Biofilm development within a larval rearing tank of the tropical rock lobster, Panulirus ornatus

The role of bacterial biofilms in disease processes is becoming increasingly recognised in both clinical and environmental settings. Biofilm development within a rearing tank of the tropical rock lobster Panulirus ornatus was studied to evaluate if the biofilm is a reservoir for potentially pathogenic bacteria that cause mass larval mortalities. Within a 5000 L larval rearing tank, fiberglass microscope slides were systematically distributed during a standard rearing attempt to assess biofilm development. Culture-based counts for two media types, TCBS and Marine Agar (MA), demonstrated increased bacterial densities until days 11 and 13 respectively. For both media types, a drop in the plate counts was followed by a subsequent increase towards the end of the experiment. Scanning electron microscopy (SEM) confirmed that cell densities decreased between days 13 and 17, most likely due to sloughing of the biofilm into the water column. SEM images revealed distinct changes in dominant morphologies reflecting a succession of bacterial populations. A dynamic succession of microbial species during biofilm development was also demonstrated using denaturing gradient gel electrophoresis (DGGE) profiling of bacterial 16S rRNA genes in combination with statistical ordination analysis. Prominent changes in the DGGE profiles coincided with the decrease in bacterial numbers observed by SEM and plating on MA between days 13 and 17. Fluorescence in situ hybridization (FISH) identified α-Proteobacteria as being numerically abundant in the biofilm. This was supported by results from DGGE analysis, which retrieved only sequences affiliated with α- and γ-Proteobacteria. DGGE bands affiliated with Vibrio became dominant towards the end of the larval run (days 21 to 24). A Vibrio harveyi strain isolated from the biofilm late in the larval rearing trial (day 24) demonstrated increased larval mortality in small scale phyllosoma survival studies. The detection of Vibrionaceae at the end of the larval trial coincided with mass phyllosoma mortality and show that the biofilm is a reservoir for potentially pathogenic bacteria.