V. Mazzei

Ligia italica (Isopoda, Oniscidea) as Bioindicator of Mercury Pollution of Marine Rocky Coasts

In this study, we evaluated the possible role of Ligia italica as a bioindicator for the monitoring of heavy metals pollution in the suppralittoral zone of marine rocky coasts. Between 2004 and 2011 specimens of L. italica were collected along the Eastern Sicilian coasts from sites known for their high pollution levels as they are near to an area where in September 2001 a refinery plant discharged into the sea some waste containing Hg. Other specimens were collected from the Vendicari Natural Reserve located about 30 miles from the polluted sites and used as control area. On a consistent number of animals, the concentration in toto of As, Cd, Cr, Hg, Ni, Pb, V, was determined by Atomic Absorption Spectrometry. On other animals, investigations were carried out in order to check for ultrastructural alterations of the hepatopancreas, that is the main metals storage organ in isopods. Results revealed the presence, in the animals collected in 2004 from the polluted sites, of considerable concentrations of Hg and of lower concentrations of other metals such as As, Pb and V. The Hg bioaccumulation resulted in remarkable ultrastructural alterations of the two cellular types (B and S cells) in the epithelium of the hepatopancreas. Surprisingly, a moderate amount of Hg was also found in specimens collected in 2004 from the Vendicari Natural Reserve, proving that the Hg pollution can also spread many miles away. Animals collected from the polluted sites in the following years showed a progressively decreasing Hg content, reaching very low levels in those from the last sampling. Also, the ultrastructural alterations found in the hepatopancreas of the animals from the last sample were quite irrelevant. In conclusion, Ligia italica can represent a good bioindicator and the ultrastructure of the hepatopancreas could be used as ultrastructural biomarker of heavy metals pollution in the supralittoral zones.

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.

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.

Isopods of the genus Ligia as potential biomonitors of trace metals from the gulf of California and pacific coast of the Baja California peninsula.

Supralittoral and high intertidal coastal zones are exposed to pollution from both marine and terrestrial sources and undergo higher deposition rates than the subtidal zone. It is therefore important to identify organisms for this section of the coastal area that can be tolerant to contaminants. The aim of this study was to determine if supralittoral isopods of the genus Ligia can be used as biomonitors, since they are abundant and widely distributed. For this purpose, concentrations of trace elements were determined in Ligia isopods in toto from 26 locations across the Gulf of California and Pacific coast of the Baja California peninsula, which were collected during the summers of 2009 and 2010. The concentrations of trace elements followed the order of; Zn≥Cu>As>Cd>Pb>Hg. Elevated concentrations of copper (up to 1010 μg/g) were detected in Ligia from Santa Rosalía (SRo), a locality where industrial mining of copper has historically occurred. Industrial and municipal sewage discharges appear to have contributed to the high concentrations of zinc (326 μg/g) and lead (144 μg/g) found in organisms from Guaymas location. The high mercury concentration in organisms from Mazatlán (M) (2.01 μg/g) was associated with a thermoelectric plant. Natural sources of metals were also detected; coastal upwelling appears to be associated with high cadmium concentrations in Ligia from Punta Baja (PB) (256 μg/g) in the Pacific coast, whereas hydrothermal vents may have contributed to high concentrations of arsenic at Ensenada (E) (61 μg/g). Our results suggest that Ligia isopods reflect the natural and anthropogenic inputs of trace metals in the environment and could potentially be used as biomonitor organisms of the intertidal rocky shores of the Gulf of California and Pacific coast.

Metallothioneins and heat shock proteins 70 in Armadillidium vulgare (Isopoda, Oniscidea) exposed to cadmium and lead.

