Cytochemical investigations on tunic morphogenesis in the sea peach Halocynthia papillosa (Tunicata, Ascidiacea) 2: demonstration of proteins

In a previous paper, cellulose fibres were demonstrated in the larval, the metamorphosing, and the juvenile tunics. In this paper we used cytochemical methods and X-ray microanalysis to obtain additional information on tunic morphogenesis in Halocynthia papillosa. The chemical composition of the tunic evolves with its structural complexity. The larval and juvenile fibres are shown to be structurally and chemically different. While neither proteins nor glycosaminoglycans seem to be associated with the larval fibres, the juvenile fibres consist of a cellulose core wrapped in a sheath of tannophilic proteins. Patches of glycosaminoglycans line their longitudinal axes. In the course of metamorphosis, the cuticle undergoes profound modifications in regions of spine morphogenesis. Granular material that was previously called fibro-granular material (Lübbering et al., 1993) is essential to the formation of cuticular plates and spines. During metamorphosis, this material accumulates in epidermal granules and is discharged into the tunic. It crosses the fundamental layer of the tunic and reaches the cuticle. Our results strongly suggest that this material consists of proteins rich in cysteine and hydrophobic amino acids.     

A spectroscopic assessment of cellulose and the molecular mechanisms of cellulose biosynthesis in the ascidian Ciona intestinalis

Tunicates are the only animal group known to synthesize cellulose. The current hypothesis is that a horizontally acquired cellulose synthase gene of bacterial origin might have contributed to the establishment of this unique trait. Cellulose biosynthesis in tunicates thus provides an opportunity to understand how a foreign gene was assimilated into a new genome to establish a new trait. Because little is known of the molecular mechanisms underlying cellulose biosynthesis, we set up a practical assessment of cellulose in the ascidian tunicate Ciona intestinalis. We first demonstrated and characterized cellulose in the tunic of adult specimens by chemical purification and by subsequent scanning electron microscopic observation and X-ray diffractometry. Next, we showed that Fourier transform infrared spectroscopic microscopy (FTIR microscopy) can be used to assess cellulose in the small tunic of individual larval specimens without chemical purification. Using FTIR microscopy together with a blastomere isolation technique, we demonstrated that cellulose biosynthesis occurred cell-autonomously in the animal hemisphere of an embryo where the future epidermis, the known site of cellulose biosynthesis, will arise. We combined FTIR microscopy with morpholino antisense oligonucleotide-mediated gene knockdown technology to generate a reverse genetic system to identify genes involved in cellulose biosynthesis. FTIR microscopy was thus able, in combination with current research resources, to contribute to cellular and molecular investigations of animal cellulose biosynthesis.     

Evidence for the role of the glomerulocyte in cellulose synthesis in the tunicate,Metandrocarpa uedai

Summary The tunicate,Metandrocarpa uedai, contains a large quantity of cellulose; however, it is not known how and where the cellulose is synthesized. Based on evidence from electron diffraction and conventional thin-sectioning for electron microscopy, this study shows that the glomerulocyte is involved in the synthesis of cellulose. The bundles of microfibrils in the glomerulocyte as well as the tunic were identified as cellulose I using selected area electron diffraction analysis. The diffraction pattern of cellulose in the glomerulocyte was similar to that from the tunic, suggesting that the crystallization of cellulose already is initiated in the glomerulocyte. The diameter of cellulose microfibrils, both in the glomerulocyte and the tunic was the same, about 16 nm. These results suggest that the glomerulocyte is the most probable site for the synthesis of cellulose in the tunic ofM. uedai. Using thin-sectioning techniques, a series of observations showed that individual microfibrils are primarily assembled in structures tentatively identified as vacuole-like structures, then they are bundled by a tapering region within the vacuole-like structures. These bundles of microfibrils are deposited in a continuously circular arrangement. The microtubules are oriented parallel to the bundles of microfibrils at the tapering vacuole-like structure, and they may be involved in the tapering of these structures (perhaps controlling the shape). This study also provides the first account for the involvement of a vacuole-like structure in the synthesis of cellulose microfibrils among living organisms.     

