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.     

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.     

Flagellar structure of the gametes of Ectocarpus siliculosus (Phaeophyta) as revealed by negative staining

Summary From light microscopic observations of the mating reaction in the marine brown alga Ectocarpus siliculosus it can be deduced that the foremost part of the front flagellum of the male gamete plays an important role in establishing initial sexual contact. The flagellar apparatus of both male and female isogametes of this organism has been studied electron microscopically using negative staining technique, with special attention focused onto the whiplash tips (acronema). The fine structure of this terminal flagellar appendage, which is very long (about 10 m) and extremely sensitive to mechanical stress, is identical in structure in both, the tinsel and the smooth (hind) flagellum. It is characterized by a continuation of only the two central microtubules of the flagellar shaft which appear cross-bridged to each other and are ensheathed by the plasma membrane. Although the two types of gametes are different in their functions, differences in the fine structure of their flagellar apparatus were not detected.     

Ultrastructure of the spermatozoon of the earthworm as revealed by negative staining

This is the first demonstration of an apparent acrosome reaction by the use of negative staining (phosphotungstic acid) and electron microscopy. It is also the first report of such a reaction in spermatozoa of an oligochaete annelid (Lumbricus terrestris). The morphology of the negatively stained unreacted acrosomal complexes is entirely comparable to that seen in sectioned material. After reaction, a short straight filament is extruded and the compartmentalization seen in unreacted spermatozoa is no longer visible. In the flagellar axoneme there are short cross-bridges linking the two central singlet microtubules at 150–170 Å intervals. Two fibrous elements are also present, forming, with the singlets, a tetragon. All four of these structures apparently change their elastic properties after incubation in distilled water for two hours, so that instead of following the doublet microtubules in gentle coils, the central complex components fall as a unit in jagged configurations.     

Properties of the myosin molecule revealed by negative staining

Aspects of the structure of myosin molecules from striated muscle as revealed by negative staining are reviewed. These include curvature and domain structure in the heads and flexibility in the tail and its connection to the heads. The effects of various factors on observed structure of the molecule are discussed, including radiation damage, removal of the light polypeptide chains, elevations in temperature, and variations in bathing medium composition.     

A new species of land planarian from Madeira (Platyhelminthes, Turbellaria, Tricladida)

A new species of terrestrial planarian from Madeira, Kontikia bulbosa sp. nov. , is described. The genus Kontikia is redefined and its scope and status reviewed.     

Ultrastructural alterations of the mitochondrial ATPase in the calcium paradox as revealed by negative staining

We have used a simple negative staining technique to study the structural alterations of mitochondria from biopsies of hearts subjected to the calcium paradox and treatment with diltiazem, a calcium channel blocker. A significant (P less than 0.05) decrease in the number of spheres on the mitochondrial membranes occurs during the calcium paradox (58.0 +/0 4.1/micrometer vs. control 80.5 +/- 6.5). Treatment with diltiazem prevented the loss of spheres from mitochondrial membranes during the calcium paradox (75.5 +20 5.0 micrometer). We found that this negative staining technique can be used for quick assessment of the condition of mitochondria in biopsies from normal and pathological organs.     

The First Record of the Microturbellarian Macrostomum longituba Papi 1953 (Platyhelminthes,Rhabditophora) in Russian Freshwater Fauna

Biology Bulletin - Macrostomum longituba (Platyhelminthes, Rhabditophora, Macrostomorpha), formerly known as a representative of the Mediterranean brackish water complex, is recorded for the first...     

Morphometry of the eyes in regenerant of genus Dugesia (Platyhelminthes, Turbellaria, Dugesiidae).

The photodocumentation technique was used weekly to observe until the end of the regenerative process of eyes in three species of Dugesia. Morphometric data based on the measurement of 96 samples of the species: Dugesia tigrina, D. schubarti, and D. anderlani were analyzed. A comparative analysis was made through graphic representation of statistical parameters of the following morphometric characters: the length of the transversal and longitudinal axes of the macula alba, pigment spot, and ocular opening. An interesting feature appeared in D. schubarti: the average of ocular opening during the regenerative process is bigger than in the original samples.     

