Lepeophtheirus salmonis: characterization of prostaglandin E2 in secretory products of the salmon louse by RP-HPLC and mass spectrometry
Introduction
Numerous compounds isolated from the secretions of terrestrial arthropod parasites have demonstrated prolonged parasitic feeding (anticoagulants), increased blood flow to the site of attachment (vasodilation) and/or evasion of host immune responses (immunomodulators) (Balashov, 1984; Bowman et al., 1996; Mans et al., 2002; Ribeiro, 1995; Wikel et al., 1994). Of these compounds a group of well-studied cyclic fatty acid derivatives with vasodilatory and immunomodulatory capabilities, known as prostanoids, have been discovered in the saliva of many Ixodid tick species such as Boophilus microplus, Amblyomma americanum, and Ixodes dammini (Bowman et al., 1995; Inokuma et al., 1994; Ribeiro et al., 1985). Prostaglandin E2 (PGE2) is the most commonly found prostanoid in parasitic secretions and is not just limited to ectoparasites but also found in endoparasitic infections with the protozoan, Entamoeba histolytica (Belley and Chadee, 1995) and the fluke, Fasciola hepatica (Ali et al., 1999). Prostaglandin E2 has a myriad of effects on host cells and tissues including, but not limited to, vasodilation (Williams and Higgs, 1988), decreased proliferation of lymphocytes targeted to the source (Papadogiannakis et al., 1984), decreased adhesion of lymphocytes to endothelial cells (To and Schrieber, 1990), and favoring T-helper cell type 2 (Th2) responses over Th1 lymphocyte responses through the down regulation of IL-2 and IFN-γ (Betz and Fox, 1991).
With respect to arthropod parasites of aquatic animals, very little is known about the nature of their host–parasite interactions, however, it is reasonable to expect similar mechanisms. Sea lice is a term used primarily for marine parasitic copepods that belong to the well studied genera Lepeophtheirus and Caligus. Of all of the sea lice species, the salmon louse, Lepeophtheirus salmonis has received the most extensive study due to its importance as a disease-causing agent of wild and farmed salmonids (Johnson et al., in press; Pike and Wadsworth, 1999). This parasite is responsible for indirect and direct losses in aquaculture in excess of US$100 million annually (Johnson et al. in press).
Lepeophtheirus salmonis feeds on host mucus, skin, and blood of salmonids belonging to the genera, Onchorynchus, Salmo, and Salvelinus (Berland and Margolis, 1983; Brandal et al., 1976). The attachment and feeding activities of sea lice result in lesions that vary in their nature and severity depending upon: the species of sea louse, their abundance, the developmental stages present, host species and a variety of environmental parameters such as water temperature. On most host species, the attachment and feeding activities result in little if any host tissue response (Johnson and Albright, 1992a, Johnson and Albright, 1992b; Kabata and Cousens, 1976). The lack of host tissue response to L. salmonis, as well as to many other species of sea lice has lead to the suggestion that sea lice produce compounds that modulate host immune responses. L. salmonis is known to secrete compounds such as proteases onto the surface of its hosts that may aid in feeding and/or avoidance of host immune responses (Fast et al., 2002, Fast et al., 2003; Firth et al., 2000; Ross et al., 2000). The goal of this study was to determine if secretions of L. salmonis contain prostanoids. To achieve this goal we used a modification of the techniques used to isolate secretory products from terrestrial arthropod parasites and subjected these secretions to RP-HPLC and LC–MS.
Section snippets
Lepeophtheirus salmonis collection and incubation
Adult male and female L. salmonis were collected from farmed Atlantic salmon held at various seawater net cage sites in Atlantic Canada and Maine at various times throughout the year (Table 1). Following collection, animals were washed with sterile seawater and maintained off hosts in sterile seawater for differing periods of time prior to incubation with dopamine. The majority of incubations were conducted after 24 h off the host due to the inability of conducting temperature-controlled
Results
Incubation of L. salmonis in SSW containing DA (0.1–1.0 mM) resulted in the production of SEPs as evidenced by the presence of proteases in the incubation media (Fig. 1). There was little evidence of any protease activity in SEPs obtained in the absence of DA. Having determined that dopamine stimulates the production of SEPs, we further examined this material for the presence of eicosanoids.
Dopamine from the incubation medium was removed from each sample during the water and ethanol washes of
Discussion
We have demonstrated the presence of protease activity in samples after topical DA stimulation. These proteases are the same as those reported produced by L. salmonis in the presence of host mucus (Fast et al., 2003). This is taken as evidence of DA stimulating the production of SEPs. The presence of PGE2 in both the SEP and SP samples indicates that PGE2 is actively secreted and not excreted into the incubation media.
We have confirmed the presence of PGE2 in dopamine-induced secretions of L.
Acknowledgements
The authors thank Dr. John Burka, Jennifer Ramsey, and other UPEI-AVC staff, Heritage Salmon Ltd., Stolt Sea Farms, Holly Beaman, Dr. Fred Whoriskey, and Jonathan Carr of the Atlantic Salmon Federation for all their assistance in obtaining parasite samples. This work was funded as part of an Industrial Post Graduate Scholarship from NSERC and the Microtek International collaboration with the Institute for Marine Biosciences. The manuscript is NRCC Publication #42390.
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