Maria Violetta Brundo

Cellular biomarkers in the mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) from Lake Faro (Sicily, Italy)

Abstract Lake Faro (Sicily, Italy) is a natural confined brackish environment particularly subject to anthropogenic impact, resulting in a mixture of xenobiotic substances, i.e. heavy metals and polycyclic aromatic hydrocarbons (PAHs), and characterised by low hydrodynamics. In order to assess the water quality status of this pond, a multi-biomarker approach was applied on mussel Mytilus galloprovincialis (Lamarck, 1819) both inhabiting the lake and from a control site (Goro). Different biomarkers were investigated on mussel digestive glands and gills, including biomarkers of exposure (cytochrome P450 4, CYP4), neurotoxicity (acetylcholinesterase, AChE; choline acetyltransferase, ChAT), general stress (lysosomal membrane stability, LMS), and genotoxicity (micronucleus and comet assays). The results suggest significant responses related to the selected area. A statistically significant inhibition (P < 0.0001) of CYP4 in the digestive gland, and of AChE and ChAT in the gills, was found in specimens collected from Faro compared with the control. The lysosomal membrane stability of mussels from Lake Faro was lower than the reference site, while the DNA damages were significantly higher in mussels from the brackish area compared to the control. This study represents the first time the quality status of Lake Faro is assessed using a multi-biomarker approach on the mussel M. galloprovincialis, which proved to be suitable to identify the effects of environmental pollutants at molecular and cellular levels.

Effects of "in vivo" exposure to toxic sediments on juveniles of sea bass (Dicentrarchus labrax).

Aquatic ecosystems are affected by all the impacts generated by a variety of anthropogenic activities present along coastal environments. The sediment compartment is the final receptor of water-insoluble pollutants, acting both as a sink and as a source of pollutants to the water column, and affecting both nektonic and benthic organisms. The aim of this study is to assess the impact of metals in the sediments collected from two sites in the petrochemical area between Augusta and Priolo (SR, Sicily, Italy) on gills of Dicentrarchus labrax. This was done to enhance the scarce knowledge on the bioavailability of metals bound to sediment and their capacity to interact with the bioindicator species. Various sublethal endpoints were assessed such as histopathological lesions, metallothioneins (MTs) and molecules involved in the homeostasis pathways by immunolocalization and RT-PCR. In the specimens exposed to sediments, the data suggested a reduction of gill cell membrane permeability, which could result in altered osmotic balance and gas exchange. Further, an increase of MT expression was detected, consisted the involvement of this protein in detoxification of toxic non-essential metals. The findings of this study demonstrate that a subchronic test, conducted by using sensitive and sub-lethal endpoints, in combination with chemical analyses, is a powerful tool for early identification of environmental hazards associated with contaminated sediments.

Modification of graphene oxide by laser irradiation: a new route to enhance antibacterial activity.

The antibacterial activity and possible toxicity of graphene oxide and laser-irradiated graphene oxide (iGO) were investigated. Antibacterial activity was tested on Escherichia coli and shown to be higher for GO irradiated for at least three hours, which seems to be correlated to the resulting morphology of laser-treated GO and independent of the kind and amount of oxygen functionalities. X-ray photoelectron spectroscopy, Raman spectroscopy, dynamic light scattering and scanning electron microscopy (SEM) show a reduction of the GO flakes size after visible laser irradiation, preserving considerable oxygen content and degree of hydrophilicity. SEM images of the bacteria after the exposure to the iGO flakes confirm membrane damage after interaction with the laser-modified morphology of GO. In addition, a fish embryo toxicity test on zebrafish displayed that neither mortality nor sublethal effects were caused by the different iGO solutions, even when the concentration was increased up to four times higher than the one effective in reducing the bacteria survival. The antibacterial properties and the absence of toxicity make the visible laser irradiation of GO a promising option for water purification applications.

First data on trace elements in Haliotis tuberculata (Linnaeus, 1758) from southern Italy: Safety issues.

