Histological sections of growth anomalies (GA) only identified in blister-affected samples embedded in LR white resin.

<p>A) and d) Survey sections stained with toluidine blue. B) and e) Sections stained with acridine orange for the detection of bacteria, as visualised by red fluorescence. In this case, red fluorescence is attributed to autofluorescence of host nuclei (e.g. mucocyte nuclei). C) and f) Nigrosin staining, targeting necrotic tissues. Block arrow indicates epithelium separated from underlying tissue, revealing a cleft (represented by the asterisk). Dashed arrow highlights growth from a centralised area. Scale bars = 10 µm.</p

Microbial analysis of <i>Plectropomus leopardus</i> samples; a) Bacterial 16S rRNA gene fingerprints (represented on Denaturing Gradient Gel Electrophoresis) of fish mucus (SWB) and tissue samples (TSU), standardised for gel-to-gel comparison using BioNumerics; b) resin embed histological section of a healthy fish, stained with the general DNA stain 4′6-diamidino-2-phenylindole (DAPI); c) histological section of the lesion on a diseased fish stained with DAPI, both showing no bacteria within the dermis suggesting the bacteria present in (a) are localised within the mucus layer not within the tissues.

<p>Scale bars = 10 µm.</p

Lesions were present on approximately 15% of the sampled population of <i>Plectropomus leopardus</i>; a) affected individual showing <10% coverage of body surface; b) <i>P. leopardus</i> with almost complete coverage >90%; c) healthy tissue under light microscope and d) the lesion.

<p>Scale bars = 20 µm.</p

Representative histological sections from healthy (a, b and c) and blister-affected (d, e and f) <i>Echinopora lamellosa</i> tissues embedded in LR white resin.

<p>Blister sections were taken at the blister-affected interface. A) and d) Survey sections stained with toluidine blue (E = epidermis, G = gastrodermis and Me = mesoglea). B) and e) Sections stained with acridine orange for the detection of bacteria, as visualised by red fluorescence. In this case, red fluorescence is attributed to autofluorescence of symbiotic algae nuclei and coral mucus (Mu). C) and f) Nigrosin staining, targeting necrotic tissues. Scale bars = 10 µm.</p

Microscopic images of <i>Plectropomus leopardus</i> tissues; a) Scanning Electron Micrograph (SEM) of the healthy tissue; b) SEM of the lesion.

<p>MGC = mucus goblet cells, M = mucus. c) Light microscope image of a healthy scale and d) light microscope image of a diseased scale, showing disorganisation of natural melanin patterns seen in (c). Scales bars = 10 µm.</p

Transmission Electron Micrographs of different samples of <i>P. leopardus</i> exhibiting; (a) healthy tissue showing the two cell types (A & B) associated in the dermis along the collagenous basal membrane (CBM).

<p>Cell A shows localisation of melanosomes and Cell B shows absence of melanosomes in the same area. (b) Lesion showing disorganisation of pleomorphic cells (A & B) with an increase in number and spread of melanosomes. This lesion is an example of a <i>P. leopardus</i> suffering from stage II melanoma, where the melanosomes are restricted to the dermis. Scale bars = 10 µm.</p

Histological section of LR white resin embedded samples of healthy and diseased <i>Plectropomus leopardus</i>; a) Healthy section stained with toluidine blue; b) lesion stained with toluidine blue; c) healthy section stained with melanin specific stain Masson-Fontana; d) lesion stained with Masson-Fontana; e) Transmission Electron Micorgraph (TEM) of healthy section; f) TEM of lesion; g) higher magnification of TEM in (e); h) higher magnification of TEM in (f).

<p>Scale bars for (a–f) = 10 µm; scale bars for (g) and (h) = 2 µm. D = dermis (cologne of stroma), E = epithelium, M = melanosome, N = cell nucleus, CBM = caliginous basal membrane. Double headed arrows shows thickening of the integument, characteristic of laboratory induced-melanomas in the <i>Xiphophorus</i> model.</p