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Glucocorticoid receptors on and in a unicellular organism, Cryptobia salmositica
International Journal for Parasitology 44 (2014) 205–210
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International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara
Glucocorticoid receptors on and in a unicellular organism, Cryptobia salmositica Mao Li ⇑, Patrick T.K. Woo Department of Integrative Biology, University of Guelph, Guelph, ON N1G 2W1, Canada
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Article history: Received 15 August 2013 Received in revised form 26 October 2013 Accepted 28 October 2013 Available online 11 December 2013 Keywords: Glucocorticoid receptor (GR) Protozoan Cryptobia salmositica Cortisol RU486 In vitro culture Host–parasite interaction
a b s t r a c t This is the first report to our knowledge that demonstrates a functional steroid hormone receptor in a protozoon. The study used Cryptobia salmositica, a pathogenic haemoflagellate found in salmonid fishes. It has been previously shown that cortisol and dexamethasone (a synthetic glucocorticoid) enhanced the multiplication of C. salmositica under in vitro conditions indicating the presence of glucocorticoid receptors on/in the parasite. Also, the glucocorticoid receptor antagonist, mifepristone (RU486), inhibited the stimulatory effect of the two glucocorticoids on parasite multiplication. In the present study, we used an antibody (produced in a rabbit against glucocorticoid receptor protein) agglutination test and confocal microscopy with immunohistofluorescence staining to demonstrate cortisol-glucocorticoid receptor-like protein receptors on the plasma membrane and in the cytoplasm of the parasite. In two in vitro studies, the addition of 50 ng ml 1 of RU486 was more effective in inhibiting parasite replication in cultures with 7,000 parasites ml 1 than in cultures with 14,000 parasites ml 1. Also, 100 ng ml 1 of RU486/ml was more effective than 50 ng ml 1 in inhibiting parasite multiplication in the 14,000 parasites ml-1 cultures. These in vitro studies indicate that the number of binding sites on/in the parasite is finite. The findings may be important in future studies especially on steroid receptor signalling pathways and dissection of ligand–receptor interactions, and for evaluating the adaptations that develop in pathogens as part of the host–parasite interaction. Crown Copyright Ó 2013 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc. All rights reserved.
1. Introduction Cryptobia salmositica is a pathogenic unicellular haemoflagellate present in salmon (Oncorhynchus spp.) collected from streams on the Pacific Coast of North America; the parasite is transmitted by the freshwater leech, Piscicola salmositica. Briefly, the pathogen has been cloned and attenuated using serial culture in minimum essential medium (MEM) supplemented with foetal bovine serum (FBS). A single dose of the attenuated strain protects 100% of several juvenile and adult salmonid species from cryptobiosis and mortality. Also, the vaccine has no detectable bioenergetic cost to the fish and does not affect growth of juvenile fish. The vaccine and the pathogenic strains have been used extensively to study the biology of C. salmositica and its host–parasite relationships (Woo, 2003, 2012), resulting in the development of protective strategies against the pathogen and disease (Woo, 2010). Outbreaks of cryptobiosis with high mortalities (in pre- and postspawning salmonids) have occurred in hatcheries and sea cages, and approximately 50% of gravid salmon from some streams brought into hatcheries die from the disease (Woo, 2003, 2012). ⇑ Corresponding author. Tel.: +1 519 824 4120; fax: +1 519 767 1656. E-mail addresses: [email protected], [email protected] (M. Li).
