Lepeophtheirus salmonis: characterization of prostaglandin E2 in secretory products of the salmon louse by RP-HPLC and mass spectrometry

https://doi.org/10.1016/j.exppara.2004.04.001Get rights and content

Abstract

Lepeophtheirus salmonis is an ectoparasitic copepod that causes serious disease outbreaks in both wild and farmed salmonids. As the relationship between L. salmonis and its hosts is not well understood, the current investigation was undertaken to investigate whether any immunomodulatory compounds could be identified from secretions of L. salmonis. By incubating live L. salmonis adults with the neurotransmitter dopamine in seawater, we were able to obtain secretions from the parasite. These were analyzed by RP-HPLC column, as well as LC–MS. L. salmonis secretions contained a compound with the same retention time and mass of PGE2. The identity of this compound as PGE2 was confirmed by MS—in source dissociation. The concentrations of PGE2 in L. salmonis secretions ranged from 0.2 to 12.3 ng/individual and varied with incubation temperature and time kept off the host. Prostaglandin E2 is a potent vasodilator and thought to aid in parasite evasion from host immune responses. This is the first reported evidence of prostaglandin production in parasitic copepod secretions and its implications for the host–parasite relationship are discussed.

Index Descriptors and Abbreviations: prostaglandin (PG), sea lice, copepod, Lepeophtheirus salmonis, immunomodulation, secretory/excretory product (SEP), secretory products (SPs), dopamine, liquid chromatography–mass spectrometry (LC–MS), reverse phased-high performance liquid chromatography (RP-HPLC)

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.

References (62)

  • J.M.C. Ribeiro et al.

    Amblyomma americanum: characterization of salivary prostaglandins E2 and F by RP-HPLC/bioassay and gas chromatography–mass spectrometry

    Experimental Parasitology

    (1992)
  • U.F. Schade et al.

    Differential protective activities of site specific lipoxygenase inhibitors in endotoxic shock and production of tumor necrosis factor

    International Journal of Immunopharmacology

    (1991)
  • G.B. Schoeler et al.

    Ixodes scapularis: effects of repeated infestations with pathogen-free nymphs on macrophage and T lymphocyte cytokine responses of BALB/c and C3H/HeN mice

    Experimental Parasitology

    (1999)
  • C.J. Secombes et al.

    The effect of eicosanoids on rainbow trout, Oncorhynchus mykiss, leucocyte proliferation

    Veterinary Immunology and Immunopathology

    (1994)
  • A. Terragno et al.

    High performance liquid chromatography and UV detection for the separation and quantitation of prostaglandins

    Prostaglandins

    (1981)
  • S.K. Wikel et al.

    Tick-induced modulation of the host immune response

    International Journal of Parasitology

    (1994)
  • P. Yang et al.

    Quantitative high-performance liquid chromatography/electrospray ionization tandem mass spec analysis of 2- and 3-series prostaglandins in cultured tumor cells

    Analytical Biochemistry

    (2002)
  • Y.S. Balashov

    Interactions between blood-sucking arthropods and their hosts, and its influence on vector potential

    Annual Review of Entomology

    (1984)
  • H.A. Ball et al.

    Role of thromboxane, prostaglandins and leukotrienes in endotoxic and septic shock

    Intensive Care Medicine

    (1986)
  • S. Bell et al.

    The distribution of exocrine glands in Lepeophtheirus salmonis and Caligus elongatus (Copepoda: Caligidae)

    Contributions to Zoology

    (2000)
  • A. Belley et al.

    Eicosanoid production by parasites: from pathogenesis to immunomodulation?

    Parasitology Today

    (1995)
  • B. Berland et al.

    The early history of `Lakselus' and some nomenclatural questions relating to copepod parasites of salmon

    Sarsia

    (1983)
  • M. Betz et al.

    Prostaglandin E2 inhibits production of Th1 lymphokines but not of Th2 lymphokines

    Journal of Immunology

    (1991)
  • P.A. Bjorn et al.

    The physiological effects of salmon lice infection on sea trout post-smolts

    Nordic Journal of Freshwater Research

    (1997)
  • P.O. Brandal et al.

    Host blood: a major food component for the parasitic copepod Lepeophtheirus salmonis Kröyer 1838 (Crustacea: Caligidae)

    Norwegian Journal of Zoology

    (1976)
  • Bricknell, I.R., Bron, J.E., Cook, P., Adamson, K., 2003. Is blood an important component of the diet of sea lice? In:...
  • M.A.A. Crawford

    A background to essential fatty acids and their prostanoid derivatives

    British Medical Bulletin

    (1983)
  • Dalton, J., O'Neill, S., 2002. Immunomodulatory mechanisms expressed by Fasciola hepatica. In: Proceedings of the 10th...
  • M.D. Fast et al.

    Susceptibility of rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar and coho salmon Oncorhynchus kisutch to experimental infection with sea lice Lepeophtheirus salmonis

    Diseases of Aquatic Organisms

    (2002)
  • M.D. Fast et al.

    Enzymes released from Lepeophtheirus salmonis in response to mucus from different salmonids

    Journal of Parasitology

    (2003)
  • B.R. Ferreira et al.

    Successive tick infestations selectively promotes a Th2 cytokine profile in mice

    Immunology

    (2001)
  • Cited by (57)

    • Salmon louse rhabdoviruses: Impact on louse development and transcription of selected Atlantic salmon immune genes

      2018, Developmental and Comparative Immunology
      Citation Excerpt :

      This may be a response to the virions directly, or the result of an altered saliva composition. The salmon louse salivary gland is expected to secrete substances that dampens the host immune response, however; it is also likely to secrete digestive enzymes as trypsins and astacins (Fast et al., 2003, Fast et al., 2004, Fast et al., 2007, Unpublished data). Generally, viruses that regulate transcription or translation induce a decrease in host protein synthesis, including the rabies virus (Komarova et al., 2007; Walsh et al., 2013).

    • Prostaglandins E<inf>2</inf> and D<inf>2</inf>–regulators of host immunity in the model parasite Diphyllobothrium dendriticum: An immunocytochemical and biochemical study

      2017, Molecular and Biochemical Parasitology
      Citation Excerpt :

      However, there are some data on concentrations in other host-parasite systems. Different parasites with different strategies of immune evasion produce varying concentrations of PGE2 (E1) from 0.14 to 12.34 ng·mg−1 [29,60,61]. Our previous studies demonstrated in vitro effects of PGE2 on leucocytes from sticklebacks (Gasterosteus aculeatus) infected and not infected with the cestode Schistocephalus solidus [9].

    View all citing articles on Scopus
    View full text