Two bacterial strains (DY05(T) and 47666-1) were isolated in Queensland, Australia, from diseased cultured crustaceans Panulirus ornatus and Penaeus monodon, respectively. On the basis of 16S rRNA gene sequence identity, the strains were shown to belong to the Harveyi clade of the genus Vibrio. Multilocus sequence analysis using five housekeeping genes (rpoA, pyrH, topA, ftsZ and mreB) showed that the strains form a monophyletic group with 94.4% concatenated sequence identity to the closest species. DNA-DNA hybridization experiments showed that strains DY05(T) and 47666-1 had 76% DNA similarity to each other, but <70% to their closest neighbours Vibrio harveyi LMG 4044(T) (< or =55%), Vibrio campbellii LMG 11216(T) (< or =52%) and Vibrio rotiferianus LMG 21460(T) (< or =46%). Strains DY05(T) and 47666-1 could be differentiated from their relatives on the basis of several phenotypic characteristics. The major fatty acids were C(15:0) iso 2-OH and/or C(16:1)omega7, C(16:0), C(18:1)omega7 and C(14:0). Based on the polyphasic evidence presented here, it can be concluded that strains DY05(T) and 47666-1 belong to the same novel species of the genus Vibrio, for which the name Vibrio owensii sp. nov. is proposed. The type strain is DY05(T) (=JCM 16517(T)=ACM 5300(T)).

bThe 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. Major steps toward the commercialization of closed life cycle aquaculture production of the ornate spiny lobster (Panulirus ornatus) have been reported recently (36), yet nutritional deficits (41) and mortality caused by bacterial disease (5) remain major constraints to hatchery productivity. Mass mortalities of larvae (phyllosomas) are often associated with enteric vibriosis, an infection of the midgut gland (hepatopancreas) caused by Vibrio species (6, 52).Members of the genus Vibrio are natural marine inhabitants, playing important roles in nutrient cycling and forming associations with zooplankton (49). Microhabitat preferences and ecological selection may be key factors in the speciation of vibrios (20), and intensive aquaculture systems are thought to select for bacterial virulence, including traits that enhance infectivity and transmission (29,33). Accordingly, many Vibrio species are pathogenic to cultured crustacean zooplanktonic larval forms, including the three closely related species Vibrio harveyi (32, 35), V. campbellii (17, 44), and the recently described V. owensii (8).It is of paramount importance to the development of efficient disease management strategies that pathogens be identified by using experimental infection models that provide information on infection routes and infection dynamics (38). Recently, microorganisms engineered to express fluorescent proteins (FP) have significantly increased the understanding of invasive pathways and infection dynamics of pathogens, including V. anguillarum, Aeromonas hydrophila, and Edwardsiella tarda in fish models (10, 24, 30) and V. harveyi in abalone (50). Panulirus sp. p...

With recent technologies making it possible for commercial scale closed life-cycle aquaculture production of spiny lobster (Panulirus ornatus) comes a strong impetus to further understand aspects of lobster health. The gut microbiome plays a crucial role in host health, affecting growth, digestion, immune responses and pathogen resistance. Herein we characterise and compare gut microbiomes across different developmental stages (6-7 days post-emergence [dpe], 52 dpe and 13 months post-emergence [mpe]) and gut regions (foregut, midgut and hindgut) of cultured P. ornatus juveniles. Gut samples were analysed using 16S rRNA next-generation sequencing. Core gut microbiomes of P. ornatus comprised the phyla Tenericutes and Proteobacteria. Within class Gammaproteobacteria, families Pseudoalteromonadaceae and Vibrionaceae were dominant members across the majority of the gut microbiomes. Characterisation of bacterial communities from 13 mpe lobsters indicated that the hindgut microbiome was more diverse and compositionally dissimilar to the foregut and midgut. The bacterial composition of the hindgut was more similar among younger juveniles (6-7 dpe and 52 dpe) compared to 13 mpe lobsters. This is the first study to explore gut microbiomes of spiny lobster juveniles. We demonstrate that the composition of the gut microbiome was shaped by gut region, whereas the structure of the hindgut microbiome was influenced by developmental stage.

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.

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Shell (cuticular) disease manifests in various forms and affects many crustaceans, including lobsters. Outbreaks of white leg disease (WLD) with distinct signs of pereiopod tissue whitening and death have been observed in cultured larvae (phyllosomas) of ornate spiny lobster Panulirus ornatus , eastern rock lobster Sagmariasus verreauxi , and slipper lobster Thenus australiensis . This study aimed to characterise and identify the causative agent of WLD through morphological and molecular (16S rRNA gene and whole genome sequencing) analysis, experimental infection of damaged/undamaged P. ornatus and T. australiensis phyllosomas, and bacterial community analysis (16S rRNA gene amplicon sequencing) of P. ornatus phyllosomas presenting with WLD during an outbreak. Bacterial communities of WLD-affected pereiopods showed low bacterial diversity and dominant abundance of Aquimarina spp. compared to healthy pereiopods, which were more diverse and enriched with Sulfitobacter spp. 16S rRNA gene Sanger sequencing of cultures from disease outbreaks identified the dominant bacterial isolate (TRL1) as a Gram-negative, long non-flagellated rod with 100% sequence identity to Aquimarina hainanensis . Aquimarina sp. TRL1 was demonstrated through comparative genome analysis (99.99% OrthoANIu) as the bacterium reisolated from experimentally infected phyllosomas presenting with typical signs of WLD. Pereiopod damage was a major predisposing factor to WLD. Histopathological examination of WLD-affected pereiopods showed masses of internalised bacteria and loss of structural integrity, suggesting that Aquimarina sp. TRL1 could enter the circulatory system and cause death by septicaemia. Aquimarina sp. TRL1 appears to have important genomic traits (e.g., tissue-degrading enzymes, gliding motility, and aggregate-promoting factors) implicated in the pathogenicity of this bacterium. We have shown that Aquimarina sp. TRL1 is the aetiological agent of WLD in cultured Palinurid and Scyllarid phyllosomas and that damaged pereiopods are a predisposing factor to WLD.

Lobsters have an open circulatory system with haemolymph that contains microorganisms even in the healthy individuals. Understanding the role of these microorganisms becomes increasingly important particularly for the diagnosis of disease as the closed life-cycle aquaculture of the spiny lobster Panulirus ornatus nears commercial reality. This study aimed to characterise haemolymph responses of healthy cultured P. ornatus juveniles at control (28 °C) and elevated (34 °C) temperatures. This was assessed by measuring immune parameters (total granulocyte counts, total haemocyte counts, clotting times), and culture-independent (pyrosequencing of haemolymph DNA) and culture-dependent (isolation using nonselective growth medium) techniques to analyse bacterial communities from lobster haemolymph sampled on days 0, 4 and 6 post-exposure to the temperature regimes. Elevated temperature (34 °C) affected lobster survival, total granulocyte counts, and diversity, load and functional potential of the haemolymph bacterial community. Pyrosequencing analyses showed that the core haemolymph microbiome consisted of phyla Proteobacteria and Bacteriodetes. Overall, culture-independent methods captured a higher bacterial diversity and load when compared to culture-dependent methods, however members of the Rhodobacteraceae were strongly represented in both analyses. This is the first comprehensive study providing comparisons of haemolymph bacterial communities from healthy and thermally stressed cultured juvenile P. ornatus and has the potential to be used in health monitoring programs.

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