The heavy metals bioaccumulation capability in Armadillidium vulgare feeded with chestnut leaves contaminated with various sublethal concentrations of Cd and Pb, was evaluated under laboratory conditions. The metal concentration found in the hepatopancreas of treated animals, as measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS), affected the expression and localization of MT and HSP70 as shown by immunohistochemical and western blotting analysis. The Cd content of the animals treated with the various concentrations of the metal has been always higher than that of chestnut leaves contaminated. The accumulation of Pb was, instead, always modest compared to the content of the chestnut leaves. The immunohistochemical investigation in hepatopancreas tissue of animals treated with increasing concentrations of Cd and Pb, by using the anti-MT and anti-HSP70 antibodies, has provided a response clearly positive even if differentiated in relation to the metal and concentration tested. In particular, a positive response to anti-MT antibody was detected in B and S cells nuclei and S cells cytoplasm; the localization of HSP70 was particularly intense at the cell surface. Western blotting analysis showed significant up-regulation of the expression (about 2.6 fold) of HSP70 proteins in the hepatopancreas of animals exposed to highest Pb concentrations respect to control. Moreover, samples exposed to higher Cd and Pb concentrations showed a higher expression of MT (3.2 fold and 4 fold respectively) compared to control. In summary, our data beyond to clearly demonstrate for the first time the expression of MT in terrestrial isopods, suggest that A. vulgare would be a suitable organism for assessing Cd and Pb exposure in environments threatened by metal pollution as suggested by the modulation of the biomarkers MT and HSP70.

Presence of F-actin in sperm head of Armadillidium peraccae (Isopoda, Oniscidea)

Sperm of Armadillidium peraccae have been examined with cytochemical and immunocytochemical methods for fluorescence and electron microscopic visualization of cytoskeleton components. Sperm incubation in an antibody anti-β-tubulin shows only the presence of two centrioles located in the cytoplasmic region above the nucleus; no other microtubules are present in the sperm head. Instead, fluorescence microscopy of sperm incubated in FITC-phalloidin allowed to detect the presence of a large amount of F-actin in the apical region of the sperm head. The incubation of ultrathin sections of sperm embedded in Lowicryl K4M with a phalloidin-gold complex allowed a more precise localization of F-actin in the amorphous part of the acrosome and in the cytoplasmic region between acrosome and nucleus; F-actin is also present in the thin cytoplasmic layer between plasma membrane and nuclear envelope at the apical portion of the nucleus. Although the sperm was always found completely devoid of motility, the discovery of the presence of an actin cytoskeleton leads us to hypothesize a possible acquisition of motility by the sperm at the time of its interaction with the female gamete. Such a hypothesis is supported by what is known for ostracods whose aflagellate sperm implement a type of amoeboid movement only at the time of their interaction with the female gamete.

Effects of heavy metal accumulation on some reproductive characters in Armadillidium granulatum Brandt (Crustacea, Isopoda, Oniscidea).

The impact of heavy metal bioaccumulation on reproduction of the isopod Armadillidium granulatum was studied by exposing the animals to food contaminated with various sub-lethal concentrations of cadmium, lead and zinc salts over a period of three weeks. The analysis carried out by atomic absorption spectrometry on whole body and on isolated female and male genital systems highlighted that, although metals bioaccumulation was always concentration-dependent, it varies considerably depending on the metal (Cd>Zn>Pb) as pointed out by the respective values of the concentration factor. The heavy metals bioaccumulation has influenced in different ways the reproductive characters observed; while no significant difference was found with regard to the length of the incubation period and the number of broods--A. granulatum has an iteroparous reproductive strategy--the onset and the length of the reproductive season were negatively affected by the increase in concentration of the tested metals, in particular of Cd. The rate of gravid females, instead, was negatively affected by the bioaccumulation of Cd and Zn while in the groups treated with the highest concentrations of Pb all females produced at least one brood. The number of juveniles released from the brood pouch at the end of incubation resulted considerably higher and it was always positively correlated to the increase of the concentration of each metal, except for the highest Pb concentration. The explanation of this result, apparently anomalous, could be the object of a future research.

