Differentiation of the body wall musculature in Macrostomum hystricinum marinum and Hoploplana inquilina (Plathelminthes), as models for muscle development in lower Spiralia

Recent studies on the differentiation of the body wall musculature in a medicinal leech and in the free-living plathelminth Macrostomum hystricinum marinum, Beklemischev 1950 provide the first evidence of a complex developmental signalling pattern, possibly involving stem cells and the nervous system, in the organization of the muscle grid formed by developing myocytes. To enhance further our understanding of the ontogenetic and phylogenetic origin of such muscle grids, which consist of circular, longitudinal and diagonal muscle fibres, we have undertaken a study of muscle development in the polyclad flatworm Hoploplana inquilina Wheeler 1894 in collaboration with the Marine Biological Laboratory, Woods Hole. We have also continued our examination of the development of the body wall musculature in M. hystricinum. Both species were studied using rhodamine-phalloidin staining and transmission electron microscopy. Additional visualization of the fluorescent whole mount preparations was performed with confocal laser microscopy and digital image processing. The results of our investigation suggest that: (1) the mechanism of muscle development in H. inquilina supports the deeply rooted concept of bilateral symmetry (right and left longitudinal founder muscle), and (2) a first circular muscle in this species develops on the border between an anterior body unit and the main body; a caudalmost region is less obvious. The presence of a spiral muscle functioning as a circular muscle system of the head region points to a separate developmental mechanism for this region and the trunk. In contrast to H. inquilina, where the larval stage forces an intermediate restructuring of the musculature of the body wall before the adult body shape is finally developed, the formation of the body wall musculature of M. hystricinum already seems constrained by the adult body shape.     

Localisation of GYIRFamide immunoreactivity inMacrostomum hystricinum marinum (Plathelminthes,Macrostomida)

The distribution of GYIRFamide immunoreactivity in the nervous system ofMacrostomum hystricinum marinum has been demonstrated by an indirect fluorescence technique in conjunction with confocal scanning laser microscopy (CSLM). Immunostaining was extensive in both the central (CNS) and peripheral (PNS) nervous systems, revealing detailed information on the microanatomy of the peptidergic nervous system of this free-living plathelminth. In the CNS, immunoreactive nerve cell bodies and nerve fibres occurred in the brain and along two pairs of longitudinal nerve cords: the main nerve cords and the ventral nerve cords. In the PNS, immunostaining was prevalent in nerve cells and fibres innervating the pharynx and the gut. The employed antibody is directed against a recently characterised FMRF-amide-related peptide (FaRP), GYIRFamide, isolated from two species of the Tricladida,Dugesia tigrina andBdelloura candida. Phylogenetically, GYIRFamide represents the most ancient neuropeptide thus far identified within the Bilateria     

Immunoreactivity to a specific echinoderm neuropeptide in the nervous system of the flatworm Macrostomum hystricinum marinum (Turbellaria,Macrostomida)

Immunocytochemical characterization of the neuropeptides FMRF-amide and serotonin (5-HT) is a well-known method successfully applied to demonstrate nervous-system morphology in several platyhelminths (see Wikgren & Reuter, 1985, and Reuter, 1988, for review). We have immunolabeled whole-mount preparations of Macrostomum hystricinum marinum Rieger from cultures (see Rieger et al., 1988) with anti-SALMF-amide, an antibody specific for the C-terminal pentapeptide sequence of the neuropeptide GNFSALMF-amide recently isolated from echinoderms (source M. Thorndyke, England). Immunoreactivity to SALMF-amide gave a more detailed picture of the nervous system of M. hystricinum than FMRF-amide. Conventional light microscopy (Luther, 1905) shows this nervous system to consist of a bilobed brain, a pharyngeal nerve-ring system, a posterior commissure, and two main ventrolateral nerve cords. Immunostaining reveals, in addition, two thin paired longitudinal nerve strands and fine subepithelial and submuscular nerve nets. Anti-SALMF-amide labels a distinct class of neurons, causing the main lateral longitudinal cords and pharyngeal nerve-ring system to appear more filamentous than with other techniques. Recent fine-structural investigations on the nervous system of Macrostomum hystricinum marinum revealed several axon types with characteristic vesicles and neurotubules (D. R. pers. obs.). Partly supported by FWF grant P7816.     