Reproductive migration in salmon and reproduction itself are stressors that result in elevated cortisol levels in fish (Schreck et al., 2001; Norris and Hobbs, 2006) and increased susceptibility to infectious pathogens. It was shown that in vivo implantation of cortisol into rainbow trout (Oncorhynchus mykiss) significantly increased Cryptobia parasitaemias in infected fish by decreasing the host’s immune response (Woo et al., 1987). More recently, Li et al. (2013) showed that both cortisol and the glucocorticoid analogue, dexamethasone, significantly enhanced in vitro multiplication of C. salmositica, while the glucocorticoid receptor (GR) antagonist, mifepristone (RU486), neutralised the enhancing effects of glucocorticoids. These observations suggest that C. salmositica may contain GR-like proteins that are involved in the interaction of the parasite with glucocorticoid ligands. The present study used a GR antibody agglutination test and confocal microscope immunohistofluorescence (IHF) methodology to determine whether GR-like proteins are present in the unicellular flagellate and if so, where they are located. 2. Materials and methods The C. salmositica used for the study were originally isolated from its leech vector found on spawning coho salmon
0020-7519/$36.00 Crown Copyright Ó 2013 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc. All rights reserved. http://dx.doi.org/10.1016/j.ijpara.2013.10.006
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(Oncorhynchus kisutch) on Vancouver Island, Canada (Woo, 1978). The parasite was cloned, maintained by serial culture in MEM + FBS, subpassaged in rainbow trout (O. mykiss), and cryopreserved at 90 °C (Woo, 2003). 2.1. Experiments to demonstrate GRs on C. salmositica 2.1.1. Agglutination test Parasites cultured in MEM + FBS were centrifuged at 12,000g for 20 min at 4 °C. The parasite pellet was resuspended in Cold-Blooded Vertebrate Ringer’s solution (CBVR) and washed three times in CBVR. After the last wash, the pellet was vortexed in 0.5 ml of CBVR and resuspended to approximately 1 107 parasites ml 1. Three microlitres of parasite suspension (1 107 parasites ml 1) were mixed with 3 ll of polyclonal IgG antibody (produced in rabbit and heat inactivated at 37 °C) against rainbow trout GR (Sathiyaa and Vijayan, 2003) on a clean chilled glass slide, covered with a wax-sealed coverslip and placed on ice. The control was 3 ll of heat inactivated naïve rabbit serum and 3 ll of parasite suspension (1 107 parasites ml 1). Various dilutions (undiluted, 1/2 and 1/4 in CBVR) of the trout GR antibody and naïve rabbit control serum were mixed with the parasite suspension to give overall dilutions of 1/2, 1/4 and 1/8 respectively. The slides were examined for parasite agglutination every 20 min under a light microscope. 2.1.2. In situ IHF staining Cryptobia salmositica does not survive and multiply in MEM unless FBS is present to satisfy the nutrient requirements of the parasite (Li and Woo, 1991). Since the FBS contained some cortisol, it was anticipated that the parasites from the control medium (without additional cortisol) would also stain weakly for GRs. 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 (1 mg ml 1 in ethanol) and added to MEM + FBS as described previously (Li et al., 2013). Briefly, parasites from cultures were exposed to cortisol-enriched medium (MEM + FBS + 25 ng ml 1 of cortisol) for 18 h at 10 °C. Parasites from cortisol-enriched medium and control medium (MEM + FBS) were isolated by centrifugation at 12,000g for 20 min at 4 °C, then washed three times in CBVR and resuspended in 0.5 ml of CBVR. After the last wash, smears of the parasite were made on chilled clean slides, fixed for 10 min at 22 °C in 2% buffered paraformaldehyde, rinsed three times in PBS and used for IHF or kept moist at 4 °C until needed. The in situ IHF staining protocol of Madan et al. (2007), and modified by Li et al. (2012) was used. The test was repeated on at least six separate occasions using rabbit polyclonal IgG antiserum (against trout GRs). The antibody has been used in at least three previous studies (Sathiyaa and Vijayan, 2003; Alderman et al., 2012; Li et al., 2012). Fluorescence images were taken using a Zeiss LSM 410 confocal microscope (Olympus America, New York, USA); objective (100) at 405 nm wavelength for DAPI, and at 488 nm for FITC. The fluorescent dye was conjugated to the secondary antibody of Alex Fluor⁄ 488 donkey anti-rabbit IgG (H + L) (Invitrogen, Burlington, ON, Canada). The laser confocal microscope was set at the optimal setting for imaging by adjusting the laser intensity for the background. All images were captured and stored as TIFF files. Z-stack images by Z-scan were saved as multiview live files and analysed using the Zeis LSM software package (ImagePro, NY, USA). 2.2. The GR antagonist, RU486, was used to confirm the apparent finite numbers of GRs that were present on/in C. salmositica RU486 (Sigma–Aldrich, St. Louis, USA) was dissolved in absolute ethanol to produce a stock solution (1 mg ml 1 in ethanol) and
