Regeneration of ciliary comb plates in the ctenophore Mnemiopsis leidyi. i. morphology

Regeneration of missing body parts in model organisms provides information on the mechanisms underlying the regeneration process. The aim here is to use ctenophores to investigate regeneration of their giant ciliary swimming plates. When part of a row of comb plates on Mnemiopsis is excised, the wound closes and heals, greatly increasing the distance between comb plates near the former cut edges. Video differential interference contrast (DIC) microscopy of the regeneration of new comb plates between widely separated plates shows localized widenings of the interplate ciliated groove (ICG) first, followed by growth of two opposing groups of comb plate cilia on either side. The split parts of a new plate elongate as their bases extend laterally away from the ICG widening and continue ciliogenesis at both ends. The split parts of a new plate grow longer and move closer together into the ICG widening until they merge into a single plate that interrupts the ICG in a normal manner. Video DIC snapshots of dissected gap preparations 1.5–3‐day postoperation show that ICG widenings and/or new plates do not all appear at the same time or with uniform spacing within a gap: the lengths and distances between young plates in a gap are quite variable. Video stereo microscopy of intact animals 3–4 days after the operation show that all the new plates that will form in a gap are present, fairly evenly spaced and similar in length, but smaller and closer together than normal. Normal development of comb plates in embryos and growing animals is compared to the pattern of comb plate regeneration in adults. Comb plate regeneration differs in the cydippid Pleurobrachia that lacks ICGs and has a firmer mesoglea than Mnemiopsis. This study provides a morphological foundation for histological, cellular, and molecular analysis of ciliary regeneration in ctenophores. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.     

Gap junctions suggest epithelial conduction within the comb plates of the ctenophore Pleurobrachia bachei

Intercellular gap junctions occur between the ciliated cells that make up the comb plates of the ctenophore Pleurobrachia. Similar junctions are found within the ciliated grooves which run from the apical organ to the first plate of each comb row, as well as throughout the endoderm of the meridional canals. Gap junctions were not found in the ectodermal tissue between the comb rows. The distribution of junctions suggests that excitation conduction within the ciliated grooves, comb plates and meridional canal endoderm may be epithelial.     

Calcium control of ciliary reversal in ionophore-treated and ATP- reactivated comb plates of ctenophores

Previous work showed that ctenophore larvae swim backwards in high-KCl seawater, due to a 180 degrees reversal in the direction of effective stroke of their ciliary comb plates (Tamm, S. L., and S. Tamm, 1981, J. Cell Biol., 89: 495-509). Ion substitution and blocking experiments indicated that this response is Ca2+ dependent, but comb plate cells are innervated and presumably under nervous control. To determine whether Ca2+ is directly involved in activating the ciliary reversal mechanism and/or is required for synaptic triggering of the response, we (a) determined the effects of ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and Ca2+ on the beat direction of isolated nerve-free comb plates dissociated from larvae by hypotonic, divalent cation-free medium, and (b) used permeabilized ATP- reactivated models of comb plates to test motile responses to known concentrations of free Ca2+. We found that 5 microM {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and 10 mM Ca2+ induced dissociated comb plate cells to beat in the reverse direction and to swim counterclockwise in circular paths instead of in the normal clockwise direction. Detergent/glycerol-extracted comb plates beat actively in the presence of ATP, and reactivation was reversibly inhibited by micromolar concentrations of vanadate. Free Ca2+ concentrations greater than 10(-6)M caused reversal in direction of the effective stroke but no significant increase in beat frequency. These results show that ciliary reversal in ctenophores, like that in protozoa, is activated by an increase in intracellular free Ca2+ ions. This allows the unique experimental advantages of ctenophore comb plate cilia to be used for future studies on the site and mechanism of action of Ca2+ in the regulation of ciliary motion.     

