Cortisol and dexamethasone increase the in vitro multiplication of the haemoflagellate, Cryptobia salmositica, possibly by interaction with a glucocorticoid receptor-like protein

International Journal for Parasitology 43 (2013) 353–360

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Cortisol and dexamethasone increase the in vitro multiplication of the haemoflagellate, Cryptobia salmositica, possibly by interaction with a glucocorticoid receptor-like protein Mao Li a, John F. Leatherland b, Patrick T.K. Woo a,⇑ a b

Department of Integrative Biology, University of Guelph, Guelph, ON, Canada N1G 2W1 Department of Biomedical Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1

a r t i c l e

i n f o

Article history: Received 16 August 2012 Received in revised form 19 November 2012 Accepted 21 November 2012 Available online 20 December 2012 Keywords: Cryptobia salmositica Haemoflagellate In vitro culture Cortisol Dexamethasone Mifepristone Glucocorticoids

a b s t r a c t Cryptobia salmositica is a pathogenic haemoflagellate of Pacific salmon, Oncorhynchus spp., on the west coast of North America. The in vitro multiplication of the parasite was significantly enhanced by the addition of cortisol (within a range consistent with physiological levels in salmonid fishes; 10–50 ng ml 1) to the culture medium (MEM supplemented with FBS). However, higher cortisol concentrations (100 and 200 ng ml 1) either had no enhancing effects or resulted in lower replication rates compared with the controls. The synthetic glucocorticoid, dexamethasone (Dex), also stimulated the replication of the parasite and mifepristone (RU486), a synthetic steroid that has glucocorticoid receptor (GR) antagonist properties, inhibited the stimulatory actions of both cortisol and Dex, when added to the medium at a concentration of 100 ng ml 1 co-culture with cortisol or Dex. Furthermore, the dose-dependent effects of glucocorticoids (cortisol and Dex) on the multiplication of the haemoflagellate were correlated with the initial size of the inocula. The study revealed a novel relationship between the parasite and its host, in which the host’s cortisol is used by the parasite to enhance its replication. Also, since C. salmositica responds to both native and synthetic glucocorticoids and to the GR antagonist, RU486, and exhibits a biphasic (hormetic) response to the amount of cortisol in the medium, we propose that the glucocorticoid exerts its effects via an interaction with GR-like proteins in C. salmositica that are functionally similar to those present in vertebrate cells. Crown Copyright Ó 2012 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc. All rights reserved.

1. Introduction Disease outbreaks cause significant financial losses to the aquaculture industry, and numerous environmental stressors are considered to be the major contributors to the outbreaks of diseases in fish (e.g. Woo, 2003, 2010; Alvarez-Pellitero, 2008; Price et al., 2010; Baldwin et al., 2011). One example of this is the infection of wild populations of migrating sexually mature Pacific salmonid fishes (Oncorhynchus spp.) with the parasitic haemoflagellate, Cryptobia salmositica, which is associated with high mortalities (Woo, 2003, 2007, 2012; Currie and Woo, 2008). The clinical signs of cryptobiosis include anorexia, exophthalmia, general edema, distension of the body cavity with ascites, splenomegaly and a microcytic hypochromic anaemia (Woo, 1979, 2003). Cryptobia salmositica is transmitted by the freshwater leech (Piscicola salmositica) in streams/rivers and it is considered to be an important pathogen in semi-natural and intensive salmon culture facili⇑ Corresponding author. Tel.: +1 519 824 4120x53581; fax: +1 519 767 1656. E-mail address: [email protected] (P.T.K. Woo).

ties on the Pacific coast of North America (Bower and Margolis, 1984; Bower and Thompson, 1987). Outbreaks of cryptobiosis associated with high mortality are found in mature (pre- and post-spawning) salmon in freshwater hatcheries and sea cages. Also, approximately 50% of gravid salmon brought into hatcheries from some streams in the USA died from the disease (Woo, 2003, 2012). In the Fraser River drainage in British Columbia, Canada, the prevalence of the infection in sexually mature salmon returning to fresh water is low in September but increases to approximately 100% by December, and many infected fish die before spawning (Bower and Margolis, 1984). Even in uninfected fish, the migratory and reproductive events are stressful, and leads to an increase in the secretion of stress-related steroids, such as cortisol. It was widely assumed that the combination of reproductive stress together with the stress associated with infectious diseases in fish would trigger a chronic increase in plasma cortisol concentration, which would depress the hosts’ immune system functions, making them more susceptible to infectious diseases (Vallée et al., 1997; Griffin and Ojeda, 2000; Schreck et al., 2001; Norris and Hobbs, 2006; Pérez et al., 2007; Yada et al., 2007). This theory is

