Characterisation of the physical and chemical properties influencing bacterial epibiont communities on benthic gelatinous egg masses of the pulmonate Siphonaria diemenensis

The ability of sessile benthic egg masses to deter or prevent epibiosis is essential to the success of species that employ this life-history strategy. This study characterised the physical structure and bacterial communities on the surface of egg masses from the Siphonariid mollusc Siphonaria diemenensis (Quoy & Gaimard, 1833). Egg masses at the veliger stage of development were collected from two intertidal sites in the Gulf St. Vincent, South Australia. Physical structure was assessed using a combination of light microscopy and scanning electron microscopy. Egg mass surfaces were characterised by wave-like elevations 1-3. μm apart, fouled only by cocci, and longitudinal ridges (5-20. μm) fouled by a diversity of microorganisms and dense exopolymeric substance. Bacteria from the surface of egg masses and adjacent rock substratum were then isolated using standard culture procedures. The biochemical profiles of the isolates were used, along with Gram stain and visual morphological observations, to identify the bacteria. Eight species of bacteria were isolated and the composition of culturable epibiont communities from the egg mass was found to be significantly different from those found on the adjacent substrata. One species of bacterium on egg masses exhibited antibacterial activity in mixed culture and was identified as Bacillus psychrodurans using PCR of the partial 16S rRNA gene and sequence alignment on the GenBank database. Chemical extraction was performed on 'clean' and 'fouled' eggs and antibacterial activity was assessed against the marine pathogen Vibrio harveyi using the disc diffusion assay. Extracts from the cleaned egg masses were found to inhibit the growth of V. harveyi, whilst the fouled egg masses and extracts from the epibionts showed no antibacterial activity. However, extracts from the supernatant and cell pellet from the cultured B. psychrodurans exhibited antibacterial activity against V. harveyi and two human pathogens, Staphylococcus aureus and Escherichia coli. The results obtained in this study suggest that the surfaces of S. diemenensis egg masses are selective towards coccoid bacteria, which may result from a combination of physical structure and chemical antimicrobial properties, with further competitive interactions possibly occurring between the epibionts post settlement.