Ciliary motility: the components and cytoplasmic preassembly mechanisms of the axonemal dyneins

Motile cilia and flagella are organelles, which function in cell motility and in the transport of fluids over the surface of cells. Motility defects often result in a rare human disease, primary ciliary dyskinesia (PCD). Cell motility depends on axonemal dynein, a molecular motor that drives the beating of cilia and flagella. The dyneins are composed of multiple subunits, which are thought to be preassembled in the cytoplasm before they are transported into cilia and flagella. Axonemal dyneins have been extensively studied in Chlamydomonas. In addition, analyses of human PCDs over the past decade, together with studies in other model animals, have identified the conserved components required for dynein assembly. Recently also, the first cytoplasmic component of dynein assembly, kintoun (ktu), was elucidated through the analysis of a medaka mutant in combination with human genetics and cell biology and biochemical studies of Chlamydomonas. The components of dynein and the proteins involved in its cytoplasmic assembly process are discussed.     

Ciliary filter-feeding structures in adult and larval gymnolaemate bryozoans

Abstract. Ciliary filter-feeding structures of gymnolaemate bryozoans—adults of Flustrellidra hispida and Alcyonidium gelatinosum , larvae of Membranipora sp.—were studied with SEM. In F. hispida and A. gelatinosum , the distal part of each tentacle has a straight row of stiff laterofrontal cilia which carry out "ciliary sieving" to capture suspended food particles that are subsequently transported downward towards the mouth by tentacle flicking; both structure and function resemble those of stenolaemate tentacles. The proximal part of the tentacle and of the ciliary ridge of a cyphonautes larva have strikingly similar structures, except that the laterofrontal cells are monociliate in the adults and biciliate in the larvae. The laterofrontal cells of the tentacles are arranged in a zigzag row and their cilia form two parallel rows, a frontal and a lateral row. The latter probably forms the sieve of stiff filter cilia in front of the water-pumping lateral cilia, whereas the frontal row appears to be held close to the frontal ciliary band of the tentacle. The biciliate laterofrontal cells of the cyphonautes larva have the cilia arranged in similar rows. The detailed morphological similarities between the ciliary bands of adult and larval filtering structures suggest that the feeding mechanisms are similar, contrary to what has been previously thought.     

Two distinct compartments of a ctenophore comb plate provide structural and functional integrity for the motility of giant multicilia

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Ciliary reversal inParamecium: Temperature dependence of K+-induced excitability decrease and of recovery

Summary The duration of K⁺-induced reversed swimming ofParamecium caudatum was used as a measure of excitability. The time course of excitability decrease following an exposure to high [K⁺]_o and of the subsequent recovery in a solution devoid of K⁺ was measured at various temperatures.When the temperature was increased during the exposure to K⁺ the loss of excitability increased with a Q₁₀ of ca. 1.3 (Fig. 2). The recovery of excitability was enhanced with a Q₁₀ of 2.0–3.0 (Fig. 4). The renormalization of ciliary beat in K⁺-rich medium showed Q₁₀-values between 2.1 and 2.5 (Fig. 5).The observed alterations in excitability are interpreted in terms of passive and active Ca²⁺ fluxes. Ca channels in the excitable membrane are assumed to inactivate as a result of increased [Ca²⁺]_i. Recovery of excitability is thought to be mediated by active Ca²⁺ extrusion.I would like to thank Mrs. A. Müller-Holtkamp, who carried out the experiments, for her invaluable help. This work is part of the research program SFB 160 of the Deutsche Forschungsgemeinschaft.     

Phosphorylation of axonemal proteins in Chlamydomonas reinhardtii.

