Francesca Carella

Morphological features of the inflammatory response in molluscs

Over the last few years, there has been a large increase in studying the biology and pathology of molluscs, predominantly in addressing the molecular patterns involved in their immune-mediated and inflammatory responses. Conversely, the literature-based diagnostic criteria concerning the morphology of the above phenomena still involves pathogenetic confusion and conflicting terminology. A comparison of bibliographic resources, such as the Abridged Glossary of Terms Used in Invertebrate Pathology and the National Status manual for molluscan histopathological examination and analysis from the NOAA, have revealed variability in the definitions of superimposable lesions, emphasising the need for further efforts in establishing standard terminology and methodologies in this field of study. This review suggests some possible solutions for overcoming the use of parallel terminologies in diagnosing inflammation in molluscs and also highlights conflicting features requiring further discussion.

The metallothionein genes of Mytilus galloprovincialis: genomic organization, tissue expression and evolution.

Recently, increasing interest has been directed to the study of metallothioneins (MTs), which are small proteins that are able to bind metal ions. The induction of MT synthesis after exposure to metal or other environmental contaminants in a large number of aquatic invertebrates makes these proteins good biomarkers in water monitoring programs. Within bivalves, the species Mytilus galloprovincialis and Mytilus edulis represent model organisms for these types of studies, as well as for molecular studies regarding the expression and characterization of MT encoding genes. In the present paper, we focused on the genomic characterization, evolutionary, and tissue-expression analyses of the MT-10, MT-10 Intronless, and MT-20 genes in M. galloprovincialis. The comparison of the genomic sequences showed the presence of long nucleotide stretches within the introns of the MT genes that are conserved between M. galloprovincialis and M. edulis. These non-coding conserved sequences may contain regulatory motifs. Real-Time RT-PCR experiments revealed that, at the basal conditions, the MT-10 and MT-10 Intronless genes are expressed at levels considerably higher than the MT-20 gene, mainly in the digestive gland and gill tissue. The strong induction of the MT-20 gene expression detected in a field-collected sample is associated with the up-regulation of both the MT-10 and MT-10 Intronless genes. Evolutionary analysis revealed signals of localized positive selection that, together with the tissue-expression data, support a possible functional diversification between the MTs encoded by the MT-10 and MT-10 Intronless genes.

Comparative pathology in bivalves: Aetiological agents and disease processes

Comparative pathology as a scientific discipline studies animal diseases in relation to their aetiology, pathogenesis and prognosis. Among the main aspects of this discipline, regressive changes, host defense responses with pathological implications and progressive changes, represent the majority of the possible responses of cells and tissues to pathogens and exposure to chemicals. One of the most persistent issues in the field of invertebrate pathology is the variability in terminology and definition, which has led to confusion in scientific communication. The aim of this paper is to provide an overview of the pathological basis of bivalve disease (defensive, regressive and progressive phenomena) and contribute to the standardised terminology for bivalve molluscan disease in the context of comparative pathology.

Histological, Histochemical and Morphometric Changes of Splenic Melanomacrophage Centers (Smmcs) in Sparicotyle-Infected Cultured Sea Breams (Sparus aurata)

Diseases caused by parasites are much more frequently described in cultured fish, which suffer from artificial conditions and numerous stress factors. This study investigates the histological, histochemical and morphometric modifications of splenic melanomacrophage centers (SMMCs) infected by Sparicotyle chrisophrii (Monogenea, ectoparasite of the gills) in sea breams (Sparus aurata), cultured in floating cages in the Gulf of Gaeta (Italy). Infected fish swam near the water surface, showing severe signs of anemia. Several spleens were collected from both healthy and dead fish (70–100 gr. body weight). A spleen histopathology was evaluated by using traditional stainings, such as Haematoxylin and Eosin (HE), Periodic Acid-Schiff reaction (PAS), Perls reaction for haemosiderin and Schmorls reaction for lipofuscins. Furthermore, SMMCs morphometry was performed on PAS-stained sections to study 7 morphometric parameters [Mean SMMCs profile area (MPA), Mean SMMCs maximum diameter (Media), Mean SMMCs minimum diameter (media), Mean SMMCs diameter (Dia), Mean SMMCs Perimeter (P), Mean SMMCs Form Factor (FF) and Mean SMMCs number per square millimeter of spleen tissue (MN)]. A light microscope of HE stained sections of spleen revealed a dramatic increase in the size and number of SMMCs in parasitized animals. Morphometric data illustrated statistically significant differences (p < 0.01) of all studied parameters between healthy and diseased fish. This study emphasizes the importance of using histopathological investigations to unravel the complex biological host/parasite interaction, which results in systemic lesions affecting reared marine species.

Cytomorphology and PCNA expression pattern in bivalves Mytilus galloprovincialis and Cerastoderma edule with haemic neoplasia

Haemic neoplasia (HN) is a pathologic condition reported in several bivalve species in different geographic areas. In this study we describe the cytomorphological features and the proliferative behaviour, assessed by the proliferating cell nuclear antigen (PCNA), of HN in common cockle Cerastoderma edule and Mediterranean mussel Mytilus galloprovicialis. In mussels the presence of at least 5 types of atypical haemocytes was detected, including A- and B-type cells, previously described in M. edulis and Mytilus sp., with predominance of A-type cells in early phases of the disease and B-type cells in more advanced stages. PCNA immunostaining was positive for 97 to 100% of the neoplastic cells, with both cytoplasmic (A cells) and nuclear patterns (B cells). Conversely, in C. edule there was no distinctive morphological cell sub-population, and staining atypical haemocytes with PCNA (range 93 to 100%) showed nuclear expression in early phases of disease and cytoplasmic expression in more advanced stages. The above findings suggest distinct histo-pathogenetic pathways for HN in mussels and common cockles.

