Haemolymph microbiome of the cultured spiny lobster Panulirus ornatus at different temperatures

Abstract

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.

Figure 1

Experimental design showing sample groups and sampling parameters.

Figure 2

(A) Survival rate (%), (B) total granulocyte counts (cells mL⁻¹), (C) total haemocyte counts (cells mL⁻¹) and (D) clotting time (s) of P. ornatus juveniles exposed to 28 °C or 34 °C. Each bar represents mean + SEM, n = 6 (except clotting time on days 4 and 6 with n = 4).

Figure 3

Principal coordinate analysis plots based on (A) Bray Curtis index, (B) weighted UniFrac and (C) unweighted UniFrac distance methods showing similarity in haemolymph sequence libraries of P. ornatus juveniles. Key: 28C0d: 28 °C 0 dpe; 28C6d: 28 °C 6 dpe; 34C6d: 34 °C 6 dpe.

Figure 4

(A) Core microbiome analysis based on relative abundance and sample prevalence of bacterial OTUs grouped by phyla from haemolymph samples of juvenile P. ornatus. (B) Venn diagram showing shared and unique OTUs in haemolymph samples of juvenile P. ornatus. Key: 28C0d: 28 °C 0 dpe; 28C6d: 28 °C 6 dpe; 34C6d: 34 °C 6 dpe.

Figure 5

Comparisons of relative abundance (culture-independent) and actual abundance (culture-dependent) of OTUs in haemolymph sample libraries of juvenile P. ornatus at (A) phylum, (B) class, (C) family and (D) genus levels. Key: 28C0d: 28 °C 0 dpe; 28C6d: 28 °C 6 dpe; 34C6d: 34 °C 6 dpe; 28C6d128: cultured colonies from 28 °C 6 dpe; 34C6d178: cultured colonies from 34 °C 6 dpe; NA: not assigned to bacterial taxa based on classification methods used in this study.

Figure 6

Functional diversity profiling of culture-independent OTUs in haemolymph samples of juvenile P. ornatus based on KEGG metabolism using PICRUSt. Key: 28C0d: 28 °C 0 dpe; 28C6d: 28 °C 6 dpe; 34C6d: 34 °C 6 dpe.

Figure 7

Bacterial load in the haemolymph of P. ornatus juveniles exposed to 28 °C and 34 °C based on culture-independent (rpoB gene) technique. Each bar is mean + SEM, n = 6.