Are there differences between proseriates and lower flatworms in ultrastructure of the nervous system?

The ultrastructure of the CNS (central nervous system) in three species of the Proseriata — Archiloa unipunctata (O. Fabricius), Bothriomolus balticus (Meixner), and Promonotus schultzei (Meixner) — was studied and compared to that of the lower flatworms Stenostomum leucops (Catenulida) and Microstomum lineare (Macrostomida) in material available from our previous studies. Conventional electron microscopical fixation was used for A. unipunctata and B. balticus; the tannic-acid-incubation method (TARI), a method that reveals especially nonsynaptic exocytotic release of neuronal substances, was applied in study of P. schultzei.In general, the ultrastructural features of neurons and nerve fibers were similar in proseriates and lower flatworms. A striking feature common to both groups was the secretory appearance of all neurons. A significant difference between them is the occurrence, in the proseriates, of wrapping of neurons by glial cells. Heterogeneity in the population of neuronal vesicles and in structure of synapses in the proseriates are probably advanced characters; by contrast, S. leucops has relatively homogeneous vesicles and simple synapses. A gradual advancement from the state in the catenulids through that in the macrostomids to the proseriates seems to be reflected in the differentiation of synapses and the variability of neuronal vesicles. This probably reflects differences in functional demands but also evolutionary advancement.     

Catecholamines demonstrated by glyoxylic-acid-induced fluorescence and HPLC in some microturbellarians

The microturbellarians Stenostomum leucops (Catenulida), Microstomum lineare (Macrostomida), Promonotus schultzei (Proseriata) and Gyratrix hermaphroditus (Rhabdocoela) were subjected to the glyoxylic-acid-induced-fluorescence method to reveal catecholaminergic parts of their nervous systems. Histofluorescence indicative of catecholamines was evident in nerve cells and fibers of all investigated species. The patterns of distribution were compared to those of other neuroactive substances. The catecholaminergic fluorescence was at least partly confined to separate neuronal subsets in S. leucops and M. lineare and probably also in P. schultzei. In S. leucops, the presence of dopa and dopamine was demonstrated by high-pressure liquid chromatography. The occurence of catecholamines in these representatives of four orders of microturbellarians suggests that catecholamines arose early in the evolution of flatworms and underscores the importance of dopaminergic substances for the function of early nervous systems.     

Characterization of the first mitochondrial genome of a catenulid flatworm: Stenostomum leucops (Platyhelminthes)

The first complete mitochondrial genome of a catenulid, Stenostomum leucops, was characterized. Illumina sequencing and 90 813 reads were utilized in the assembly, producing one contig with an average coverage of 1118×. The length of this genome is 15 742 bp with 12 protein‐coding, two rRNA and 22 tRNA genes. Although the atp8 gene is absent in other Platyhelminthes, a highly divergent putative atp8 gene was found in S. leucops. In contrast to other Platyhelminthes, the mitochondrial genes of S. leucops are encoded on both strands. The gene order in the S. leucops mitogenome is very divergent from those observed in other Platyhelminthes, showing only small blocks of synteny. With AAA as the codon for lysine S. leucops shows the probable plesiomorphic condition, whereas Rhabditophora possess the derived GAA. This evolutionary transition is correlated with changes in the respective anticodons in trnK. It remains unclear whether the absence of the D arm and loop in trnS1 is a convergence in Catenulida and Neodermata.     

Substance P immunoreactivity in sensory structures and the central and pharyngeal nervous system of Stenostomum leucops (Catenulida) and Microstomum lineare (Macrostomida)

Immunoreactivity (IR) obtained by monoclonal antibodies to substance P (SP) was studied in the asexually reproducing microturbellarians Stenostomum leucops and Microstomum lineare. The IR pattern was studied by confocal and ordinary fluorescence microscopy. In both species, IR occurs in the brain in peripheral cells, neuropilar fibres, in longitudinal cords and in the pharyngeal nervous system. The IR patterns reveal neuroanatomical details not observed with other neuroactive substances. In both species, immunopositive cells send fibers to the ciliary pits. In M. lineare, additional fibres run to more frontally located sensory structures. In S. leucops, two pharyngeal nerve rings are visualized. The pharyngeal nerve ring close to the surface associated with symmetrical immunopositive cell pairs is demonstrated for the first time, while the deeper-lying pharyngeal nerve ring has been previously demonstrated by antibodies to the molluscan cardioactive peptide FMRF-amide. Two cells with strong IR are connected by short fibres to the pharyngeal nerve ring in M. lineare. In the developing new individuals, i.e., the zooids of M. lineare, IR to SP is first revealed in nerve fibres growing out from parental lateral nerve cords towards the centre of the worm where the new brain commissure will appear. Immunopositive cells in the brain periphery and close to the developing ciliary pits appear later. Simultaneous staining by antibodies to SP and 5-HT shows that IR to SP appears later than IR to 5-HT.     

