Scanning Electron Microscopy of Budding and Metamorphosis in Discophrya collini (Root)*

SYNOPSIS. Budding and metamorphosis in the suctorian ciliate, Discophrya collini, have been investigated by scanning electron microscopy. The adult body form, tentacles, stalk, and attachment disk are described. A field of depressions or small pits was observed in the pellicle of adult suctorians in the early stages of bud formation. These pits deepen and coalesce until one large pore, the birth pore, remains. Cilia protrude through the pore, and as eversion of the bud proceeds the meridional arrangement of the larval ciliation is evident. After eversion is completed, a pronounced division furrow is found between the adult and soon-to-be-released swarmer. The stalk-forming region is seen on swarmers. Metamorphosing swarmers produce tentacles upon settling before any indication of ciliary resorption. Resorption of cilia and change in body form occur progressively with the production of the attachment disk and stalk.     

Expression of a novel receptor tyrosine kinase gene and a paired-like homeobox gene provides evidence of differences in patterning at the oral and aboral ends of hydra

Axial patterning of the aboral end of the hydra body column was examined using expression data from two genes. One, shin guard, is a novel receptor protein-tyrosine kinase gene expressed in the ectoderm of the peduncle, the end of the body column adjacent to the basal disk. The other gene, manacle, is a paired-like homeobox gene expressed in differentiating basal disk ectoderm. During regeneration of the aboral end, expression of manacle precedes that of shin guard. This result is consistent with a requirement for induction of peduncle tissue by basal disk tissue. Our data contrast with data on regeneration of the oral end. During oral end regeneration, markers for tissue of the tentacles, which lie below the extreme oral end (the hypostome), are detected first. Later, markers for the hypostome itself appear at the regenerating tip, with tentacle markers displaced to the region below. Additional evidence that tissue can form basal disk without passing through a stage as peduncle tissue comes from LiCl-induced formation of patches of ectopic basal disk tissue. While manacle is ectopically expressed during formation of basal disk patches, shin guard is not. The genes examined also provide new information on development of the aboral end in buds. Although adult hydra are radially symmetrical, expression of both genes in the bud's aboral end is initially asymmetrical, appearing first on the side of the bud closest to the parent's basal disk. The asymmetry can be explained by differences in positional information in the body column tissue that evaginates to form a bud. As predicted by this hypothesis, grafts reversing the orientation of evaginating body column tissue also reverse the orientation of asymmetrical gene expression.     

Asexual reproduction in the suctorianDiscophrya collini

Summary Discophrya collini reproduces asexually through the formation of a ciliated swarmer by evaginative budding. This process is initiated by the repeated replication of a single subcortical kinetosome to form a kinetosome field. The epiplasm of the multilayered cortex covering this field becomes reduced in thickness and the whole cortex invaginates to produce an internal embryonic cavity. The kinetosomes become organised into rows, and each produces a cilium which projects into the cavity. On completion of the embryonic cavity its walls are extruded through the cavity opening to form an external ciliated swarmer connected to the parent by a thin bridge of cytoplasm. It is suggested that this evagination is induced by a rapid breakdown of supporting microtubules in the cavity wall and the subsequent hydrostatic pressure exerted by the body cytoplasm. The connecting bridge shows no specialised ultrastructural features and separation of swarmer from parent probably is achieved by the active movement of the swarmer. The cytoplasm of the swarmer is similar in structure to that of the adult cell but contains a number of primordia of tentacle axonemes. The infraciliature resembles that of other suctorian swarmers. On settling, the cilia of the swarmer are lost, at least some by resorption, a stalk may be secreted and the axoneme primordia are extended to form functional tentacles.     

