Comparative myoanatomy of cycliophoran life cycle stages

The metazoan phylum Cycliophora includes small cryptic epibionts that live attached to the mouthparts of clawed lobsters. The life cycle is complex, with alternating sexual and asexual generations, and involves several sessile and free‐living stages. So far, the morphological and genetic characterization of cycliophorans has been unable to clarify the phylogenetic position of the phylum. In this study, we add new details on the muscular anatomy of the feeding stage, the attached Prometheus larva, the dwarf male, and the female of one of the two hitherto described species, Symbion pandora. The musculature of the feeding stage is composed of myofibers that run longitudinally in the buccal funnel (two fibers) and in the trunk (variable number of fibers). The mouth opening is lined by a myoepithelial ring musculature. A complex myoepithelial sphincter is situated proximal to the anus. In the attached Prometheus larva, three longitudinal sets of myofilaments run dorsally, laterally, and ventrally along the entire anterior‐posterior body axis. The muscular architecture of the dwarf male is complex, especially close to the penis, in the posterior part of the body. An X‐shaped muscle structure is found on the dorsal side, whereas on the ventral side, longitudinal muscles and a V‐shaped muscle structure are present. These muscles are complemented by additional dorsoventral muscles. The mesodermal muscle fibers attach to the cuticle via the epidermis in all life cycle stages studied herein. The musculature of the female is similar to that of the Pandora larva of Symbion americanus and includes dorsoventral muscles and longitudinal muscles that run in the dorsal and ventral body region. Overall, our results reveal striking similarities in the muscular arrangement of the life cycle stages of both Symbion species. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Three‐dimensional reconstruction of the musculature of various life cycle stages of the cycliophoran Symbion americanus

Cycliophora is a very recently described phylum of acoelomate metazoans with a complex life cycle and a phylogenetic position that has been under debate ever since its discovery in 1995. Symbion americanus, which lives attached to the mouthparts of the American lobster, Homarus americanus, represents the second species described for the phylum. Aiming to increase the morphological knowledge about this cryptic clade, the present study describes the muscle arrangement of the feeding stage, the attached Prometheus larva with the dwarf male inside, the free living male, the Pandora larva, and the chordoid larva of S. americanus using actin staining and confocal laser scanning microscopy. 3D reconstructions of the muscular systems are presented. In the feeding stage, circular muscles compose the buccal funnel aperture. In addition, a pair of muscles runs longitudinally in the buccal funnel. A complex sphincter was found just proximally to the anus, and six longitudinal muscles run from the trunk constriction (“neck”) in basal direction. The musculature of the larval stages and the dwarf male is very complex and includes longitudinal muscles that run dorsally and ventrally. In addition, we found dorso‐ventral muscles. The male has a complex posterior muscle apparatus in the vicinity of the penis. In this stage, X‐ and V‐shaped structures were identified on the dorsal and the ventral side, respectively. Pandora and chordoid larvae possess additional circular muscles. We discuss our findings with respect to muscle elements of other metazoan groups and the chordoid larva of Symbion pandora. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

Serotonin immunoreactivity in the nervous system of the Pandora larva, the Prometheus larva, and the dwarf male of Symbion americanus (Cycliophora)

Cycliophora is a recently described phylum of enigmatic metazoans with a very complex life cycle that includes several sexual and asexual stages. Symbion pandora and Symbion americanus are the only two cycliophoran species hitherto described, of which morphological and genetic knowledge is still deficient to clarify the phylogenetic position of the phylum. Aiming to increase the database on the cycliophoran neural architecture, we investigated serotonin immunoreactivity in the free swimming Pandora larva, the Prometheus larva, and the adult dwarf male of S. americanus. In the larval forms, serotonin is mainly expressed in a ring-shaped pattern at the periphery of the antero-dorsal cerebral ganglion. Additionally, several serotonergic perikarya emerge from both sides of the cerebral ganglion. Thin neurites project anteriorly from the cerebral ganglion, while a pair of ventral longitudinal neurites emerges laterally and runs along the anterior-posterior body axis. Posteriorly, the ventral neurites fuse and extend as a posterior projection. In the dwarf male, serotonin is found mainly in the commissural neuropil of the large anterior cerebral ganglion. In addition, serotonin immunoreactivity is present in the most anterior region of the ventral neurites. Comparative analysis of spiralian nervous systems demonstrates that the neuroanatomy of the cycliophoran larval stages resembles much more the situation of adult rather than larval spiralians, which may be explained by secondary loss of larval structures and heterochronic shift of adult components into the nervous system of the Pandora and the Prometheus larva, respectively.     

