Loretta Guidi

New insights into placozoan sexual reproduction and development.

Unraveling animal life cycles and embryonic development is basic to understanding animal biology and often sheds light on phylogenetic relationships. A key group for understanding the evolution of the Metazoa is the early branching phylum Placozoa, which has attracted rapidly increasing attention. Despite over a hundred years of placozoan research the life cycle of this enigmatic phylum remains unknown. Placozoa are a unique model system for which the nuclear genome was published before the basic biology (i.e. life cycle and development) has been unraveled. Four organismal studies have reported the development of oocytes and one genetic study has nourished the hypothesis of sexual reproduction in natural populations at least in the past. Here we report new observations on sexual reproduction and embryonic development in the Placozoa and support the hypothesis of current sexual reproduction. The regular observation of oocytes and expressed sperm markers provide support that placozoans reproduce sexually in the field. Using whole genome and EST sequences and additional cDNA cloning we identified five conserved sperm markers, characteristic for different stages in spermatogenesis. We also report details on the embryonic development up to a 128-cell stage and new ultrastructural features occurring during early development. These results suggest that sperm and oocyte generation and maturation occur in different placozoans and that clonal lineages reproduce bisexually in addition to the standard mode of vegetative reproduction. The sum of observations is best congruent with the hypothesis of a simple life cycle with an alternation of reproductive modes between bisexual and vegetative reproduction.

Morphology of Xenodasys (Gastrotricha) : the first species from the Mediterranean Sea and the establishment of Chordodasiopsis gen. nov. and Xenodasyidae fam. nov

During a survey of the Italian marine meiofauna, several specimens of the rare gastrotrich genus Xenodasys were found in a submarine cave along the Ionian coast of Apulia. The ¢nding represents the ¢rst record of the genus for the Mediterranean Sea and reinforces the consideration of marine caves as habitats of high naturalistic value. The specimens, analysed using diierent microscopy techniques, showed a new species, named Xenodasys eknomios. Scanning electron microscopy, unveiling the astonishing morphology of this unusual gastrotrich, indicates that, due to technical artefacts, light microscopy may generate unreal features, which in the past may have led to the misinterpretation of the anatomical traits of these creatures. Transmission electron microscopy indicated that the ‘Seitenfu« sschen’, are genuine elements of the adhesive apparatus, in contrast with previous investigation, which attributed an exclusive sensorial function to these organs. Confocal laser scanning microscopy, combined with actin-binding £uorochromes, revealed muscular elements in a region where originally the muscular chordoid organ was reported for gastrotrich species belonging to the genus Chordodasys. A taxonomic revision of the species currently allocated to the genus Xenodasys led to the establishment of Chordodasiopsis gen. nov. to integrate the former Xenodasys (¼Chordodasys) antennatus and to the drafting of emended diagnosis of the genus Xenodasys. An overview of the high-rank systematization of these genera is also provided, with the establishment of Xenodasyidae fam. nov. to allocate both Xenodasys and Chordodasiopsis.

Comparative sperm ultrastructure of Neodasys ciritus and Musellifer delamarei, two species considered to be basal among Chaetonotida (Gastrotricha)

The spermatozoa of two species supposed to be basal to Gastrotricha Chaetonotida, Neodasys ciritus and Musellifer delamarei, were studied in order to supply further elements to the understanding of sperm evolution in Chaetonotida, a group in which a fully parthenogenetic reproduction is dominant. Two considerably different sperm patterns were found: the spermatozoon of N. ciritus has a simple, conical acrosome, a short, condensed nucleus, few conventional mitochondria randomly arranged along the sperm head, and a 9×2+2 flagellum perpendicular to the sperm major axis. The spermatozoon of M. delamarei is a filiform cell with a simple acrosome, a partially condensed nucleus, four mitochondria at the nuclear base, and a flagellum with a 9×2+2 axoneme and large accessory fibers. Some sperm features of M. delamarei are comparable to those of Xenotrichulidae, the only other Chaetonotida producing conventional spermatozoa, whereas the sperm of N. ciritus appears different from all the other patterns known among Gastrotricha, thus knowledge of it does not help in solving the problem of the discussed phylogenetic position of the genus.

Ultrastructural analyses support different morphological lineages in the phylum placozoa Grell, 1971

The morphology and ultrastructure of 10 clonal placozoan lineages were studied. We scored several morphological characters at a cellular and intracellular level and identified a number of morphological differences among clones. Some differences appear clone specific and allow recognizing five distinct lineages based on morphological criteria only. These data will be crucial for a yet to be established placozoan systematics. Furthermore, we here describe three new diagnostic morphological characters for Placozoa: a new structure in the upper epithelium, called “concave disc,” two distinct subpopulations of fiber cells, and especially small cells in the body margin. Besides the fiber cells appear to be arranged in several layers forming a complex, three‐dimensional net not previously described. We also describe the marginal cells as the formerly suggested potential stem‐cell type. The basic morphology is revised. J. Morphol., 2011.

Reproductive system and spermatozoa of Paraturbanella teissieri (Gastrotricha, Macrodasyida): implications for sperm transfer modality in Turbanellidae

Abstract. The morphology of the reproductive apparatus of several species of Turbanellidae, which are sequential hermaphrodites, has been studied for a comparison with that of other Gastrotricha Macrodasyida, which are simultaneous hermaphrodites. The common structural plan of the genital system of Turbanellidae includes two testes which extend into two sperm ducts turning anteriorly and fusing in a midventral pore, two ovaries with oocytes maturing in a cephalic direction and only one accessory organ, a seminal receptacle, provided with an external pore. A possible sperm transfer modality alternative to that described in the literature is advanced. Spermatological characters of Paraturbanella teissieri have been compared with those of the two Turbanella species studied up to date. Turbanellidae share with other Macrodasyida the general model of spermatozoon, but are the only representatives of this taxon known so far which have sperm devoid of the striated cylinder around the axoneme. Both the structure of the reproductive apparatus and the fine morphology of the spermatozoa of Turbanellidae species agree with the evolutionary view, recently supported by morphological and molecular data, which puts this taxon on a separate clade, early divergent from the stem lineage of Macrodasyida.