In order to determine whether microtubular proteins of flagellar axonemes were phosphorylated, cells of Chlamydomonas reinhardtii were grown in medium containing [32P]orthophosphate for several generations. Only one (alpha subunit) of the two tubulin polypeptides separated by Na dodecyl-SO4-polyacrylamide gel electrophoresis appeared labeled, as detected by autoradiography of the dried gel. 3H- and 32P-labeled alpha tubulin subunit purified by preparative Na dodecyl-SO4-polyacrylamide gel electrophoresis and Na dodecyl-SO4-hydroxyapatite chromatography contained about 0.2 mol of phosphate per mol of polypeptide. Upon partial acid hydrolysis, radioactivity could be accounted for as serine and threonine phosphate. By altering the conditions of the Na dodecyl-SO4-polyacrylamide gel electrophoresis is was possible to resolve the purified alpha-tubulin subunit into five or more components: a major band comprising approximately 65% of the total mass, not phosphorylated, and four or more minor bands comprising together 35% of the mass. Among the minor components at least two were phosphorylated.     

The role of axonemal components in ciliary motility

1. The axoneme is the detergent-insoluble cytoskeleton of the cilium. 2. All axonemes generate movement by the same fundamental mechanism: microtubule sliding utilizing ATP hydrolysis during a mechanochemical cycling of dynein arms on the axonemal doublets. 3. Structure, fundamental biochemistry and physiology of the axoneme are conserved evolutionarily, but the phenotypes of beating movements and the responses to specific cytoplasmic signals differ greatly from organism to organism. 4. A model of asynchronous dynein arm activity--the switch point hypothesis--has been proposed to account for cyclic beating in the face of unidirectional sliding. The model suggests that the diversity of beat phenotype may be explicable by changes in the timing of switching between active and inactive states of doublet arm activity. Evidence of axonemal splitting in arrested axonemes provides new support for the hypothesis.     

Preparation of liposomes using the phase reversal method without sonication

A simple procedure for preparation of liposomes based on the phase reversal method but differing from the original technique by the absence of ultrasonic treatment, has been developed. Using this method, the effects of NaCl concentration used for the preparation of liposomes from a mixture of neutral and charged lipids, on the inner liposome volume were studied. The experimental data suggest that NaCl concentration may be regarded as a basic criterion determining the size of liposomes in many routine techniques used for the preparation of liposomes from a mixture of neutral and charged lipids.     

The physiology of feeding in the ctenophore Pleurobrachia pileus

Ciliary organelles play important roles in many of the locomotory and sensory functions of ctenophores. This paper reviews published work on the physiology and behavior of ciliary-based feeding in Pleurobrachia pileus and presents recent preliminary work on the conduction pathway controlling this behavior. The feeding response can now be described in terms of specific physiological events at most levels of organization.     

The development of bioluminescence in the ctenophore Mnemiopsis leidyi

The photocytes of the ctenophore Mnemiopsis have a discontinuous distribution along the radial canal between the sites where the comb plate cilia cells are located on the side of the canal which contains the testes. They are separated from the lumen of the canal by a population of gastric cells. Cytologically these cells are characterized by a condensed nucleus and cytoplasm which stains lightly with basophilic dyes.The ability of the ctenophore embryo to produce light appears at the developmental stage when the comb plate cilia first begin to grow out. At this stage four light-producing areas are present; each area corresponds to one quadrant of the adult animal. At the sites of light production, a population of cells can be identified that have some of the cytological properties of the photocytes of the adult animal. Within 8–10 hr after light production begins there is a 10-fold increase in the amount of light produced by an embryo and a cytological maturation of its photocytes; during this time period there is no increase in photocyte number. At about the time the embryo begins to feed, each light-producing region splits into two regions, each of which corresponds to a radial canal.During the process of embryogenesis the photocyte cell lineage is first segregated from non-photocytes at the differential division which gives the 8-cell stage embryo. The M macromere lineage goes on to form photocytes, but the E macromere lineage does not. The M macromeres form a micromere at the aboral pole of the embryo at each of the next two cleavages; during these cleavages the potential for photocyte differentiation continues to segregate with the M macromeres. During the division which gives the 64-cell stage the M macromeres divide equally; the potential for photocyte differentiation segregates with the M macromeres nearest the oral-aboral axis. M macromeres which are isolated from the embryo at the 8-, 16-, or 32-cell stage of development will continue to cleave as though they were part of a normal embryo and differentiate to form photocytes.The events that are responsible for the differential division during the formation of the 8-cell stage embryo have been studied by centrifuging eggs to produce fragments of different cytoplasmic composition. Egg fragments which contain only cortical cytoplasm differentiate comb plate cilia cells, but do not produce photocytes. Cortical fragments with a small amount of yolk differentiate comb plate cilia cells and photocytes. Both the M and E macromeres from cortical fragments with no yolk produce comb plate cilia. Only M macromeres containing yolk form photocytes; if an M macromere forms photocytes it does not form comb plate cilia.     