Vertical transmission of photosymbionts in the colonial ascidian Didemnum molle: the larval tunic prevents symbionts from attaching to the anterior part of larvae

Morphological processes in the vertical transmission of photosymbionts were investigated in the Prochloron-bearing ascidian Didemnum molle. Prochloron cells were found exclusively in the common cloacal cavity of the colony, attached mainly to the tunic lining of the cavity wall. Oocytes were found in the abdominal region of each zooid, but no Prochloron cells were associated with this stage. During embryogenesis, embryos moved into the tunic core of the colony and were always separated from Prochloron cells in the cloacal cavity by the tunic matrix, until they hatched out from the tunic core. In swimming larvae, Prochloron cells covered the surface of the posterior half of the larval trunk, whereas a thin larval tunic layer covered the anterior half, where no Prochloron cells were found. The tunic of the posterior half of the larval trunk had many folds that enfolded the Prochloron cells and may be adhesive in order to acquire Prochloron cells from the mother colony. The thin larval tunic layer is probably not adhesive and protects the anterior half of the trunk from interference by Prochloron cells with sensory receptors and adhesive organs.     

Test cell migration and tunic formation during posthatching development of the larva of the ascidian, Ciona intestinalis

Morphological changes in the tunic layers and migration of the test cells during swimming period in the larva of the ascidian, Ciona intestinalis , were observed by light and electron microscopy. The swimming period was divided into three stages. In stage 1, further formation of juvenile tunic layer started only in the larval trunk and neck region. In stage 2, the layer became swollen in the ventral and dorsal sides of the neck region and in stage 3, the swelling expanded backward. Concomitantly with these changes, the outermost larval tunic layer (outer cuticular layer), which had been formed before hatching, also swelled in the neck region in stage 2 and formed two humps in stage 3, although the layer did not change in the tail region during the swimming period. Test cells that were present over the entire larval tunic layer in stage 1 began to move from the surface of the fin toward that of the side of the body in stage 2, and finally gathered to form six bands running radially from the anterior end to the posterior end of the trunk region and aligned along the lateral sides of body in the tail region in stage 3. In electron microscopic observations, pseudopodia protruding from the test cells invaded the larval tunic, following which they extended proximate to the juvenile tunic in the trunk region. In the tail region, which had no juvenile tunic layer as that described, the pseudopodia invaded and remained adjacent to the surface of the epidermis or the sensory cilia protruded from the epidermis. Metamorphosis of the larvae, further tunic formation, degradation of adhesive papilla, attachment of larva to the substratum and tail resorption commenced after these morphological changes occurred. The possible role of the test cells in metamorphosis is discussed.     

Behavior and cellular morphology of the test cells during embryogenesis of the ascidian Halocynthia roretzi

During the early stages of embryogenesis of the ascidian Halocynthia roretzi the test cells creep exclusively on the inner surface of the chorion. Concomitant with elongation of the embryonic tail, however, the test cells begin to gather around the embryo and finally cover the whole embryo. The time at which the test cells surround the embryo almost coincides with that of initiation of larval tunic formation. Scanning electron microscope observations revealed that the test cells extend numerous cytoplasmic processes or pseudopodia. During larval tunic formation, the test cells compose a net by intertwining their filopodia, and the cell net covers the whole embryo.     

Ascidian larval tunic: Extraembryonic structures influence morphogenesis

Summary The larval tunic of Corella inflata is composed of two cuticular layers, extracellular filaments and ground substance. It lies outside the epidermis and most of it is known to be produced by the epidermis. The dorsal, ventral and caudal fins are specialized parts of the tunic that are essential for larval locomotion. The following hypothesis was tested: Morphogenesis of the larval fins is dependent upon the presence of extraembryonic structures (test cells, chorion or follicle cells) before completion of the late tail bud stage of development. We tested this by dechorionating embryos of Corella inflata and Ascidia paratropa. The operation removes all extraembryonic structures. It was performed mainly on neurula, early tail-bud and late tail-bud stages.Fin formation is inhibited when neurulae are dechorionated but not when late tail-bud or older embryonic stages are dechorionated. Dechorionated neurulae produce all of the major components of the tunic (cuticular layers, filaments and ground substance) but they are unable to form functional fins. At the time of dechorionation, in all experiments, the embryos had no fins.Removal of the follicle cells does not inhibit fin formation. The test cells are known to secrete granular ornaments that attach to the surface of the tunic. The fibrous, acellular chorion may serve to contain the test cells and their products or products of the embryo that are not firmly attached. The test cells may induce or control the morphogenesis of the larval fins in ascidians before the late tail-bud stage of development. We suggest ways of testing this hypothesis and an alternative hypothesis.     

Lumichrome. A larval metamorphosis-inducing substance in the ascidian Hhalocynthia roretzi.

