Temperature-induced changes to the microbiome of the haemolymph in the cultured spiny lobster Panulirus ornatus

The closed life-cycle aquaculture production of the spiny lobster (Panulirus ornatus) is on the verge of commercial reality; hence the need for the development of health management strategies. Lobsters have an open circulatory system in which the haemolymph is inhabited by microorganisms that could be used as indicators of health under various culture conditions. The aim of this study was to characterise the responses (total haemocyte and granulocyte counts, clotting times) and microbiome (culture-dependent and -independent analysis) of the haemolymph of healthy cultured P. ornatus juveniles under two thermal regimes; control (28 °C) and elevated (34 °C). Haemolymph was sampled on days 0, 4 and 6 post-exposure. Next-generation sequencing (NGS) of 16S rRNA genes showed that the core haemolymph microbiome consisting of phyla Proteobacteria and Bacteriodetes was not affected by temperature. However, small but significant shifts in the abundance of certain bacterial groups were detected following exposure to 34 °C after 6 d. This included an increase in family Rhodobacteraceae and decrease in phylum Planctomycetes, and families Flavobacteriaceae, Alteromonadaceae and Cohaesibacteraceae when compared to control lobsters. These observations were commensurate with a significant decrease in total granulocyte counts. Overall, culture-independent methods captured a higher bacterial diversity and load with respect to culture-dependent methods, yet members of the Rhodobacteraceae (Alphaproteobacteria) were strongly represented in both analyses. Other haemolymph responses (total haemocyte counts, clotting times) and the haemolymph bacterial load as determined by qPCR of the rpoB gene (55 - 13076 cell equivalents mL-1) and colony forming units (0 - 3190 CFU mL-1) were not associated with changes in temperature. This is the first comprehensive study on bacterial communities in the haemolymph of P. ornatus and highlights the importance of microbiome analyses in identifying microbial shifts that may impact lobster health.