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

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

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