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supported by observations of high pre-spawning mortality of gravid Pacific salmon with high haemoflagellate infections upon arrival at the spawning grounds (Woo, 2003, 2012). Similarly, in vivo experimental studies with rainbow trout (Oncorhynchus mykiss) suggest a correlation between glucocorticoid suppression of immune system function and increasing propagation of the haemoflagellate (Woo et al., 1987). Also, sexually mature trout infected experimentally with C. salmositica were markedly more susceptible to cryptobiosis (significantly higher parasitaemias and mortalities) than juvenile fish infected with similar parasitic doses (Currie and Woo, 2008). In addition, trout implanted with exogenous cortisol had significantly higher parasitaemias relative to controls (Woo et al., 1987). However, as will be discussed in the following paragraphs, there is compelling evidence from in vitro studies which suggest that the increased C. salmositica parasitaemia might involve more than a depression of the host’s immune system. Currie and Woo (2008) showed that sexually mature rainbow trout infected experimentally with C. salmositica were much more susceptible to cryptobiosis (significantly higher parasitaemias and mortality) than infected juvenile fish. The addition of 17b-estradiol, the predominant estrogen in female salmonid fish, at levels that were in the physiological (or higher) range to the culture medium containing the haemoflagellate did not enhance parasite multiplication. However, the addition of a small volume of plasma (0.1 ml of plasma in 5 ml of Minimum Essential Medium (MEM) or even 0.05 ml in 5 ml of MEM) from sexually mature male or female O. mykiss significantly increased in vitro parasite multiplication. Also, plasma from females was more potent than that from males (Currie and Woo, 2007). A similar enhancing response was found following the addition of 10 ng ml 1 of cortisol to C. salmositica cultures (Woo, 2008, unpublished data). These findings suggest there is a direct action of cortisol on the replication of the parasite. The present study was undertaken to further determine the involvement of the host stress hormone in parasite replication and the possible mechanism of action of the glucocorticoid on the propagation of the haemoflagellate. In vertebrates, the action of cortisol is through the activation of its receptor, the glucocorticoid receptor (GR), to maintain homoeostasis by a stress response such as an immune response. The GR is a member of the nuclear receptor superfamily that includes proteins that have specific affinity for a range of ligands, including various steroids, thyroid hormones, prostaglandins and retinoids, and when activated by their ligands, the receptors act as transcription factors. Although receptors of this superfamily are ubiquitous in representatives of all living metazoan phyla (Sáez et al., 2010), they have not yet been identified in protozoans, plants, fungi or algae (García et al., 2003). Further, with regard to the GR, the only evidence of its presence is in the chordate line; the gene is thought to have appeared for the first time in a primitive cephalochordate ancestor (Baker, 2004). The current consensus is that nuclear receptor proteins are not present in protozoans; however, there are characteristic responses of parasitic protozoans to steroids including cortisol that are consistent with the involvement of steroid receptors similar to that found in vertebrate cells (Escobedo et al., 2005). For example, adrenal hormones have profound effects on parasite numbers and the size of gametocytes of Plasmodium falciparum, and cortisol stimulates Entamoeba histolytica to proliferate under in vitro conditions in a dose-dependent manner (Maswoswe et al., 1985; Escobedo et al., 2005; Carrero et al., 2006). However, the mechanism(s) by which cortisol can enhance the propagation of parasites remains to be elucidated. The current study was undertaken to further examine the relationship between the in vitro propagation of C. salmositica and glucocorticoids, specifically to determine whether there is evidence of GR receptors that mediate mechanisms of glucocorticoid action. To do this we examined the effects of cortisol and the synthetic

