Sara Ferrando

Observations of crypt neuron-like cells in the olfactory epithelium of a cartilaginous fish

A new receptor neuron (RN) type was recently described in bony fish olfactory epithelium (OE): the crypt receptor neuron. This name is due to its main feature: the presence, at the apical part, of a deep invagination into which cilia protrude. The presence of this receptor neuron type is well documented in different species of bony fishes but it has never been described in cartilaginous fishes. In this study we demonstrate that crypt neuron-like cells are present in the olfactory epithelium of the elasmobranch Scyliorhinus canicula (Linnaeus, 1758). Histological observations allowed us to detect the presence of a few egg-shaped cells, characterized by a crypt like zone; alpha-tubulin immunoreactivity suggested the presence of cilia in the same area; fluorocrome conjugated lectin bindings suggested a distinctive mucus composition inside the presumptive crypt. The possible presence of crypt neuron-like cells in chondrichthyes would represent an interesting common feature between bony and cartilaginous fishes.

Developmental abnormalities and changes in cholinesterase activity in sea urchin embryos and larvae from sperm exposed to engineered nanoparticles

The objective of this study is to examine the toxicity of engineered nanoparticles (NPs) that are dispersed in sea water by using an in vivo model. Because many products of nanotechnology contain NPs and are commonly used and well-established in the market, the accidental release of NPs into the air and water is quite possible. Indeed, at the end of their life cycle, some NPs are inevitably released into waste water and can reach marine ecosystem and affect the organisms there. Although there are few data on the presence of NPs in the marine environment, our awareness of their potential impact on environmental and organismal health is growing. Shallow-water benthonic organisms such as sea urchins provide planktonic larvae as a trophic base for finfish juveniles and are exposed to water from estuaries and precipitation. Such organisms can therefore be directly affected by NPs that are dispersed into those media. We evaluated the effects of exposure to different concentrations of nanosilver, titanium oxide and cobalt NPs on the sperm of the sea urchin Paracentrotus lividus by analyzing the functionality and the morphology and biochemistry of the first developmental stages of the sea urchin. Sperm were exposed to sea water containing suspensions of NPs ranging from 0.0001 mg/L to 1 mg/L. Fertilization ability was not affected, but developmental anomalies were identified in embryos from the gastrula to pluteus stages, including morphological alterations of the skeletal rods. In addition, the enzymatic activity (cholinesterase, ChE) of the larvae was measured. Acetylcholinesterase (AChE) and propionylcholinesterase activity (PrChE) was affected in all of the exposed samples. The results did not vary consistently with the concentration of NP, but controls were significantly different from exposed samples. Exposure of sea urchin to these NPs may cause neurotoxic damage, and the altered ChE activity may be involved in skeletogenic aberrations. In conclusion, the sea urchin represents a suitable and sensitive model for testing the toxicity and effects of engineered NPs that are dispersed in sea water.

Toxicity and transfer of metal oxide nanoparticles from microalgae to sea urchin larvae

Nanoparticles (NPs) contained in commercial products are released and enter into the aquatic ecosystem, posing serious possible risks to the environment and affecting the food chain. Therefore, investigating the potential toxicity of NPs on aquatic organisms has become an important issue. This study assessed the toxicity and trophic transfer of metal oxide NPs from marine microalgae (Cricosphaera elongata) to the larvae of the sea urchin Paracentrotus lividus. Larvae (24 h old) were fed on 2000 cell mL−1 48 h of microalgae contaminated with 5 mg L−1 of several metal oxide NPs (SiO2, SnO2, CeO2, Fe3O4) for 15 days. Larval viability and development were monitored from the 4-arm stage to the 8-arm pluteus stage. A significant decrease in survival was observed in larvae fed with microalgae exposed to SiO2 and CeO2 NPs. Abnormal development, characterised by skeletal degeneration and altered rudiment growth, was observed in all larvae fed with contaminated NP algae. Our findings revealed that SiO2 and CeO2 NPs exerted a toxic effect in the trophic interaction analysed, by reducing sea urchin larval viability, and all metal oxide NPs induced toxicological effects. In conclusion, metal oxide NPs may enter the food chain and become bioavailable for marine organisms, affecting their development.

