Biotechnologies to evaluate the environmental status: New test organisms in ecotoxicology and histopathological and molecular biomarkers in natural population

Marine animals inhabiting polluted coastal waters nearness urbanised areas may be affected by a complex of toxic substances of domestic and industrial origin. Campania region with its main Naples city and Caserta province are italian areas with high pollutant impact (Maselli et al., 2010; Senior and Mazza, 2003; Imperato et al., 2003. In order to assess the biological effects of this condition, mussels belonging to Mytilus galloprovincialis species were collected in 12 rearing farms distributed from northern to southeast part of Campanian coast from January 2008 to July 2010 in A and B zone, repartition for mollusks rearing (Dlg. 854/2004); at the same time, from October 2008 to December 2009, sampling of three different species of mollusks occupying different ecological niche (M. galloprovincialis, Tapes philippinarum and Patella caerulea) were conducted at Volturno River Estuary. Tissue-level biomarkers including Defensive phenomena (Haemocyte clusters, Granuloma, Perintestinal inflammation, interstitial inflammation, Brown cells), Regressive phenomena (gonadal atresia and digestive tubules atrophy), Progressive phenomena (gonadal and haemic neoplasia), were recorded and measured in term of prevalence and intensity and related to the different sites. A total of 31 pathological parameters were recorded in the reared animals along Campanian coast, comprehended different inflammatory response, in some case related to pathogens or as apparent non-specific reaction and regressive changes represented by digestive gland epithelial thinning, often accompanied by intense haemocyte reaction. Moreover, in three areas, cases of Disseminated Neoplasia (DN) presented peculiar cellularity, monomorphism or polymorphism, were observed with a prevalence of 10%, usually few represented in the literature in M. galloprovincialis. Different kind of pathogens (Steinhausia mytilovum, Marteilia refringens, Nematopsis sp., Proctoecoes maculatus) and symbionts (Eugymnanthea inquilina, Ancystrum mytili, Rinkodid-like phyllopharingea protozoa) have been recorded, some of them observed for the first time in the area (Carella et. al., 2010; Carella et al., in press). Prevalence of recorded lesions was equally distributed, did not show a clear temporal trend, present almost in all the samples and abundant in both A and B zones. Defensive phenomena such as non-specific inflammatory responses and regressive changes are useful tools to assess environmental quality in pollution monitoring programmes (Couch, 1985; Au, 2004; Kim et al., 2007). On the basis of our observation, we have identified areas with detrimental conditions that, for current law (Dlg. 854/2004) are considered appropriate for mollusk designated to food consumption; this areas, evaluated using these pathological parameters, based on recognized international standard, wouldn’t be considered anymore appropriate, because of stressful environmental condition. On regarding on Volturno River estuary mollusk population, we have described different gonadal lesions in sampled animals. In particular, about the limpet (Patella coerulea), the majority of males collected at January 2008 showed unusual gonadal disorders, such as early change of sex, pathological hermaphroditism and germinoma (Carella et al., 2009). At the end of the year, limpets population of the estuary decline to finally disappear. Literature suggests these findings could be related to some gonadal alteration, in both humans (Testicular Disgenesis Syndrome -TDS), and animals (lower vertebrates and invertebrates) exposed to Endocrine Disruptor (ED) chemicals; these are heterogeneous classes of chemical pollutants acting upon the endocrine system, which adversely affect reproduction and development of aquatic organisms, causing negative changes in community structure, abundance, diversity and composition. In order to confirm our pathogenetic hypothesis, adult limpets (Patella caerulea) at different stages of gonadal development were exposed, for 30 days, to xenoestrogen 4-Nonylphenol (1,100,200 μg/L) and estrogen 17β estradiol (1,100 μg/L). Results showed females in more advanced phases of development than corresponding controls, while males exhibited active proliferating oocytes. In one male individual, exposed to 17β estradiol 100 μg/L, a case of intersex was recorded. In order to follow male and female gonad development during experiments, a GnRH probe has been produced on the basis of GnRH aminoacidic sequence in the genome of Lottia gigantea gastropod. In summary, we have provided data on health status of animals on Campanian coast throughout an integrative study of tissue-level biomarkers with natural and experimental measures, helpful to support the present conclusions. Moreover, such data suggest the prosobranch limpet Patella coerulea could be a reliable and effective model for EDc exposure and GnRH molecule as a new possible biomarker for such substances.