed to be protein are present in the cytoplasm. These protein accumulations are not seen in neighboring cells, suggesting that protein synthesis is especially high in the idioblasts.In older crystal cells, layers of wall material are deposited on the wall between the two crystals of the pair and towards the cell wall adjacent to the mesophyll. Not only does the original wall become thickened but a new wall develops at the border of the crystal vacuole. Eventually this wall material becomes continuous and the crystal becomes, on two sides, directly connected with the wall.     

EFFECTS OF LIGHT INTENSITY ON DIVISION RATE,STIMULABLE BIOLUMINESCENCE AND CELL SIZE OF THE OCEANIC DINOFLAGELLATES DISSODINIUM LUNULA,PYROCYSTIS FUSIFORMIS AND P. NOCTILUCA12

Preadapted cultures were grown in a 12:12 LD cycle at a series of light intensities under cool-white, fluorescent lamps. Pyrocystis fusiformis Murray maintained high division rates at low light intensities at the expense of cell size. In contrast, Dissodinium lunula (Schuett) Taylor had relatively lower division rates at low light intensities with little concomitant decrease in size. The response of P. noctiluca Murray was intermediate between these two species. For all three, cell numbers did not increase above an intensity of 5–10 μEin·m^?2·sec^?1 and division rate was saturated at ca. 30, 60, and 60μEin·m^?2·sec^?1 for P. fusiformis, P. noctiluca, and D. lunula, respectively. The capacity for stimulable bioluminescence was saturated at light intensities of 0.15 μEin·m^?2·day in short-term (2-day) experiments. In cultures of P. fusiformis and P. noctiluca, maintained for at least one month at lower intensities than needed to saturate division rate, a decrease in the capacity for stimulable bioluminescence was accompanied by a reduction in cell size. Our results suggest that cell size and bioluminescent capacity may prove to be a potentially useful indication of the history of exposure of natural populations of Pyrocystis spp. to ambient intensities.