cortisol analog, dexamethasone (Dex), on (i) the in vitro multiplication of varying numbers of parasites (from 2,000 to 30,000 parasites per ml) in the culture medium. In nature, the number of parasites inoculated into a fish is highly variable and in part is dependent on the number and size of infected leeches feeding on the fish; and (ii) whether these responses could be ameliorated by the addition of a GR antagonist, mifepristone (RU486) to cultures. RU486 is a synthetic steroid that is an antagonist of GR function in vertebrate cells and tissues, and it has been used extensively for investigations into the molecular nature of GR function in vertebrates. 2. Materials and methods 2.1. Parasites The haemoflagellate, C. salmositica, was originally isolated from its leech vector, P. salmositica, found on spawning coho salmon (Oncorhynchus kisutch) on Vancouver Island, Canada (Woo, 1978). The parasite was cloned and its biology and host-parasite relationships studied extensively. Cryptobia salmositica has been maintained by serial culture in MEM supplemented with FBS, subpassaged in fish, cryopreserved and stored at 90 °C (Woo, 2003, 2012). The pathogen has also been attenuated during serial in vitro culture at 10 °C in MEM (Woo and Li, 1990) and it has been used routinely as an experimental vaccine to study various aspects of the host-parasite relationships including the mechanism of protective immunity (Woo, 2010, 2012). 2.2. Preparation of cortisol-enriched MEM Cortisol (hydrocortisone or 11b, 17a, 21-trihydoxypregn-4-ene3, 20-dione (Sigma Chemical Co., MO., USA) was dissolved in absolute ethanol to produce a stock solution of 1 mg ml 1. A stock cortisol-enriched medium was prepared by adding 1 ll of the stock ethanolic cortisol solution to 1 ml of MEM supplemented with approximately 22% FBS serial dilutions of the stock cortisol medium with MEM (cortisol-enriched medium) and were then prepared to ensure the final ethanol concentration in the culture medium was less than 0.01% by volume. The culture medium (pH 7.2) was made up according to Li and Woo (1991) and it contained 22% heat-inactivated FBS (at 37 °C in a water bath for 45 min), an essential component of the medium. Consequently, MEM would also contain some cortisol and corticosteroid-binding globulin (amounts not known) which were present in FBS; however, this would be the same in both control (with no added cortisol) and experimental (cortisol-enriched) media. Also, for consistency, the same batch of FBS (Fisher Canada, cat. # 12483020) was used throughout the present study. Preparations of culture medium and parasite inoculations were made aseptically in a Biological Safety Cabinet (Canadian Cabinets, Ottawa, Canada). 2.2.1. Determination of an effective range of cortisol concentrations on parasite multiplication A series of cortisol-enriched MEM solutions (concentrations 10, 50, 100, 200 ng ml 1 of cortisol) was prepared by serial dilution of the stock cortisol medium (1 lg ml 1) with MEM; the control medium consisted of MEM alone. Nine ml of the serially diluted cortisol-enriched media were dispensed into each 25 ml culture flask (Falcon, NY, USA). Each culture flask (with 9 ml of medium) was inoculated with 1 ml of 20,000 parasites (the final parasite number was 2,000 parasites per ml) with four replicates for each cortisol concentration and cultures were maintained at 10 °C. The number of parasites in each flask was counted weekly between 5 and 11 weeks after

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the start of the experiment. Briefly, the culture flask was vortexed and 50 ll of the culture was taken aseptically using a sterile pipette, dispensed into the haemocytometer chambers and allowed to settle for 1 min. The numbers of C. salmositica were counted in all eight of the white blood cell squares of the haemocytometer chambers under a light microscope at 10  16 magnification. Each replicate was sampled twice; if dilution was necessary (when parasite numbers were high), the final calculation would include the dilution factor using the equation: total parasites per ml (N) = (total counts of eight squares  dilution factor  103)/0.8 (Archer, 1965). 2.2.2. Short-term effects of the optimal cortisol concentrations on parasite multiplication (large inocula) Based on the preliminary study described in Section 2.2.1, the effective range of cortisol concentration was determined to be between 10 and 50 ng ml 1; a series of the effective cortisol concentrations (0, 10, 25, 50 ng ml 1) was carried out in the study on short-term effects on high number of parasites. One ml containing 300,000 parasites was inoculated aseptically into each culture flask with 9 ml of medium (the final parasite number was 30,000 parasites per ml) in four replicates and maintained at 10 °C. The cultures were sampled at 3-day intervals until 12 days postexposure (PE); the number of parasites was determined using a haemocytometer as described previously. 2.2.3. Effects of the effective cortisol concentrations on parasite multiplication (small inocula) A similar series of cortisol concentrations (as described in Section 2.2.2) were prepared into which 10,000 parasites per ml were transferred and cultured at 10 °C. The number of parasites was determined weekly from 2 to 5 weeks PE using a haemocytometer as described previously. 2.3. Effects of the cortisol analogue, Dex and GR antagonist, mifepristone (RU486) on parasite multiplication RU486 or Dex (Sigma–Aldrich, St. Louis, USA) were dissolved in absolute ethanol and diluted with MEM to their final concentrations separately or in combination, at which point the ethanol in the final medium was less than 0.001% by volume. One ml of parasite suspension was inoculated into 9 ml of each culture medium in a 25 ml culture flask (four replicates). The number of parasites in each medium was counted weekly from 2 to 5 weeks PE. 2.3.1. The suppressive action of RU486 on the stimulatory effects of Dex and cortisol (at 25 ng ml 1concentration) on the multiplication of C. salmositica in vitro In this study, one ml of parasite suspension (200,000 parasites ml 1) was inoculated into 9 ml of the combination medium (RU486 at 100 ng ml 1 with either 25 ng ml 1 Dex or 25 ng ml 1 cortisol); the medium alone (MEM) was the normal control, and medium with added cortisol (at 25 ng ml 1) was the positive control. Parasite enumeration began at 2 weeks PE until 5 weeks PE. 2.3.2. The dose-additive effects of Dex and cortisol (at 10 ng ml 1 concentration) on the multiplication of C. salmositica and the modulating action of RU486 on the effects of glucocorticoids in vitro This study was designed to determine the dose-additive effects of glucocorticoids on parasite replication, and the inhibitory action of RU486 on the effects of Dex and cortisol on parasites. Media with 10 ng ml 1 of Dex or cortisol, or a combination of cortisol and Dex (10 ng ml 1 of each) were made separately; 100 ng ml 1 of RU486 was added to each medium, and 9 ml of the combined culture media was dispensed in a flask, each with four replicates. One ml of 130,000 parasites ml 1 was inoculated into each flask

