The Australian ornate rock lobster, Panulirus ornatus has been identified as a good candidate for commercialisation as an aquaculture species due to the high demand for the animal on overseas markets and a short larval lifecycle compared to other Palinurid lobsters. However like most cultured crustaceans, disease, in particular bacterial diseases, are currently major constraints on the successful larval culture of the animal. This study represents the first detailed investigation of the microbial diversity within a larval rearing system of the ornate rock lobster P. ornatus. The study has particularly concentrated on the microbial communities within the water column in addition to bacteria associated with cultured and wild P. ornatus larvae (phyllosoma) using both culture-based and molecular microbiological techniques. An insight into the microbial dynamics of the larval rearing system is provided, which combined with the microbial community overview, has allowed more effective microbial management regimes to be implemented, subsequently improving larval survival.
The microbial population of the water column was dynamic and highly diverse, consisting largely of marine microorganisms affiliated with the classes α and γ-Proteobacteria and the Division Bacteroidetes. A small number of opportunistic pathogens were also retrieved, consisting mainly of Vibrio species, however it is unclear whether these organisms within the water column are involved in the larval mortalities observed. Several Bacillus sp. strains were isolated from the water column and there is potential to use these as probiotics to benefit phyllosoma survival.
Extensive colonisation of PIII phyllosoma by filamentous bacteria was observed following scanning electron microscopy analysis of phyllosoma and the majority of these bacteria were later confirmed as Thiothrix sp. following fluorescent in-situ hybridisation analysis. This colonisation is predicted to have negative effects on the health of early stage phyllosoma by hindering their ability to moult and feed. As a result, the animals are likely to be susceptible to infection by pathogenic bacteria such as Vibrio spp. A diverse collection of Vibrio species featured prominently in culture-based and molecular analyses of cultured phyllosoma including potential pathogenic strains, such as Vibrio harveyi and Vibrio campbellii. As Thiothrix sp. is a sulphide-oxidising bacteria, the lowering or removal of sulphide compounds within the larval rearing system is a potential means of control. At present, ozonation of all incoming larval rearing water is serving as an effective Thiothrix sp. control mechanism.
The microbial diversity associated with cultured and wild phyllosoma differed markedly when compared using molecular based clone library analysis. The cultured phyllosoma microbial community was dominated by γ-Proteobacteria, principally comprising a large proportion of Vibrio and Pseudoalteromonas species, with many likely candidates responsible for compromising phyllosoma health and causing larval mortalities. In contrast, wild phyllosoma were largely dominated by α-Protebacterial organisms, such as Sulfitobacter sp. The wild environment potentially promotes phyllosoma health through the presence of bacteria, such as Roseobacter sp. that possess favourable attributes for phyllosoma survival. By incorporating such potentially probiotic bacteria into the larval rearing of phyllosoma it may be possible to enhance phyllosoma health and subsequently improve larval survival.
Whole phyllosoma survival assays were undertaken to identify both potential pathogenic and probiotic strains isolated during the course of this study. Bacteria were inoculated either directly to the water column or through an Artemia sp. feed vector and phyllosoma survival monitored to measure improved or reduced larval survival. No statistically significantly increased mortality (pathogenic strains) or improved survival (probiotic strains) were identified in these animal assays. An absence of water exchanges, with progressively increasing organic load comprising phyllosoma health within all treatments was identified as one flaw that may have caused the negative results. However candidate bacterial strains were identified for further study including a potentially probiotic Vibrio alginolyticus strain. It is also possible that the Vibrio sp. strains tested during our experiments were not pathogenic, an argument supported by the absence of haemolysin production in all isolates tested. The use of probiotic bacterial strains to improve survival in cultured P. ornatus phyllosoma has potential and is an area that currently requires further research.
It is envisaged that an Australian aquaculture industry for P. ornatus will be viable in due time, however at present further research is required into the monitoring and removal of pathogenic Vibrio spp., through the use of such techniques as real time PCR, bacteriophage therapy, quorum sensing inhibition and probiotics. In addition, the development of improved nutritional sources to aid phyllosoma health and promote a robust immune defence for the animal are required if a successful commercial aquaculture industry for the species is to be established.