Expression of synapsin and co-localization with serotonin and RFamide-like immunoreactivity in the nervous system of the chordoid larva of Symbion pandora (Cycliophora)

Abstract. Cycliophora is one of the most recently described metazoan phyla and hitherto includes only two species: Symbion pandora and Symbion americanus . With a very complex life cycle, cycliophorans are regarded as an enigmatic group with an uncertain phylogenetic position, although they are commonly considered lophotrochozoan protostomes. In order to extend the database concerning the distribution of immunoreactive substances in the free-swimming chordoid larva of S. pandora , we investigated synapsin immunoreactivity using fluorescence-coupled antibodies in combination with confocal laserscanning microscopy. Moreover, we analyzed the co-localization patterns of synapsin, serotonin, and RFamide-like immunoreactivity in the chordoid larva by 3D imaging technology based on the confocal microscopy image stacks. Synapsin is expressed in large parts of the bilobed anterior cerebral ganglion including anterior and dorsal projections. Two pairs of ventral neurites run longitudinally into the larval body of which the inner pair shows only weak, scattered synapsin immunoreactivity. In addition, a lateral synapsin immunoreactive projection emerges posteriorly from each ventral longitudinal axon. Double immunostaining shows co-localization of synapsin and serotonin in the cerebral ganglion, the outer and the inner ventral neurites, and the anterior projections. Synapsin and RFamide-like immunoreactivity co-occur in the cerebral ganglion, the outer ventral neurites, and the dorsal projections. Accordingly, the cerebral ganglion and the outer ventral neurites are the only neural structures that co-express the two neurotransmitters and synapsin. The overall neuroanatomical condition of the cycliophoran chordoid larva resembles much more the situation of adult rather than larval life cycle stages of a number of spiralian taxa.     

External morphology of the cycliophoran dwarf male: a comparative study of Symbion pandora and S. americanus

Cycliophora is a recently described phylum to which only two species have been assigned so far, Symbion pandora and S. americanus. The cycliophoran life cycle is complex and alternates between asexual and sexual stages. Although not recognized as an entirely independent free-swimming stage when the phylum was first described, the dwarf male has a remarkably complex bodyplan albeit its very small size (approx. 30–40 μm in length). Aiming to increase the knowledge on the gross morphology of the cycliophoran dwarf male, specimens from S. pandora and S. americanus were analyzed by scanning electron microscopy. In both species, anterior and ventral ciliated fields, as well as paired lateral sensorial organs, were identified, thus confirming previous observations. However, new details are described herein such as the penial pouch that encloses the penis. We compare our findings on both Symbion species with the data currently available on other metazoan dwarf males.     

Immunocytochemistry of the nervous system and the musculature of the chordoid larva of Symbion pandora (Cycliophora)