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with 9 ml of the medium and parasite numbers enumerated at 2 weeks PE until 5 weeks PE. 2.4. Statistical analysis The data of parasite numbers per ml were analyzed using oneway ANOVA followed by Holm-Sidak method (SigmaStat version 2.0, SPSS Inc., Chicago, IL USA) for multiple comparisons versus the control group to examine for the effects of the treatments at each sample time. If the tests for normality or equal variance failed, Kruskal–Wallis’s one-way ANOVA on ranks was used, followed by Dunn’s (multiple comparisons versus control group) or the Student–Newman–Keuls pair-wise multiple comparison method. The Student’s t test was used to examine for differences between means of the treatment and control within each time of sampling. A probability level of P < 0.05 was considered to be significant for all statistical tests. 3. Results 3.1. Effects of cortisol on the in vitro multiplication of C. salmositica 3.1.1. The effective range of cortisol concentrations on parasite multiplication In this low inoculum study (2,000 parasites ml 1 initially), the significant increases in parasite numbers were evident in the 10 ng ml 1 cultures from 5 weeks PE until 11 weeks PE (P < 0.05 at 5 weeks PE, <0.01 thereafter); the stimulatory effects of 50 ng ml 1 cultures were seen at 7 and 8 weeks PE (P < 0.05); 100 and 200 ng ml 1 significantly suppressed multiplication from 5 weeks PE until 11 weeks PE (P < 0.05 at 5, 6 weeks PE; <0.01 thereafter). By 11 weeks PE, a marked biphasic effect of cortisol concentration on parasite multiplication was evident, in which the low concentration of cortisol (10 ng ml 1) stimulated parasite multiplication, whereas the higher doses of cortisol (100 and 200 ng ml 1) had significant depresssive effects on parasite multiplication; cortisol at a concentration of 50 ng ml 1 had no stimulatory or suppressive effect (Fig. 1A). 3.1.2. Effects of cortisol on the multiplication of parasites over a 12 day period (high parasite inocula) A cortisol concentration of 10 ng ml 1 significantly (P < 0.05) enhanced parasite multiplication 3–6 days PE, and cortisol in the medium at 25 ng ml 1 significantly (P < 0.01) enhanced stimulation from 3 to 12 days PE. Fifty nanogram per millilitre of cortisol significantly (P < 0.05) stimulated parasite multiplication from 3 to 9 days PE compared with controls, but there were no significant effects after 12 days PE when the parasite numbers were very high in the cultures (Fig. 1B). 3.1.3. Effects of cortisol on the multiplication of parasites over a 5 week period (low parasite inocula) There were significant replication-stimulating effects relative to the controls of cortisol at 10 and 25 ng ml 1 from 3 to 5 weeks PE. There were no detectable effects of cortisol at 50 ng ml 1 on parasite multiplication at 2 and 3 weeks PE; however, the parasite numbers in this treatment group were significantly higher than the controls at 4 and 5 weeks PE (P < 0.05 and <0.01, respectively; Fig. 1C). 3.2. Effects of the cortisol analogue (Dex) and the GR antagonist (RU486) on parasite multiplication In this part of study, the stimulatory effects of a low level of cortisol on the parasite multiplication (positive controls) were

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Weeks Post-Exposure Fig. 1. Effects of cortisol concentrations on in vitro multiplication of the haemoflagellate, Cryptobia salmositica. Data are shown as means ± S.E.M. (n = 4). (A) Identifying an effective range of cortisol concentrations (10–200 ng ml 1) on parasite multiplication 5–11 weeks post exposure (PE). ⁄Indicates significant increases (P < 0.05) in parasite numbers between the cortisol cultures and control cultures (no cortisol); #indicates significant decreases (P < 0.05) in parasite numbers between the cortisol cultures and control cultures. (B) The short-term effects of cortisol levels in the effective range (10–50 ng ml 1) on parasite multiplication from large initial inocula from 3–12 days PE. (C) The effects of cortisol levels in the effective range (10–50 ng ml 1) on parasite multiplication from low initial inocula from 2–5 weeks PE. The same letter (a, b, c) indicates that there are no significant differences in the parasite numbers between cortisol cultures and control cultures (P < 0.05). NSD, non-significant difference.