Metamorphosis in Tokophrya infusionum; an Electron-Microscope Study

SYNOPSIS. In Tokophrya infusionum metamorphosis from a ciliated swimming embryo to a sessile organism with a stalk, disc, and tentacles lasts only 3 minutes. The remarkable speed of meta-morphosis was clarified by an electron-microscope study of embryos before and during metamorphosis. Ultrathin sections have revealed that the embryo has at the anterior end of the body a number of specialized structures, such as dense bodies containing the precursor material for the disc and stalk, and microtubules which align the dense bodies into rows leading to pit-kite invaginations of the pellicle at the tip of the anterior end. At meta-morphosis the embryo settles down on this end and the precursor material is released thru the pits to the outside. At the same time the body of the embryo invaginates at this end, forming a cavity which becomes deeper and narrower until it acquires the shape of a channel. The 1st drops released from the dense bodies spread out on the substrate, forming the disc. The rest of the material, secreted into the channel, solidifies there to form the stalk. It seems obvious that the channel serves as a mold for the stalk, since after completion of the stalk the channel disappears. The stalk is structureless with no limiting membrane; it is outside the boundaries of the cell. Both the stalk and disc are extra-cellular organelles.
Of the new organelles appearing at metamorphosis, only the stalk and disc are formed de novo. The electron-microscope study disclosed that the embryo has internal parts of tentacles composed of a tube formed of microtubules. At the distal end of the microtubules is a ring of dense material. During metamorphosis the microtubules, together with the dense ring, grow out of the body, and along with them the pellicle and plasma membrane to form the external part of the tentacle.     

草鱼鳃上寄生毛管虫一新种——变异毛管虫的研究

吸管亚纲(Subclass suctoria)中,多数种类具有或长或短的炳,附着它物上营固着生活。寄生在鱼类体表、鳃丝内的毛管虫(Trichophrya)和簇管虫(Erastophrya)的种类中,至今未见报道有固着柄的代表。       

Response to insulin and the expression pattern of a gene encoding an insulin receptor homologue suggest a role for an insulin-like molecule in regulating growth and patterning in Hydra

 A gene encoding a receptor protein-tyrosine kinase closely related to the vertebrate insulin receptor has been identified in the Cnidarian Hydra vulgaris. The gene is expressed in both epithelial layers of the adult polyp. A particularly high level of expression is seen in the ectoderm of the proximal portions of the tentacles and in a ring of ectodermal cells at the border between the foot basal disk and body column. The expression pattern of the gene in asexual buds is dynamic; expression is high throughout the newly emerging bud but the area of high expression becomes restricted to the apex as the bud lengthens. When the bud begins hypostome and tentacle formation, a high level of expression appears at the bases of the emerging tentacles. Finally, a ring of high expression appears just above the foot of the bud, completing the pattern seen in the adult polyp. The presence of this receptor and its pattern of expression suggested that an endogenous molecule related to insulin plays a role in regulating cell division in the body column and in differentiation of the tentacle and foot cells in Hydra, with the switch between the two being determined by the level of the receptor. Treatment of Hydra polyps with mammalian insulin caused an increase in the number of ectodermal and endodermal cells undergoing DNA synthesis. Received: 19 April 1996 / Accepted: 5 July 1996     

Hym-301, a novel peptide, regulates the number of tentacles formed in hydra

Hym-301 is a peptide that was discovered as part of a project aimed at isolating novel peptides from hydra. We have isolated and characterized the gene Hym-301, which encodes this peptide. In an adult, the gene is expressed in the ectoderm of the tentacle zone and hypostome, but not in the tentacles. It is also expressed in the developing head during bud formation and head regeneration. Treatment of regenerating heads with the peptide resulted in an increase in the number of tentacles formed, while treatment with Hym-301 dsRNA resulted in a reduction of tentacles formed as the head developed during bud formation or head regeneration. The expression patterns plus these manipulations indicate the gene has a role in tentacle formation. Furthermore, treatment of epithelial animals indicates the gene directly affects the epithelial cells that form the tentacles. Raising the head activation gradient, a morphogenetic gradient that controls axial patterning in hydra, throughout the body column results in extending the range of Hym-301 expression down the body column. This indicates the range of expression of the gene appears to be controlled by this gradient. Thus, Hym-301 is involved in axial patterning in hydra, and specifically in the regulation of the number of tentacles formed.     

The morphology and life cycle of Ophryodendron mysidacii sp. nov. a marine suctorian epibiont on a mysid crustacean

A new species of suctorian protist epibiont of the mysid Schistomysis parkeri is described. The individuals show two types of adult form: elongated and flattened, both with 4-8 tentacular lobes. This new suctorian differs from described species of pro-Ophryodendron group by size, number of tentacular lobes, insertion of the tentacles, union of the lorica with the body, shape of the macronucleus, number of micronuclei and the lack of stalk (adult forms). The life cycle of this species is analysed and a succession pattern of its different stages is proposed.     

