Samples of Aplysina aerophoba were collected by scuba at 3-7 m depth at Pacug, Slovenia for microbiological and chemical comparison between sponge lesions and unaffected tissue on 10 diseased sponges and 10 healthy controls. To estimate disease incidence, a survey was performed along a randomly selected 15m line transect: all healthy and diseased specimens found within 0.5 m to either side were recorded: 53 Aplysina aerophoba colonies were recorded of which, 22 presented visible disease symptoms (black patches, white necrotic tissue or exposed skeletal fibres).DGGE was used to explore the similarity in microbial communities between healthy and diseased samples. A total of 65 unique bands were used to compare community similarity between samples, of these, 24 were sequenced to obtain phylogenetic detail. 16S rRNA gene sequencing disclosed bacterial phyla with representatives of the Alpha-, Gamma-, Delta- and Epsilon-proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chloroflexi, Firmicutes and Cyanobacteria. The Bacteroidetes (13, 19 and 21) and Epsilonproteobacteria (20 and 24) were only detected in diseased sponges.A more comprehensive phylogenetic comparison between the diseased and healthy samples using clone sequence data showed considerable differences proportions of the microbial community composition. Rarefaction analysis was performed to determine the number of unique bacterial clones as a proportion of the estimated total diversity.To determine if any eukaryotic organisms were specifically associated with the lesions on Aplysina aerophoba, DGGE was conducted with universal eukaryotic primers. A large number of bands were detected, but a single band was present in all diseased samples (with the exception of D2) and absent from the unaffected tissue of diseased sponges and healthy controls. 18S rRNA gene sequencing of this band revealed 96% sequence homology to the Harpactic copepod, Tisbe furcata.A maximum-likelihood phylogenetic tree was constructed from analysis of all 16S rRNA gene sequences retrieved from DGGE analysis. HPLC-UV-MS was used to acquire chemical profiles of healthy, diseased and unaffected portions of diseased Aplysina aerophoba tissue.

The study aimed to understand the microbiology associated with 'Aplysina Black Patch Syndrome' and to determine if shifts in sponge metabolite production occur with disease.