The turbellaria fauna of a small water reservoir in Central Russia

The Turbellaria fauna has been studied in the Uvod’ Reservoir. A total of 18 species have been found representing three orders: Catenulida, Macrostoimida, and Neorhabdocoela. The patterns of Turbellaria distribution in the reservoir have been analyzed. The most abundant species have been determined: Stenostomum leucops Duges, 1828, Microstomum lineare (O.F. Müller, 1774), Bothromesostoma essenii M. Braun, 1885, and Mesostoma lingua (Abildgaard, 1789).     

The organization of the nervous system in Plathelminthes. The neuropeptide F-immunoreactive pattern in Catenulida,Macrostomida, Proseriata

The organization of the nervous system of Archilopsis unipunctata Promonotus schultzei and Paramonotus hamatus (Monocelididae, Proseriata) and Stenostomum leucops (Catenulida) and Microstomum lineare (Macrostomida) was studied by immunocytochemistry, using antibodies to the authentic flatworm neuropeptide F (NPF) (Moniezia expansa). The organization of the nervous system of the Monocelididae was compared to that of the nervous system of Bothriomolus balticus (Otoplanidae), a previously studied species of another family of the Proseriata. The results show that the main nerve cords (MCs), independent of lateral or ventral position in the Monocelididae and the Otoplanidae, correspond to each other. The study also confirms the status of the lateral cords as main cords (MCs) in S. leucops and M. lineare. Common for MCs in the members of the investigated taxa are the following features: MCs consist of many fibres, originate from the brain and are adjoined to 5-HT-positive neurons. In Monocelididae and Otoplanidae, the MCs additionally have the same type of contact to the pharyngeal nervous system. Also common for both proseriate families is the organization of the two lateral nerve cords, with weaker connections to the brain, and the pair of dorsal cords running above the brain. The organization of the minor cords differs. The Monocelididae have a pair of thin ventral cords forming a mirror image of the dorsal pair. Furthermore, an unpaired ventral medial cord connecting medial commissural cells was observed in P. schultzei. Marginal nerve cords, observed in Otoplanidae, are absent in Monocelididae. All minor nerve cords are closely connected to the peripheral nerve plexus. The postulated trends of condensation of plexal fibres to cords and/or the flexibility of the peripheral nerve plexus are discussed. In addition, the immunoreactivity (IR) pattern of NPF was compared to the IR patterns of the neuropeptide RFamide and the indoleamine, 5-HT (serotonin). Significant differences between the distribution of IR to NPF and to 5-HT occur. 5-HT-IR dominates in the submuscular and subepidermal plexuses. In the stomatogastric plexus of M. lineare, only peptidergic IR is observed in the intestinal nerve net. The distribution of NPF-IR in fibres and cells of the intestinal wall in M. lineare indicates a regulatory function for this peptide in the gut, while a relationship with ciliary and muscular locomotion is suggested for the 5-HT-IR occurring in the subepidermal and submuscular nerve, plexuses. In M. lineare, the study revealed an NPF- and RFamide-positive cell pair, marking the finished development of new zooids. This finding indicates that constancy of these cells is maintained in this asexually reproducing and regenerating species.     

Neurohistochemistry in molluscan species: focus on extracellular matrix

Carbohydrate polymers with acid?Cbase properties of extracellular matrix (ECM) have a critical impact on guidance, migration and synaptic transmission. The present study showed acid?Cbase properties of ECM stained with histochemical techniques. The central nervous system (CNS) of mature terrestrial slug Semperula maculata and freshwater bivalve Lamellidens corrianus was selected to visualize sulphated and carboxylic strong acid groups. Protein and carbohydrate molecules were found scattered in three different regions, viz. periganglionic sheath, along with the basement lamina, interperikaryal space including the perineuronal net, and neuropil, in both the molluscan species. Sulphated proteoglycans were abundant in periganglionic sheath of S. maculata and also occurred in the neuropil of L. corrianus. The interperikaryal space contained carboxyl groups along with hyaluronic acid, and neuropil contained abundant glycogen moiety in both species. Comparing the SDS?CPAGE of the neural tissue extracts revealed protein molecules that were found in the same fraction. The ECM of neuronal cells from S. maculata and L. corrianus presented different kinds of glycoproteins and proteoglycans, which showed its morphological as well as habitual differentiation.     

