title: Insights into the immune system of the ornate spiny lobster Panulirus ornatus
Creators: name: Nguyen, TAT
Publisher: name: University of Tasmania
Year: 2019

description: Panulirus ornatus is a lobster species of high commercial value and commonly cultured using wild-caught seedstock in the Asia-Pacific region. The practice of harvesting wild seedstock places enormous pressure on the sustainability of wild fisheries, which can be alleviated through closed life-cycle culture. The University of TasmaniaNexus Aquasciences Pty. Ltd. (UNA) has recently succeeded in the development of technologies for commercial-scale closed-life cycle aquaculture production of P. ornatus. However, further gains in productivity will be linked to more robust health monitoring strategies, including a better understanding of the immune responses of lobsters across ontogenesis and against pathogens. As is the case for other invertebrates, lobsters lack a true adaptive immune system, yet an expanse of emerging studies propose that invertebrate immunity is more complex than once thought. The primary aims of this study were to identify immune-related genes from the transcriptome of P. ornatus, map the expression of these immune genes across ontogenesis, and investigate aspects of immunological memory following exposure to pathogens. High throughput Illumina sequencing was performed on cDNA libraries constructed from the hepatopancreas, haemocytes and gills of healthy P. ornatus juveniles. A total of 165,341 transcripts were assembled from the host global transcriptome data, representing 97.2% completion based on BUSCO assessment. Annotated contigs showed high homology to Insecta, Malacostraca and Merostomata sequences, indicating a strong evolutionary relationship among the Arthropoda. Annotated transcripts related to immunity were divided into three categories: pathogen recognition receptors (PRRs), immune regulatory pathways, and immune effectors. Phylogenetic analysis and predicted domain architecture confirmed that P. ornatus immune-related proteins are highly conserved and closely related to homologs of other crustaceans, particularly components associated with Toll, IMD, and JAK/STAT pathways. The study provides the first detailed analysis of molecular components of the P. ornatus immune system and will be foundational to the further study of molecular mechanisms associated with Palinurid lobster immune systems. Changes in immune gene expression were further investigated in 12 developmental stages across P. ornatus ontogenesis (phyllosoma, puerulus and juvenile). A total of 265 immune-related genes spanning 54 immune families were detected by profiling transcripts produced by RNA-seq. Following clustering of immune-related genes into 10 expression patterns, it was found that PRRs, immune effectors and signalling pathways were expressed differently across ontogenesis. PRRs and coagulation pathways were largely expressed over the 12 developmental stages investigated, while the expression of crustin was phyllosoma-specific and the expression of HSP70, TNFα, defensin, peroxidase and lysosome were metamorphosis–specific. The factors impacting the differential expression of these immune genes were assumed to involve physiological changes (moulting, metamorphosis) and associated microbiological invasion threats, and ecological shifts (diet, salinity). Specific immune priming was investigated in P. ornatus as a type of immunological memory that is functionally equivalent to acquired immunity in vertebrates. Lobster juveniles were primed with either sterilised sodium chloride (control) or inactivated inoculums of Yersinia ruckeri or Vibrio harveyi. After 7 days, all lobsters were injected with a 1:1 mixture of Y. ruckeri or V. harveyi. Plasma and haemocytes were subsequently sampled after 30 minutes for further analysis of antibacterial, phagocytotic, agglutinin activity as well as expression of immune-related genes. The level of specificity in lobster immune responses against a particular pathogen was firstly demonstrated through antibacterial activity assays. Plasma from lobsters primed with either V. harveyi or Y. ruckeri significantly inhibited the growth of V. harveyi or Y. ruckeri respectively when compared to other treatments. Further, genes encoding antimicrobial peptides (eg. defensin, ALF, ProPO, astakine) were upregulated in lobsters primed with either V. harveyi or Y. ruckeri when compared to control lobsters. Moreover, higher levels of ALF and defensin mRNA were found in lobsters primed with V. harveyi compared to lobsters primed with Y. ruckeri, while conversely a higher expression of lysozyme was found in lobsters primed with Y. ruckeri when compared to those primed with V. harveyi. However, significantly higher phagocytic activity was detected in the control group in comparison to the group primed with V. harveyi. There were no differences in agglutination activity across treatment groups. The duration and mechanistic drivers of immunological memory in P. ornatus were examined. Lobster juveniles received either no injection (naïve) or primary and secondary injections of phosphate buffered saline (control) or formalin-inactivated V. harveyi. Haemocytes and plasma were sampled 1 day and 7 days after the primary and secondary injections. The antibacterial activity of plasma taken from lobsters exposed to V. harveyi twice was higher than in lobsters that received V. harveyi injection and then PBS injection. Antibacterial activity lasted at least 7 to 14 days upon exposure to V. harveyi. Observations of the antibacterial assays were supported by the higher expression of immune-related genes (i.e. defensin, HSP, IL-16 and TNFα) in lobsters that had received primary and secondary injections of V. harveyi compared to naïve lobsters. P. ornatus histone 3 and 4 (H3 and H4) were characterised at the molecular and protein level for post-translational modifications (i.e. acetylation) and putative roles in governing immunological memory in invertebrates. A higher acetylation of total histone H3 and H4 was detected in lobsters injected with bacteria twice when compared to that of naïve lobsters. Taken together, these studies show evidence for the presence of specific immunity in P. ornatus where histone acetylation may be a core mechanism driving immunological memory in spiny lobsters.