Olga I. Raikova

An immunocytochemical and ultrastructural study of the nervous and muscular systems of Xenoturbella westbladi (Bilateria inc. sed.)

Abstract The phylogenetic position of the Xenoturbellida is highly disputed. Are they primitive flatworms? Are they related to Deuterostomia? Do they form a sister taxon to other Bilateria? Are they bivalve molluscs? In order to provide more data for this discussion, a study of the nervous system of Xenoturbella westbladi and its relation to the musculature was performed, using 5-HT and FMRFamide immunocytochemistry, TRITC-conjugated phalloidin fluorescence for staining of F-actin filaments, confocal scanning laser microscopy and transmission electron microscopy. The nervous system comprises solely an intraepidermal net of nerve cells and processes. No ganglia or any other internal nervous structures could be detected. No evidence of 5-HT- or FMRFamide-immunoreactive innervation below the subepidermal membrane complex was obtained. The 5-HT and FMRFamide immunoreactivity occurs in separate sets of neurones. On the ultrastructural level, three types of neurones were observed: (1) the predominating ”light” neurones, (2) the smaller ”dark” neurones and (3) the bipolar sensory neurones bearing a single cilium with a long bipartite rootlet. Non-synaptic, paracrine, release sites are common and synapses are inconspicuous. In the layer of epidermal cells, close to the lateral furrow, F-actin filaments were observed. They reach from the basal membrane to the surface. The organisation of the nervous system appears very simple. Our results are compatible with the hypothesis of Xenoturbellida forming a sister taxon to Bilateria. No evidence was obtained for inclusion of X. westbladi in either the Mollusca or Plathelminthes.

An endocrine brain? The pattern of FMRF-amide immunoreactivity in Acoela (Plathelminthes)

In order to solve the problem of whether the Acoela have a brain of a common flatworm type, an immunocytochemical study was performed of the pattern of FMRF-amide in the nervous system of four species of Acoela, Anaperus biaculeatus, Childia groenlandica, Antinoposthia beklemischevi and Mecynostomum sp. In all species a FMRF-amide positive bilobed brain-like structure lacking neuropile was observed. This brain is composed of large multipolar nerve cells with short processes. The FMRF-amide immunoreactivity appears as spots in the cytoplasm. Short lateral branches originating from the periphery of the brain were observed in Anaperus biaculeatus. No immunoreactive nerve cords were detected. The results indicate that the brain-like structure of Acoela is not homologous with the brains of other Plathelminthes and implies an independent evolution of Acoela.

Cleavage in Nemertoderma westbladi (Nemertodermatida) and its phylogenetic significance

Recent phylogenetic analyses of ribosomal and protein coding nuclear genes place the marine worms within the Nemertodermatida as one of the oldest lineages among the bilaterian animals. We studied the early embryonic cleavage in Nemertoderma westbladi to provide the first account of nemertodermatid early development. Live embryos were studied with interference microscopy and fixed embryos were either sectioned or studied with confocal laser scanning microscopy. Initially the divisions in the embryo are radial, but then micromeres are shifted clockwise generating a spiral pattern. The four-cell stage is characterized by duets of macromeres and micromeres and thus resembles the duet cleavage reported from members of the Acoela. However, subsequent stages differ from the acoel duet pattern and also from quartet spiral cleavage. The optimization of the cleavage pattern on current phylogenetic hypotheses with Nemertodermatida and Acoela as early bilaterian branches is discussed.

Acoel spermatozoa: ultrastructure and immunocytochemistry of tubulin

Acoel spermatozoa are filiform and contain two parallel axonemes, which do not show the trepaxonematan 9 + ‘1’ pattern, but instead, another kind of 9 + ‘1’ pattern, or a 9 + 0 or 9 + 2 pattern. Spermatozoa have either cortical singlet microtubules or central microtubules. Identification of these groups of microtubules and recognition of homologies between species is difficult with electron microscopy. In addition to conventional electron microscopy, indirect immunofluorescence of tubulin was performed on three species (Symsagittifera schultzei, Symsagittifera psammophila, and Actinoposthia beklemischevi). This technique facilitated understanding of the general morphology of the filiform spermatozoon and of the arrangement of the microtubular organelles along its length. We have found that different monoclonal antibodies (anti-alpha-, anti-alpha-acetylated- and anti-beta-tubulin) can distinguish distinct subcellular populations of microtubules. The axonemes were labelled by the three antibodies in all species. The cortical microtubules (in Actinoposthia beklemischevi) were labelled by the three antibodies. The central microtubules (in Symsagittifera schultzei and S. psammophila) were labelled with the anti-beta-tubulin antibody and not labelled by the anti-alpha- and anti-alpha-acetylated-tubulin. Similar experiments were performed on other Platyhelminthes and indicated that immunocytochemistry of spermatozoa may provide new characters for phylogenetic studies.

