Ultrastructure of the frontal organ in Convoluta and Macrostomum spp.: significance for models of the turbellarian archetype

Present models of turbellarian evolution depict the organism with a frontal organ — a complex of glands whose necks emerge at the anterior tip of the body — and therefore imply that this organ is homologous throughout the Turbellaria. However, comparisons of representatives of the Acoela and Macrostomida, two putatively primitive orders of the Turbellaria, show that frontal organs in these two are not similar in ultrastructure or histochemistry. The acoel Convoluta pulchra had a prominent cluster of frontal mucous glands whose necks emerged together in a frontal pore at the exact apical pole of the organism, and an array of smaller glands of at least five other types opened at the anterior end, separately from and ventral to this pore. The frontal organs (Stirndrüsen) of two species of Macrostomum on the other hand, comprised an array of discretely emerging necks of at least two gland types including one with rhabdiform (rhammite) and one with globular mucous secretion granules neither of which emerge at the apical pole. In neither species did the organ appear to be sensory. Our findings indicate a low probability of homology between the frontal glands of the Acoela and Macrostomida.     

Physiological regeneration of the digestive parenchyma in Convoluta convoluta and Oxyposthia praedator (Turbellaria,Acoela)

Autoradiography has been applied to two acoel turbellarians, Convoluta convoluta and Oxyposthia praedator, to determine the distribution and fate of proliferative cells. In C. convoluta, mitotic figures and nuclei that labelled with [³H]thymidine could be observed in the peripheral parenchyma but not in the middle zone of the central parenchyma. The time required for regeneration of physiologically competent digestive cells was about 10–15 days. In O. praedator, mitotic figures (in metaphase and telophase) were observed in the peripheral parenchyma while none were found in the epidermis either in untreated animals or after treatment with colchicine. Mitotic figures were found only rarely in the central parenchyma and only in its marginal zone. Autoradiographs of O. praedator demonstrated [³H]thymidine incorporation into both the nuclei and the cytoplasm of peripheral parenchymal cells. In the central parenchyma, no nuclei with primary labelling were observed. The digestive parenchyma of the acoels is regarded as a unique histological system involving both specialized cells of the central parenchyma and stem cells located in the peripheral parenchyma.     

On the phylogenetic significance of sagittocysts and copulatory organs in acoel turbellarians

Viewed by SEM and TEM, sagittocysts of Convoluta bifoveolata Mamkaev, 1971, and needles of C. sagittifera Ivanov, 1952, have the same structure. Both are capsule-form extrusomes containing a protrusible needle. Only seven similar species of convolutimorph acoels symbiotic with green algae and C. sagittifera, without algae, possess extrusomes of this peculiar and complicated type. The sagittocyst is a clear synapomorphy of all these species. A sacciform ciliated antrum lacking a seminal vesicle is also characteristic of these species and also of three Japanese species of green (algae-symbiotic) convolutimorph acoels lacking sagittocysts. We suggest schemes of the possible evolution of male and female copulatory organs to provide a basis for better using such organs as phylogenetic characters. We regard the formation of a ciliated sacciform antrum as an independent evolutionary trend. This conclusion forms the basis for establishing the separate family Sagittiferidae. Species of this family seem to have originated in the West Pacific.     

The system of epidermal ciliary rootlets in Turbellaria

Summary The rootlets of the kinetic cilia form patterns of different types in the different turbellarian subgroups (cf. Rieger 1981). In the Acoela a rather complex system of ciliary rootlets is found in the epidermis (Dorey 1965; Hendelberg & Hedlund 1973; Bedini & Papi 1974). In the acoel Childia groenlandica (Levinsen) the four rootlets of each cilium make contact with those of adjacent cilia at two levels (Hendelberg & Hedlund 1974). Distinct granules are found in the interior of the main rootlets (Hendelberg & Hedlund 1974; Bedini & Papi 1974, Fig. 16) and basal bodies (Silveira 1972; Hendelberg & Hedlund 1974) of the epidermal kinetic cilia of acoels. Similar granules, probably of identical structure, can be seen in nemertodermatids, in the same positions (Tyler & Rieger 1977, Figs. 3 & 6). Such granules were studied in C. groenlandica with histochemical methods adapted for electron microscopy. Like Silveira (1972) I found the granules of the basal bodies to be Thiéry-positive, and thus evidently to be made up of or at least to contain polysaccharide material. The granules of the main rootlets were also found to be Thiéry-positive (Hendelberg 1976). Digestion experiments (Hendelberg & Hellmén 1978 and unpublished results) strongly support the concept that the granules are glycogen beta-particles.We know that cilia can function as kinetic organelles without any rootlets. But we are still uncertain about the function of the rootlets when occurring. Most probably they form an anchorage, a function which may be favoured by branching rootlets making contact with each other. Another function which has been discussed is the transmittance of impulses regulating the ciliary beat. Glycogen granules represent an energy deposit. The functional implication of these granules in the interior of the ciliary rootlets and basal bodies is not clear. However, the observations raise the question of how energy is transmitted to the cilia. Are the ciliary rootlets, when occurring, involved? This question will be further discussed, with references, in a future full report on the digestion experiments (to be published elsewhere).     