To date, the phylum Cycliophora comprises only one described extant species of acoelomate marine invertebrates, Symbion pandora. Adult specimens live commensally on the mouthparts of the Norwegian lobster, Nephrops norvegicus. Its complicated life cycle includes an asexually produced Pandora larva and a sexually produced chordoid larva. Despite detailed TEM investigations and its inclusion in recent molecular phylogenetic analyses, cycliophoran relationships still remain enigmatic. In order to increase the morphological database, I investigated the anatomy of the nervous system and the musculature of the chordoid larva by applying fluorescence-coupled antibodies against the neurotransmitters serotonin and FMRFamide, as well as FITC-coupled phalloidin to label filamentous F-actin, in combination with confocal laser scanning microscopy. The FMRFamidergic nervous system shows a bilobed anterior ganglion and one pair of ventral nerve cords, while serotonin is distributed in a scattered pattern in the anterior ganglion. In addition, there are two pairs of ventral serotonergic nerves, of which the inner pair fuses with the outer nerve cords in the posterior third of the larva. The musculature comprises an outer layer of six units of circular body wall muscles, several helicoid muscle fibers, a set of paired longitudinal muscles that span the entire anterior-posterior axis of the larva, and a few oblique muscle strands. Furthermore, an anterior muscle complex and one pair of posterior muscles are present. The chordoid organ consists of a number of distinct subunits that are each formed by a dense layer of circular muscle fibers.The overall arrangement of the oblique and longitudinal muscles as well as the body wall musculature in the chordoid larva of Symbion pandora exhibits similarities with the condition found in certain rotifers. This is congruent with some recent phylogenies based on 18S rRNA sequences but additional morphological, developmental, and molecular data are needed to clarify the phylogenetic relationships of Cycliophora.     

The chordoid larva of Symbion pandora (Cycliophora) is a modified trochophore

Developmental and free-living stages of the chordoid larva of the cycliophoran species, Symbion pandora Funch and Kristensen 1995, were studied using light and electron microscopy. In the free-living stage of the larva, about 200 μm long, four ciliated areas are found: two anterior bands, a ventral ciliated field, and a posterior unit on the ventral side of the foot. The nervous system consists of a dorsal brain and a pair of ventral longitudinal nerves. A gut is absent. A pair of protonephridia, each with a single multiciliated terminal cell and at least one duct cell, is present. Nephridiopores are not localized. A pair of corsal ciliated organs is posterior to the brain. The homology between these and the apical organ of a trochophore larva is discussed. A distinctive longitudinal rod, the chordoid organ, consists of vacuolized cells with circular myofilaments. The organ is comparable to a similar structure in gastrotrichs. In the discussion of the phylogenetic position of Cycliophora among protostomians, important morphological observations that are described in the present study indicate that, despite some dissimilarities, the chordoid larva is a modified trochophore. © 1996 Wiley-Liss, Inc.     

Microanatomy and Development of the Dwarf Male of Symbion pandora (Phylum Cycliophora): New Insights from Ultrastructural Investigation Based on Serial Section Electron Microscopy

Cycliophorans have a complex life cycle that involves several sexual and asexual stages. One of the sexual stages is the 40 μm-long dwarf male, which is among the smallest free-living metazoans. Although the dwarf male has a highly complex body plan, this minute organism is composed of a very low number of somatic cells (~50). The developmental processes that give rise to this unique phenotype are largely unknown. Here we use high resolution serial block face—scanning electron microscopy to analyze the anatomy and morphogenesis of three cycliophoran dwarf males at different developmental stages ranging from internal bud to mature male. The anatomical and morphological features of the mature dwarf male stage reported here largely correspond to those reported in earlier studies. Interestingly, the organs that typically characterize the anatomy of the mature dwarf male, e.g., muscles, brain, testis and glands, are already formed in the young male. However, there are striking differences between the mature male and young male stages at the level of cellular architecture. Thus, while the young male stage, like the internal bud stage, possesses approximately 200 nucleated cells, the mature male stage comprises only around 50 nucleated cells; muscle and epidermal cells of the mature male lack nuclei. Moreover, the total body volume of the mature male is only 63% of the body of the young male implying that the maturation of the young male into a mature male involves a marked reduction of internal body volume, mainly by massive nuclei loss. Our comparative analysis of these dwarf male specimens reveals unprecedented insight into the striking morphological and developmental differences that characterize these highly miniaturized male stages both at the level of body organization and at the level of cellular ultrastructure.     

