Sachiko Ishida

To be or not to be a flatworm : the acoel controversy

Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa - the Catenulida, the Acoelomorpha and the Rhabditophora - have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives.

Localization of mitochondrial ribosomal RNA on the chromatoid bodies of marine planarian polyclad embryos.

Electron‐dense cytoplasmic structures, referred to as chromatoid bodies, are observed in the somatic stem cells, called neoblasts, and germline cells in adult planarians. Although it has been revealed that the chromatoid bodies morphologically resemble germline granules in Drosophila and Xenopus embryos, what essential role it plays in the planarian has remained unclear. In the present study, to examine whether chromatoid bodies in planarian embryos are responsible for germline formation, the presence and behavior of chromatoid bodies during embryogenesis were examined. Mitochondrial large ribosomal RNA and mitochondrial small ribosomal RNA were used as candidate markers for components of the chromatoid body. Starting from the fertilized egg, extramitochondrial signals of both RNA (mtrRNA) were observed. At the ultrastructural level, mtrRNA were localized on the surface of the chromatoid bodies. At subsequent stages, the signals of mtrRNA were observed in certain restricted blastomeres that contribute to the formation of larval structures. The signals gradually decreased from the gastrula stage. These results suggest that the chromatoid bodies associated with mtrRNA in embryogenesis are not germline granules. The chromatoid bodies of blastomeres may be concerned with the toti‐ or pluripotency and cell differentiation as proposed in adult planarian neoblasts.

Eggshell formation in polyclads (Turbellaria)

Eggshell formation in polyclads was studied by means of transmission electron microscopy and histochemistry. Shell-forming granules (SFG) in the egg, as well as secretions of shell glands (SGS), play roles in eggshell formation. As the oocytes pass through the portion of the female tract where the shell glands open, they are surronded by a two-layered envelope of SGS. This envelope prevents the dispersion of SFGs discharged after oviposition, and its inner layer participates in eggshell formation with the SFGs. In Pseu-dostylochus sp., most SFGs consist of five parts. Similarities in staining between the parts of the SFGs and the parts of the eggshell indicate that discrete parts of the shell are derived from specific SFG components. Hardening of the eggshell and egg-plate matrix takes place through primary tanning of a sclerotin-like protein.

Analytical studies of the ultrastructure and movement of the spermatozoa of freshwater triclads

The spermatozoa in the testis of three species of triclad turbellarians showed slightly different morphologies from spermatozoa in the seminiferous tubule and ovovitelline duct, and we suggest these differences relate to maturation of the spermatozoa. Glycogen granules could be found in the flagella between the central core and the peripheral doublets of spermatozoa in the seminiferous tubule and ovovitelline duct but only rarely in spermatozoa in the testis, and connections between a third arm of the doublets and the flagellar membrane were detectable only in those spermatozoa in the ovovitelline duct and seminiferous tubule. The movement of spermatozoa also appeared to change with capacitation: spermatozoa isolated from the spermiducal vesicle swam with either the anterior process or the head leading; those within the ovovitelline duct appeared to move with the anterior process leading. The flagellum, with the so-called ‘9 + 1’ axonemal pattern, was observed to divide into nine branches toward its distal end; the central cylinder of the axoneme did not continue into the branches. An acrosome has not been observed in planarian spermatozoa. Rod-like structures and a dense mass lying in the anterior process and suspected of containing an acrosomal substance, could not be shown to produce acrosome-like proteolytic activity.

Detection and Changes in Levels of Testosterone During Spermatogenesis in the Freshwater Planarian Bdellocephala brunnea

Abstract It was reported recently that vertebrate-type steroids exist and control reproduction in several groups of invertebrates, including molluscs. Sexually reproductive freshwater planarians of the species Bdellocephala brunnea have a limited breeding season in their natural habitat. This phenomenon suggests that some endogenous reproductive hormones might play a role in vivo. However, to date, sex steroids such as androgen, estrogen, and progesterone have not been found in planarians. The goal of the present study was to determine whether androgen is present in sexual planarians such as B. brunnea. The presence of testosterone was detected by high-pressure liquid chromatography and, in sexually reproductive individuals in which no seminal vesicles were visible, the level of testosterone was about twice than that in individuals with visible seminal vesicles. An enzyme-linked immunosorbent assay revealed that the levels of testosterone during terminal spermatogenesis were three times higher than during the spermatocyte-building phase. Our results indicate that sexually reproductive freshwater planarians such as B. brunnea might have vertebrate-type steroids and show variation in testosterone levels during spermatogenesis.

Studies on the speciation of Japanese freshwater planarian Polycelis auriculata based on the analysis of its karyotypes and constitutive proteins

Various kinds of chromosomal polymorphisms or karyotypic variations are found in the Japanese freshwater planarian Polycelis auriculata. Within this species, there are found worms whose chromosome numbers are 2n = 6, 10, 11, 12 and others, and 3x = 6 and 9. There are some which have cells with triploidy and tetraploidy complements (3x = 6 & 4x = 8), and others which have cells with triploidy and hexaploidy complements (3x = 6 & 6x = 12). These worms with such varied karyotypes are usually found in separate habitats, though occasionally they occur together. Electrophoretic analysis of the proteins extracted from the karyotipically different worms which belong to three different local populations shows some dissimilarity in the constitutive proteins according to their karyotypic differences. The results obtained suggest that this species is still in the process of speciation or chromosomal evolution.