Ultrastructure and tubulin immunocytochemistry of the copulatory stylet-like structure in Childia species (Acoela)

One of the main characters used in acoel taxonomy is the male copulatory organ. Despite this, ultrastructural studies of this structure are scarce. We studied the ultrastructure of the copulatory organ in eight species of acoels belonging to the taxon Childia. Members of Childia possess a well-developed conical or cylindrical stylet-like structure composed of needles. Immunogold cytochemistry of tubulin was used to determine the composition of the needles. Stylet-like structures of Childia species at the ultrastructural level are basically similar. Stylet needles show intracellular differentiations. As shown both by ultrastructural and immunocytochemical methods, the stylet needles, in all species studied, are composed of long, parallel microtubules, either tightly packed or polymerized. We report unusual polymerization of microtubules, resulting in formation of a honeycomb-like structure in cross section. Variations of ultrastructure among Childia species include numbers and arrangement of stylet needles, shape of needles, needle compactness, microtubule polymerization, direction of stylet growth, and presence/absence of different types of granules. The stylet-like structures are homologous within Childia, but are likely to prove nonhomologous with the other needle-like structures found in acoel copulatory organs. Stylets in Platyhelminthes are not homologous with stylet-like structures in acoels.     

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.     

Correlation of fluorescence and electron microscopy of F-actin-containing sensory cells in the epidermis of Convoluta pulchra (Platyhelminthes: Acoela)

Phalloidin‐stained whole mounts of acoel turbellarians show brightly fluorescing club‐shaped structures distributed over the epidermis and concentrated especially at the anterior and posterior tips of the body. By correlating electron micrographic images and fluorescence images of Convoluta pulchra, these structures can be seen to be sensory receptors with a central cilium surrounded by a collar of microvilli. The other candidate for showing fluorescence in the epidermis, namely gland necks, can be ruled out since their distribution is too dense to resemble the distribution of the fluorescent structures seen here. The collared sensory receptors were inserted between epidermal cells, and each bore a central cilium surrounded by a collar of 6–18 microvilli and an additional centrally positioned 2–7 microvilli of which 2 or 3 were associated with a modified rootlet called the swallow’s nest. Confocal scanning laser microscopy resolved the core of actin filaments within the microvilli of the collar and their rootlet‐like connections to the base of the sensory cell. Such receptors could also be identified by fluorescence microscopy in several other species of acoel turbellarians.     

Back in time: a new systematic proposal for the Bilateria

Conventional wisdom suggests that bilateral organisms arose from ancestors that were radially, rather than bilaterally, symmetrical and, therefore, had a single body axis and no mesoderm. The two main hypotheses on how this transformation took place consider either a simple organism akin to the planula larva of extant cnidarians or the acoel Platyhelminthes (planuloid-acoeloid theory), or a rather complex organism bearing several or most features of advanced coelomate bilaterians (archicoelomate theory). We report phylogenetic analyses of bilaterian metazoans using quantitative (ribosomal, nuclear and expressed sequence tag sequences) and qualitative (HOX cluster genes and microRNA sets) markers. The phylogenetic trees obtained corroborate the position of acoel and nemertodermatid flatworms as the earliest branching extant members of the Bilateria. Moreover, some acoelomate and pseudocoelomate clades appear as early branching lophotrochozoans and deuterostomes. These results strengthen the view that stem bilaterians were small, acoelomate/pseudocoelomate, benthic organisms derived from planuloid-like organisms. Because morphological and recent gene expression data suggest that cnidarians are actually bilateral, the origin of the last common bilaterian ancestor has to be put back in time earlier than the cnidarian-bilaterian split in the form of a planuloid animal. A new systematic scheme for the Bilateria that includes the Cnidaria is suggested and its main implications discussed.     

Acetylcholinesterase activity in the developing and regenerating nervous system of the acoel Symsagittifera roscoffensis

Bery, A. and Martínez, P. 2010. Acetylcholinesterase activity in the developing and regenerating nervous system of the acoel Symsagittifera roscoffensis. —Acta Zoologica (Stockholm) 92 : 383–392. The use of the cholinergic system is widespread in the animal kingdom. It controls different processes, including reproduction and neural transmission. However, its evolutionary history is not yet well understood. For instance, the role played by the cholinergic system in the nervous system of basal bilaterian taxa, where the first signs of architectural complexity appear, is still unknown. Here, we describe the structure of the cholinergic system during the development and regeneration of the acoel flatworm Symsagittifera roscoffensis, using acetylcholinesterase (AchE) activity as a marker. In this species, AchE activity is observed at all developmental stages, including in the early embryos. The juvenile and adult patterns reveal the presence of a complex nervous system that includes three pairs of longitudinal neurite bundles, which are connected to an anterior centralized mass of neurons and neural processes formed by two pairs of connectives and four commissures. The power of the technique also allows the detection of newly born neurons as they are incorporated into the growing nervous system (during regeneration).     

Frontal organs in the Acoelomorpha (Turbellaria): Ultrastructure and phylogenetic significance

Using characters discernible through electron microscopy, we redefine the organ traditionally identified as the frontal organ in acoelomorph turbellarians as being a collection of two to several large mucus-secreting glands whose necks emerge together through a frontal pore at the exact apical pole of the body, i.e. at the point where the pattern of epidermal ciliary rootlets converges. Representatives that we have studied of each of the acoel families Paratomellidae, Diopisthoporidae, Solenofilomorphidae, Convolutidae, Otocelidae, and Mecynostomidae, as well as a representative of the Nemertodermatida, have such glands. Up to five additional types of glands that open anteriorly outside of the frontal pore, some of which are indistinguishable from glands of the general body wall, could be seen in the nemertodermatid, in Hesiolicium inops (Paratomellidae), and in representatives of the latter four acoel families. In Paratomella, three different types of glands open in diffuse fashion in a frontal glandular complex reminiscent of that in the Macrostomida.Sensory elements near the frontal pore appear to be independent of the gland necks, and so the organ cannot be considered a sensory organ.The frontal organ, as described above, appears very likely to be homologous within the Acoelomorpha, and represents another strong (although unrooted) autapomorphy for this line of turbellarian evolution.