Structure and development of Austramphilina elongata Johnston, 1931 (Cestodaria: Amphilinidea)

The life cycle and structure of the larva of Austramphilina elongata using light-microscopy, scanning and transmission electron microscopy are described. Eggs are round and non-operculate. Larvae hatch in freshwater and penetrate through the cuticle of juvenile crayfish, Cherax destructor, and of freshwater shrimps, Paratya australiensis and Atya (= Atyoida) sp., shedding their ciliated epidermis. In the last two hosts, development to the infective stage does not occur. In crayfish, larvae grow and reach the infective stage. Turtles, Chelodina longicollis, become infected by eating infected crayfish. Larvae penetrate through the oesophageal wall of the turtle and migrate toward the coelom, where maturation occurs. The free-swimming larva has a syncytial epidermis which covers most of the body except for the posterior region bearing the hooks. It is loosely attached to a thin underlying tegument, which is connected to ‘insunk’ nucleated cell bodies. It forms a thick surface layer in the posterior region. There are three flame cells on each side of the body and two postero-lateral excretory pores. There are no lateral flames. The weir apparatus of the flame cell has the structure typical of parasitic platyhelminths. The smaller capillaries have a smooth surface, that of the terminal ducts is covered by numerous microvilli. Three types of penetration glands open anteriorly. There are five pairs of hooks; one median ‘normal’, two submedian halberd-shaped, and two lateral serrate. Hook are not lost, they are arranged around the gonopore of the adult. Frontal glands opening into the proboscis were found in the anterior part of the body in all stages examined. Infective stages in crayfish have developing reproductive organs and ducts. The tegument of the adult has many microvilli.     

Crucial stages of embryogenesis of R. arvalis: Part 1. Linear measurements of embryonic structures

Investigations of individual variability have allowed us to reveal the crucial (= nodal) stages in embryogenesis of the moor frog (Rana arvalis Nills.). These crucial stages are: the late gastrula stage (stages 18-20), the hatching stages (stages 32-33) and, apparently, early metamorphosis (stage 39). Moreover, we have found that each embryonic structure passes through its specific crucial stages. For example, stage 34 is crucial for the trait "tail width" but is internodal for all other embryonic traits. At this stage, larva passes from an attached to a free-swimming life style. We also found considerable differences between the different frog populations in the the level of developmental variability. These differences were associated with internodal developmental stages.     

Crucial stages of embryogenesis of <Emphasis Type="Italic">R. arvalis</Emphasis>: Part 1. Linear measurements of embryonic structures

Investigations of individual variability have allowed us to reveal the crucial (=nodal) stages in embryogenesis of the moor frog (Rana arvalis Nills.). These crucial stages are: the late gastrula stage (stages 18–20), the hatching stages (stages 32–33) and, apparently, early metamorphosis (stage 39). Moreover, we have found that each embryonic structure passes through its specific crucial stages. For example, stage 34 is crucial for the trait “tail width” but is internodal for all other embryonic traits. At this stage, larva passes from an attached to a free-swimming life style. We also found considerable differences between the different frog populations in the the level of developmental variability. These differences were associated with internodal developmental stages.     

Ultrastructural Observations on the Dwarf Male of Bonellia viridis (Echiura)

The organization of the dwarf male of Bonellia viridis was studied by electron microscopy. The epidermis is formed by two types of epithelial cells: the majority are multiciliated cells; highly vacuolated, non-ciliated cells are less abundant. The body wall musculature consists of an outer circular, a diagonal, and a longitudinal layer. As a unique feature in coelomate spiralians it was found that the perikarya of all muscle cells are located internal to the entire contractile muscular layer. The muscles are solitary myocytes embedded in extracellular matrix. Masses of secretory and indifferent cells occur inside the muscles. Two types of secretory cells were distinguished. Both of them apparently undergo holocrine secretion. A complete lining of thin peritoneal cells delimits the body cavity. Also, the gut and sperm sac have a complete peritoneal lining. The coelomic lining of the gut is a single-layered myoepithelium, that of the sperm sac a pseudo-stratified myoepithelium. The vas deferens was seen to be ciliated. The entrance of the sperm sac is formed by a ciliated funnel that leads into the reservoir by means of a thin, ciliated canal. The existence of repeated transverse nerves and of four longitudinal nerve cords is described for the first time.     