Variability of the female genital system morphology and of sperm storage in terrestrial isopods (Isopoda, Oniscidea)

Abstract In females of Isopoda Oniscidea, the genital system displays a remarkable variability of its morphological and functional organization as possible adaptation to different strategies of sperm storage. In all the oniscidean species, the sperm received from the male during mating are temporarily stored in the bursa copulatrix, a chitinous pouch of the oviduct. In 27 of the 32 species we studied, the sperm that remain in the bursa copulatrix after the fertilization of eggs of the first oviposition are transferred into the seminal receptacle, where they can be stored for a variable time. In these species, the seminal receptacle is a small kidney-shaped cup, localized at the insertion of the oviduct into the ovary. Only in two species of the genus Trichoniscus, in which the oviduct is very short, is the bursa copulatrix modified to form a large lateral diverticle. Instead, in three species of the family Halophilosciidae, Halophiloscia couchii, Halophiloscia hirsuta and Stenophiloscia glarearum, the ovary is consistently shorter, while the seminal receptacle is greater; after mating, both seminal receptacle and ovary appear completely filled with sperm. Finally, in two species of the family Tylidae, Tylos europaeus and Helleria brevicornis, the female genital system lacks specialized structures for sperm storage, and every oviposition requires a mating for the eggs fertilization. The authors present some hypotheses to explain the variability of the female genital system morphology and the sperm storage strategies.

Spermiophagic activity in the female genital system of some species of terrestrial isopods (Crustacea, Halophilosciidae).

The females of some species of the family Halophilosciidae receive in the course of mating a quantity of sperm considerably redundant with respect to the number of eggs that can be fertilized; this is possible thanks to the peculiar morpho-functional organization that characterizes their genital system and that allows them to store the sperm not only in the great seminal receptacle but also within the ovary. While most of the sperm stay free in the lumen of the seminal receptacle, a part of those present in the ovary undergoes a process of capture by the follicular cells with consequent internalization within endocellular cavities. This process concerns exclusively the immotile tail, that characterizes the peculiar spermatozoon of the isopods and which is essentially of proteic nature. After their capture the sperm tails undergo a gradual process of digestion, which seems to be apparently realized without the intervention of lysosomes. The possible role of this spermiophagic activity might be to represent a significant trophic paternal investment aimed at improving the fitness of the female and of the offsprings.

Testis follicles ultrastructure of three species of terrestrial isopods (Crustacea, Isopoda Oniscidea).

The aim of the research, carried out on three species of terrestrial isopods - Armadillidium granulatum, Halophiloscia hirsuta and Trichoniscus alexandrae - is to bring a first consistent contribution to the knowledge of the ultrastructural organization of the testis follicles. The testis follicles are seat of a remarkable dynamic activity of their cell components (somatic cells and germ cells) that results in a continuous variation, related to the trend of spermatogenesis, of their morphology, organization and of the relationships between the two cell populations. The somatic cells, known in literature as follicular cells, nurse cells or Sertoli cells, are arranged at the periphery of the follicle to form an epithelial layer of variable thickness resting on a thin basal lamina in turn surrounded by a discontinuous network of muscle cells. In A. granulatum and H. hirsuta, two types of Sertoli cells are present: a first type, the nurse cells, envelop the spermatids in cavities within their cytoplasm and through their secretion activity play a fundamental role in the formation of the spermatophores; moreover, they phagocytizes the residual cytoplasm of spermatids. A second type of Sertoli cells shows features that leave clearly identify its supporting role to the spermatophores in formation. In T. alexandrae, instead, only one type of Sertoli cells, the nurse cell, is present, whose features are widely superimposable to those observed in the other two species. Moreover, two septa of Sertoli cells depart from the periphery of the testis follicle to constitute an articulated compartmentalization of the follicle itself, probably targeted to realize at its inside a series of microenvironments functionally diversified in order to meets the needs of the different stages of the spermatogenic cycle.