Growth factors in asexually reproducing catenulida and macrostomida (Plathelminthes)?

Summary The occurrence and localization of immuno-reactivity (IR) to epidermal growth factor receptor (EGF-r), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), revealed by the present study, indicate growth-factor-like substances in the asexually reproducing flatworms,Stenostomum leucops andMicrostomum lineare. IR to all three antibodies occurs in the nervous system. Differences in the number of positive cells and intensity of IR during asexual development of new individuals were observed. By confocal scanning laser microscopy, immunopositive growth cones of nerve fibres were seen in developing zooids, and weakly or unstained perikarya were observed in close contact with the positive nerve fibres. Antibodies to the growth factors EGF-r, EGF and bFGF as well as to the neuroactive substances 5-HT and RF-amide had a negative influence on the growth and asexual reproduction of culturedS. leucops. No significant differences in the influence of antibodies to growth factors and antibodies to the neuroactive substances were observed.     

Fine Structural Observations on the Turbellarians Stenostomum sp. and Microstomum lineare with Special Reference to the Extracellular Matrix and Connective Tissue Systems

Employing transmission electron microscopy, observations were made on epidermis, muscle cells and connective tissue systems, with special emphasis on extracellular matrix components (ECM), in two rather primitive turbellarians: Stenostomum sp. (Catenulida) and Microstomum lineare (Macrostomida). In Stenostomum the only ECM components found are basal laminae, predominantly situated subepidermally. In Microstomum ECM is well developed and connective tissue filaments abundant in conspicuous extracellular spaces. It is uncertain whether basal laminae exist. The finding of basal lamina structures as the only ECM component present in Stenostomum makes it now possible to establish a complete ECM and connective tissue hierarchy in turbellarians, ranging from a purely cellular type with no ECM present to systems dominated by ECM and very similar to loose connective tissue in vertebrates. Comparative aspects of ECM and connective tissue systems in turbellarians are discussed in addition to the difficulties and ambiguities regarding definition and nomenclature of basal matrices as basal laminae and subepidermal membranes.     

Stem cells in microturbellarians

Summary A combination of microscopical, immunocytochemical, and autoradiographic techniques were employed to study stem cells and their fates during asexual reproduction and regeneration in two microturbellarians,Microstomum lineare (Macrostomida) andStenostomum leucops (Catenulida). Special attention was paid to the development of the immunoreactivity (IR) to FMRF/RF-amide and 5-HT in differentiating nerve cells.Asexual reproduction inM. lineare andS. leucops occurs by paratomy, i.e., fragmentation after completed differentiation of the new organs. Regeneration, on the other hand, involves a combination of morphallactic and epimorphic processes without the formation of a regeneration blastema. The only cells incorporating tritiated thymidine ([³H]T) were the mesenchymal and gastrodermal neoblasts, which proliferate continuously replenishing the population of stem cells available for growth, asexual reproduction and regeneration. These proliferative cells occurred in two ultrastructurally different forms, differing from each other only by the presence or absence of ciliar basal bodies in the cytoplasm. Few differentiated cells were labeled in the head piece after completed regeneration. A greater amount of labeled differentiated cells were, however, observed postpharyngeally in the first zooid as well as in zooids having developed during the same time (i.e., 20–45 h after the treatment with [³H]T). Furthermore, many labeled cells were still undifferentiated at that time or just in the beginning of the differentiation process. It can therefore be concluded that neoblasts function both as reserve cells and as functional stem cells for all differentiated cell types in these worms. IR to FMRF/RF-amide neuropeptides was not observed in nerve cells differentiating from neoblasts until the occurrence of dense-core vesicles in their cytoplasm. Due to methodological difficulties only weak or no IR to 5-HT could be traced in the nervous system of the asexual and regenerating worms.Abbreviations ICC Immunocytochemical - IR immunoreactivity - [³H]T tritiated thymidine     

