Francesca Simoncelli

Ecology: The proximate cause of frog declines?

Arising from: J. A. Pounds et al. 439, 161–167 (2006)10.1038/nature04246; Pounds et al. replyPounds et al. argue that global warming contributes to amphibian declines by encouraging outbreaks of the chytrid fungus Batrachochytrium dendrobatidis. Although our findings agree with the climate-linked epidemic hypothesis, this pathogen is probably not the only proximate factor in such cases: in the Trasimeno Lake area of Umbria in central Italy, for example, the water frog Rana lessonae first declined in the late 1990s, yet chytridiomycosis was not observed until 2003 (refs 5, 6). Here we show that the chytrid was common there throughout 1999–2002, in a previously unknown form that did not cause disease. We therefore think that the focus by Pounds et al. on a single pathogen is hard to justify because the host–parasite ecology is at present so poorly understood.

The effects of copper on actin and fibronectin Organization in Mytilus galloprovincialls haemocytes

The effects of copper on actin and fibronectin organization in Mytilus galloprovincialis haemocytes were studied. The Cu2+ exposure of mussels caused severe perturbations in haemocyte actin and fibronectin organization with respect to non-exposed organisms. Cytoskeletal actin was analysed by indirect immunofluorescence, using an antitotal actin monoclonal antibody, and by rhodamine-conjugated phalloidin. The majority of haemocytes from Cu(2+)-exposed mussels displayed a round morphology, with short and blunt filopodia; they lacked the polarized phenotype which was typical in control samples. The cytoskeleton alteration, more evident after phalloidin staining, resulted in the disappearance of filamentous actin. The actin cortical meshwork also appeared disorganized. The cytoskeletal morphology studied by transmission electron microscopy after negative staining of Triton X-100-treated haemocytes confirmed these observations. The structural organization of actin when analysed by Western blotting showed a larger number of Triton-soluble actin pools in treated mussel haemocytes. Fibronectin was studied by indirect immunofluorescence using a polyclonal antiserum directed against mussel fibronectin. In treated mussels, fibronectin appeared to be strongly disorganized and its levels decreased in both haemocytes and haemolymph. The mechanism(s) of the copper-induced alterations on actin and fibronectin organization in mussel immunocytes is discussed.

Evidence of Batrachochytrium dendrobatidis Infection in Water Frogs of the Rana esculenta Complex in Central Italy

Batrachochytrium dendrobatidis (phylum Chytridiomycota, order Chytridiales) is the causative organism of chytridiomycosis in amphibians, a disease associated with their population decline worldwide. In this work, we report a cutaneous infection in water frogs of the Rana esculenta complex in agricultural areas of Umbria, central Italy. Histological, immunohistochemical, ultrastructural, and molecular analyses demonstrated for the first time the presence of the Batrachochytrium dendrobatidis in this complex; to date, no association between the presence of chytrid fungal infection and mortality has been found, to our knowledge. However, the presence of Batrachochytrium dendrobatidis infection in the water frogs of the Rana esculenta complex is of concern because the frogs could act as a reservoir species and contribute to the decline of less resistant species.

The Pathogen of Frogs Amphibiocystidium ranae Is a Member of the Order Dermocystida in the Class Mesomycetozoea

ABSTRACT The pathogen of frogs Amphibiocystidium ranae was 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 Dermocystidium spp., Rhinosporidium seeberi, and Sphaerothecum destruens in 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. ranae from both Rana esculenta and Rana lessonae was closely related to all of the above organisms. Our phylogenetic analysis placed this pathogen of frogs as the sister group to the genus Dermocystidium and closely related to Rhinosporidium. These data strongly supported the placement of the genus Amphibiocystidium within the mesomycetozoeans, which is in agreement with the phenotypic features that A. ranae shares with the other members of this class. Interestingly, during this study Dermocystidium percae did not group within the Dermocystidium spp. from fish; rather, it was found to be the sister group to Sphaerothecum destruens. This finding suggests that D. percae could well be a member of the genus Sphaerothecum or perhaps represents a new genus.

Bioaccumulation of organochlorine pesticides in frogs of the Rana esculenta complex in central Italy

Concentrations of commonly used organochlorine pesticides (OCPs) were determined in tissues of 23 adult and 24 larval water frogs of two coexisting species (Rana lessonae and the hemiclonal hybrid R. esculenta) and in the water of their breeding pond in an agricultural zone in Umbria, central Italy, where increased occurrence of infectious diseases and distinctly oversized tadpoles were recently observed. The concentrations of OCP in tissues of both species were lower than those in the water of their breeding pond, except for DDT, which was more concentrated in adult frogs than in pond water (bioaccumulation factor 7 for R. lessonae, 15 for R. esculenta). Total OCP concentration and adult body weight were positively correlated for both species, which is consistent with bioaccumulation. In accord, adults contained higher OCP concentrations than tadpoles. Oversized tadpoles had higher OCP concentrations than normal tadpoles. Mean OCP concentrations in individual organs were about an order of magnitude higher than those in whole-frog homogenates. They were highest in brain, higher in ventral than in dorsal skin, and moderately high in ovaries; transmission of bioaccumulation loads to the next generation is therefore possible. The observed OCP concentrations appear too low to directly cause mortality in water frogs, but effects of cumulative exposure to low-level pollutants and their synergistic interactions with the effects of other natural and anthropogenic environmental stressors are unknown.