Immunolocalization of G-protein alpha subunits in the olfactory system of the cartilaginous fish Scyliorhinus canicula.

In the olfactory and vomeronasal systems of vertebrates, the morphology of the receptor neurons, the receptor gene family they express, the G‐protein coupled with the receptor (in particular the G‐protein alpha subunit), and their projection to the olfactory bulb are correlated. Much information about this complicated system have been collected in different groups, but nothing is known about Chondrichthyes. In this work, the presence and distribution of immunoreactivity for different types of G‐protein alpha subunit (Gαo, Gαq and Gαs/olf) were investigated in the olfactory mucosa and olfactory bulb of the shark Scyliorhinus canicula. Only Gαo‐like immunoreactivity was detected in the olfactory mucosa and bulb, both in tissues and homogenates. Its distribution was partially similar to that found in other vertebrates: it was localized in the microvillous receptor neurons, in numerous axon bundles of the fila olfactoria, in the stratum nervosum and in the most of glomeruli in the stratum glomerulosum. No immunoreactivity was instead observed in the crypt neurons, the second type of olfactory neurons present in cartilaginous fish. The projections of crypt neurons to olfactory bulb probably correspond to the few ventrally‐located glomeruli which were negative to the antiserum against Gαo. These data suggest, in S. canicula, different olfactory neuron types send projections to the olfactory bulb with a segregated distribution, as observed in other vertebrates. Anat Rec, 2009.

Apoptosis, cell proliferation and serotonin immunoreactivity in gut of Liza aurata from natural heavy metal polluted environments: preliminary observations.

In the present paper, the effect of natural environment non-lethal heavy metal concentration on cell renewal of Liza aurata intestinal epithelium, was studied by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling) method and anti-PCNA (proliferating cell nuclear antigen) immunohistochemistry, in order to detect, respectively, apoptosis and cell proliferation. In addition, the presence and distribution of the cell renewal regulator, serotonin, was immunohistochemically investigated. In order to reduce variability, only immature specimens were considered. The results indicated that in the control specimens from non-polluted areas, the PCNA immunoreactive nuclei of the proximal intestinal epithelium were only located at the bottom of the intestinal folds, together with a few TUNEL-positive nuclei, and goblet mucous differentiated cells. In the specimens from polluted areas, the number of PCNA immunoreactive cells was greatly enhanced, and they extended along the mid portion of the intestinal folds; the number of TUNEL-positive nuclei was enhanced as well, but they were almost exclusively detected in the third apical portion of the intestinal folds. Serotonin immunoreactive nerve elements were more frequently detected in the intestinal wall of L. aurata specimens from polluted areas, and besides that, some serotonin immunoreactive endocrine cells were also present. Variations in distribution and frequency of TUNEL-positive nuclei, PCNA immunoreactive nuclei, and serotonin immunoreactivity put in evidence an alteration of cell renewal with an enhancement of cell proliferation, probably leading to morphological intestinal fold changes.

Appearance of Crypt Neurons in the Olfactory Epithelium of the Skate Raja clavata During Development

Crypt neurons are olfactory receptor cells located in the olfactory epithelium of fishes. They exhibit a peculiar and well‐recognizable morphology, although their odorant specificity is still unknown. Data on their appearance during development are few and far between. This study set out to identify the time at which crypt neurons appeared in the skate, Raja clavata, using histological and immunohistochemical methods. For this purpose, embryos and juveniles at different stages of development, from 13 weeks after laying (11 weeks before hatching) to 24 weeks after hatching, were examined. The crypt neurons were identified on a morphological basis. An anti–α‐tubulin antibody and two lectins (wheat germ agglutinin and peanut agglutinin) were used to highlight morphological details. The olfactory marker protein was detected by immunohistochemistry, because this protein is a marker of neuronal maturity in vertebrates. The crypt neurons could be detected by their morphology at 15 weeks after laying and became strongly olfactory marker protein immunoreactive 22 weeks after laying. Although involvement of crypt neurons in reproductive behavior has been inferred in various studies on bony fishes, their early presence in skate embryos and juveniles may suggest that they are not exclusively involved in sexual behavior. Anat Rec, 290:1268‐1272, 2007.