The brain of the Nemertodermatida (Platyhelminthes) as revealed by anti-5HT and anti-FMRFamide immunostainings

The taxa Nemertodermatida and Acoela have traditionally been considered closely related and classified as sister groups within the Acoelomorpha Ehlers 1984 (Platyhelminthes). Recent molecular investigations have questioned their respective position. In this study, the 5-HT and FMRFamide immunoreactivity (IR) in the nervous system of two nemertodermatids, Nemertoderma westbladi and Meara stichopi, is described. The 5-HT immunoreactive pattern differs in the two nemertodermatids studied. In M. stichopi, two loose longitudinal bundles of 5-HT-immunoreactive fibres and an basi-epidermal nerve net were observed. In N. westbladi the 5-HT-IR shows a ring-shaped commissural structure, different from the commissural brain of acoels. In both nemertodermatids, FMRFamide immunoreactive nerve fibres followed the 5-HT-immunoreactive fibres. It is demonstrated that the Nemertodermatida have neither a 'commissural brain' structure similar to that of the Acoela, nor a 'true', ganglionic brain and orthogon, typical for other Platyhelminthes. The question of the plesiomorphic or apomorphic nature of the nervous system in Nemertodermatida cannot yet be answered. The neuroanatomy of the studied worms provides no synapomorphy supporting the taxon Acoelomorpha.     

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.     

Notes on the biology of the freshwater planarian Dugesia bengalensis (Platyhelminthes Turbellaria Tricladida)

Dugesia bengalensis was described by Kawakatsu (Kawakatsu et al., 1983) from specimens collected in West Bengal. We have been studying populations from many different localities in Santiniketan and adjoining areas of West Bengal and can provide additional biological information.The species is hermaphroditic, and its breeding season was found to occur usually between October and March when the winter temperature falls below 25 °C. Outside of the breeding season, D. bengalensis is capable of asexual reproduction by binary fission (Mahapatra et al., 1987).Development of the reproductive organs appeared to be from neoblasts and other mesenchymal cells and, therefore, to be like that typical of other triclads with the exception that some of the neoblasts used for the reproductive tissue appeared to be derived from the intestinal region (Ghosh, 1988; cf. Teshirogi, 1986).During copulation, the partners were oriented in the same direction and not in a head-to-tail position as has been reported (Hyman, 1945) for some planarians.The oval, stalked cocoons were laid in marshy places, and during the period of summer (usually from April to June) they lay dormant in the sandy soil until the onset of the monsoon rains. Then, typically three or four months after they were laid, the cocoons hatched to yield three or four young, a remarkably low number for freshwater triclads (cf Ball & Reynoldson, 1981).     

Nuclei in the plasmodium of Intoshia variabili (Orthonectida) as revealed by DAPI staining

DAPI staining of wholeamounts was used to reveal the parasitic plasmodium of the orthonectid Intoshia variabili in its host, the turbellarian Macrorhynchus crocea. The nuclei of the parasite differ drastically from those of the host in size, morphology, and the estimated DNA content. Our findings indirectly support the idea that the orthonectid plasmodium is a distinct parasitic organism, rather than modified host cells.     

A method for the preparation of high-molecular-weight DNA from marine and freshwater triclads (Platyhelminthes, Turbellaria)

Although the last decade has seen much activity devoted to the phylogeny of the Platyhelminthes, such activity has relied in the main on traditional anatomical, ultrastructural, and developmental data. Extension of these studies to the molecular level has been impeded by the lack of a reliable method for the isolation of DNA from these organisms, especially marine triclads whose DNA is particularly difficult to isolate using methods currently used with other eukaryotic or prokaryotic organisms. We report here the details of a method that results in the rapid isolation of relatively pure DNA in good yields from small amounts of material. The method involves the treatment of the animals with high concentrations of NaDodSO4 followed by phenol extraction, resulting in a product that is an excellent substrate for restriction endonuclease digestion and Southern blotting.     

A new species of land planarian from Northern Ireland (Platyhelminthes: Turbellaria)

The anatomy of Kontikia andersoni sp. nov. is described, compared to other species of the genus, and its taxonomic position briefly discussed.