Lorenzo Gallus

Toxic effects of harmful benthic dinoflagellate Ostreopsis ovata on invertebrate and vertebrate marine organisms

Harmful benthic microalgae blooms are an emerging phenomenon causing health and economic concern, especially in tourist areas. This is the case of the Mediterranean Sea, where Ostreopsis ovata blooms occur in summer, with increasing regularity. Ostreopsis species produce palytoxin (PTX) and analogues, and a number of deaths directly associated with the ingestion of PTX contaminated seafood have been reported. PTX is considered one of the most toxic molecules occurring in nature and can provoke severe and sometimes lethal intoxications in humans. So far in temperate areas, O. ovata blooms were reported to cause intoxications of humans by inhalation and irritations by contact. In addition, invertebrate mass mortalities have been reported, possibly linked to O. ovata blooms, although other causes cannot be ruled out, such as oxygen depletion or high seawater temperature. In order to improve our knowledge about the direct toxicity of this species on invertebrate and vertebrate marine organisms, we performed an ecotoxicological screening to investigate the toxic effects of different concentrations of O. ovata (cultured in the laboratory and sampled in the field during blooms) on crustaceans and fish as model organisms. Artemia salina, Tigriopus fulvus, and Amphibalanus amphitrite larvae and juveniles of the sea bass Dicentrarchus labrax were used as model species. Toxic effects associated with cultured O. ovata cells were investigated using a crossed design: testing two different temperatures (20 and 25 °C), four different cell concentrations, and four treatments (untreated O. ovata culture, filtered and resuspended algal cells, growth medium devoid of algal cells, and sonicated algal cells). The results indicate that the toxicity of cultured O. ovata is related to the presence of living O. ovata cells, and that this effect is amplified by temperature. Furthermore, both tests with laboratory cultured algae and field sampled cells pointed out that A. salina is the most sensitive species even at concentrations below the Environmental Alarm Threshold set by the Italian Ministry of Health. Some possible explanations of such sensitivity are discussed, taking into account evidence of O. ovata cells ingestion and the activity of its toxins on the Na⁺/K⁺-ATPase.

Involvement of Acetyl Choline in Settlement of Balanus amphitrite

The aim of the present study was to investigate the presence and distribution of cholinergic molecules in Balanus amphitrite cyprids and their possible involvement in settlement and adhesion. Acetylcholinesterase (AChE, the lythic enzyme of acetylcholine) activity was detected, for the first time, by biochemical and histoenzymological methods, in the thoracic muscles, gut wall and cement gland. The immunodetection of choline acetyltransferase-like (ChAT) molecules in the same area and in the neuropil of the central nervous system suggests the presence of a cholinergic innervation, and the involvement of acetylcholine in muscular contraction and cement gland exocytosis. The binding of FITC-conjugate f -bungarotoxin in the cement gland cells confirms the latter hypothesis. Acetylcholine involvement in the settlement process was also investigated by laboratory tests employing cholinergic antagonists and agonists. An increase of available acetylcholine due to the partial inhibition of AChE activity produced an increase in cyprid settlement. The data presented support the hypothesis that acetylcholine has a neurotransmitter/neuromodulator role in settlement and adhesion of barnacle cyprids.

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.

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.

Endocytosis of GABAB receptors modulates membrane excitability in the single-celled organism Paramecium.

GABAB receptors modulate swimming behavior in Paramecium by inhibiting dihydropyridine-sensitive Ca2+ channels via G-proteins. Prolonged occupancy of GABAB receptors by baclofen results in a decrease in GABAB receptor functions. Since changes in the number of cell-surface GABAA receptors have been postulated to be of importance in modulating inhibitory synaptic transmission in neurons, we have studied the cell-surface expression and maintenance of GABAB receptors in P. primaurelia. In this study, we use immunostaining in electron and confocal microscopy to demonstrate that constitutive internalization of GABAB receptors in P. primaurelia is mediated by clathrin-dependent and -independent endocytosis. Indeed, GABAB receptors colocalize with the adaptin complex AP2, which is implicated in the selective recruitment of integral membrane proteins to clathrin-coated vesicles, and with caveolin 1, which is associated with uncoated membrane invaginations. Furthermore, when endocytosis is blocked with hypertonic medium, cytosol acidification, filipin or with a peptide that disrupts the association between amphiphysin and dynamin, the effect of baclofen on swimming is increased. These results suggest that GABAB receptor endocytosis into clathrin-coated and -uncoated vesicles represents an important mechanism in the modulation of swimming behavior in Paramecium.

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.

Detection of cholinesterase activities and acetylcholine receptors during the developmental cycle of Dictyostelium discoideum

Previous results of our study carried out on Dictyostelium discoideum showed the presence in single-cell amoebae of both cholinesterase (ChE) activity, able to hydrolyze the propionyl thiocholine iodide substrate, and “true” acetylcholinesterase (AChE) activity, similar to that of Electrophorus electricus . These activities are sensitive to anti-ChE agents, and to the inhibitors classically used to discriminate different ChEs. In this work, we have investigated the presence of ChE activities and of both acetylcholine (ACh) nicotinic and muscarinic receptors, during the developmental cycle of D. discoideum . The spectrophotometric evaluation showed that slugs displayed the highest enzyme activities, AChE being the main activity, while fruiting bodies exhibited the lowest. The presence of AChE activity was cytochemically detected in all the cell populations of the developmental cycle, except in the fruiting bodies where it was localized exclusively to the level of the spores. Molecules recognized by the anti-butyrylcholinesterase antibody were detected in single-cell amoebae, first fingers, slugs, and culminants, except in the fruiting bodies where they were found exclusively in the spore walls. The ACh nicotinic receptors were cytochemically identified in single-cell amoebae, slugs, and spores; however, molecules immunologically related to the ACh muscarinic receptors were not present in the spores.