Alternative modes of asexual reproduction inTrichoplax adhaerens (Placozoa)

Summary Hollow swarmers are budded off at the dorsal surface ofTrichoplax and are covered by dorsal epithelium. Their inner cavity is lined with the flagellated cells of the ventral epithelium. There is no indication that the fiber cells included between the epithelia take any part either in morphologenesis or the separation of the bud from the mother animal. The early primordium forms in the interspace. A single layer of cells derived from both epithelia surrounds a cavity filled with granular matter that stains like proteins. The latter is used up during the floating phase of the swarmers that may last for a week. After settling at the bottom, the hollow sphere opens at one point. The concave ventral epithelium gradually flattens as more cells become incorporated in it. The latter form new flagella and flagellar pits. More frequently found than swarmers are small spherical forms that are unable to float and possess a distinct polarity. Their upper half is covered by dorsal epithelium and their lower half by ventral epithelium. Large fiber cells are in the center. Their site and mode of formation is unknown. Rarely observed are dorsal stolons whose bulbous end flattens upon touching the substrate. Since they are totally covered by the flat cells of the dorsal epithelium, they may have to undergo a transformation, like the hollow swarmers, to bring the ventral epithelium into contact with the substrate.     

Patterns of ciliary currents in Atlantic reef corals and their functional significance

Patterns of ciliary currents of 35 species of Atlantic reef corals are described and compared with currents of Pacific corals. Observations were made during the day and at night, during feeding and without food. There is a basic pattern of ciliary currents common to both Atlantic and Pacific species. In all but the family Agaricidae currents flow off the oral disk and up or out between the tentacles. In the centre of the disk region currents flow towards the mouth or the peristome. On the polyp stalk or column there was considerable variation between species in both Atlantic and Pacific forms. In some species currents flow downwards toward the coenosarc while in others, current pass up the stalk towards the tentacles.
In the Atlantic Agaricidae there may be an inward flow towards the mouth, an outward flow or a unidirectional flow across the corallum. The patterns of flow depend upon the state of contraction of the polyps or the shape and proximity of adjacent polyps.
No ciliary current reversal was observed in Atlantic species. Ciliary currents are functional as a cleansing mechanism and facilitated the ability of mucus nets and strands to gather particles.     

VARIATION IN HYDRA'S TENTACLE NUMBERS AS A FUNCTION OF TEMPERATURE

Chlorohydra uiridissima whose tentacle number is altered at different temperatures, was studied to see how other developmental variables changed as a function of temperature. The results suggest that temperature is instrumental in establishing the size of bud and tentacle primordia, but the number of primordia present may play a limiting role.

Animals were cultured at 18, 23 and 28°C and shifted between the extreme temperatures. Large animals with 8 tentacles, small animals with 5 tentacles, and intermediate animals with 6 and 7 tentacles served as parents. Buds and parents were monitored daily and scored for numbers of buds and tentacles.

Temperature, not parental size, determined the size of the buds. At the lower temperature buds were produced more slowly and initiated less frequently, but occurred in greater numbers per parent and had more tentacles than at the higher temperatures. The duration of bud development also increased at lower temperature, but at the lowest temperature the duration of bud development was not correlated with tentacle numbers on buds.

Changes in the frequency of bud initiation and the duration of bud development induced by changing temperature did not parallel changes in the number of tentacles produced on buds. Animals shifted from 18°C to 28°C underwent rapid increases in the rate of bud initiation and rapid shortening in the duration of bud development, while animals shifted from 28°C to 18°C underwent equally rapid changes in the opposite directions. The number of tentacles produced on buds, however, changed slowly to that characteristic of buds acclimated to the new temperatures. The frequency of bud initiation and the duration of bud development, therefore, do not determine tentacle number.

The number of tentacles already present seems to limit possibilities for adding new tentacles. Parents with five tentacles were especially likely to undergo upward changes in their tentacle number while parents with eight tentacles were resistant to such changes.     