First detection of olfactory marker protein (OMP) immunoreactivity in the olfactory epithelium of a cartilaginous fish.

Olfactory marker protein (OMP) is a protein expressed in the mature olfactory and vomeronasal neurons of many vertebrates, such as mammals, amphibians and bony fishes. Aim of this work was to investigate the OMP expression in the olfactory epithelium of the shark Scyliorhinus canicula (Linnaeus, 1758), by immunohistochemistry (IHC). Immunoreactivity was detected in the olfactory receptor neurons, in the crypt neurons and in the nerve fibers below the epithelium. Although very little is known about the OMPs function, its involvement in synaptogenesis, transduction cascade, neurogenesis and development of olfactory system has been suggested. The present work shows for the first time OMPs presence in a cartilaginous fish.

G protein alpha subunits in the olfactory epithelium of the holocephalan fish Chimaera monstrosa

Receptor neurons in the olfactory and vomeronasal epithelia of vertebrates have dendritic specialization that is correlated to the receptor gene family they express and the G protein coupled with that receptor (in particular the G protein alpha subunit). There are not very many data in the literature about the morphological and molecular features of the olfactory epithelium of Chondrichthyes. In this work, the presence and distribution of different types of G protein alpha subunits (Galpha(o), Galpha(q) and Galpha(olf)) were investigated in the olfactory epithelium of the holocephalan Chimaera monstrosa using immunohistochemistry. Only Galpha(o)-like immunoreactivity was detected in the microvillous receptor neurons and in numerous axon bundles of the fila olfactoria. These preliminary data are in agreement with studies showing G protein alpha subunits in elasmobranchs and support the data present in the literature about putative odorant receptor families in the sequenced genome of the holocephalan Callorhinchus milii.

The compensatory growth in juveniles of sea bass: gastric distributive pattern of molecules regulating metabolism.

The aim of this work was to investigate the distribution of regulative molecules in the stomach of juvenile Dicentrarchus labrax during compensatory growth, using immunohistochemical methods. Antisera against galanin, neuropeptide Y, ghrelin, leptin, and serotonin were used on fasted and refed D. labrax. The results show a characteristic distributive pattern for the sought molecules in fish refed after 35 days of fasting, with a high increased presence of both ghrelin and leptin.

Cell proliferation and apoptosis in the olfactory epithelium of the shark Scyliorhinus canicula.

To date, no study has been published on cell renewal in the olfactory epithelium of Chondrichthyes. Our work aimed at detecting proliferating cells (by Proliferating Cell Nuclear Antigen - PCNA immunohistochemistry) and apoptotic cells (by terminal uridine deoxynucleotidyl transferase nick end labeling method) in the olfactory epithelium of the shark Scyliorhinus canicula. PCNA immunoreactivity and mitotic figures were localized almost exclusively at the basal and apical thirds of the epithelial thickness. Double immunofluorescence for PCNA and OMP (a marker of mature olfactory neurons) showed that PCNA immunoreactivity is lacking in mature olfactory neurons, with the exception of crypt neurons. Crypt neurons, a cell type peculiar to fish, often showed PCNA immunoreactivity in the nucleus and may be involved in repair processes. The role of PCNA in mature crypt neurons requires further investigation to be clarified. Apoptosis was observed in sensory neurons and in basal cells. Our data highlight the presence of cell proliferation at different levels within the epithelium and the occurrence of apoptosis in both mature and proliferating cells.