Ciliary feeding structures and particle capture mechanism in the freshwater bryozoan Plumatella repens (Phylactolaemata)

Abstract. In contrast to marine bryozoans, the lophophore structure and the ciliary filter‐feeding mechanism in freshwater bryozoans have so far been only poorly described. Specimens of the phylactolaemate bryozoan Plumatella repens were studied to clarify the tentacular ciliary structures and the particle capture mechanism. Scanning electron microscopy revealed that the tentacles of the lophophore have a frontal band of densely packed cilia, and on each side a zigzag row of laterofrontal cilia and a band of lateral cilia. Phalloidin‐linked fluorescent dye showed no sign of muscular tissue within the tentacles. Video microscopy was used to describe basic characteristics of particle capture. Suspended particles in the incoming water flow, set up by the lateral ‘pump’ cilia on the tentacles, approach the tentacles with a velocity of 1–2 mm s^‐1. Near the tentacles, the particles are stopped by the stiff sensory laterofrontal cilia acting as a mechanical sieve, as previously seen in marine bryozoans. The particle capture mechanism suggested is based on the assumed ability of the sensory stiff laterofrontal cilia to be triggered by the deflection caused by the drag force of the through‐flowing water on a captured food particle. Thus, when a particle is stopped by the laterofrontal cilia, the otherwise stiff cilia are presumably triggered to make an inward flick which brings the restrained particle back into the downward directed main current, possibly to be captured again further down in the lophophore before being carried to the mouth via the food groove. No tentacle flicks and no transport of captured particles on the frontal side of the tentacles were observed. The velocity of the metachronal wave of the water‐pumping lateral cilia was measured to be ～0.2 mm s^‐1, the wavelength was ～7 μm, and hence the ciliary beat frequency estimated to be ～30 Hz (～20 °C). The filter feeding process in P. repens reported here resembles the ciliary sieving process described for marine bryozoans in recent years, although no tentacle flicks were observed in P. repens. The phylogenetic position of the phylactolaemates is discussed in the light of these findings.     

Sex-role reversal of a monogamous pipefish without higher potential reproductive rate in females

In monogamous animals, males are usually the predominant competitors for mates. However, a strictly monogamous pipefish Corythoichthys haematopterus exceptionally exhibits a reversed sex role. To understand why its sex role is reversed, we measured the adult sex ratio and the potential reproductive rate (PRR), two principal factors influencing the operational sex ratio (OSR), in a natural population of southern Japan. The adult sex ratio was biased towards females throughout the breeding season, but the PRR, which increased with water temperature, did not show sexual difference. We found that an alternative index of the OSR (Sf/Sm: sex ratio of 'time in') calculated from the monthly data was consistently biased towards females. The female-biased OSR associated with sex-role reversal has been reported in some polyandrous or promiscuous pipefish, but factors biasing the OSR differed between these pipefish and C. haematopterus. We concluded that the similar PRR between the sexes in C. haematopterus does not confer reproductive benefit of polygamous mating on either sex, resulting in strict monogamous mating, and its female-biased adult sex ratio promotes female-female competition for a mate, resulting in sex-role reversal.     