团头鲂寄生吸管虫一新种,双泡毛管虫的研究

描述了寄生在团头鲂(Megalobrama amblycephala)鳃上的一吸管虫新种,双泡毛管虫。活体无色透明至淡黄色,正面观为卵圆形至长椭圆形,稍扁平;侧面观为棒状或香肠状,常附着在鳃小片上。身体的表膜中有不明显的细小颗粒。吸管指状,一束,着生在虫体前端,一般有4—8根,最多达23根,收缩时其表面出现明显的螺旋纹8—11个。两个伸缩泡,交替地进行伸缩,彼此间隔约为10μm,位于吸管基部的胞质中。大核短杆状或椭圆形,核膜明显,染色质均匀。小核圆球形。成虫和幼虫没有固着柄,游泳幼虫的纤毛带宽6.0—7.0μm,由7—11行纤毛纹组成,无锥形的钻孔器。       

Extracellular Formation of Body and Tunic Spicules in the New Zealand Solitary Ascidian Pyura pachydermatina (Urochordata, Ascidiacea)

The New Zealand ascidian Pyura pachydermatina has a 7–10 cm long body at the end of a stalk up to 1 m long and 1–2 cm in diameter. Two different spicule types are present: dumbbell-shaped spicules of calcite in the fibrous tunic that covers the body and stalk, and antler-shaped spicules of amorphous calcium carbonate in the soft body tissues. Both types form extracellularly within a closed compartment surrounded by an epithelium of sclerocytes. In adults the tunic spicules form in 2–3 weeks in the lumen of the tunic blood vessels, as determined by calcein uptake studies. They add mineral only while surrounded by the sclerocyte epithelium, which is anchored to the vessel wall. Ultimately the sclerocytes rupture at one or more leading points on the spicule. The blood vessel epithelium also becomes very thin at these points and either ruptures or the cells separate. allowing the spicules to migrate out into the tunic. The sclerocytes degenerate and the blood vessel closes behind the migrating spicule, thus maintaining the vessel's integrity. Tunic spicules accumulate in the subcuticular region of the stalk, but the outermost layer of tunic covering the body is periodically sloughed off along with some spicules. This gives the "neck" between body and stalk a flexibility that allows it to orient to currents, and prevents an accumulation of epizoic organisms on the body. The antler spicules form within blood sinuses of the body tissues. The mineral and organic material are arranged in concentric layers. In the branchial sac, oral tentacles, gut and endostyle, where antler spicules occur most densely, the branches interlock, providing support to the soft tissues. They are of many sizes and apparently remain where they form, increasing in number and size throughout the animal's lifespan.     

Wnt signaling in hydroid development: ectopic heads and giant buds induced by GSK-3beta inhibitors

In Hydractinia, a colonial marine hydroid representing the basal phylum Cnidaria, Wnt signaling plays a major role in the specification of the primary body axis in embryogenesis and in the establishment of the oral pole during metamorphosis. Here we report supplementing investigations on head regeneration and bud formation in post-metamorphic development. Head and bud formation were accompanied by the expression of Wnt, frizzled and Tcf. Activation of Wnt signaling by blocking GSK-3beta affected regeneration, the patterning of growing polyps and the asexual formation of new polyps in the colony. In the presence of lithium ions or paullones, gastric segments excised from adult polyps showed reversal of tissue polarity as they frequently regenerated heads at both ends. Phorbol myristate acetate, a known activator of protein kinase C increased this effect. Global activation of the Wnt pathway caused growing polyps to form ectopic tentacles and additional heads along their body column. Repeated treatment of colonies evoked the emergence of many and dramatically oversized bud fields along the circumference of the colony. These giant fields fell apart into smaller sub-fields, which gave rise to arrays of multi-headed polyps. We interpret the morphogenetic effects of blocking GSK-3beta as reflecting increase in positional value in terms of positional information and activation of Wnt target genes in molecular terms.     