Unique patterns of longitudinal body-wall musculature in the Acoela (Plathelminthes): the ventral musculature of Convolutriloba longifissura

The ventral musculature of Convolutriloba longifissura (Acoela) has been studied using electron microscopy and fluorescently labeled whole mounts to demonstrate filamentous actin. Attention was directed to the reorganization and renewal of musculature during asexual reproduction and the adaptation of muscle sets for special predatory behavior. Three ventral subepidermal muscle layers could be distinguished in adult C. longifissura: (1) outer circular muscles that encircle the body, (2) intermediate modified longitudinal muscles with concentric pattern around the mouth and V-shaped orientation in the posterior part of the animal, and (3) inner special pore muscles with radial alignment fanning out from the mouth. Additionally, a few very fragile muscles were found at the anterior margin of the animal. The anterior ventral muscle system built a funnel with the mouth opening as organizing center. The special radial muscles and the antagonistically concentric muscles are perfectly adapted to catch prey in such a way that the funnel is put over the prey to press it through the mouth into the digestive syncytium. Convolutriloba longifissura shows a unique way of asexual reproduction by a two-step fission which results in three individuals. Immediately after separation from the mother animal, daughter individuals are missing the concentric and the radial muscle sets around the mouth completely, but within 30 h these sets are renewed for the most part. Two to three days after separation, the mouth opening is visible and the animals move for capturing prey. The peculiar course of longitudinal muscles in C. longifissura with concentric rings anteriorly and a V-shape muscle layer posteriorly shows that the pattern of body-wall musculature in such basal Plathelminthes as the Acoela may be highly modified from the original pattern of longitudinal and circular muscles.     

Organization and differentiation of the body-wall musculature in Macrostomum (Turbellaria,Macrostomidae)

We studied the body-wall musculature, its ECM (extracellular matrix), and the junctional complexes between muscle cells and between muscle cells and ECM in Macrostomum hystricinum marinum Rieger, 1977, using Nomarski-contrast and electron microscopy. Differentiation of these body-wall components was followed by monitoring embryonic stages at 52%, 64%, and 82% of the time between egg-laying and hatching and with study of the hatchling and adult stages. For comparison, the body-wall musculature of other macrostomidans has been examined in conventional light-histological sections.Muscles form a grid of longitudinally, diagonally, and circularly oriented fibers beneath the epidermis in M. hystricinum marinum and this orientation of cells can be found already in embryos at 64% development. Younger embryos at 52% development show no muscle differentiation. The ECM forms a net-like arrangement that apparently envelops the individual muscle cells. Characteristic knob-like thickenings of the ECM occur at the base of the epidermis. Muscle cells attach to each other, to the epidermis, and to other cell types through hemidesmosome-like junctions at thickenings of the ECM in the adult and hatchling stages; no true desmosomes exist between muscle cells. Gap junctions occur commonly between longitudinal muscles of adult specimens and between perikarya of muscle cells in embryos at 64% and 82% development.More comparative studies are needed to determine the systematic value of presence or absence of the diagonal muscle fibers in the body wall of turbellarians.     

Differentiation of the body wall musculature in Macrostomum and Hoploplana (Turbellaria,Platyhelminthes)

Using the Phalloidin-Rhodamine flourescence-labelling technique for F-actin, we have studied the development of the body wall musculature in Macrostomum hystricinum marinum and in thepolyclad Hoploplana inquilina. The structure of the muscle grid in the freshly hatched Macrostomum (see also Rieger & Salvenmoser, 1991) and the young larva of Holplana served as reference systems for the embryonic development of the body wall musculature. In Macrostomum muscle fiber differentiation starts around 60% of developmental time between egg-laying and hatching, and in Hoploplana around 80% of embryonic development.In Macrostomum, early stages show TV-antenna-like arrangements of one longitudinal and several circular fibers. In Hoploplana our preliminary results show a particularly large, longitudinal fiber on either side of the body. These primary longitudinal fibers may serve as a founder cell for other longitudinal fibers and as spatial guides for the circular muscles. Similar founder cells have been reported during early muscle differentiation in leeches (Jellies & Kristan, 1988; Jellies, 1990). In Hoploplana, a special muscle system is present at the outset under the apical organ. It consists of what seems to be a spirally toranged fiber — when seen in head-on view — and of two additional fibers crossing this spiral, from the later developing posterior to the anterior lobe.TEM-studies of embryos of Macrostomum suggest that the longitudinal nerve cords represent an important guide during early differentiation of the pattern within the body wall musculature. Young stages of myoblasts can be identified along the main lateral nerve cord. Commonly, the myoblasts are seen to alternate with young neurons in their position along the nerve cord. Embryonic stages of Macrostomum hystricinum marinum were obtained from our cultures (Rieger et al., 1988). Immediately prior to fixation (Paraformaldehyde, Stephanini's fixative) the eggshells were punctured with tungsten needles. We noted some variability of developmental time for certain embryonic stages, which we cannot explain. Developmental stages of Hoploplana inquilina were collected at the Marine Biological Laboratory, Woods Hole, MA, USA according to the procedure outlined in Boyer (1987) and Boyer (1989). They have been timed in relation to normal developmental time to an early Müller's larva at about 100 hours.     

