Figure 2. Detection and characterization of specific and non-specific fluorescence in situ hybridization (FISH) probe binding to target bacteria (BA and Bac), granular gland cells (Gc), and spirocysts (Sp) using Cy3-labelled FISH probes (A–C, E, F, H, I) and Hematoxylin and Eosin (H&E) staining of coral tissue sections (D, G).

Non-specific binding of EUB338 (A) and nonEUB338 (B) FISH probes to granular gland cells and lack of auto-fluorescence of these structures in probe-free treatments (C) is demonstrated through serial tissue sections. Detailed granular gland cell morphology within the gastrodermis is shown in H&E stained (D) and EUB338 FISH-hybridized (E) tissue sections. Non-specific binding of EUB338 FISH probes to spirocysts within epidermal cells (F) was detected in tissue sections and was particularly prevalent in coenosarc and tentacle regions of polyps. Detailed spirocyst morphology is shown through H&E staining (G). A bacterial aggregate within the calicoblastic layer is shown near to non-specific binding signals of granular gland cells (H) in healthy coral tissues hybridized with EUB338 FISH probes. Bacteria assemblages were detected within necrotic tissues associated with WS disease using EUB338 FISH probes (I). Scale bars represent 50 µm in (A–C) and 10 µm in (D)–(I). Abbreviations: Gc, granular gland cell; Sp, spirocysts; Symb, Symbiodinium; BA, bacterial aggregation; Bac, bacterial assemblages.