Expression and developmental regulation of the Hydra-RFamide and Hydra-LWamide preprohormone genes in Hydra: evidence for transient phases of head formation

Hydra magnipapillata has three distinct genes coding for preprohormones A, B, and C, each yielding a characteristic set of Hydra-RFamide (Arg-Phe-NH2) neuropeptides, and a fourth gene coding for a preprohormone that yields various Hydra-LWamide (Leu-Trp-NH2) neuropeptides. Using a whole-mount double-labeling in situ hybridization technique, we found that each of the four genes is specifically expressed in a different subset of neurons in the ectoderm of adult Hydra. The preprohormone A gene is expressed in neurons of the tentacles, hypostome (a region between tentacles and mouth opening), upper gastric region, and peduncle (an area just above the foot). The preprohormone B gene is exclusively expressed in neurons of the hypostome, whereas the preprohormone C gene is exclusively expressed in neurons of the tentacles. The Hydra-LWamide preprohormone gene is expressed in neurons located in all parts of Hydra with maxima in tentacles, hypostome, and basal disk (foot). Studies on animals regenerating a head showed that the prepro-Hydra-LWamide gene is expressed first, followed by the preprohormone A and subsequently the preprohormone C and the preprohormone B genes. This sequence of events could be explained by a model based on positional values in a morphogen gradient. Our head-regeneration experiments also give support for transient phases of head formation: first tentacle-specific preprohormone C neurons (frequently associated with a small tentacle bud) appear at the center of the regenerating tip, which they are then replaced by hypostome-specific preprohormone B neurons. Thus, the regenerating tip first attains a tentacle-like appearance and only later this tip develops into a hypostome. In a developing bud of Hydra, tentacle-specific preprohormone C neurons and hypostome-specific preprohormone B neurons appear about simultaneously in their correct positions, but during a later phase of head development, additional tentacle-specific preprohormone C neurons appear as a ring at the center of the hypostome and then disappear again. Nerve-free Hydra consisting of only epithelial cells do not express the preprohormone A, B, or C or the LWamide preprohormone genes. These animals, however, have a normal phenotype, showing that the preprohormone A, B, and C and the LWamide genes are not essential for the basic pattern formation of Hydra.     

Giant forms of the suctorian ciliateDiscophrya collini

Summary Discophrya collini subjected to high levels of feeding onParamecium caudatum developed giant forms in culture. These take several forms: a single enlarged cell, a giant with attached normal cells or attached giants with normals. All the cells possess functional tentacles. The giant cells show qualitative and quantitative macronuclear changes and an abnormally thickened epiplasm containing membraneous profiles and other aberrant structures. These cells contain kinetosome fields and brood pouches identical to those found during normal swarmer production. It is suggested that the giant complexes are formed by the normal production of swarmers but a failure in their release from the adult, perhaps attributable to the abnormal epiplasm, results in their subsequent metamorphosisin situ. The abnormal epiplasm could be produced by the deposition of myelin body food residues from the cytoplasm. The initial induction of gigantism itself may be related to disruption of the normal growth-division cycle similar to that experienced during natural senescence. Possible mechanisms of this disruption and differences with other suctoria are discussed.This work was supported by the J. S. Dunkerley Research Fellowship in Protozoology.     

Development of the tentacular apparatus in sipunculans (Sipuncula): I. Thysanocardia nigra (Ikeda, 1904) and Themiste pyroides (Chamberlin, 1920)

The development and arrangement of the tentacular apparatus of Thysanocardia nigra (Ikeda, 1904) and Themiste pyroides (Chamberlin, 1920) are described and illustrated using scanning electron microscopy. In T. nigra, the tentacular apparatus is composed of two crowns: the nuchal arc enclosing the nuchal organ and a crown of numerous oral tentacles arranged in U-shaped festoons. In early juveniles, two dorsal horn-like protrusions develop into the first, or primary, pair of tentacles of the nuchal arc. The second pair of tentacles of the nuchal arc develops dorsolaterally on the bases of the primary tentacles. Two ventrolateral lobes of the oral disk grow and become subdivided by the longitudinal ciliary groove into anlages of one set of dorsal and one set of ventral tentacles, thus forming a first oral festoon. Later, a pair of dorsolateral lobes develop between the first festoons and the nuchal arc to form a second pair of oral festoons. The third and following pairs of oral festoons develop in the dorsolateral growth zones lateral to the borders of the nuchal arc, where they meet the oral crown. The growing festoons extend down the oral disk and run alongside the head. A new oral tentacle appears directly at/on the base of the previous tentacle, thus giving rise to a typical sympodium with an alternating arrangement of tentacles. In T. pyroides, a second pair of tentacles develops from two ciliary lobes that are ventrolateral outgrowths of the circumoral ciliary field around the terminal mouth opening. The third pair of tentacles appears from the dorsolateral lobes at the base of primary tentacles, between the first two pairs of tentacles. These six tentacles determine the position of six main stems of the tentacular apparatus designated the first tentacles in the corresponding stems. The second tentacle in every stem appears as a ventrolateral outgrowth at the base of the first tentacle. The third and following tentacles in the stem are developed between the two previous tentacles according to a sympodial pattern. In both species, the distinct sympodial pattern in the arrangement of tentacles in the tentacular apparatus is well evidenced by the outlines of the ciliary oral grooves. The branched stems of T. pyroides may be homologized structurally and functionally to the oral festoons of T. nigra. J. Morphol. (c) 2006 Wiley-Liss, Inc.     