Microtubular System During Spermiogenesis and in the Spermatozoon of Convoluta saliens (Platyhelminthes, Acoela): Tubulin Immunocytochemistry and Electron Microscopy

Spermiogenesis and the spermatozoon were studied in Convoluta saliens, an acoel platyhelminth, by transmission electron microscopy, labelling of nuclei and immunocytochemistry of tubulin with various antibodies. Spermiogenesis involves formation of a long spermatid shaft containing two axonemes. It is established that the nucleus, after a stage of elongation, does not migrate up to the distal extremity of the spermatid, and that the centriolar derivatives are located at the distal extremity of the shaft. This contrasts with the parasitic Platyhelminthes. The mature spermatozoon, 180 μm in length, comprises a nuclear region, 50 μm in length, and a cytoplasmic region, with a short region of overlap. The cytoplasmic region contains two lateral axonemes with a 9 + 2 pattern of microtubules, granules of two different sizes, and two rows of longitudinal microtubules in the center. Each row consists of 5–6 singlet microtubules, with links between them. Whereas the two axonemes are labelled by antibodies against alpha, acetylated‐alpha, and beta tubulin, the microtubule rows are labelled only by the anti‐beta‐tubulin antibody. This suggests that acetylation does not occur in this part of the cytoskeleton, and that the epitope recognized by the anti‐alpha‐tubulin antibody (DM1A) is different in these units. Mol. Reprod. Dev. 52:74–85, 1999.

Ultrastructural and immunocytochemical investigation of acoel sperms with 9+1 axoneme structure: new sperm characters for unraveling phylogeny in Acoela

Acoel sperm characters proved useful in deciphering acoel taxonomy. The phylogenetic value of sperm characters in closely related sub-groups or in a monophyletic taxon has not yet been assessed. We have investigated sperm ultrastructure in seven members of the monophyletic taxon Childia sensu (Tekle et al. J Zool Sys Evol Res 43(1):72–90, 2005) and in their closest relatives, the Mecynostomidae (four taxa). All members of Childia examined show little variation in their sperm ultrastructure. The common characters of Childia taxa are: 9 + 1 axoneme structure, the presence of six distal cytoplasmic microtubules in the absence of axial or cortical ones, long nucleus and extensive nucleus–flagella overlap. We have identified a new set of cytoplasmic microtubules lying in the centriolar end of the sperm cell, distal microtubules. The origin and phylogenetic significance of this character is discussed. The types and arrangement of cytoplasmic granules could be used as phylogenetic characters at a low taxonomic level. A loose membrane amorphous core type of granule was found to be a synapomorphy for the following clade within the taxon Childia: C. crassum + C. groenlandica + C. vivipara + C. brachyposthium + C. macroposthium. Sausage shaped granules are plesiomorphic among the taxa examined. The rest of the granule characters were found to be homoplasious. Sperm ultrastructural characters have again proven their concordance with molecular phylogeny. The only morphological synapomorphies known for the sister taxa Childia–Mecynostomidae, in the molecular phylogeny, are characters derived from sperm ultrastructure: distal microtubules arranged in two groups of three microtubules each and a 9 + 1 axoneme structure. The spermatozoa of Childia and Mecynostomidae show 9 + 1 axoneme configuration, seemingly similar to the 9 + ‘1’ axoneme pattern of the Platyhelminthes—Trepaxonemata. Using electron-microscope immunocytochemistry, we have demonstrated that, unlike the central cylinder of trepaxonematans, the central cylinder of the 9 + 1 axonemal pattern in acoels is immunoreactive to tubulin and contains a single central microtubule. Therefore, the 9 + 1 patterns in acoels and trepaxonematans are homoplasious.

Oogenesis in Actinoposthia beklemischevi (platyhelminthes, acoela): an ultrastructural and cytochemical study

The oogenesis of the acoel Actinoposthia beklemischevi can be divided into a previtellogenic and a vitellogenic stage. Maturing oocytes are surrounded by accessory cells (a.c.) that produce electrondense granules, the content of which is released into the space between the oocyte and a.c. and gives rise to a thin primary egg envelope. The a.c. may also contribute to yolk synthesis by transferring low molecular weight precursors to the oocyte. Two types of inclusion are produced in maturing oocytes. Type I inclusions are small, roundish granules produced by the Golgi complex. They have a proteinaceous non-polyphenolic content which is discharged in the intercellular space and produce a thicker secondary egg envelope. Type I inclusions represent eggshell-forming granules (EFGs). Type II inclusions are variably sized globules progressively changing their shape from round to crescent. They appear to be produced by the ER, contain glycoproteins and remain scattered throughout the cytoplasm in large oocytes. Type II inclusions represent yolk. The main features of oogenesis in Actinoposthia are: (a) EFGs have a non-polyphenolic composition; (b) the egg envelope has a double origin and is not sclerotinized; (c) yolk production appears to be autosynthetic. The present ultrastructural findings are compared with those from other Acoelomorpha and Turbellaria.

Ultrastructure of the ciliary pits in the Geocentrophora group (Platyhelminthes, Lecithoepitheliata)

The ultrastructure of the paired lateral ciliary pits in several endemic species of Geocentrophora from Lake Baikal and in one cosmopolitan species, G. baltica, has been compared and the possible functional significance is discussed. The pit is composed of two distinctive parts; the bottom of the pit is an extensive sensitive area, filled with uni-and biciliary sensory receptors with reduced rootlets and numerous neurotubules. The walls of the pit are formed by several large ‘dark cells’, characterized by a dark cytoplasm with numerous mitochondria, a large nucleus, intracellular canaliculi, basal infoldings of the cell membrane, glycogen granules and a varying number of cilia. A protruding, densely ciliated ridge occurs along the anterior wall of the pit. The cilia have a strengthened rootlet system and seem to provide a strong water current into the pit. Dark cell processes penetrate the basement membrane of the pit and come into the vicinity of large cells with a cytoplasm similar to that of the ‘dark cells’ of the pit. These large cells in their turn come close to the terminal parts of the protonephridial canals, containing a weir. Smaller protonephridial capillaries without a weir seem to open directly into the pit lumen. The morphological data obtained suggest that the ciliary pit in not only a sensory structure, but plays a part in osmoregulation and ion exchange as well.