Quantitative histopathology of the Mediterranean mussel (Mytilus galloprovincialis L.) exposed to the harmful dinoflagellate Ostreopsis cf. ovata

Ostreopsis cf. ovata is a benthic dinoflagellate that produces palytoxin-like compounds that adversely affect both marine vertebrates and invertebrates and are reported to be responsible for human intoxication in aerosol form. In this work, a histopathological analysis accompanied by quantitative evaluation of tissue injury in mussels (Mytilus galloprovincialis) exposed to O. cf. ovata cells under natural and experimental conditions, provided baseline data on the health status of the mussels in terms of defensive and regressive pathological changes. We recorded a total of 15 health parameters in the digestive system, muscle, kidney and gills in mussels exposed to O. cf. ovata both in the laboratory and at sea. Animals exposed to different concentrations of O. cf. ovata cells (300, 500 and 1000cellsml(-)(1)) for 48h showed activation of the inflammatory response, which increased with the cell concentration, mainly characterized by haemocyte aggregates actively enclosing the algae, while mussel mortality was also recorded in some cases. Moreover the use of image analysis for the evaluation of digestive tubule damage revealed a pronounced increase in the lumen in terms of its area, perimeter and circularity, with a shift in a high percentage of tubules from an adsorbing profile to an atrophic profile. Animals collected from the natural environment during a summer bloom of O. cf. ovata in the Gulf of Naples (Italy) showed comparable lesions in terms of types and severity. This is the first quantitative study assessing damage to the digestive epithelia in terms of lumen modifications in mussels exposed to O. cf. ovata. The presented methodology provides a new technique for automating the evaluation of epithelial tubule modifications. Our results highlight the importance of monitoring the presence of O. cf. ovata in this area, taking into account the effects on the residing marine species.

The Gonadotropin Releasing Hormone (GnRH)-like Molecule in Prosobranch Patella caerulea: Potential Biomarker of Endocrine-disrupting Compounds in Marine Environments

It has been reported that endocrine disrupter compounds (EDCs) interfere with the endocrine system, mimicking the action of sex steroid hormones in different species of mollusks. Prosobranchs are frequently used as a reliable bioindicator to evaluate EDC exposure. In this article, we evaluate the effects of the xenoestrogen 4-n-nonylphenol (NP) in the prosobranch gastropod Patella caerulea, which exhibits protandrous hermaphroditism as its reproductive strategy. We isolated a partial sequence of a GnRH-like molecule from the gonads of Patella caerulea. The deduced amino acid sequence is highly homologous to that reported for the Lottia gigantea GnRH. Patella caerulea GnRH (pGnRH) mRNA expression is widespread in both male and female germ lines during gametogenesis. We suggest pGnRH as a novel biomarker for the early assessment of presence of EDCs and monitoring short and long-term impacts on Patella caerulea community structure.

Nocardiosis in Mediterranean bivalves: first detection of Nocardia crassostreae in a new host Mytilus galloprovincialis and in Ostrea edulis from the Gulf of Naples (Italy).

In this work M. galloprovincialis and O. edulis specimens were surveyed for a pathological study in the Gulf of Naples (Mediterranean sea, Campania Region, southern Italy). Clusters of Nocardia sp.-like cells were observed in histological slides. PCR amplification, sequencing and in situ hybridization were carried out in order to corroborate Nocardia species identification for both hosts. Blast results showed a 99% of maximum identity with Nocardia crassostreae sequences in Genbank. This is the first report of N. crassostreae in the new host M. galloprovincialis and, in a new area, the Mediterranean Sea.

A case of germinoma in a limpet (Patella coerulea) (Patellogastropoda)

Germinoma is a gonadal neoplasm originating from progenitor cells in germinal epithelium. Frequently described in some populations of bivalve molluscs, to our knowledge, germinoma has never been reported in gastropods so far. In this paper we describe the histopathological findings of some atypical cellular masses, originating in the undifferentiated germ cell layer in the male gonads of a limpet (Patella coerulea), whose morphological appearance resembled that of a germ cell tumor. The abnormal, and independent growth with no evidence of maturation of the undifferentiated and atypical germ cells, the limited number of follicles involved (n<10%) and the absence of tissue invasion, supported a diagnosis of Stage 1 germinoma.

Disease and Disorders of Freshwater Unionid Mussels: A Brief Overview of Recent Studies

The use of aquatic invertebrates in biomedical research and as environmental sentinels has dramatically grown in recent decades, with an increased need in understanding of comparative pathology. The Unionids freshwater mussels are a group of worldwide distributed bivalves residing small ditches and ponds, lakes, canals and rivers, often used as animal test in eco-toxicological studies. Once one of the most abundant bivalve molluscs in ancient rivers around the world, now many of them are declining in many countries and consequently are nearly extinct in many areas. The causes of this decline are not fully understood but alteration and degradation of the freshwater habitat seemed to play a central role. To date, link causality to the observed losses during episode of mussel die-offs has been more difficult to establish, and disease and pathogen presence have been scarcely considered. In this article we provide a brief overview of unionids freshwater mussel conservation status, also describing reported diseases and pathogens and illustrating a few relatively well-documented studies.