Comparative morphology of the bursal nozzles in acoels (Acoela, Acoelomorpha)

Systematics of the Acoela is particularly difficult because of the paucity of readily discernible morphological features. In other soft-bodied worms, sclerotized structures, such as copulatory stylets, provide important characters that can be seen in whole mounts, but acoels generally lack such features. Among the few sclerotized structures in acoels are bursal nozzles-tubiform outlets on the seminal bursae that are believed to be conduits (spermatic ducts) through which allosperm are transported to the oocytes. Early classifications of the Acoela used features of the female reproductive system, including bursal nozzles, for distinguishing major groups, but the current system essentially ignores them as too plastic to provide higher-level distinctions. We used confocal and electron microscopy to further characterize bursal nozzles in five acoel species, and found all composed of actin-reinforced extensions of stacked, flat mesenchymal cells. In Notocelis gullmarensis, Aphanostoma bruscai, and Daku woorimensis, the nozzle is a stiffened region of the same cells forming the wall of the bursa. By contrast, in Wulguru cuspidata cells forming the nozzle are distinct from those of the bursa. The so-called bursal cap of A. bruscai and D. woorimensis has small sclerotized disjunct units within it, also composed of stacked, flat, actin-reinforced cells. The nozzle of W. cuspidata, prominent like that of other convolutid acoels, is relatively complex, its actin-reinforced cells sandwiched with secretory cells and its base bearing a "sorting apparatus" of egg-shaped cells that send narrow processes inside the spermatic duct. Cases of sperm inside the nozzle corroborate its assumed role in reproduction. Whereas most nozzles sit at the end of the bursa facing the ovary, in species of Pseudmecynostomum and purportedly in a few other acoels, they sit between the female pore and the bursa, constituting what we call a vaginal nozzle. All bursal nozzles of acoels show a common ground pattern indicating common ancestry, but certain features discerned through electron and confocal microscopy show promise of providing synapomorphies for grouping some species.     

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. This revised version was published online in July 2006 with corrections to the Cover Date.     

A new viviparous acoel Childia vivipara sp. nov. with observations on the developing embryos, sperm ultrastructure, body wall and stylet musculatures

A free‐living viviparous acoel, Childia vivipara sp. nov., from the Gullmar fjord of the Swedish coast is described. The new species is assigned to the taxon Childia based on histological, ultrastructural and molecular sequence similarities. All available molecular markers (18S rRNA, 28S rRNA and histone H3) and several morphological characters, obtained using transmission electron microscopy and confocal scanning laser microscopy of whole mount specimen stained with TRITC‐labelled phalloidin, support the placement of C. vivipara in the taxon Childia. Childia vivipara and other Childia species share the following morphological synapomorphies: well‐developed copulatory organs built of tightly packed stylet needles, proximal part of the stylet inserted into the seminal vesicle, reversed body‐wall musculature, absence of ventral diagonal muscles, presence of dorsal diagonal muscles, and presence of ventral straight longitudinal muscles between frontal pore and mouth, 9 + 1 sperm axoneme structure, six distal sperm cytoplasmic microtubules, and extensive overlap of axonemes and nucleus. The new species can be easily distinguished from other Childia species by its viviparous mode of reproduction and single curved stylet. Observations on late embryonic development based on the oldest developing embryos are discussed.     

Revision of the Childiidae (Acoela), a total evidence approach in reconstructing the phylogeny of acoels with reversed muscle layers

The Childiidae sensu Dörjes 1968 comprises the acoel worms characterized by a cone‐shaped penis with muscular or sclerotized elements. Based on differences in body‐wall musculature arrangement, Hooge (2001) recently restricted the family to the genus Childia Graff, 1910 and placed the remaining genera to his new family Actinoposthiidae Hooge 2001 . This rearrangement has been questioned ( Raikova et al. 2004 ). We reconstructed the phylogeny of the Childiidae sensu Dörjes 1968 by means of a total evidence analysis including Histone H3, 28S rDNA and new 18S rDNA sequences, as well as 50 morphological characters. New characters of the muscular system and copulatory organs discovered through confocal laser scanning microscopy of phalloidin‐stained specimens are included in the phylogenetic analysis. A total of 12 taxa (nine ingroup and three outgroup) were used in the parsimony analysis of the 18S data set, which was aligned with different parameters for a sensitivity analysis, and the combined data set (18S + 28S + H3 + morphology). Incongruence in the node support of the groups among the four partitions was very low in the total evidence tree; except for the H3 partition. The conflict observed in the H3 partition is likely due to large homoplasy observed in the synonymous alternatives at both first and third codon positions. All data partitions demonstrated that Actinoposthia beklemischevi Mamkaev 1965 , and the newly defined taxon Childiidae (comprising Childia and Paraphanostoma Westblad 1942 ) are not close relatives. The monophyly of Childia and Paraphanostoma is strongly supported by both the 18S and 28S data partitions. Our study also reveals additional apomorphies uniting Childia with Paraphanostoma from body‐wall musculature, statocyst muscles and male copulatory organ. Muscular system, statocyst muscles, male copulatory organ and nervous system characters proved to be the best characters for taxonomic delimitations of subtaxa within the Childiidae, whereas the seminal bursa (a frequently used character in the taxonomy of Acoela) was highly homoplastic. We also described the body‐wall musculature of six Paraphanostoma species, which is characterized by the reversed arrangement of the longitudinal and circular muscle layers, and by the absence of diagonal muscles on the ventral side of the body and the presence of two types of diagonal muscles on the dorsal side. Childia groenlandica (Levinsen, 1879) is nested among the Paraphanostoma species in our total evidence tree, so we synonymize Paraphanostoma with Childia; all former members of Paraphanostoma are transferred to Childia.     