This study evaluated for the first time the concentrations of 10 metals in wild specimens of abalone, Haliotis tuberculata (Ht) (Linnaeus, 1758) from three sites along the southern Italian coast: Gulf of Catania (CT), the Northern Coast of Messina (ME) and the harbor of Villa San Giovanni (VSG). The species is commonly found in the area and has significant commercial value. Additionally, it is long lived, thus suitable as bioindicator of the environmental monitoring. The potential human health risks due to consumption of Ht have been assessed by estimated average daily intake (EDI) and target hazard quotient (THQ) of metals, respectively. In particular arsenic (As), cadmium (Cd), copper (Cu), chromium (Cr), iron (Fe), lead (Pb), manganese (Mn), mercury (Hg), selenium (Se) and vanadium (V) were quantified in the edible tissue of specimens by acid digestion of the samples and ICP-MS determination. The highest concentrations were found in CT sample area for most metals analyzed. Mean values for Pb, Cd and Hg were lower than the maximum levels (MLs) set for bivalve mollusks by Regulation (CE) no. 1881/2006 in all sites, and average intake values below the risk levels for human consumption.

Morphological and ultrastructural organization of the male genital apparatus of some Aphididae (Insecta, Homoptera)

Ultrastructural investigations on the amphigonic reproductive mechanisms in Aphidoidea (Homoptera, Sternorryncha) species, of which little is known in the literature, can provide useful information on their reproductive biology. Morphological and ultrastructural investigations were carried out on the reproductive tract and on spermatozoa from sexually mature males of five species belonging to three subfamilies of Aphidoidea. The organization of the reproductive tract and of spermatozoa appears simple and similar in the examined species. Each testis consists of three follicles containing many cysts arranged in a progressive order of maturation from the distal to the proximal tract; spermiogenesis only occurs in sexually mature males, ending with the organization of sperm bundles. Gametes are neatly arranged in each bundle and kept together by a cap showing a particular organization. The distal tract of each spermiduct is enlarged and full of gametes; close to the testis the two spermiducts merge together, except in Drepanosiphum platanoidis where the spermiducts run independently. The various tracts of each examined gland showed no peculiar differences either in the same species or among the species. Some interesting hypotheses are proposed in this work about the probable roles of the investigated structures in the reproductive mechanisms of these insects.

Bioaccumulation of cadmium and lead and its effects on hepatopancreas morphology in three terrestrial isopod crustacean species.

This study was designed to compare cadmium (Cd) and lead (Pb) bioaccumulation in three species of oniscidean isopods - Armadillidium granulatum Brandt, Armadillidium vulgare (Latreille) and Porcellio laevis Latreille which were exposed for three weeks to a contaminated diet, and to determine the morphological and ultrastructural changes in hepatopancreas. Metal accumulation, determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), was linearly associated with the exposed concentration and was a function of the metal and the species tested. All three species accumulated lower levels of Pb than Cd. A. vulgare accumulated the largest concentration of Pb, especially at the higher doses, whereas P. laevis showed the greatest Cd accumulation, and the highest Cd concentration was lethal for all exposed species. The highest concentrations of Pb and Cd induced significant changes both in the general morphology of tubules and in the ultrastructural organization of epithelial cells in hepatopancreas. Some Pb/Cd induced alterations include: brush border disorganization; reduction of the basal labyrinth formed by the plasma membrane; condensation of some cytoplasm areas and of chromatin; rough endoplasmic reticulum and mitochondrial alterations; increase of secondary lysosomes and of type B granules in S cells. Some of the ultrastructural changes observed overlap with those induced by prolonged starvation, whereas others can be useful biomarkers of heavy metal toxicity. This study has confirmed that in terrestrial isopods, the accumulation of the different metals occurs in a species-specific manner; therefore ecological monitoring and assessment studies should consider each species individually. The research has confirmed that in the terrestrial isopods the accumulation of the different metals occurs in a species-specific way; therefore each species should first be evaluated in view of its employ in biomonitoring programs.