Larval Development in Discinisca (Inarticulate Brachiopod)

Shell-less Discinisca larvae of 2–3 p.c. (pairs of cirri)and small shelled larvae of 4 p.c. stages, hitherto undescribed,form a growth series with those previously described. The shellfirst formed during early 3 p.c. or early 4 p.c. stage. In swimmingthese young larvae did not rotate about their longitudinal bodyaxis, unlike larger larvae. In some larvae pigment granulesaggregated in the anterolateral stomach wall, forming "eye spots,"which are not comparable to the sensory eye spots of articulatelarvae. The order of appearance of embryonic setae and larvalsetae was described. The role of the former in floatation andin protective response was suggested. In recent brachiopod ontogeny there is an evolutionary simplificationfrom the presumably primitive condition in lingulids with shelledembryo, shelled larva with statocysts, long planktotrophic existenceand well developed trocholophe with continuous budding of cirrito 8-20 pairs; to the discinids with setiferous, shell-lessembryo, shelled larva with statocysts, shorter planktotrophicexistence and larval trocholophe with a maximum of 4 cirruspairs; and finally to the articulates with setiferous, shell–lessembryo and larva with no statocysts, no differentiated cirriand short free-swimming existence.     

Lecithotrophic development and metamorphosis in the Indo-West Pacific brittle star Ophiomastix venosa (Echinodermata: Ophiuroidea)

Summary

The larval development of the ophiocomid ophiuroid Ophiomastix venosais described using SEM. The gastrula transforms into a uniformly ciliated early larva which progressively changes into a lecithotrophic late premetamorphic larva with a continuous bilateral ciliated band. This stage is short-lived and equivalent to a highly reduced ophiopluteus. Comparisons between O. venosa and other ophiuroid species whose development has been investigated suggest that, whatever the developmental mode (lecithotrophic or planktotrophic), a pluteus stage always occurs in ophiuroids with planktonic development. Two metamorphic stages were identified, the late metamorphic larva differing from the early one by the closure of the larval mouth. The appearance of the permanent mouth marks the end of the metamorphosis. The postlarva still possesses remnants of larval features. The transformation of the reduced ophiopluteus into a barrel-shaped metamorphic larva with transverse ciliated bands, a vitellaria larva, is followed. The possible occurrence of a unique type of metamorphic larva in non-brooding ophiuroids is discussed. Verification of this, however, needs further SEM investigations on metamorphic larva from species having “regular” planktotrophic development.     

Ultrastructure and morphology of the cycliophoran female

Knowledge on the morphology of the cycliophoran female has mostly been based on observations of immature females in brood chambers of feeding stages. With the use of light‐ and transmission electron microscopy, the morphology and ultrastructure of the free and fully mature female of Cycliophora is described now for the first time. The external morphology is characterized by a ciliation consisting of an anteroventral ciliated field, a posterior ciliated tuft, and four sensory structures extending anteriorly from the anteroventral ciliated field. In addition, a small ciliated structure in the midventral region is interpreted as a round‐shaped gonopore. Internally, a bilateral cerebral ganglion is situated in the anterior region and a large oocyte is located medially in the body. Several glands are present anteriorly, while posteriorly a pair of glands is associated with the ciliated tuft. Dorsal and ventral longitudinal muscles, as well as, dorsoventral muscles are identified by electron microscopy. Muscle fibers attach to the endocuticle via the epidermis, by means of attachment fibers. An unknown endosymbiont is present throughout the body of the female. We discuss the functional implications of the morphological and ultrastructural aspects of the cycliophoran female. Finally, we compare this life cycle stage with that fromother phyla, suggested as phylogenetically close. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