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confirmed in both 10 and 25 ng ml 1 cultures in the two independent incubations. The parasite numbers in both cortisol treatment groups were significantly (P < 0.05) higher at 3 and 4 weeks PE compared with controls (Fig. 2A and B), except for the 25 ng ml 1 cultures at 4 weeks PE, which were higher but not significant (P = 0.18) (Fig. 2A). It was noted that the initial parasite numbers determined whether there were significant differences among treatments when parasite numbers became high at 4–5 weeks PE (compared with Fig. 1B and C).

3.2.2. Antagonistic effects of RU486 (100 ng ml 1) on the stimulatory actions of cortisol and Dex (at a concentration of either 10 or 25 ng ml 1) on parasite multiplication RU486 at a concentration of 100 ng ml 1 significantly (P < 0.05 for cortisol and P < 0.01 for Dex) suppressed the stimulation of both cortisol and Dex (at a concentration of 25 ng ml 1) on parasite multiplication at 3 and 4 weeks PE (Fig. 2A). At 5 weeks PE, where the parasite numbers were high (>2.5  106 ml 1) there was no significant difference between the control, cortisol and Dex treatment groups, but in both culture media of cortisol and Dex in combination with RU486, the RU486 treatments significantly (P < 0.05) suppressed parasite multiplication (Fig. 2A). With the 10 ng ml 1 of cortisol and Dex media, cortisol significantly (P < 0.05) increased parasite numbers at 2, 3 and 4 weeks PE (Fig. 2B); moreover the co-incubation of the glucocorticoids with RU486 eliminated the stimulatory effect of cortisol on parasite replication (Fig. 2B). In contrast, Dex at 10 ng ml 1 did not significantly effect multiplication of the parasite. At 5 weeks PE of this experiment, there were no apparent effects of cortisol or Dex,

3.2.1. Cortisol analogue (Dex) and the additive effects of glucocorticoids As for cortisol, Dex at a concentration of 25 ng ml 1 exerted a significant (P < 0.05) stimulatory effect on parasite multiplication (Fig. 2A); at a concentration of 10 ng ml 1, Dex had no detectable effects on parasite multiplication. However, a combination of Dex and cortisol (each at the 10 ng ml 1 concentration) had a significant (P < 0.05) effect on the multiplication of the parasite compared with controls from 2 to 5 weeks PE (Fig. 2B).

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Weeks Post-Exposure Fig. 2. Effects of cortisol (Cort) analog (dexamethasone, Dex) and antagonist (mifepristone, RU486) on the in vitro multiplication of the haemoflagellate, Cryptobia salmositica. Data are shown as means ± S.E.M. (n = 4). (A) Cortisol (25 ng ml 1) or Dex (25 ng ml 1) with or without RU486 (100 ng ml 1) added to the medium 2–5 weeks post exposure (PE). (B) Shows Cort (10 ng ml 1) or Dex (10 ng ml 1) or Cort + Dex (10 ng ml 1 of each) with or without RU486 (100 ng ml 1) added to the parasite medium from 2–5 weeks PE. The same letter (a, b, c, d) indicates that there are no significant differences in the parasite numbers amongst Dex, Cort or combinations with RU486 cultures and control cultures (no Cort) (P < 0.05).

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and with or without RU486, on parasites (Fig. 2B); however, the additive effects of cortisol + Dex on the parasites were significantly (P < 0.05) depressed by the presence of RU486 in the culture medium (Fig. 2B).