It has long been known that metamorphosis of ascidian larvae is induced by exposure to adult tunic extract or larval-conditioned seawater. However, such a natural 'inducer' has not been identified, probably due to its very low concentration in organisms. Here we have succeeded in isolating the same metamorphosis-inducing substance from the larvae, the larval-conditioned seawater, and the adult tunic of the ascidian Halocynthia roretzi. Structural analysis revealed that this substance was identical to lumichrome. Lumichrome was active toward H. roretzi larvae, but inactive toward another ascidian larvae, suggesting that lumichrome is species-specific. Riboflavin (vitamin B2), from which lumichrome might be derived from, was found to be inactive in induction of larval metamorphosis. In addition, it was demonstrated that lumichrome is localized predominantly in the basal region of the adhesive organ and the posterior part of the larval trunk. Thus, we propose that lumichrome functions as a natural inducer for larval metamorphosis in H. roretzi. This is the first natural metamorphosis-inducing substance to be identified in ascidians.     

A new cellulosic structure, the tunic cord in the ascidianPolyandrocarpa misakiensis

Summary A specialized structure of tunic cord inPolyandrocarpa misakiensis is investigated by electron microscopy. The tunic cord is a cord-like coiled structure of 5–30 m in diameter and 0.1–9.0 mm in length. The tunic cords originate and elongate from the dorsal tunic, and their termini have a swollen and ornamented structure. Scanning and transmission electron micrographs and the electron diffractogram show that the tunic cords are composed of bundled microfibrils of cellulose I with high crystallinity. The tunic cord is completely surrounded by single-layered epidermal cells, which have been found as the site of cellulose biosynthesis. A number of tunic cords are connected to the internal tunic of the siphon by forming eyelet structures at their termini. These observations suggest that the tunic cords act as a connector between dorsal and internal tunic of the siphon.     

Lineage relationship of direct-developing melanocytes and melanocyte stem cells in the zebrafish

Previous research in zebrafish has demonstrated that embryonic and larval regeneration melanocytes are derived from separate lineages. The embryonic melanocytes that establish the larval pigment pattern do not require regulative melanocyte stem cell (MSC) precursors, and are termed direct-developing melanocytes. In contrast, the larval regeneration melanocytes that restore the pigment pattern after ablation develop from MSC precursors. Here, we explore whether embryonic melanocytes and MSCs share bipotent progenitors. Furthermore, we explore when fate segregation of embryonic melanocytes and MSCs occurs in zebrafish development. In order to achieve this, we develop and apply a novel lineage tracing method. We first demonstrate that Tol2-mediated genomic integration of reporter constructs from plasmids injected at the 1-2 cell stage occurs most frequently after the midblastula transition but prior to shield stage, between 3 and 6 hours post-fertilization. This previously uncharacterized timing of Tol2-mediated genomic integration establishes Tol2-mediated transposition as a means for conducting lineage tracing in zebrafish. Combining the Tol2-mediated lineage tracing strategy with a melanocyte regeneration assay previously developed in our lab, we find that embryonic melanocytes and larval regeneration melanocytes are derived from progenitors that contribute to both lineages. We estimate 50-60 such bipotent melanogenic progenitors to be present in the shield-stage embryo. Furthermore, our examination of direct-developing and MSC-restricted lineages suggests that these are segregated from bipotent precursors after the shield stage, but prior to the end of convergence and extension. Following this early fate segregation, we estimate approximately 100 embryonic melanocyte and 90 MSC-restricted lineages are generated to establish or regenerate the zebrafish larval pigment pattern, respectively. Thus, the dual strategies of direct-development and MSC-derived development are established in the early gastrula, via fate segregation of the two lineages.     

Tunic morphogenesis in the sea peach (Halocynthia papillosa,Ascidiacea, rochordata): Cellular events,the initiation of twisted fibrous arrangements and spine formation

Summary— The adult tunic of the sea peach (Halocynthia papillosa) shows a high degree of organisation. Tunic morphogenesis was monitored from the onset of tunic secretion until juveniles reached the age of 3 months. While some characteristics of the adult tunic are still missing, like certain types of intratunical cells and striated bodies, its main features have already developed by this time. Crucial events take place at or soon after the onset of metamorphosis (stage M 0). Cuticular spines cover the external surface of the juvenile. At least two types of intratunical cells enter the tunic and the fibrous material adopts a three-dimensional twisted helicoidal architecture. The initiation of this helicoidal arrangement of fibrils directly after stage M 0 is discussed regarding accompanying developmental events. The existence of cells that penetrate the outer compartment of the tunic at the end of larval life is reported for the first time.     