The HfaB and HfaD adhesion proteins of Caulobacter crescentus are localized in the stalk

The differentiating bacterium Caulobacter crescentus produces two different cell types at each cell division, a motile swarmer cell and an adhesive stalked cell. The stalked cell harbours a stalk, a thin cylindrical extension of the cell surface. The tip of the stalk is decorated with a holdfast, an adhesive organelle composed at least in part of polysaccharides. The synthesis of the stalk and holdfast occur at the same pole during swarmer cell differentiation. Mutations in the hfaABDC gene cluster had been shown to disrupt the attachment of the holdfast to the tip of the stalk, but the role of individual genes was unknown. We used lacZ fusions of various DNA fragments from the hfaABDC region to show that these genes form an operon. In order to analyse the relative contribution of the different genes to holdfast attachment, mutations were constructed for each gene. hfaC was not required for holdfast attachment or binding to surfaces. The hfaA and hfaD mutants shed some holdfast material into the surrounding medium and were partially deficient in binding to surfaces. Unlike hfaA and hfaB mutants, hfaD mutants were still able to form rosettes efficiently. Cells with insertions in hfaB were unable to bind to surfaces, and lectin binding studies indicated that the hfaB mutants had the strongest holdfast shedding phenotype. We determined that HfaB and HfaD are membrane-associated proteins and that HfaB is a lipoprotein. Purification of stalks and cell bodies indicated that both HfaB and HfaD are enriched in the stalk as compared to the cell body. These results suggest that HfaB and HfaD, and probably HfaA, serve to anchor the holdfast to the tip of the stalk.     

Somatic tissue–male germ cell barrier in Hydra viridis (hydrozoa,coelenterata)

In Hydra viridis, cordons of male germ cells lie in gonadal compartments, which are enlarged spaces between the elongated and “spongy” epidermal cells. The germ cells are surrounded by these cells, except for small areas where the interstitial cells and spermatogonia are in direct contact with the mesoglea. Cells from both epidermis and gastrodermis project cytoplasm into the mesoglea, where they contact each other and form trans-mesogleal bridges. The latter exhibit gap junctions, which are particularly abundant at the spermary region. Here, the mesoglea is thinner then elsewhere in the body. Both epithelia are joined by septate junctions toward their apical ends, which are totally impermeable to horseradish peroxidase (HRP). HRP gained entry to the cells of both epithelia by pinocytosis. Incorporation into the cells was high at the basal disk, in the tentacles, and in the mesoglea in the lower part of the body stalk. The tracer was never found within the gonadal space of the testis during spermatogenesis. In mature spermaries during spermiation, tracer-filled intracellular vacuoles fused with the gonadal spaces as the thin cytoplasmic columns of the epidermal cells ruptured; HRP thus gained access to the germ cells. During spermatogenesis, germ cells of Hydra viridis are in a closed compartment. The barrier that controls the access of metabolites to the germ cells is formed by epidermal cells, thinned-out mesoglea, and numerous transmesogleal interepithelial bridges. The presumed role of the barrier is the control of the environment (1) where interstitial cells are differentiating into spermatogonia and meiosis occurs and (2) in which ripe spermatozoa are kept immotile until spermiation.