4. Discussion In nature the number of parasites inoculated into a fish is highly variable and this is in part dependent on the number and size of infected leeches feeding on a fish. Consequently, we wanted to determine the effects cortisol might or might not have on varying numbers of parasite. Our results confirm that enhancement of parasite multiplication is not affected by the initial number of parasites in the medium. However, the sampling times have to be varied as we have to wait longer for some cultures (e.g. with low inoculum size) to build up so that our estimates of parasite numbers are more accurate. The present study provides robust evidence of an effect of glucocorticoids (cortisol and Dex) on the in vitro replication of C. salmositica. At concentrations of cortisol in the medium (10– 50 ng ml 1), the glucocorticoids stimulated replication (Fig. 1), whereas at higher levels (100 and 200 ng ml 1) cortisol exerted a suppressive action on parasite multiplication (Fig. 1A). Further, the inhibitory effects of the GR antagonist, RU486, on the cortisoland Dex-stimulated actions on parasite replication suggest that the activation of a GR-like protein in the parasite’s response to cortisol action was possibly involved (Fig. 2A and B). In the glucocorticoid dose-dependent studies, the initial parasite numbers were also a factor that determined whether there was a significant effect of cortisol concentrations on the specific initial parasite multiplication in vitro (Fig. 1C; Fig. 2A and B). Short-term in vitro cultures showed cortisol at 10 ng ml 1 stimulated parasite multiplication from 3 to 6 days PE, and cortisol exposure up to 50 ng ml 1 had significant stimulatory effects on parasite multiplication (Fig. 1B). The combination of Dex and cortisol at 10 ng ml 1 was found to have additive stimulatory effects on parasite replication (Fig. 2B). The results agree with an earlier dose-dependent study on the effects of cortisol on the in vitro replication of E. histolytica (Carrero et al., 2006). In the current study, these cortisol levels (Fig. 1) were well within the plasma cortisol concentration range of stressed (30–200 ng ml 1) and non-stressed (1.0–100 ng ml 1) rainbow trout (Schreck et al., 2001; Norris and Hobbs, 2006), and well within the range of plasma cortisol levels measured in females (70–650 ng ml 1) from a range of Oncorhynchus spp. during sexual maturation (Caldwell et al., 1991; Kubokawa et al., 1999; Carruth et al., 2000; Koldjaer et al., 2004; McConnachie et al., 2012). However, it should be emphasized that the plasma cortisol concentrations obtained from most of these studies were of total plasma cortisol concentration and the proportion of cortisol (the so called ‘free’ hormone) that would be available to the parasite in sexually mature fish of this genus is much lower. Unfortunately, the actual values are known for only rainbow trout (45% of the total cortisol concentration of 20–25 ng ml 1 is present as ‘free’ steroid in females (10–12 ng ml 1) and 22% of 20– 25 ng ml 1 in males (<3 ng ml 1) (Caldwell et al., 1991)) and male sockeye salmon (Oncorhynchus nerka) (20% of approximately 400 ng ml 1 (80 ng ml 1) (Barry et al., 2001)). Based on these values, the lower range of cortisol exposures in the present study are similar to those found in sexually maturing salmonid fishes in the wild. Also, the culture medium with 22% FBS likely contained corticosteroid-binding globulin which would bind a percentage of the cortisol and thus lower the biologically available cortisol. However, this does not affect the results of the present study because the same base medium was used for both the controls (no added cortisol) and cortisol-enriched media.

The lower range of cortisol levels (10–50 ng ml 1) used in the study stimulated parasite multiplication whereas the higher concentrations used (100 and 200 ng ml 1) did not. The decrease in number at 11 weeks PE was probably because the cultures contained large numbers of dying and/or dead parasites (non-motile and rounded forms). A previous in vivo study showed higher parasitaemias in rainbow trout implanted with 70 ng ml 1 of cortisol than in the control (sham) 4 weeks after the infection; this was attributed only to depression of the host’s immune system by the cortisol (Woo et al., 1987). In light of our current in vitro study, we now suggest that the higher parasite numbers in cortisol-implanted fish (Woo et al., 1987) were due to the depression of the host immune system by cortisol which then allowed the parasite to multiply more rapidly, and the ability of the parasite to utilize the cortisol as a reproductive hormone to promote its own multiplication. This suggestion may also apply to other pathogen-host interactions because it has been shown that stressed animals, including humans, are often more susceptible to infectious diseases and a stressed host environment is more feasible for parasite survival (Escobedo et al., 2005; Pérez et al., 2007; Dubansky et al., 2011; current study). However, if cortisol is directly utilized by the parasite its mode of action is currently not clear; this is an area for further investigations. In the present in vitro study, the growth responses of the haemoflagellate to cortisol concentrations exhibit a hormetic pattern, with low hormone levels eliciting an enhancing response on replication while there was no increase in replication at the higher cortisol concentrations (Fig. 1A). Similar hormetic patterns of responses have been shown in in vitro fish embryo cell proliferation (e.g., Li et al., 2010, 2012). The biphasic patterns also exist in the growth and proliferation of mammalian stem cells or cell lines (e.g., Bellows et al., 1987; Derfoul et al., 2006; Phillips et al., 2006; Stewart et al., 2008; Du et al., 2009a,b). In addition, these patterns are consistent with the in vivo responses of the immune system of fishes to the glucocorticoid. Exposure of fishes to acute stressors stimulates an acute ‘spike’ in plasma cortisol concentration that is an essential physiological response that enables the animal to counter the effect of the stressor. Part of that response is the stabilization of the inflammatory responses, and the stimulation of the animal’s immune response to mount a defense against infectious organisms by increasing cytokine production (Wendelaar Bonga, 1997; Contreras-Sánchez et al., 1998; Mommsen et al., 1999; Schreck et al., 2001; Guerriero and Ciarcia, 2006). However, chronic increases result in sustained high plasma cortisol concentrations that suppress the animal immune system, inhibit growth and reproduction, and impose an allostatic (metabolic) load that collectively compromise the animal’s homeostatic regulatory systems (e.g., Schreck et al., 2001; Pérez et al., 2007; Leatherland et al., 2010; Schreck, 2010). This biphasic characteristic of cortisol, whereby a low acute exposure to the hormone has a physiologically enhancing effect, whereas chronic higher exposures exert suppressive actions, is a concept that is well established in both the disciplines of endocrinology and toxicology (Calabrese and Baldwin, 2002; Mattson, 2008). We suggest the hormetic phenomenon demonstrated in the present study would also occur with other parasites; however, further studies are needed to confirm this suggestion. The synthetic glucocorticoid, Dex, is used widely as a pharmaceutical agent because it has a greater potency than the native steroids, probably because it is more resistant to catabolism by the liver and therefore has a longer half-life in the circulation (Griffin and Ojeda, 2000; Braun et al., 2009; Du et al., 2009a,b). The synthetic steroid, RU486, is an antagonist of several steroid hormone receptors, including progesterone receptors (PRs) and GRs; it has also been used widely to study GR function in vertebrates (Vijayan et al., 1994, 2003; Du et al., 2009a,b; Oakley and Cidlowski, 2011; Alderman et al., 2012; Li et al., 2012). In the present study, these