Characterization and immunocytochemical localization of actin and fibronectin in haemocytes of the musselMytilus galloprovincialis

SummaryCell-extracellular matrix interactions are recognized to be important for human leucocyte functions, including chemotaxis and phagocytosis. These activities depend on a reorganization of the microfilament actin (F-actin) promoted by fibronectin, one of the major components of extracellular matrices. Although invertebrate haemocytes are, in many aspects, similar to the human granulocyte-monocyte-macrophage cell lineage, actin and fibronectin have not been well studied in these cells. Consequently, the characterization and structural organization of actin and fibronectin in mussel (Mytilus galloprovincialis) haemocytes was investigated using Western blotting analysis, indirect immunofluorescence and immunoelectron microscopy. Actin was immunocharacterized by an anti-total actin monoclonal antibody. Fibronectin was immunocharacterized by an autologous polyclonal antiserum directed against the protein of mussel haemolymph. Actin was mainly localized along the peripheral cytoplasm of the haemocyte. The distribution of the F-actin microfilaments was assayed with Rhodamine-labelled phalloidin. F-actin was associated mainly with stress-fibres of spreading haemocytes and with microspikes at the adhesion sites. The labelling by the anti-fibronectin antiserum of the haemocyte rough endoplasmic reticulum vesiles, revealed by immunoelectron microscopy, suggests that these cells are involved in fibronectin biosynthesis. Gold particles were also present along the outer surfaces of the cell plasma membrane and its protrusions. Mussel fibronectin was localized immunohistochemically at the adhesion sites and in the extracellular matrix fibrils. The relationships between fibronectin and the actin cystoskeleton inMytilus galloprovincialis haemocytes are discussed.

Molecular characterization and expression of a heat-shock cognate 70 (Hsc70) and a heat-shock protein 70 (Hsp70) cDNAs in Rana (Pelophylax) lessonae embryos

Heat-Shock Protein 70 (HSP70) is a class of highly conserved proteins which is involved in essential functions as molecular chaperones and in the acquired tolerance processes. In this work, two cDNAs encoding a constitutive Hsc70 and an inducible Hsp70 from the water frog Rana (Pelophylax) lessonae, belonging to the Rana (P.) esculenta complex of central Italy, have been isolated and characterized. The two cDNA clones, named Rl-Hsc70 and Rl-Hsp70, encode 646 and 640 amino acid proteins respectively, which present extremely conserved functional domains characteristic of cytosolic members of the HSP70 family. Comparative studies of the amino acid sequences showed that Rl-Hsc70 and Rl-Hsp70 had the highest homology with constitutive and inducible HSP70 members of other amphibian species. The phylogenetic analysis also demonstrated a separate clustering of the Rl-Hsc70 and Rl-Hsp70 with constitutive and inducible members from other vertebrate species. Heat-inducibility assays performed during embryogenesis showed that the two isolated mRNAs displayed different expression profiles. Rl-Hsp70 was induced only in heat shock-treated embryos, whereas Rl-Hsc70 transcript levels, which were constitutively modulated in non-stressed embryos, did not increase following the heat treatment. In situ hybridization experiments demonstrated that both transcripts showed a tissue-specific enrichment in the central nervous system and in the somites.

Molecular characterization and expression of a divergent α-tubulin in planarian Schmidtea polychroa

We report the cloning and sequencing of a cDNA from planarian Schmidtea polychroa (Platyhelminthes, Turbellaria, Tricladida) encoding for an unusual tubulin isoform (SpTub-1) which is specifically expressed in testis. Sequence comparison of SpTub-1 with other known tubulins reveals that it has the highest homology with alpha-tubulins, even though the analysis of the molecular features shows that this isoform is significantly divergent. Hybridization of SpTub-1 to restriction-digested genomic DNA to Southern blotting produced a multiple banding pattern indicating that in planarian, a tubulin multigene family exists. Using in situ hybridization, we showed that the transcript is specifically detectable in planarian testis, suggesting that it may play a role in spermatogenesis.

Fibronectin from the hemolymph of the marine bivalve Mytilus galloprovincialis. Purification, immunological characterization and immunocytochemical localization

A gelatin-binding protein of 215 kDa subunit molecular weight was isolated from the hemolymph of Mytilus galloprovincialis. Using Western blotting analysis the protein reacted with polyclonal antiserum but not with monoclonal antibody against human plasma fibronectin. The rabbit antiserum prepared utilizing the isolated protein from Mytilus as the immunogen strongly reacted with the same gelatin-binding protein and also with human, bovine and chicken plasma fibronectin. The antiserum is suitable for cytochemical localization of fibronectin-like molecule in mussel tissues using either immunoperoxidase or immunogold staining. The data reported here indicate that the gelatin-binding protein purified from Mytilus displays molecular and immunological properties similar to those of vertebrate fibronectin.

Actin isoforms in amphioxus Branchiostoma lanceolatum

Abstract 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 β-cytoplasmic actin (anti-β actin), a monoclonal antibody against sarcomeric actins (anti-αSR-1), and a monoclonal antibody specific for α-smooth actin (anti-αSM-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-β actin showed positive staining mainly in non-muscle cells, anti-αSR-1 labelled dorsolateral myotomal muscles, and anti-αSM-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.