Production of cell- and tissue-specific monoclonal antibodies in the freshwater planarian Phagocata vivida

To provide a tool for studying regeneration in planarians, we have produced monoclonal antibodies against a variety of cells and tissues of the freshwater planarian Phagocata vivida (Ijima et Kaburaki). We obtained five kinds of monoclonal antibodies specific, respectively, to 1) the excretory system, 2) nerve cells, 3) rhabdoid-forming cells and body-surface mucus, 4) gastrodermal and epidermal cells, and 5) male germ cells and epidermis.

Genetic Divergence of Japanese Turbellarians, Studied by Comparisons of Partial 18S rRNA Gene Sequences. I. On Representatives of Dendrocoelidae (Platyhelminthes: Tricladida: Paludicola)

Abstract This study obtained 18S rDNA sequence data to investigate the extent of genetic divergence in the Japanese dendrocoelid group, and applied the data to the estimation of phylogenetic relationships and taxonomy. The sequence data, consisting of 558 base positions in an aligned data set, were obtained by using the polymerase chain reaction and direct sequencing. For comparative analysis, the 18S rRNA gene sequence data on dendrocoelids from different distant geographical places (Europe, Lake Baikal in Siberia, Kamchatka) published previously were used. Such analysis showed that: (1) Bdellocephala species from Japan, Kamchatka and Lake Baikal are closely related ; (2) Japanese species classified within the genus Dendrocoelopsis do not form a single cluster of closely related organisms; (3) Dendrocoelopsis ichikawai differs minimally from Bdellocephala baicalensis (from Lake Baikal), reliably groups with all Bdellocephala, and should therefore be classed with this genus. Taxonomic conclusions. (1) The genus Dendrocoelopsis should be revised after molecular typification of European and American representatives. (2) The current generic status of D. ichikawai based on the presence of a well-developed penis papilla should be reconsidered. (3) Sequence divergence amongst representatives of B. brunnea indicates that representatives should be closely examined for the presence of morphological characters by which they might be distinguished.

Further survey of chromosomal polymorphisms in the freshwater planarian Polycelis auriculata

Although the chromosomal number of Polycelis (Seidlia) auriculata Ijima et Kaburaki is basically 2n = 6, this species shows remarkable chromosomal polymorphisms in different populations. Among worms collected at 30 stations in the central part of Japan’s Main Island and in Hokkaido, we found five new karyotypes, considered to be variations of tetraploidy and diploidy: 4n?fis1. + 1 = 3SM + 2A + 3T1 + 2T2 + 4M = 14, 4n?fis2.(B) = 2SM + 2A + 4T1 + 2T2 + 4M + (0-2B) = 14 - 16, 4n?fis2. + 1 = 2SM + 2A + 4T1 + 2T2 + 5M = 15, 4n?fis2.-2 = 2SM + 2A + 4T1 + 4M = 12, and 2nM- = 2SM + 2A = 4 (where SM = submetacentric; A = acrocentric; T1 and T2 = telocentric; M = metacentric; B = B-chromosome; fis1. and fis2. = occurrence of one or two centric fissions, respectively; and M- = deletion of a metacentric chromosome). These karyotypes exist singly in one individual or as various mixoploids within the same individual. No individual having only karyotype 2nM- has yet been found. This brings the total number of known karyotypes in P. auriculata to 17; these can be arranged in an order that we believe reflects their evolution.

Light and electron microscopic studies of the intestinal epithelium in Notoplana humilis (Platyhelminthes, Polycladida): the contribution of mesodermal/gastrodermal neoblasts to intestinal regeneration.

Some free-living flatworms in the phylum Platyhelminthes possess strong regenerative capability that depends on putative pluripotent stem cells known as neoblasts. These neoblasts are defined based on several criteria, including their proliferative capacity and the presence of cellular components known as chromatoid bodies. Polyclads, which are marine flatworms, have the potential to be a good model system for stem cell research, yet little information is available regarding neoblasts and regeneration. In this study, transmission electron microscopy and immunostaining analyses, using antibodies against phospho-histone H3 and BrdU, were used to identify two populations of neoblasts in the polyclad Notoplana humilis: mesodermal neoblasts (located in the mesenchymal space) and gastrodermal neoblasts (located within the intestine, where granular club cells and phagocytic cells are also located). Light and electron microscopic analyses also suggested that phagocytic cells and mesodermal/gastrodermal neoblasts, but not granular club cells, migrated into blastemas and remodeled the intestine during regeneration. Therefore, we suggest that, in polyclads, intestinal regeneration is accomplished by mechanisms underlying both morphallaxis (remodeling of pre-existing tissues) and epimorphosis (de novo tissue formation derived from mesodermal/gastrodermal neoblasts). Based on the assumption that gastrodermal neoblasts, which are derived from mesodermal neoblasts, are intestinal stem cells, we propose a model to study intestinal regeneration.