Life cycle and postembryonic development of Oochoristica anolis (Cyclophyllidea: Linstowiidae)

Gravid proglottids of Oochoristica anolis from naturally infected anole lizards, Anolis carolinensis, were placed in covered Petri dishes with laboratory-reared beetles, Tribolium confusum and Tenebrio molitor. After maintenance at 25 C, metacestodes developed in 29 of 61 T. confusum (48%), but in none of 5 T. molitor. Beetles contained from 1 to 22 metacestodes (means = 3.3), which were fully developed by day 40 postexposure. A primary lacuna was never observed, but the possibility of its presence could not be ruled out without histological study. No cercomer was formed and metacestodes retained larval hooks throughout development. Scolices were invaginated at removal from the hemocoel, but usually evaginated quickly in Ringer's. On day 60 postexposure, metacestodes were fed by stomach tube to 5 anoles, 2 lacertid lizards (Podarcis muralis) and 2 mice. Worms developed only in anoles, 3 of which were infected upon examination. Oncospheral hooks were present in worms after 7 days development in the lizard; a median excretory pore was present at the posterior tip of all stages examined, including the terminal mature proglottid of a worm after 105 days in a lizard. Scolex growth rate was linear throughout metacestode and adult development, but growth rate in body length was diphasic, punctuated by change of hosts, associated with strobilization. Attempts to establish parenteral infections in anoles were unsuccessful. Present data constitute the most complete life history study thus far for any species of Oochoristica.     

The functional morphology of the musculature of squid (Loliginidae) arms and tentacles

The arms and tentacles of squid (Family Loliginidae: Sepioteuthis sepioidea (Blainville), Loligo pealei (LeSueur), Loligo plei (Blainville), Loliguncula brevis (Blainville)) do not possess the hardened skeletal elements or fluid-filled cavities that typically provide skeletal support in other animals. Instead, these appendages are made up almost entirely of muscle. It is suggested here that the musculature serves as both the effector of movement and as the skeletal support system itself. High-speed movie recordings were used to observe prey capture by loliginid squid. Extension of the tentacles (1 pair) during prey capture is probably brought about by contraction of transverse muscle fibers and circular muscle fibers. Contraction of longitudinal muscle fibers causes retraction of the tentacles. Torsion of the tentacles during extension may be the result of contraction of muscle fibers arranged in a helical array. The inextensible but manipulative arms (4 pairs) may utilize a transverse muscle mass to resist the longitudinal compression caused by contraction of the longitudinal muscles which bend the arms. A composite connective tissue/muscle helical fiber array may twist the arms.     

Comparative morphology of statocysts in the Plathelminthes and the Xenoturbellida

The general fine-structural organization of statocysts in Catenulida, Nemertodermatida, Acoela, Proseriata, Lurus (Dalyellioida), and Xenoturbella are summarized. In lithophorous (statocyst-bearing) members of the Catenulida, the statocysts exhibit a few parietal cells and one or several movable statoliths within a spacious intracapsular cavity. Statocysts in the Nemertodermatida have several parietal cells and two lithocytes, each equipped with one statolith, whereas those of the other acoelomorphan taxon, the Acoela, always have two parietal cells and one movable lithocyte. The statocysts of lithophorous members of the Proseriata represent more sophisticated systems: each has two clusters of accessory cells in addition to several parietal cells and a voluminous lithocyte in which the statolith is movable. In catenulids and proseriates, processes of outer neurons penetrate the capsule of the statocyst, whereas such innervations have not been found in the Nemertodermatida and Acoela. I conclude that the different types of statocysts have evolved independently within the Plathelminthes. Xenoturbella displays an intraepidermal statocyst with many monociliary parietal cells and several mobile cells (lithocytes) within the central cavity of the statocyst. Each of these mobile cells carries a statolith-like structure and one prominent cilium. The statocyst of Xenoturbella does not correspond to any type of plathelminth statocyst.     

