Abstract
Haemic neoplasia (HN) in bivalves has been reported in association with mass mortality events in various species of molluscs. The aim of this work was to quantify the nuclear morphometry and DNA content of neoplastic cells of mussels Mytilus galloprovincialis affected by HN using nuclear densitometry in Feulgen-stained preparations. The results were also compared with a population of normal mussel haemocytes. We captured 256 images of 3 different neoplasia stages and 120 images of normal haemocytes; thus, a total of 120,166 nuclei were analysed. We extracted 21 morphological parameters from normal and neoplastic nuclei. Eighteen of these parameters were different (P<0.05). Among those (expressed in pixel units—inter-pixel distance of 0.45 micrometres—as: normal vs. neoplastic) nuclear area (117.1±94.1 vs. 423.1±226.9), perimeter (44.9±14.0 vs. 79.0±21.3) and (IOD) integrated optical density (13.47±34.5 vs. 177.1±150.8) were relevant features to discriminate between normal and neoplastic cells. Those differences allowed identifying two distinctive populations of neoplastic nuclei, occasionally in the same individuals at a given phase of the disease. Moreover, neoplastic haemocytes in less extended lesions showed a ploidy value of 6.2 n along with the presence of a second population of circulating cells with a DNA content of 10.7n. In samples with moderate disease only one peak at 7n was observed. Finally, in more severe conditions, a further ploidy peak of 7.8n was recorded, accompanied by a shallow but broad peak of 31n. This latter extreme value is thought to be due to the presence of giant multinucleated cells where individual nuclei overlap in space and cannot be discerned individually. Computer-based imaging allowed the direct visualization of the cell populations and simultaneous collection of ploidy data as well as morphological features of nuclei.
Highlights
Nuclear morphology is an important feature associated with cellular function and disease
In order to add new insights in the biology of this interesting neoplasia, we focused on Haemic neoplasia (HN) in M. galloprovincialis and used a morphometric approach involving light microscopy to 1) describe the nuclear morphology and quantify DNA contents of the neoplastic cells of mussels affected by HN in Feulgen-stained histological preparations; 2) compare the obtained data with normal haemocytes; and 3) assess the data in the context of the disease progression according to staging
Four cases were at the intermediate level of neoplasia characterized by small isolated scattered foci of both A and B cells distributed in different percentages in gills, visceral mass, mantle and kidney (Fig 3A and 3C)
Summary
Nuclear morphology is an important feature associated with cellular function and disease. In relation to cell types, there has been reported the existence of the so-called A and B cell subpopulations in individuals affected with HN in different mussel species (e.g. M_edulis and M. galloprovincialis) under both light and electron microscopy in contrast to flow cytometry [13, 33,34] These cells, characteristic of haemic neoplasia in mussel species, are discernible by their peculiar morphology [13, 33,34], their role in disease pathogenesis have not been clarified. In order to add new insights in the biology of this interesting neoplasia, we focused on HN in M. galloprovincialis and used a morphometric approach involving light microscopy to 1) describe the nuclear morphology and quantify DNA contents of the neoplastic cells of mussels affected by HN in Feulgen-stained histological preparations; 2) compare the obtained data with normal haemocytes; and 3) assess the data in the context of the disease progression according to staging
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