Neural crest cell migration in relation to extracellular matrix organization in the embryonic axolotl trunk

Temporal and regional aspects of early neural crest cell migration in relation to extracellular matrix (ECM) organization and distribution in the embryonic axolotl trunk were studied by light microscopy, TEM, and SEM. The dominating structure of the interstitial ECM is a complex network of fibrils, which are indicated by ruthenium red staining to consist of collagen in association with ruthenium red-positive components, probably including glycosaminoglycans. The ECM fibrils, which are largely used as substratum for locomotion by the crest cells, have a temporally and regionally specific organization and distribution. Increase in ECM fibrils on the neural tube, ahead of the crest cell front, is correlated with initiation of crest cell emigration, and it is suggested that the fibrils may stimulate this process by providing a suitable substratum for cell locomotion. An increase in ECM fibrils in extracellular spaces surrounding the crest cell population is correlated with an expansion of these spaces and with progressing crest cell migration into them. It is proposed that the spatial organization of the ECM fibrils influences crest cell shape and orientation during early migration.     

Ultrastructure of the subepidermal musculature of Xenoturbella bocki, the adelphotaxon of the Bilateria

 Xenoturbella bocki is the only species of the high-ranked taxon Xenoturbellida. The species lives on marine mud bottoms at a depth of 20–120 m and moves extremely slowly by ciliary gliding. Nevertheless it possesses a well-developed body wall musculature with outer circular muscles, a prominent layer of inner longitudinal muscles and radial muscles that extend from the outer circular myocytes to the musculature surrounding the gastrodermis. The longitudinal myocytes are not compact cells, but form fascicles of fibrils running parallel to each other. Fine cytoplasmic cords connect the fibres of a cell to each other and with its nuclear region. The muscles are embedded within a sometimes expansive extracellular matrix (ECM) that lacks any fibrillar components. All muscle cells display conspicuous and numerous cytoplasmic extensions that are intermingled with each other. Tight coupling between adjacent cell membranes is not found, but zonula adhaerens-like junctions exist. Fibrils belonging to different myocytes, but also fibrils of the same cell, are coupled by such cytoplasmic extensions. Circular, radial and at least the peripheral longitudinal myocytes display cell-matrix connections with the internal lamina, a component of the subepidermal ECM. This internal lamina projects down into the centres of the fascicles with longitudinal muscle fibrils and forms extensive attachment zones with the muscle cells, reminiscent of focal contacts. For the ingestion of food, X. bocki opens the simple mouth pore and protrudes the aciliated gastrodermis. The body wall musculature is responsible for this protrusion and also for the withdrawal of the gastrodermis. In the past, possible phylogenetic kinships with the Acoelomorpha (Plathelminthes) or the Enteropneusta and Holothuroidea were discussed, but, on the basis of all information available, X. bocki is hypothesized to be the sister taxon of the Bilateria. Accepted: 2 April 1997     

Extracellular matrix regulation of fibroblast function: redefining our perspective on skin aging

The dermal extracellular matrix (ECM) comprises the bulk of skin and confers strength and resiliency. In young skin, fibroblasts produce and adhere to the dermal ECM, which is composed primarily of type I collagen fibrils. Adherence allows fibroblasts to spread and exert mechanical force on the surrounding ECM. In this state, fibroblasts display a “youthful” phenotype characterized by maintenance of the composition and structural organization of the dermal ECM. During aging, fibroblast-ECM interactions become disrupted due to fragmentation of collagen fibrils. This disruption causes loss of fibroblast spreading and mechanical force, which inextricably lead to an “aged” phenotype; fibroblasts synthesize less ECM proteins and more matrix-degrading metalloproteinases. This imbalance of ECM homeostasis further drives collagen fibril fragmentation in a self-perpetuating cycle. This article summarizes age-related changes in the dermal ECM and the mechanisms by which these changes alter the interplay between fibroblasts and their extracellular matrix microenvironment that drive the aging process in human skin.     