Argentophilic structures of the miracidia and cercariae of Philophthalmus distomatosa n. comb. from Israel

The morphology and patterns of distribution of the argentophilic structures of miracidia and cercariae of Philophthalmus distomatosa n. comb. are described. The epidermal plate arrangement of the vast majority (94.2%) of miracidia studied conformed to the formula 6:8:4:2 = 20. The rest (5.8%) displayed the following patterns of arrangement of epidermal plates: 6:7:4:2 = 19; 6:6:4:2 = 18; and 6:4:4:2 = 16. Twenty papillalike structures were observed on the terebratorium. They were arranged along 1 axis, in 3 groups. Most commonly, 16 papillae were present on the body, located between epidermal plates of the first and second rows. The eye spots were located dorsally, near interepidermal space S1. The numbers, arrangements, and locations of the excretory pores varied. Patterns of distribution of the tegumentary papillae of P. distomatosa n. comb. cercariae are given, including those of the cephalic regions, periacetabular region, and tail. Argentophilic structures of the mentioned larval stages and of other parameters clearly distinguishing between the presently described species and those of P. lucipetus from Israel are summarized. On the basis of comparison of the presently described larval stages and those of other Melanoides tuberculata-associated larval stages of Philophthalmus in Israel and Jordan, the possibility exists that a third species occurs in Israel.     

The gelatinous extracellular matrix facilitates transport studies in kelp: visualization of pressure-induced flow reversal across sieve plates

Background and Aims In vascular plants, important questions regarding phloem function remain unanswered due to problems with invasive experimental procedures in this highly sensitive tissue. Certain brown algae (kelps; Laminariales) also possess sieve tubes for photoassimilate transport, but these are embedded in large volumes of a gelatinous extracellular matrix which isolates them from neighbouring cells. Therefore, we hypothesized that kelp sieve tubes might tolerate invasive experimentation better than their analogues in higher plants, and sought to establish Nereocystis luetkeana as an experimental system.Methods The predominant localization of cellulose and the gelatinous extracellular matrix in N. luetkeana was verified using specific fluorescent markers and confocal laser scanning microscopy. Sieve tubes in intact specimens were loaded with fluorescent dyes, either passively (carboxyfluorescein diacetate; CFDA) or by microinjection (rhodamine B), and the movement of the dyes was monitored by fluorescence microscopy.Key Results Application of CFDA demonstrated source to sink bulk flow in N. luetkeana sieve tubes, and revealed the complexity of sieve tube structure, with branches, junctions and lateral connections. Microinjection into sieve elements proved comparatively easy. Pulsed rhodamine B injection enabled the determination of flow velocity in individual sieve elements, and the direct visualization of pressure-induced reversals of flow direction across sieve plates.Conclusions The reversal of flow direction across sieve plates by pressurizing the downstream sieve element conclusively demonstrates that a critical requirement of the Münch theory is satisfied in kelp; no such evidence exists for tracheophytes. Because of the high tolerance of its sieve elements to experimental manipulation, N. luetkeana is a promising alternative to vascular plants for studying the fluid mechanics of sieve tube networks.     

Localization and identification of catechol compounds in the ctenophore mnemiopsis leidyi

1. The ctenophore Mnemiopsis leidyi, was examined for monoaminergic nerves by glyoxylic acid-induced fluorescence.2. The meridional canals contained strands of fluorescent cells.3. High pressure liquid chromatography (HPLC), showed the presence of 2,3-dihydroxyphenylalanine (DOPA), 0.25–0.35 pmol/specimen, and 5-S-cysteinylDOPA, 0.3–0.85 pmol/specimen.4. Electronmicroscoplcal examination of tissues showing histofluorescence, revealed large cells (> 20 μm) containing irregular osmium-reducing vesicles, usually 100–500 nm in size. A second cell type, situated by the outside of the canal, contained dense core vesicles, 175–200 nm in diameter.5. It is suggested that the histofluorescence is caused by DOPA and 5-S-cysteinylDOPA. The presence of catecholamines is discussed.