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.     

On the development of body-wall musculature Diplostomum chromatophorum metacercariae (Trematoda: Diplostomidae)

The body-wall musculature of invasive cercariae and metacercariae of Diplostomum chromatophorum at different intervals after the penetration into the experimental intermediate host Cyprinus carpio (1, 3, 5, 6, 7, 10, 12, 20, 34, 40 days) has been investigated with the help of TEM technique. During the first 10 days after the invasion (in conditions of our experiment), the cercarial subtegumental muscle fibres degenerate. These muscles are replaced by newly formed ones. Mass differentiation of myoblasts beneath the tegument was observed in 7-10-day-old metacercariae. Obtained data indicate the metamorphosis of body-wall musculature during the morphogenesis in Diplostomum chromatophorum metacercariae.     

Morphogenesis of Pseudopallene sp. (Pycnogonida, Callipallenidae) II: postembryonic development

Pycnogonida (sea spiders) are bizarre marine arthropods that are nowadays most frequently considered as being the sister group to all other chelicerates. The majority of pycnogonid species develops via a protonymphon larva with only three pairs of limbs affiliated with the future head region. Deviating from this, the hatching stage of some representatives shows already an advanced degree of trunk differentiation. Using scanning electron microscopy, fluorescent nucleic staining, and bright-field stereomicroscopy, postembryonic development of Pseudopallene sp. (Callipallenidae), a pycnogonid with an advanced hatching stage, is described. Based on external morphology, six postembryonic stages plus a sub-adult stage are distinguished. The hatching larva is lecithotrophic and bears the chelifores as only functional appendage pair and unarticulated limb buds of walking leg pairs 1 and 2. Palpal and ovigeral larval limbs are absent. Differentiation of walking leg pairs 3 and 4 is sequential. Apart from the first pair, each walking leg goes through a characteristic sequence of three externally distinct stages with two intermittent molts (limb bud-seven podomeres-nine podomeres). First external signs of oviger development are detectable in postembryonic stage 3 bearing three articulated walking leg pairs. Following three more molts, the oviger has attained adult podomere composition. The advanced hatching stages of different callipallenids are compared and the inclusive term "walking leg-bearing larva" is suggested, as opposed to the behavior-based name "attaching larva". Data on temporal and structural patterns of walking leg differentiation in other pycnogonids are reviewed and discussed. To facilitate comparisons of walking leg differentiation patterns across many species, we propose a concise notation in matrix fashion. Due to deviating structural patterns of oviger differentiation in another callipallenid species as well as within other pycnogonid taxa, evolutionary conservation of characteristic stages of oviger development is not apparent even in closely related species.     

Ecdysteroid titres during postembryonic development of Sarcophaga bullata (Sarcophagidae: Diptera)

The level of ecdysteroids in Sarcophaga bullata was determined by radioimmunoassay (RIA) from the time of larviposition (0 hr) until adult eclosion. Five distinct peaks of ecdysteroid activity were recorded. The first two, which occurred midway through the duration of the stadia (14 and 30 hr, respectively), resulted in larval/larval moults (24 and 44 hr). The third peak of ecdysteroid activity commenced at 131 hr and was associated with formation of the white prepuparium. The fourth peak was sustained over a long time period (from 79 hr post pupariation to 120 hr) and resulted in pupal/adult apolysis and the definition of the adult form. The last elevation of the ecdysteroid titre at approx. 160 hr post pupariation) was associated with the synthesis and secretion of adult cuticle.     

Visualizing the postembryonic development of Sarcophaga peregrina (flesh fly) by NMR microscopy

The postembryonic development of the flesh fly was studied using high resolution nuclear magnetic resonance imaging. Because this development occurs in a puparium, this process cannot be observed directly using standard histological techniques. The remodelling of histolysing larval tissues to developing imaginal tissues including the yellow body, a transient alimentary structure, and the integration of the developed adult structure were revealed in the images. Most surprisingly it was found that a large gas space that forms in the central region of the prepupa moves to the dorso-anterial region in less than 5 min due to the larval–pupal apolysis together with separation of the developing pupal epidermis from the puparium.     