On the fine structure of the cerebral ganglion of Sagitta (Chaetognatha)

Summary In this study the fine structure of the cerebral ganglion of Sagitta setosa (Chaetognatha) is investigated. The ganglion is flat and superficially positioned dorsally, below the basal lamina of the cephalic epidermis. It is surrounded by a specifically differentiated sheath. This sheath is made up of cells, which are interpreted as representing glial cells, and can be divided into an outer and an inner zone. The outer zone is composed of flat sheath cells with pale nuclei and few organelles. The inner zone consists of densely packed, extremely thin lamellar cellular processes. These attenuated lamellae, which still contain cytoplasm, resemble the myelin sheath of vertebrate axons. The intercellular space between the lamellae contains electron-dense material. In the sheath specialized intercellular contacts occur. The inner zone of this sheath extends at definite points into the centre of the ganglion and separates a zone of perikarya from the neuropil, as well as the single perikarya from each other. The perikarya are relatively uniform and do not form a cortex, but are concentrated mainly in lateral parts of the cerebral ganglion. Within the neuropil are axonal endings which have synaptic contacts with several postsynaptic elements. These anatomical findings are discussed with respect to their functional significance.     

Role of the extracellular matrix in epithelial morphogenesis

The extracellular matrix (ECM) plays an essential role in organizing tissues, defining their shapes or in presenting growth factors. Their components have been well described in most species, but our understanding of the mechanisms that control ECM remodeling remains limited. Likewise, how the ECM contributes to cellular mechanical responses has been examined in few cases. Here, I review how studies performed in C. elegans have brought several significant advances on those topics. Focusing only on epithelial cells, I discuss basement membrane invasion by the anchor cell during vulva morphogenesis, a process that has greatly expanded our knowledge of ECM remodeling in vivo. I then discuss the ECM role in a novel mechanotransduction process, whereby muscle contractions stimulate the remodeling of hemidesmosome-like junctions in the epidermis, which highlights that these junctions are mechanosensitive. Finally, I discuss progress in defining the composition and potential roles of the apical ECM covering epidermal cells in embryos.     

The importance of non-pollen plant parts as food sources for the common blossom thrips, Frankliniella schultzei

Like other flower thrips, Frankliniella schultzei Trybom (Thysanoptera: Thripidae) feeds on pollen. However, the influence of a pollen diet on the life history of F. schultzei may not be as significant as reported for other thrips species. Frankliniella schultzei was reared successfully and with low mortalities (20%) on Wax Mallow, (Malvaviscus arboreus Cav.) plant part diets. Development times and fecundity on a petal diet were not significantly different from that on a pollen diet. Fecundity on a diet combining M. arboreus pollen, petal and leaf tissues was significantly higher than those individually containing these tissues. In laboratory choice tests, F. schultzei females encountered petal most often of the three plant parts. Pollen and leaf were encountered with similar frequencies. A significantly higher proportion of petal encounters (0.8) resulted in feeding than did pollen encounters (0.5). Few leaf encounters (0.1) were followed by feeding. Adult and larval F. schultzei were found in M. arboreus flowers but not on leaves. All parts of the flower were inhabited and not just the pollen-bearing petal apices and anthers. We propose that feeding on pollen within M. arboreus flowers is just one of many influences on the life history of F. schultzei and suggest that this may extend to other thrips species/host-plant combinations.     

Mating Behavior and Species Status of Host-Associated Populations of the Polyphagous Thrips, <Emphasis Type="Italic">Frankliniella schultzei</Emphasis>

The thrips taxon, Frankliniella schultzei Trybom, could possibly comprise a complex of unrecognised cryptic species. We therefore made a range of reproductive behavioral observations on F. schultzei individuals from different host-associated populations to investigate their species status. We first described the mating behavior of F. schultzei taken from flowers of a primary host species, Malvaviscus arboreus. The pattern of male-female interaction was complex and protracted, with several behavioral steps being performed. These steps were similar for all mating pairs. Most females responded to male presence before physical contact, suggesting that male pheromones may be involved. We then compared mating behaviors, brood sizes and sex ratios of F. schultzei pairs derived from different host plant species. All pairs in crosses regardless of their original host plant species produced female offspring, indicating successful mating. Frequencies of behaviors were similar for all pairs. Only those crosses involving F. schultzei individuals from Erythrina crista-galli flowers differed from the general pattern in pre-copulation and copulation times. The females from E. crista-galli also produced significantly fewer offspring than females from other crosses. Finally we used traps baited with males from two populations, but set among flowers of one host plant species, to test for differential attraction of females. We found no evidence that the different host-associated populations investigated differ from one another functionally in their sex pheromone system. However, these results do not yet discount the possibility of cryptic species within the taxon F. schultzei. Populations of F. schultzei on many other host species need to be investigated from this perspective.     

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.