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pharmaceutical agents are used (i) to determine whether the response of C. salmositica to Dex is similar to its response to cortisol; if that is the case it will indicate the response is not cortisolspecific; and (ii) to determine whether the stimulatory action of cortisol (and Dex) on the parasite’s proliferation is inhibited by RU486; if so, that will suggest that cortisol (and Dex) are acting via interaction with GR-like proteins. Both of these responses occur in C. salmositica which strongly suggest an interaction between the host’s cortisol and the parasite’s cellular signaling pathways (Fig. 2). This will probably be via GR-like proteins and the addictive effects of low concentration of glucocorticoids further provide the evidence of GR-like protein in the parasite (Fig. 2B). The best studied mode of action of cortisol on responsive cells in vertebrate cells is that of the regulation of expression of specific genes in the nucleus (Zanchi et al., 2010; Oakley and Cidlowski, 2011) and mitochondria (Du et al., 2009a,b) – the so-called genomic actions. Cortisol enters the cells and binds to GRs that are located in the cytoplasm in association with a chaperone protein complex. The attachment of cortisol causes the dissociation of the cytoplasmic GRs (cGRs) from the chaperone protein complex and allows the translocation of the glucocorticoid–cGR complex either to the nucleus via the nuclear pores or into the mitochondria. The activated GRs that enter the nucleus act as transcription factors for the regulation of expression of glucocorticoid-responsive genes. Other activated GRs that associate with other proteins in the cytoplasm are translocated into the mitochondria via transport proteins on the outer mitochondrial membrane. Once in the mitochondria, the activated GRs act as transcription factors for mitochondrial genes. However, other more rapid responses to cortisol are found that do not involve the regulation of gene expression. These include the chemical interaction of cortisol with the plasma and probably also with the outer mitochondrial membrane, resulting in changes in the transmembrane cation fluxes (Song and Buttgereit, 2006), and the binding of cortisol to G-protein linked GR receptors (mGCR) in the plasma membranes of cells; the binding of cortisol to mGCRs triggers the rapid activation of intracellular signalling cascades (Maier et al., 2005; Tasker et al., 2006; Thomas, 2012). These genomic and non-genomic actions of the host’s hormones on parasites have been proposed as factors in the host-parasite relationship (Escobedo et al., 2005). A homologue of the cellular regulation pathways that are present in vertebrates has also been identified in the blood fluke, Schistosoma mansoni, which has vertebrate-like TGF-b genes that encode for a product that interacts with the host cell function (Freitas et al., 2007, 2009; Roger et al., 2008). The interaction between parasite and host was thought to act through a putative signalling pathway that regulated the growth, development and neuroactive ligand–receptor interactions of the trematode (Freitas et al., 2007, 2009; Schistosoma japonicum Genome Sequencing and Functional Analysis Consortium, 2009). The characteristic of the GR-like receptors that have been tentatively identified in or on C. salmositica is currently under further investigation. In our present study with C. salmositica, an inhibition of cortisolstimulated cellular replication by RU486 (Fig. 2) suggests that at least one component of the cortisol activated multiplication of the parasite involves a GR-like protein. This protein may be in the cytoplasm and move to the nucleus to regulate parasite gene regulation after activation, or be present on the membrane of the parasite, thus affecting gene expression via activation of several intracellular signaling pathways as was shown for S. mansoni (Freitas et al., 2007, 2009). It is also possible that cortisol plays a role in the GR-related modulation of mitochondrial function that affects the synthesis of ATP. Moreover, at this time the possibility of an additional direct action of the steroid on the characteristics of the cell membrane of the parasite cannot be excluded. All of