Cytochemical and immunocytochemical methods for electron microscopic detection of glucose-6-phosphate dehydrogenase in brain areas.

This paper reports on protocols for the cytochemical and immunocytochemical determination of the glucose-6-phosphate dehydrogenase (G6PD) in brain areas by electron microscopy (EM). The cytochemical assay consists of a pre-embedding staining of small and flat tissue blocks, which were first mildly fixed and then floated in a staining mixture based on the reduction of tetrazolium salts by NADPH. Tissue blocks were then washed, post-fixed in OsO(4), dehydrated through graded ethanol concentrations and embedded in resin. Ultrathin sections were then obtained and observed at the EM. The immunocytochemical technique was performed on completely fixed tissues of perfused animals. After the tissue embedding in resin, ultrathin sections were obtained and treated with a primary anti-erythrocyte G6PD antibody, produced and purified in our laboratory. The immunostaining was performed with secondary gold-conjugated antibody. Gold grains were well evident by EM analysis thus revealing the G6PD protein in the subcellular compartments. These protocols are useful to detect peculiar populations of neurons which express high levels of G6PD to sustain processes of neural plasticity in some brain areas.

Glucose-6-phosphate dehydrogenase expression associated with NADPH-dependent reactions in cerebellar neurons.

This review describes the variation of glucose-6-phosphate dehydrogenase (G6PD) activity in the main neurons of the molecular and granular layers as well as in the deep nuclei of the cerebellum as observed so far by optical and electron microscopy studies. Light microscopy and semiquantitative microphotometry of histochemical staining showed that the highest G6PD activity was expressed by Purkinje cells and neurons of the deep cerebellar nuclei; the elements of the molecular layer showed a diffuse G6PD staining, while the granular layer displayed only scattered G6PD activity. Electron microscopy analysis showed that the basket and stellate cells, as well as the Golgi cells, have a remarkable G6PD activity, while in the granule cells the enzyme was barely detectable. The results show that cerebellar G6PD activity changes with different neuron types as a function of its role in sustaining NADPH dependent pathways in these cells.

Fauna Europaea: Gastrotricha.

Abstract Fauna Europaea provides a public web-service with an index of scientific names (including important synonyms) of all living European land and freshwater animals, their geographical distribution at country level (up to the Urals, excluding the Caucasus region), and some additional information. The Fauna Europaea project covers about 230,000 taxonomic names, including 130,000 accepted species and 14,000 accepted subspecies, which is much more than the originally projected number of 100,000 species. This represents a huge effort by more than 400 contributing specialists throughout Europe and is a unique (standard) reference suitable for many users in science, government, industry, nature conservation and education. Gastrotricha are a meiobenthic phylum composed of 813 species known so far (2 orders, 17 families) of free-living microinvertebrates commonly present and actively moving on and into sediments of aquatic ecosystems, 339 of which live in fresh and brackish waters. The Fauna Europaea database includes 214 species of Chaetonotida (4 families) plus a single species of Macrodasyida incertae sedis. This paper deals with the 224 European freshwater species known so far, 9 of which, all of Chaetonotida, have been described subsequently and will be included in the next database version. Basic information on their biology and ecology are summarized, and a list of selected, main references is given. As a general conclusion the gastrotrich fauna from Europe is the best known compared with that of other continents, but shows some important gaps of knowledge in Eastern and Southern regions.

Glucose-6-phosphate dehydrogenase supports the functioning of the synapses in rat cerebellar cortex.

This study investigates heterogeneous glucose-6-phosphate dehydrogenase (G6PD) expression in the rat cerebellar cortex. G6PD activity and its electrophoretic pattern, evaluated on the cerebellar homogenate, were found to be similar to those of other brain areas. However, histochemical and immunohistochemical analyses revealed that the highest expression of G6PD activity and protein was in Purkinjes cells, followed by the molecular and granular layers. Electron microscopy analysis showed that, in Purkinjes cells, the G6PD reaction products were concentrated in the neurites while in the basket cells in the cell body. The granules showed a weaker activity everywhere. The quantitative distribution of G6PD is discussed in the light of the neurochemical function of these cells.

Reproductive system of the genus Crasiella (Gastrotricha, Macrodasyida)

Crasiella diplura from Sweden and Crasiella sp. from Italy were studied alive and with transmission electron microscopy (TEM). The two species are simultaneous hermaphrodites and share the same reproductive system lay-out: paired ovaries extend along the posterior part of the intestine and join mid-dorsally, while bilateral, club-shaped testes lie at the sides of the anterior gut, extending as deferentia that fuse on the mid-ventral plane and open into a single pore; gametes mature in a caudocephalic and centripetal direction. The bulky, sac-like, frontal organ is lined by a simple epithelium and lies dorsolaterally to the intestine, on the left side of the body. The spindle-shaped caudal organ is musculo-glandular and is located ventrolaterally to the gut on the right side. It is characterized by the presence of a roughly Y-shaped internal channel that opens into two pores close to each other, which function for the intake and outlet of the (auto)sperm, respectively. The spermatozoa, which are peculiar and similar in the two species, are characterized by a long and complex acrosome consisting of four ultrastructurally distinct regions, three of which find equivalence in other gastrotrich species. The flagellum lacks a striated cylinder. Anatomy and ultrastructure enable us to hypothesize a modality of sperm transfer in Crasiella that is similar to that observed in Macrodasys.