Post-embryonic development of the two-spotted spider mite Tetranychus urticae with special reference to the differentiation of the female reproductive system

Summary

The spider mite, Tetranychus urticae passes through four active stages in its life cycle: larva, protonymph, deutonymph, adult. The duration at 23°C and 40–60% relative humidity is 10–12 days. Each of the movable stages is followed by a resting phase (= chrysalis) where moulting processes are prepared. The general internal anatomy of the developmental stages does not differ from that of the adults. The gonadial rudiment of the female larva is an undifferentiated cell mass situated between the nervous system and the anus. In the nymphochrysalis, development of oocytes starts as indicated by synaptonemal complexes occuring in cells which are interconnected via cytoplasmic bridges. Cells exhibiting extranuclear material appear during the protonymphal stage. The presumptive uterus and vagina are formed during the deutochrysalis stage and consist of an undifferentiated tube, the distal portion of which is lined by a fine cuticle. The ovary of the deutonymph contains different cell types. Oogonia and growing oocytes are found in the cranial germ region. Oocytes, nurse cells and cells with large lobed nuclei can be observed in the caudal previtellogenic region. Oocytes protrude through the ovarian surface and invade ovarian pouches covered only by the basement membrane whereby each is connected to a tri-nucleate nurse cell via a cytoplasmic bridge. Oocytes increase in size but do not form yolk droplets. The uterus and vagina are differentiated during the late deutonymphal stage and copulation may take place as indicated by the presence of sperm in the lumen of the seminal receptacle.

The results are compared to previously published information on the female reproductive system of T. urticae and discussed with reference to co-operation of ovarian cell types and their origin.     

Embryonic and larval development of the peanut worm Phascolosoma agassizii (Keferstein 1867) from the Sea of Japan (Sipuncula: Phascolosomatidea)

All stages of the embryonic and larval development of Phascolosoma agassizii from Peter the Great Bay (Sea of Japan) were studied and illustrated using light and electron microscopy. The eggs of P. agassizii have the form of an ellipsoid (long and short axes about 100 and 70?µm, respectively). Egg cleavage is typical, spiral, and unequal. Gastrulation occurs by epiboly. This species possesses two pelagic larval stages, a lecithotrophic trochophore and a planktotrophic pelagosphera. The transformation of trochophore into pelagosphera occurs 80–90?h after fertilization. After 120–180?h, the larva has developed all systems of organs characteristic of the pelagosphera and is capable of feeding. At day 10, pelagospheras can settle, for some time, on the aquarium bottom and move on a ciliated lip, collecting food with the aid of a buccal organ. In addition, the larvae periodically attach themselves to the aquarium bottom or to the surface film of the water by means of a terminal organ. The trunk of the larva elongates by enlargement of the region behind the dorsal anal opening, which is located almost in the middle of the trunk region in the 15-day old larva. In the laboratory, 1-month old larvae spend the greater part of time in the attached state. Being attached by a glandular terminal organ to the aquarium bottom, they characteristically bend the body, actively feeding on microalgae from the substratum surface. The differences in the development of P. agassizii in the isolated West-Pacific and East-Pacific populations are shown and discussed.     

Study of Specific Protein on Sex Differentiation of Momordica charantia

In Momordica charantia L. the soluble protein profile of flower bud at hermaphroditic stage and three early developmental stages (the 7th, 10th and 13th day after initial budding) of male and female flowers were analysed with capillary electrophoresis. Some specific proteins related to sex differentiation were detected. The 11 kD protein, which appeared at the 7th day of budding and existed through the three developmental stages of the female flowers with little change of content, might be an "essential protein" for the expression of female flower differentiation program. Similarly, the 30 kD protein might be an "essential protein" for the expression of the male flower differentiation.