Extracellular vesicles and post-translational protein deimination signatures in haemolymph of the American lobster (Homarus americanus).

The American lobster (Homarus americanus) is a commercially important crustacean with an unusual long life span up to 100 years and a comparative animal model of longevity. Therefore, research into its immune system and physiology is of considerable importance both for industry and comparative immunology studies. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family that catalyses post-translational protein deimination via the conversion of arginine to citrulline. This can lead to structural and functional protein changes, sometimes contributing to protein moonlighting, in health and disease. PADs also regulate the cellular release of extracellular vesicles (EVs), which is an important part of cellular communication, both in normal physiology and in immune responses. Hitherto, studies on EVs in Crustacea are limited and neither PADs nor associated protein deimination have been studied in a Crustacean species. The current study assessed EV and deimination signatures in haemolymph of the American lobster. Lobster EVs were found to be a poly-dispersed population in the 10-500 nm size range, with the majority of smaller EVs, which fell within 22-115 nm. In lobster haemolymph, 9 key immune and metabolic proteins were identified to be post-translationally deiminated, while further 41 deiminated protein hits were identified when searching against a Crustacean database. KEGG (Kyoto encyclopedia of genes and genomes) and GO (gene ontology) enrichment analysis of these deiminated proteins revealed KEGG and GO pathways relating to a number of immune, including anti-pathogenic (viral, bacterial, fungal) and host-pathogen interactions, as well as metabolic pathways, regulation of vesicle and exosome release, mitochondrial function, ATP generation, gene regulation, telomerase homeostasis and developmental processes. The characterisation of EVs, and post-translational deimination signatures, reported in lobster in the current study, and the first time in Crustacea, provides insights into protein moonlighting functions of both species-specific and phylogenetically conserved proteins and EV-mediated communication in this long-lived crustacean. The current study furthermore lays foundation for novel biomarker discovery for lobster aquaculture.

The effect of oral immuno-stimulation in juvenile carp (Cyprinus carpio L.).

The effect of a 2-week period of oral immuno-stimulation from the age of 2 or 6 weeks post-fertilisation (wpf; before and after reaching the ability to produce antibodies) onwards was investigated on various immune functions of the common carp, Cyprinus carpio. The immuno-stimulants Aeromonas salmonicida lipopolysaccharide, Yeast DNA (containing unmethylated CpG motifs) or high-M alginate (an extract of algae containing poly-mannuronic acid) were used. The effect of this treatment was studied on the kinetics of B cells in head kidney and peripheral blood leucocytes using flow cytometry, on the total plasma IgM level using ELISA, on cytokine and inducible nitric oxide synthase (iNOS) expression in the intestine, and acute phase protein expression in the liver, using real time quantitative PCR, and on exposure to Vibrio anguillarum. Oral administration of immuno-stimulants from 6 wpf resulted in decreased WCI12(+) (B) cell percentages in PBL (only after administration of LPS) and head kidney (all test groups), and a decreased total IgM level in plasma, suggesting that suppressive effects are strongly indicative of oral or juvenile tolerance. After administration from 2 wpf, the effects on WCI12(+) (B) cell percentages were less pronounced: the group fed with Yeast DNA showed higher percentages compared to the control group at 6 wpf, but lower percentages at 8 wpf. No changes were observed in the cytokine or iNOS expression levels in the intestine or acute phase protein expression in the liver. A challenge with V. anguillarum resulted in an initially higher cumulative mortality in the group fed with LPS, but lower mortality in the groups fed with Yeast DNA or high-M alginate compared to the control group, providing a provisional warning especially for the use of pathogen-derived immuno-stimulants, such as A. salmonicida LPS, in larval and juvenile fish.