New data concerning postembryonic development in Antarctic Ammothea species (Pycnogonida: Ammotheidae)

In this paper, new data on larval and postlarval stages after newly collected and museum-deposited material of six Ammothea species is provided and compared with previously known information. Different developmental stages attached to the ovigerous legs of adult males for each species were found: four stages [protonymphon (Ptn), postlarval instar 1 (PL-1), postlarval instar 2 (PL-2), and postlarval instar 3 (PL-3)] for A. carolinensis; just one (Ptn) for A. clausi and A. minor; three stages (Ptn, PL-1, PL-2) for A. bicorniculata and A. spinosa; and other three (Ptn, PL-2, PL-3) for A. longispina. In the present contribution, the external morphology of each larval and postlarval instar is described, illustrated, and discussed. The larval and postlarval development of Ammothea bicorniculata, A. carolinensis, A. longispina, and A. spinosa is characterized by (1) the eggs hatch as a protonymphon larva; (2) the larvae and subsequent postlarval stages have yolk reserves and a relatively large size (0.5–0.85 mm in length for the protonymphon); (3) the postlarvae remain on the ovigerous legs of males during several moults; (4) the spinning spine is absent; and (5) the development of walking legs is sequential. The protonymphon larva of A. clausi and A. minor is the only stage on the ovigerous legs of males, and this stage is characterized by: (1) there is no yolk reserve and it has a relatively small size (0.22–0.3 mm in length); (2) the spinning spine is present; and (3) all larval appendages have a relatively large size.     

The morphology of the intrinsic tongue musculature in snakes (Reptilia,ophidia): Functional and phylogenetic implications

Tongue musculature in 24 genera of snakes was examined histologically. In all snakes, the tongue is composed of a few main groups of muscles. The M. hyoglossus is a paired bundle in the center of the tongue. The posterior regions of the tongue possess musculature that surrounds these bundles and is responsible for protrusion. Anterior tongue regions contain hyoglossal bundles, dorsal longitudinal muscle bundles and vertical and transverse bundles, which are perpendicular to the long axis of the tongue. The interaction of the longitudinal with the vertical and horizontal muscles is responsible for bending during tongue flicking. Despite general similarities, distinct patterns of intrinsic tongue musculature characterize each infraorder of snakes. The Henophidia are primitive; the Scolecophidia and Caenophidia are each distinguished by derived characters. These derived characters support hypotheses that these latter taxa are each monophyletic. Cylindrophis (Anilioidea) is in some characters intermediate between Booidea and Colubroidea. The condition in the Booidea resembles the lizard condition; however, no synapomorphies of tongue musculature confirm a relationship with any specific lizard family. Although the pattern of colubroids appears to be the most biomechanically specialized, as yet no behavioral or performance feature has been identified to distinguish them from other snakes.     

Comparative morphology of the thorax musculature of adult Anisoptera (Insecta: Odonata): Functional aspects of the flight apparatus

Due to their unique flight mechanism including a direct flight musculature, Odonata show impressive flight skills. Several publications addressed the details of this flight apparatus like: sclerites, wings, musculature, and flight aerodynamics. However, 3D-analysis of the thorax musculature of adult dragonflies was not studied before and this paper allows for a detailed insight. We, therefore, focused on the thorax musculature of adult Anisoptera using micro-computed tomography. Herewith, we present a comparative morphological approach to identify differences within Anisoptera: Aeshnidae, Corduliidae, Gomphidae, and Libellulidae. In total, 54 muscles were identified: 16 prothoracic, 19 mesothoracic, and 19 metathoracic. Recorded differences were for example, the reduction of muscle Idlm4 and an additional muscle IIIdlm1 in Aeshna cyanea, previously described as rudimentary or missing. Muscle Iscm1, which was previously reported missing in all Odonata, was found in all investigated species. The attachment of muscle IIpcm2 in Pantala flavescens is interpreted as a probable adaption to its long-distance migration behaviour. Furthermore, we present a review of functions of the odonatan flight muscles, considering previous publications. The data herein set a basis for functional and biomechanical studies of the flight apparatus and will therefore lay the foundation for a better understanding of the odonatan flight.     

Ultrastructural observations on the development of male gametes in Allostoma sp. (Plathelminthes,Prolecithophora)

In the prosobranch snail Littorina littorea (L., 1758) the ultrastructure of the prostate gland cells (pgc) in males and the altered glandular epithelium of the pallial oviduct of females in intersex stage 3 is compared. Regarding form, structure, organelles and secretory products the pgc in males are identical with the corresponding gland cells of the females. Consequently these results demonstrate that in females of intersex stage 3 the epithelium of the pallial oviduct, which originally consists of several (3) glandular parts, is transformed into a male prostate gland.