Haemolymph and gut microbiomes of the ornate spiny lobster Panulirus ornatus

The reliance on wild stocks of ornate spiny lobster (Panulirus ornatus) for aquaculture and fisheries production will be reduced with the recent development of hatchery technology. The commercialisation of P. ornatus closed life cycle aquaculture production is nearing completion thus demanding greater understanding of various aspects of lobster health. In these early stages of sector development, each animal produced is valuable from both an economic and research standpoint, and so nonlethal sampling methods using haemolymph (blood) are preferred in health monitoring programs. Bacterial communities are indigenous to the haemolymph, a condition termed asymptomatic bacteraemia. However, dysbiosis of bacterial communities within the haemolymph can lead to invasion and proliferation of pathogens, causing disease. Limited information exists for the bacterial composition and diversity associated with the haemolymph of healthy and diseased states of P. ornatus. This knowledge gap can be filled using next generation DNA sequencing, a technical platform used to answer similar questions in the Human Microbiome Project. The primary aim of this study was to characterise haemolymph bacterial communities of healthy P. ornatus juveniles, deduce their potential functions and compare them with the gut microbiome. Next generation sequencing of bacterial 16S rRNA genes showed that the core haemolymph microbiome of juvenile P. ornatus consisted of phyla Proteobacteria and Bacteroidetes. Culture-independent analysis of bacterial communities captured a higher bacterial diversity comprising 13 phyla when compared to culture-dependent methods (2 phyla), yet members of the family Rhodobacteraceae were strongly represented in both analyses. Haemolymph bacterial loads of control lobsters ranged from 0 to 3.1 √v= 10\\(^3\\) CFU mL\\(^{-1}\\) (culturable bacteria) and from 55 to 2.8 √v= 10\\(^3\\) cell equivalent mL\\(^{-1}\\) (rpoB gene). The difference in culture-dependent and -independent analyses reinforces the limited resolving power and bias of the former method, a likely variable in the reporting of haemolymph-associated bacteria in earlier studies on lobsters. The observed prevalence and diversity of certain groups of bacteria in the haemolymph was suggestive of a number of positive functional roles including antimicrobial production and contribution to host nutrition. Juveniles thermally challenged for 6 d at 34 ¬∞C, which is 6 ¬∞C above the standard culture temperature of 28 ¬∞C, were affected in terms of survival, total granulocyte counts, bacterial diversity, bacterial load and functional potential of the haemolymph bacterial community. This study advocates nonlethal sampling and closer examination of the haemolymph microbiome for use in health monitoring programs of cultured spiny lobsters. The gut microbiome of juvenile P. ornatus across region and developmental stage was characterised and screened for similarities with the haemolymph microbiome. Next generation DNA sequencing showed the core gut microbiome consisted of phyla Tenericutes and Proteobacteria, with communities dominated by families Pseudoalteromonadaceae and Vibrionaceae. Gut region shaped the bacterial communities of 13 month post-emergence lobsters, where the hindgut community was more diverse and compositionally dissimilar to the foregut and midgut bacteria. The bacterial composition of the hindgut was influenced by developmental stage as there were more similarities among younger juveniles (6 - 7 d and 52 d post-emergence) compared to 13 month post-emergence lobsters. Bacteria identified as common to both the gut and haemolymph microbiomes included Flavobacteriaceae, Saprospiraceae, Rhodobacteraceae, Moraxellaceae, Pseudoalteromonadaceae and Vibrionaceae, suggestive of a transmission mechanism. This study was the first to comprehensively explore gut microbiomes of spiny lobster juveniles and to demonstrate that the bacteria present were influenced by gut region and developmental stage. Bdellovibrio and like organisms (BALOs) are obligate predators of other bacteria and were discovered for the first time in the present work residing in the haemolymph and gut of juvenile P. ornatus. BALOs have strong potential as biocontrol agents and alternatives to antibiotics in aquaculture, warranting further investigation into their roles in spiny lobster hosts. A combination of co-culture assays (agar and broth) and transmission electron microscopy was used to show a BALO strain (Halobacteriovorax sp.) isolated from sea water preyed upon a haemolymph-derived prey bacterium Vibrio sp. When Halobacteriovorax sp. was injected into the circulatory system of lobsters, there was no mortality and no change in immune parameter (percentage of granulocytes) or haemolymph microbiome after 3 d. However, lobsters injected with both prey and predator showed significant differences compared to other treatments (PBS control, prey only, predator only) in haemolymph bacterial community composition and reductions in plasma DNA concentration (bacterial load) after 2 d. The results indicate that BALOs are not pathogenic to juvenile P. ornatus and may assist the host in controlling bacterial population growth in the haemolymph. This study provides an extensive baseline characterisation of haemolymph and gut microbiomes of healthy cultured P. ornatus juveniles. Future comparisons with microbiomes of diseased individuals and those subject to suboptimal environmental conditions will advance the development of health management strategies for P. ornatus.