Fine structure and origin of the tunic of Perophora viridis

The fine structure of the tunic of a typical ascidian was investigated because of the cellulose-like polysaccharide known to occur in its substance. The glycoprotein mantle does contain filaments very much like plant cellulose in morphology. Tunicin filaments are 35–50 Å in diameter, often beaded, and of indeterminate length. Histochemical evidence that they are composed of cellulose is given here and past chemical and physical studies on the unusual ascidian polysaccharide are reviewed. Moreover, we present here for the first time direct autoradiographic evidence that epidermal cells are involved in the synthesis and secretion of tunicin. Tritiated glucose is immediately incorporated into the Golgi zone of epidermal cells and labeled product appears in the tunic at later intervals. The fine structure of the epidermal cell is described in detail. Unlike the rather moribund appearing vanadocyte that wanders through the tunic, the epidermal cell has well-developed cytoplasmic organelles and a large vesicular nucleus. The granular endoplasmic reticulum is abundant and the Golgi complex is highly developed. It seems likely that the lamellae and vesicles of the Golgi complex are involved in the production of the tunic sugar and that tunic proteins of as yet unknown nature are produced by the ergastoplasm. Further investigation of the ascidian mantle should be of interest because of the possibility that cellulose is a more general component of glycoprotein surface coats in animals than has heretofore been recognized.     

Resource allocation in ascidians: reproductive investment vs. other life-history traits

Abstract. To determine patterns of resource allocation among ascidians, we studied 16 colonial and solitary species. We measured investment in reproductive vs. structural material (tunic) both in terms of weight and caloric content, as well as fecundity and degree of larval complexity in the colonial species. Measurements in weight and caloric content were highly correlated in the species studied. A wide range of investment in reproduction was found. Tunic production was related to the growth form of the species, stolonic and solitary species investing less in tunic than massive species, but no significant relationship was found between investment in tunic production vs. reproduction. In colonial species we found that in species with small zooid size, the reproductive investment per zooid was significantly higher. There was a significant negative relationship between investment in reproduction and fecundity. We also found a significant relationship between reproductive investment and larval complexity. The overall trend was that species with low fecundity had large complex larvae and invested the most energy in reproduction.     

Initial stages of tunic morphogenesis in the ascidian Halocynthia: a fine structural study

Tunic morphogenesis in embryos of the ascidian Halocynthia roretzi was examined by scanning and transmission electron microscopy. For this purpose it was necessary to modify the classical embedding procedure. Soon after reaching the initial tail-bud stage, tunic deposition is initiated on the dorsal side of the embryo. As soon as the embryo is completely covered by the tunic, larval fins are formed. The test cells settle onto the embryo. At this stage only the outer cuticle and the outer tunic compartment have appeared. Tunic morphogenesis is accompanied by ultrastructural modifications of the epidermis characteristic of secreting cells. Cytochemical investigations reveal polysaccharide glycogen-like material in the lumen of epidermal lacunae and in the outer compartment of the tunic. Our observations strongly suggest that this material is stored in the lacunae and discharged into the outer compartment. The significance of fluffy osmiophilic material that appears at the early tail-bud stage and enlaces the whole embryo is discussed.     

A cellulose fiber-based diet for screwworm (Diptera: Calliphoridae) larvae

A highly absorbent cellulose fiber from recycled paper was tested and compared with a polyacrylate gelling agent, Aquatain, normally used for bulking and solidifying larval rearing medium of screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae). The absorbent fiber, when mixed with water and dietary ingredients, produced a diet medium of homogeneous texture that supported larval growth and development comparable with the standard gelled diet. Larval and pupal weights from two concentrations of cellulose fiber-based diet were significantly higher than those obtained using gelled diet. The number of pupae per tray, percentage of adult emergence, oviposition, percentage of egg hatch, and adult longevity obtained from the insects reared in the cellulose fiber-based diet were comparable or slightly better than the biological parameters recorded from flies reared in the gelled diet. Moreover, results indicate that a lesser amount of the cellulose fiber-based diet than the normal amount of gelled diet per tray would support normal larval growth. Physical properties and texture of the new diet seem to allow the larvae to move and feed more freely than they do on the semisolid gelled diet, resulting in less wasted diet. The cellulose fiber is biodegradable and inexpensive, whereas the polyacrylate gel polymer is not biodegradable and is relatively expensive. Replacing gel with cellulose fiber in the screwworm larval diet for mass rearing should result in substantial cost savings in material and labor as well as eliminating concern of environmental pollution due to diet waste disposal.     

