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'Vibrio owensii' DY05 is a serious pathogen causing epizootics in the larviculture of ornate spiny lobster 'Panulirus ornatus'. In the present study a multi-tiered probiotic screening strategy was used to identify a probiotic combination capable of protecting 'P. ornatus' larvae (phyllosomas) from experimental 'V. owensii' DY05 infection. From a pool of more than 500 marine bacterial isolates, 91 showed definitive in vitro antagonistic activity towards the pathogen. Antagonistic candidates were shortlisted based on phylogeny, strength of antagonistic activity, and isolate origin. Miniaturized assays used a green fluorescent protein labelled transconjugant of 'V. owensii' DY05 to assess pathogen growth and biofilm formation in the presence of shortlisted candidates. This approach enabled rapid processing and selection of candidates to be tested in a phyllosoma infection model. When used in combination, strains 'Vibrio' sp. PP05 and 'Pseudoalteromonas' sp. PP107 significantly and reproducibly protected 'P. ornatus' phyllosomas during vectored challenge with 'V. owensii' DY05, with survival not differing significantly from unchallenged controls. The present study has shown the value of multispecies probiotic treatment and demonstrated that natural microbial communities associated with wild phyllosomas and zooplankton prey support antagonistic bacteria capable of in vivo suppression of a pathogen causing epizootics in phyllosoma culture systems.

The type strain of 'Vibrio owensii' (DY05) was isolated during an epizootic of aquaculture-reared larvae (phyllosomas) of the ornate spiny lobster ('Panulirus ornatus'). 'V. owensii' DY05 was formally demonstrated to be the etiological agent of a disease causing rapid and reproducible larval mortality with pathologies similar to those seen during disease epizootics. Vectored challenge via the aquaculture live feed organism 'Artemia' (brine shrimp) caused consistent cumulative mortality rates of 84 to 89% after 72 h, in contrast to variable mortality rates seen after immersion challenge. Histopathological examination of vector-challenged phyllosomas revealed bacterial proliferation in the midgut gland (hepatopancreas) concomitant with epithelial cell necrosis. A fluorescent-protein-labeled 'V. owensii' DY05 transconjugant showed dispersal of single cells in the foregut and hepatopancreas 6 h postexposure, leading to colonization of the entire hepatopancreas within 18 h and eventually systemic infection. 'V. owensii' DY05 is a marine enteropathogen highly virulent to 'P. ornatus' phyllosoma that uses vector-mediated transmission and release from host association to a planktonic existence to perpetuate transfer. This understanding of the infection process will improve targeted biocontrol strategies and enhance the prospects of commercially viable larviculture for this valuable spiny lobster species.

The development of efficient probiotic application protocols for use in marine larviculture relies on comprehensive understanding of pathogen-probiont-host interactions. The probiont combination of 'Pseudoalteromonas' sp. PP107 and 'Vibrio' sp. PP05 provides additive protection against vectored 'Vibrio owensii' DY05 infection in larvae (phyllosomas) of ornate spiny lobster, 'Panulirus ornatus'. Here, fluorescently tagged strains were used to demonstrate niche specialization of these probionts in both the live feed vector organism 'Artemia' and in phyllosomas. The pathogen was vulnerable to direct interaction with PP05 in the bacterioplankton as well as in the 'Artemia' gut and the phyllosoma foregut and midgut gland. In contrast, PP107 was localized on external surfaces of 'Artemia' and phyllosomas, and direct interaction with the pathogen was limited to the bacterioplankton. While PP107 was the overall dominant ectobiont on the phyllosoma cephalothorax and inner leg segments, PP05 was the primary colonizer of outer leg segments, nutrient-rich locales that may promote ingestion during feeding. This study shows that niche specialization can contribute to the additive probiotic effect of a probiotic mixture and highlights that probiotic enrichment of 'Artemia' cultures can intercept the infection cycle of 'V. owensii' DY05 in early-stage 'P. ornatus' phyllosomas.