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these possibilities are the subject of further scrutiny in ongoing follow-up studies. In conclusion, these findings support the earlier hypotheses that C. salmositica uses a glucocorticoid (‘stress’) hormone produced by the host as a regulator of its own reproduction, and that this phenomenon may exist widely amongst parasites. The findings reveal a new notion of host-parasite interaction – that is, parasites can use the host stress hormone as a ‘reproductive’ hormone. Our in vitro study with cortisol, the glucocorticoid analogue (Dex) and the GR antagonist (RU486) results on the multiplication of C. salmositica strongly suggest a potent stimulatory action of the glucocorticoid, and an additive effect of the glucocorticoids, probably acting via the interaction with GR-like receptors that are present on the cell membrane and/or within the cell cytoplasm of the haemoflagellate. Further biochemical and molecular studies are needed to investigate the properties of GR-like proteins, and their distribution on or within C. salmositica. Acknowledgements This study and ML were supported by grants from National Science and Engineering Research Council of Canada (NSERC) to PTKW. References Alderman, S.L., McGuire, A., Bernier, N.J., Vijayan, M.M., 2012. Central and peripheral glucocorticoid receptors are involved in the plasma cortisol response to an acute stressor in rainbow trout. Gen. Comp. Endocrinol. 176, 79–85. Alvarez-Pellitero, P., 2008. Fish immunity and parasite infections: from innate immunity to immunoprophylactic prospects. Vet. Immunol. Immunopathol. 126, 171–198. Archer, R.K., 1965. Haematological Techniques for Use on Animals. Blackwell Scientific, Oxford. Baker, M.E., 2004. Co-evolution of steroidogenic and steroid-activating enzymes and adrenal and sex steroid receptors. Mol. Cell. Endocrinol. 215, 55–62. Baldwin, R.E., Banks, M.A., Jacobson, K.C., 2011. Integrating fish and parasite data as a holistic solution for identifying the elusive stock structure of Pacific sardines (Sardinops sagax). Rev. Fish Biol. Fish.. http://dx.doi.org/10.1007/s11160-0119227-5. Barry, T.P., Unwin, M.J., Malison, J.A., Quinn, T.P., 2001. Free and total cortisol levels in semelparous and iteroparous chinook salmon. J. Fish Biol. 59, 1673–1676. Bellows, C.G., Aubin, J.E., Heersche, J.N.M., 1987. Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvaria in vitro. Endocrinology 121, 1985–1992. Bower, S.M., Margolis, L., 1984. Distribution of Cryptobia salmositica, a haemoflagellate of fishes in British Columbia and the seasonal pattern of infection in a coastal river. Can. J. Zool. 62, 2512–2518. Bower, S.M., Thompson, A.B., 1987. Hatching of the Pacific salmon leech (Piscicola salmositica) from cocoons exposed to various treatment. Aquaculture 66, 1–8. Braun, T., Li, S., Sloboda, D.M., Li, W., Audette, M.C., Moss, T.J.M., Matthews, S.G., Polglase, G., Nitsos, I., Newnham, J.P., Challis, J.R.G., 2009. Effects of maternal dexamethasone treatment in early pregnancy on pituitary–adrenal axis in fetal sheep. Endocrinology 150, 5466–5477. Calabrese, E., Baldwin, L.A., 2002. Defining hormesis. Hum. Exp. Toxicol. 21, 91–97. Carrero, J.C., Cervantes, C., Moreno-Mendoza, N., Saavedra, E., Morales-Montor, J., Laclette, J.P., 2006. Dehydroepiandrosterone decreases while cortisol increases in vitro growth and viability of Entamoeba histolytica. Microbes Infect. 8, 323– 331. Caldwell, C.A., Kattesh, H.G., Strange, R.J., 1991. Distribution of cortisol among its free and protein-bound fractions in rainbow trout (Oncorhynchus mykiss): evidence of control by sexual maturation. Comp. Biochem. Physiol. 99A, 593– 595. Carruth, L.L., Dores, R.M., Maldonado, T.A., Norris, D.O., Rut, T., Jones, R.E., 2000. Elevation of plasma cortisol during the spawning migration of landlocked kokanee salmon (Oncorhynchus nerka kennerlyi). Comp. Biochem. Physiol. 127C, 123–131. Contreras-Sánchez, W.M., Schreck, C.B., Fitzpatrick, M.S., Pereira, C.B., 1998. Effects of stress on the reproductive performance of rainbow trout (Oncorhynchus mykiss). Biol. Reprod. 58, 439–447. Currie, J.L.M., Woo, P.T.K., 2007. Susceptibility of sexually mature rainbow trout, Oncorhynchus mykiss, to experimental cryptobiosis caused by Cryptobia salmositica. Parasitol. Res. 101, 1057–1067. Currie, J.L.M., Woo, P.T.K., 2008. Effects of pathogenic haemoflagellate, Cryptobia salmositica on brood fish, Oncorhynchus mykiss. Environ. Biol. Fishes 83, 355– 365. Derfoul, A., Perkings, G.L., Hall, D.J., Tuan, R.S., 2006. Glucocorticoids promote chondrogenic differentiation of adult human mesenchymal stem cell by

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