Full length articleMolecular characterization and knock-down of salmon louse (Lepeophtheirus salmonis) prostaglandin E synthase
Graphical abstract
Introduction
Salmon louse (Lepeophtheirus salmonis) is an ectoparasitic copepod of salmonid fish. Prevalence of lice and growing resistance against available chemotherapeutants is causing problems for wild and farmed salmonid hosts. The salmon louse life-cycle consists of eight stages (Hamre et al., 2013, Johnson and Albright, 1991, Schram, 1993). First, three planktonic stages (nauplius I and II and copepodid), after which the copepodid attaches to the host and start a parasitic life style going through two larval stages (chalimus I and II) and two pre-adult stages (pre-adult I and II) before the adult stage. During the parasitic stages the louse feed on host mucus, skin and blood, but only limited inflammatory response to lice infection can be detected on susceptible hosts such as Atlantic salmon (Salmo salar) (Johnson and Albright, 1992). In Atlantic salmon, salmon lice infestation leads to up-regulation of some inflammatory genes but the poor tissue response is not sufficient to expel the parasite (Braden et al., 2015, Fast, 2014, Fast et al., 2006a, Fast et al., 2006b, Skugor et al., 2008). This suggests that the salmon louse secretes substances that modulate the salmon immune response. Prostaglandin E2 (PGE2), an active vasodilator and immune modulator in higher vertebrates including fish (Fast et al., 2004, Kutyrev et al., 2014), is a common prostanoid produced by parasites (Ali et al., 1999, Belley and Chadee, 1995). PGE2 acts in an autocrine or paracrine fashion, consequently the destination of this molecule is expected to be nearby its site of production. PGE2 has also been found in parasitic secretions (Aljamali et al., 2002, Fast et al., 2007). PGE2 has been detected in excretory/secretory products from dopamine treated sea lice (Fast et al., 2004, Lewis et al., 2014), and four PGE2 receptors have been found in Atlantic salmon (Gamil et al., 2015). Moreover, PGE2 has been shown to inhibit the expression of the pro-inflammatory cytokine IL-1β and the major histocompatibility class (MHC) I and II genes important in T-cell presentation in an Atlantic salmon head kidney cell line (SHK-1) (Fast et al., 2005). However, stimulation of SHK-1 cells with salmon lice secretory/excretory products after removal of PGE2, showed that other immune-modulatory components are also likely to be present (Fast et al., 2007). Accordingly, it remains to be seen how important salmon louse produced PGE2 is for the immune response of the salmon. A high amount of PGE2 has been measured in the excretions of dissected tick salivary glands (Aljamali et al., 2002) which is in the order of magnitude to cause impairment of the mammalian immune response (Bowman, 1996). As a vasodilator, it has been speculated that PGE2 could be important to increase blood flow to the site of attachment during feeding on the host. The general role of PGE2 in immunomodulation and vasodilation is well documented and tick saliva has been shown to be a potent modulator of vasodilation with PGE2 thought to be responsible (Inokuma et al., 1994). However, direct evidence for the role of PGE2 in ticks has only been demonstrated in regulation of salivary secretions (reviewed by Bowman and Sauer, 2004) and in murine peritoneal macrophage cell culture where PGE2 affects macrophage and fibroblast migration and cytokine secretion (Poole et al., 2013).
PGE2 is synthesized from membrane-derived arachidonic acid (AA) through three enzymatic reactions (Kudo and Murakami, 2005). In higher vertebrates, Phospholipase A2 releases AA from the membrane, cyclooxygenase (COX) −1 or COX-2 converts AA to PGH2 that is further metabolized to PGE2 by the prostaglandin E2 synthase (PGES). Three isotypes of PGES are identified, membrane-bound PGES (mPGES) −1 (Jakobsson et al., 1999, Murakami et al., 2000), mPGES-2 (Tanikawa et al., 2002), and cytosolic PGES (cPGES) (Tanioka et al., 2000). These are found to be differentially expressed, contain unique structural features and preference to COX-1 and -2, giving them distinct functional properties.
Furthermore, in crustaceans like the penaeid shrimp Penaeus monodon (Wimuttisuk et al., 2013) and in Daphnia magna (Heckmann et al., 2008), genes in the biosynthetic pathway of prostaglandins have been investigated. Nine putative prostanoid biosynthesis genes were found in P. monodon and eight in D. magna including PGES and COX. In L. salmonis a gene resembling mPGES-2 has been identified (Campbell et al., 2009). Prostaglandins have been shown to affect ovary development of several crustacean species (Preechaphol et al., 2010, Reddy et al., 2004, Spaziani et al., 1995, Tahara and Yano, 2004), and measurements of prostaglandins in P. monodon ovaries have been performed (Wimuttisuk et al., 2013).
To investigate the importance of PGE2 in salmon louse infestation, we cloned and further characterized the LsPGES2 gene. This gene has previously been down regulated by RNA interference (RNAi) in salmon lice, but no further characterization of the phenotype has been published (Campbell et al., 2009, Eichner et al., 2014). Here we present the PGES2 sequence including 5′ and 3′ UTR and characterized expression of LsPGES2. Furthermore, we measure the level of PGE2 metabolites in knock-down lice to analyse the involvement of LsPGES2 in PGE2 synthesis. Finally, an infection study was carried out with LsPGES2 knock-down lice on Atlantic salmon to see if reduced LsPGES2 mRNA levels decreased the infection level of lice.
Section snippets
Salmon lice
A laboratory strain (Hamre et al., 2009) of Atlantic salmon lice (L. salmonis salmonis) (Skern-Mauritzen et al., 2014) was maintained on farmed Atlantic salmon that were hand fed on a commercial diet and reared in seawater with a salinity of 34.5 ppt and a temperature of around 10 °C. Planktonic stages of sea lice were kept in seawater from the same supply. Nauplii were obtained from hatching egg string pairs kept in single wells in a flow through system (Hamre et al., 2009). All experimental
Sequence analysis
Primers for RACE were made based on an LsPGES2 EST sequence, giving a sequence of 1819 nucleotides (nt) with an ORF of 1173 nt preceded by a 190 nt 5′-UTR and a 3′-UTR with length of 456 nt. The sequence has been submitted to GenBank (GenBank: KT013223). Three different polyadenylation sites within the 3′UTR have been found: after 1459 bp, after 1643 bp and after 1819 bp. The ORF translated into a 391 amino acids (aa) long protein, with a transmembrane helix positioned at aa 45–63 (Phobius), a
Discussion
A shorter LsPGES2 sequence, the only prostaglandin synthase identified so far in the salmon louse, can be found in GenBank (GenBank: BT077889.1) and a longer version has been described by Campbell et al. (2009). Here we have performed RACE to obtain complete 5′ and 3′ UTRs and a full length sequence of the LsPGES2 transcript has been submitted to GenBank (GenBank: KT013223). LsPGES2 consists of four exons. This is more similar to insects which have two to five exons, whereas the two crustacean
Acknowledgement
We are grateful for the invaluable help we received from Heidi Kongshaug, Teresa Cieplinska in the laboratory, from Lars Hamre and Per Gunnar Espedal for help in the wetlab and to Ewa Harasimczuk who supplied cDNA for the ontogenetic analysis. We are also grateful to Tz-Chun Guo and Amr Gamil for discussion and cooperation with the experiment, and to Kevin Glover for comments on the manuscript. This research has been funded by the Research Council Norway, SFI-Sea Lice Research Centre, grant
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