The postembryonic development of the ocellar system of Triatoma infestans Klug (Heteroptera: Reduviidae)

Simple eyes or ocelli coexist with compound eyes in many adult insects. The change in the morphology of the ocelli along the five larval instars of Triatoma infestans was studied by light and scanning electron microscopy. Our analysis showed that the development of the ocelli of these bugs occurs gradually along the larval life. The photoreceptor layer is present from the second-instar onwards. The cornea appears first at the imaginal stage and grows up to the 18-20th day after the last ecdysis, associated to an increase in the retinal mass. Findings are discussed in a comparative fashion and in relation to the functionality of the ocellar system in T. infestans.     

Transmission of cyanobacterial symbionts during embryogenesis in the coral reef ascidians Trididemnum nubilum and T. clinides (Didemnidae, Ascidiacea, Chordata)

Vertical transmission of cyanobacterial symbionts occurs in didemnid ascidians harboring Prochloron as an obligate symbiont; the photosymbionts are transferred from the parental ascidian colony to the offspring in various ways depending on host species. Although several didemnids harbor non-Prochloron cyanobacteria in their tunics, few studies have reported the processes of vertical transmission in these didemnids. Here we describe the histological processes of the transmission of cyanobacteria in two didemnids, Trididemnum nubilum harboring Synechocystis and T. clinides harboring three cyanobacterial species. In both species, the photosymbionts in the tunic of the parent colony were apparently captured by the tunic cells of the host and transferred to the embryos brooded in the tunic. The symbiont cells were then incorporated into the inner tunic of the embryo. This mode of transmission is essentially the same as that of T. miniatum harboring Prochloron in the tunic, although there are some differences among species in the timing of the release of the symbionts from the tunic cells. We suggest that the similar modes of vertical transmission are an example of convergent evolution caused by constraints in the distribution patterns of symbiont cells in the host colony.     

Biosynthesis and metabolism of sulfated glycosaminoglycans during Drosophila melanogaster development

We developed a simple methodology for labeling sulfated glycosaminoglycans (GAGs) in adult Drosophila melanogaster and studied some aspects of the biosynthesis and metabolism of these polymers during development. Adult D. melanogaster flies were fed with Na(2)(35)SO(4) for 72 h. During this period, (35)S-sulfate was incorporated into males and females and used to synthesize (35)S-sulfate-heparan sulfate (HS) and (35)S-sulfate-chondroitin sulfate (CS). The incorporation of (35)S-sulfate into HS was higher when compared to CS. In a pulse-chase experiment, we observed that (35)S-sulfate incorporated into adult female was recovered in embryos and used for the synthesis of new (35)S-sulfate-GAGs after 2 h of embryonic development. The synthesis of CS was higher than that of HS, indicating a change in the metabolism of these glycans from adult to embryonic and larval stages. Analysis of the CS in embryonic and larval tissues revealed the occurrence of nonsulfated and 4-sulfated disaccharide units in embryos, L1 and L2. In L3, in addition to these disaccharides, we also detected significant amount of 6-sulfated units that are reported here for the first time. Immunohistochemical analysis indicated that HS and CS were present in nonequivalent structures in adult and larval stages of the fly. Overall, these results indicate that (35)S-sulfate-precursors are transferred from adult to embryonic and larval tissues and used to assemble different morphological structures during development.     

Characterization of cellulose degrading bacteria from the larval gut of the white grub beetle Lepidiota mansueta (Coleoptera: Scarabaeidae)

The goal of this study is to identify and characterize the cellulose degrading microorganisms in the larval gut of the white grub beetle, Lepidiota mansueta. Thirty bacterial strains were isolated and tested for cellulolytic activity using soluble carboxymethyl cellulose (CMC) degrading assays. Of these strains, five (FGB1, FB2, MB1, MB2, and HB1) degrade cellulose. Cellulolytic activity was determined based on formation of clear zone and cellulolytic index on CMC plate media. The highest cellulolytic index (2.14) was found in FGB1. Partial 16S rDNA sequencing, morphological, and biochemical tests were used to identify and characterize the five isolates, all Citrobacter sp. (Enterobacteriaceae). This study identifies new cellulose degrading microorganisms from the larval gut of L. mansueta. The significance of identifying these strains lies in possible application in cellulose degradation.