Mode of transfer of spermatozoa in Orthoptera Tettigoniidae

A morphological and ultrastructural study was carried out on the spermatophore and spermatodoses of some species of Orthoptera Tettigoniidae. From the results concerning the spermatophore it emerged that this structure has a morphological and ultrastructural organization represented by a dilated ampulla and a peduncle or neck. From the examination of freshly deposited spermatophores and those at various time intervals thereafter, it was seen that these structures other than allowing gamete transfer, represent the site where spermatodesms, organized in the male genital tracts, undergo reorganization to acquire their definitive morphological and structural characteristics as found in the female genital tracts. The spermatodoses, in the same way as the spermatophore, represent capsules containing spermatodesms, which are originated in the spermatheca, their specific morphology seems to diversify according to the species considered. As regards their role, it is hypothesized that these structures represent a long-term conservation mechanism for spermatozoa inside the seminal receptacle.

Morphological and ultrastructural organization of the spermatheca of some Tettigoniidae (Insecta, Orthoptera)

Abstract A morphological and ultrastructural study of the spermatheca of some species of Orthoptera Tettigoniidae was carried out to understand the role of this female organ in the reproductive biology because no literature exists about it in this insect group. In all the examined species, the spermatheca is of ectodermal origin and is composed of a seminal receptacle, mainly involved in the collection and storage of the spermatozoa, and of a spermathecal duct. In both these organs, the epithelium of the wall is made up of two different cell types: cuticle-forming cells, underlying the cuticular intima, and gland cells. Both of these cell types have secretory features that differ among the species and also within the same species, in relation to the tracts examined. In particular, the ultrastructure of the distal tract of the spermathecal duct indicates a more marked secretory activity than in the other tracts of the duct. This activity is often accompanied by ultrastructural aspects, suggesting a lysis activity in both the epithelium and the upper cuticle. Based on these findings, it is hypothesized that the seminal receptacle and the spermathecal duct have different functional roles, despite having a similar general structural organization.

Structure of male accessory glands of Bolivarius siculus (fischer) (Orthoptera, Tettigoniidae) and protein analysis of their secretions.

In Tettigoniidae (Orthoptera), male reproductive accessory glands are involved in the construction of a two‐part spermatophore; one part, the spermatophylax, is devoid of sperm and considered a nuptial gift. The morphology, ultrastructure, and secretion protein content of the male reproductive accessory glands from Bolivarius siculus were investigated. Two main groups of gland tubules open into the ejaculatory duct: the “first‐order” glands, a number of large anterior tubules, and the “second‐order” glands, smaller and more numerous tubules positioned posteriorly. Along with a further subdivision of the gland tubules, we here describe for the first time an additional gland group, the intermediate tubules, which open between first and second‐order glands. The mesoderm‐derived epithelium of all glands is a single layer of microvillated cells, which can be either flattened or cylindric in the proximal or distal region of the same gland. Epithelial cells, very rich in RER and Golgi systems, produce secretions of both electron‐dense granules and globules or electron‐transparent material, discharged into the gland lumen by apocrine or merocrine mechanisms, respectively. With one exception, a unique electrophoresis protein profile was displayed by each of the gland types, paralleling their unique morphologies. To assess the contribution of different types of accessory glands to the construction of the spermatophore, the protein patterns of the gland secretions were compared with those of the extracts from the two parts of the spermatophore. All samples showed bands distributed in a wide range of molecular weight, including proteins of very low molecular mass. However, one major high molecular weight protein band (>180 kDa) is seen exclusively in extracts from the first‐order glands, and corresponds to an important protein component of the spermatophylax. J. Morphol., 2009.

Toxic Effects of Zinc Chloride on the Bone Development in Danio rerio (Hamilton, 1822)

The increase of heavy metals in the environment involves a high exposure of aquatic organisms to these pollutants. The present study is planned to investigate the effects of zinc chloride (ZnCl2) on the bone embryonic development of Danio rerio and confirm the use of zebrafish as a model organism to study the teratogenic potential of this pollutant. Zebrafish embryos were exposed to different ZnCl2 concentrations and analyzed by ICP-MS. The skeletal anomalies were evaluated to confocal microscope after staining with calcein solution and RhodZinTM-3,AM. The data show a delay in hatching compared with the controls, malformations in the process of calcification and significant defects in growth. In conclusion, the current work demonstrates for the first time the Zn toxic effects on calcification process and confirm zebrafish (Danio rerio) as suitable alternative vertebrate model to study the causes and the mechanisms of the skeletal malformations.