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ABSTRACTThe pathogen of frogs Amphibiocystidium ranaewas recently described as a new genus. Due to its spherical shape, containing hundred of endospores, it was thought to be closely related to the pathogens of fish, mammals, and birds known as Dermocystidiumspp., Rhinosporidium seeberi, and Sphaerothecum destruensin the Mesomycetozoea, but further studies were not conducted to confirm this relationship. To investigate its phylogenetic affinities, total genomic DNA was extracted from samples collected from infected frogs containing multiple cysts (sporangia) and endospores. The universal primers NS1 and NS8, used to amplify the 18S small-subunit rRNA by PCR, yielded ˜1,770-bp amplicons. Sequencing and basic local alignment search tool analyses indicated that the 18S small-subunit rRNA of A. ranaefrom both Rana esculentaand Rana lessonaewas closely related to all of the above organisms. Our phylogenetic analysis placed this pathogen of frogs as the sister group to the genus Dermocystidiumand closely related to Rhinosporidium. These data strongly supported the placement of the genus Amphibiocystidiumwithin the mesomycetozoeans, which is in agreement with the phenotypic features that A. ranaeshares with the other members of this class. Interestingly, during this study Dermocystidium percaedid not group within the Dermocystidiumspp. from fish; rather, it was found to be the sister group to Sphaerothecum destruens. This finding suggests that D. percaecould well be a member of the genus Sphaerothecumor perhaps represents a new genus.

Actin- and fibronectin-like proteins were characterized in the planarian, Dugesia lugubris s.l., by sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting analysis using antisera to vertebrate actin and fibronectin. These antisera recognized protein bands of 42 kDa and 220 kDa, respectively. In addition, the immunohistochemical distribution of both actin- and fibronectin-like material was examined by using immuno-electron microscopy. Actin-like protein was localized in myofibrils in various differentiation stages, and in the peripheral cytoplasm and lamellipodia of cells that were migrating. The fibronectin-like component was associated with the extracellular matrix in the fibrillar structures and with the surface of the migrating cells. Our data suggest that similar cellular and molecular mechanisms are involved in cell-matrix interactions and in the morphogenesis of living organisms at different evolutionary levels.

Actin is a highly conserved cytoskeletal protein that is ubiquitous in all eukaryotes. Little is known about actin expression in amphioxus, the closest living relative of the vertebrates. In the present study, involving Western blotting and indirect immunofluorescence, we report the characterization and localization of various actin isoforms in amphioxus (Branchiostoma lanceolatum) tissues. Three antibodies against vertebrate actins were used: a polyclonal antibody recognizing -cytoplasmic actin (anti- actin), a monoclonal antibody against sarcomeric actins (anti-SR-1), and a monoclonal antibody specific for -smooth actin (anti-SM-1). Western blot analysis of amphioxus extracts immunodecorated with these antibodies showed a 43-kDa-positive band co-migrating with respective controls. The amphioxus isoactin expression patterns recognized by these antibodies were similar to those of vertebrates, i.e., anti- actin showed positive staining mainly in non-muscle cells, anti-SR-1 labelled dorsolateral myotomal muscles, and anti-SM-1 stained ventral muscles. These results demonstrate that at least two muscle actins are present in amphioxus, suggesting that muscle actin gene duplication events began before vertebrate divergence from the amphioxus lineage.

In freshwater planarians the existence of at least two major actin isoforms has been demonstrated. One isoform is expressed by differentiated muscle cells and is different from vertebrate α-sarcomeric and α-smooth actin. The other isoform is recognized by a specific anti warm-blooded vertebrate α-smooth muscle actin monoclonal antibody. This latter isoform is expressed mainly in mesenchymal-like cells - generally called neoblasts - activated during regeneration and growth. To date planaria is the only invertebrate where the existence of an antigen related to α-smooth muscle actin has been demonstrated. Further studies in this field may be important to define the phylogenetic position of Platyhelminthes.

a-Smooth muscle (aSM) actin of endothermic vertebrates is selectively recognized by the monoclonal antibody anti-aSM-1. Immunoreactivity to this antibody has been shown to be localized in the NH 2-terminal sequence Ac-EEED (Chaponnier et al. 1994). Among terrestrial ectothermic vertebrates, two amphibian ( Triturus vulgaris, Rana esculenta) and three reptilian species ( Pseudemys scripta elegans, Natrix natrix, Podarcis sicula) were screened to investigate if their vascular and visceral smooth muscles were stained by anti-aSM-1. In all the specimens tested, Western-blot analysis of tissue extracts immunodecorated with anti-aSM-1 revealed a single polypeptide chain having the same electrophoretic mobility as bovine aSM actin. The binding to amphibian and reptilian tissue extracts was inhibited by the synthetic peptide Ac-EEED, but not Ac-DEED, as occurs in mammals. aSM actin expression was found in vascular and visceral smooth muscle cells of the species tested. The media of small and large blood vessels was labelled by anti-aSM-1. In the stomach and intestine the outer longitudinal and inner circular layers of the muscularis and of the muscularis mucosae were stained. In addition, myofibroblasts of the subepithelial layer were labelled. A more restricted expression of this isoactin was detected in turtle ( P. scripta elegans) visceral smooth muscle cells, which may be related to the involvement of the digestive system in respiratory activity. These data suggest that in vertebrate evolution aSM actin arose earlier than previously proposed.