Ultrastructural aspects of nervous-system and statocyst morphogenesis during embryonic development of Convoluta psammophila (Turbellaria,Acoela)

The notion that statocysts originated from an infolding of ectoderm lined by ciliated sensory cells has been challenged with evidence of capsule-limited, non-ciliary statocysts in several independent phyla. Statocysts in turbellarians primitively lack cilia and are embedded within or closely adjoined to the cerebral ganglion; they are likely to be derived from nervous tissue. We investigated the development of the simple statocyst in an acoel turbellarian, a statocyst consisting of three cells. Observations of serial TEM sections of embryos at different stages of development support the hypothesis of an inner (non-epithelial) origin of the statocyst. First, a three-cell complex is delimited by a basal lamina; it then undergoes cavitation by swelling, autophagy, and fluid secretion. The statocyst becomes discernible within the precursor ganglion cells while they still contain yolk inclusions. The two outer (parietal) cells, enclosed together by a 10-nm-thick basal lamina, arrange themselves in an ovoid of about 10 µm diameter and surround the inner statolith-forming cell. The statolith is formed later within vacuoles of the statolith-forming cell.     

Patterns of musculature as taxonomic characters for the Turbellaria Acoela

While turbellarians are generally assumed to have body-wall musculature consisting routinely of longitudinal, circular, and diagonal fibers, members of the Acoela examined by a fluorescence-microscopy technique specific for actin showed more complicated and distinctive arrangements of muscles, giving promise for better delimiting taxa within this taxonomically difficult order. Certain globose or tear-drop-shaped worms such as Convoluta pulchra and species of Pseudaphanostoma, Mecynostomum, and Otocelis, showed a complex pattern in which muscles longitudinal in the anterior half of the body arc diagonally across the posterior half; complex brushes of parenchymal muscles that cross at the level of the statocyst and arc postero-laterally also characterize these groups. The more elongate acoel Paratomella sp. was found to have musculature dominated by strictly longitudinal fibers and with relatively weak circular fibers and few fibers running diagonally to the body axis, yet the elongate mecynostomid Paedomecynostomum bruneum showed a crossing of antero-longitudinal fibers similar to that seen in the more globose Mecynostomum sp. A distinctive looping of muscles around the mouth is seen in P. bruneum and the Anaperidae. Such similarities and differences in pattern of musculature promise to provide easily recognizable characters for taxonomy of the Acoela at levels ranging from species to family. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ultrastructure and formation of the bursa mouthpiece of Philocelis cellata (Plathelminthes,Acoela)

Philocelis cellata has a strengthened bursa mouthpiece which is arranged in front of the male copulatory organ. The main components of the bursa mouthpiece are numerous ring-shaped bursa mouthpiece cells whose central parts contain strengthened elements forming a tube around the sperm duct. Each of the peripheral areas of the bursa mouthpiece cells is separated by similarly ring-shaped gap cells. The end of the bursa mouthpiece towards the bursa is formed by a so-called sorting apparatus which consists of different cells; opposite the bursa the sperm duct ends in a globe-shaped sperm vestibule. The bursa mouthpiece is differentiated successively, beginning at the distal part at the bursa and proceeding proximally.     

Spermatogenesis and the structure of the testes in Isodiametra pulchra (Isodiametridae,Acoela)

Boone M., Willems M., Claeys M. and Artois, T. 2011. Spermatogenesis and the structure of the testes in Isodiametra pulchra (Isodiametridae, Acoela). —Acta Zoologica (Stockholm) 92 : 101–108. Spermatogenesis and the structure of the testes were studied ultrastructurally in Isodiametra pulchra (Smith and Bush, Transactions of the American Microscopical Society 1991; 110: 12; Hooge and Tyler, Journal of zoological systematics and evolutionary research 2005; 43: 100). The testes are paired, compact, non‐follicular and lie dorsally and dorso‐laterally to the paired ovaries, partially enfolding them. All stages of spermatogenesis, including spermiogenesis, are described at the ultrastructural level and their spatial organization within the testes is discussed. The cells at the early stages of spermatogenesis (spermatogonia and spermatocytes) are located on the dorsal and dorso‐lateral sides of the testes, while the late stages (spermatids and filiform spermatozoa with 9+2 axonemes) lie at the ventral and inner periphery of the testes, adjacent to ovaries. All the cell types can be found both at the anterior and the posterior end of the testes. The value of the structure of the testes as a phylogenetic marker is addressed.     

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. © 1999 Wiley‐Liss, Inc.