added to MEM + FBS as described previously (Li et al., 2013). Two in vitro experiments were carried out. 2.2.1. Effects of two levels of RU486 on parasite multiplication Nine millilitres of RU486 enriched medium (MEM + FBS) were dispensed into individual 25 ml flasks with four replicates for each RU486 dosage (100 or 50 ng ml 1); an additional four flasks contained 9 ml of medium (MEM + FBS) as controls. One millilitre of parasites (140,000 parasites ml 1 in MEM + FBS) was inoculated aseptically into each flask to produced 14,000 parasites ml 1 initial inoculum cultures. 2.2.2. Effects of a single RU486 dosage on two different parasite inocula Briefly, 9 ml of RU486 enriched medium (50 ng ml 1 of RU486) were dispensed into individual 25 ml culture flasks in two groups (I and II). In Group I, each of four flasks with RU486 enriched medium (MEM + FBS) was inoculated with 1 ml of parasites (140,000) in the flask; in Group II, each of the four flasks was inoculated with 1 ml of parasites (70,000) in the medium. Nine millilitres of MEM + FBS in four replicates in groups I and II was inoculated with 1 ml of parasites as controls (140,000 and 70,000, respectively) to produce 14,000 and 7,000 parasites ml 1 initial inocula as controls, respectively. Parasite inoculations and samplings were conducted aseptically in a biological safety cabinet and parasite numbers were enumerated weekly from 2 weeks post-exposure (PE) using a haemocytometer (Archer, 1965; Li et al., 2013). 2.3. Statistical analysis Data on the effects of RU486 on parasite numbers were analysed using one-way ANOVA followed by the Holm-Sidak method for multiple comparisons; F values indicated statistical significance. If the test for normality or equal variance failed, Kruskal–Wallis’ one-way ANOVA on ranks was used, followed by Dunn’s (multiple comparisons versus controls) or the Student–Newman–Keuls pair-wise multiple comparison method. Student’s t-test was used to compare parasite numbers between the RU486 dosage experiments. For all tests, P < 0.05 was considered significant. 3. Results 3.1. GRs on and in C. salmositica 3.1.1. Demonstration of GR on the plasma membrane of the parasite using an agglutination test Parasites agglutinated rapidly (within 20 min) after the polyclonal rabbit anti-trout GR serum was added to the parasite suspension at a final dilution of 1/2 at 10 °C. At higher final dilutions of the antibody (1/4 and 1/8), agglutinations were also detected but the clumps were smaller and took longer to form (Table 1). There was no agglutination using all dilutions of the naïve rabbit serum (Table 1). In the GR antisera at 1/2 dilution, agglutinations were detected within approximately 20 min and the sizes of the clumps of parasites increased with time (Table 1). 3.1.2. In situ staining of C. salmositica GRs using an IHF test When exposed to DAPI, the parasite nuclei and kinetoplasts stained blue because they contain DNA. In immunostaining with rabbit antiserum (against rainbow trout GR), the primary trout GR antiserum was conjugated by secondary antibody (donkey anti-rabbit labelled by FITC), and produced green fluorescence under UV light in the confocal microscope. This indicates the presence of GRs, which were seen throughout the parasite’s cytoplasm.
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Table 1 Agglutination of live parasites, Cryptobia salmositica, by trout glucocorticoid receptor antibody. Live parasites were agglutinated by trout glucocorticoid receptor antiserum (final dilution, 1/2) within 20 min. At this dilution, the parasite clusters increased in size over time. The agglutination was less pronounced with increasing final dilutions of glucocorticoid receptor antiserum at 1/4 and 1/8 dilutions. There was no agglutination observed with naïve rabbit serum in any dilutions. The experiment was repeated on six separate occasions. Time (min)
20 40 60 120
Agglutination of live parasites with glucocorticoid receptor antiseruma
Agglutination of live parasites with naïve rabbit seruma
1/2
1/4
1/8
1/2
1/4
1/8
++ +++ +++ +++
– + + +
– – + +
– – – –
– – – –
– – – –
+ Indicates at least four to five parasites clustered together; ++ and +++ indicate twice and three times, respectively, of ‘‘+’’ parasites clustered together. a Final dilution when mixed with parasites.
However, in the controls, there were no such signals if the parasite was immunostained with naïve rabbit serum (Fig. 1). GR fluorescence signals from parasites in the control medium (MEM + FBS) were mostly distributed in the cytoplasm around the nucleus and kinetoplast when DAPI and GR stained images were superimposed (Fig. 1A). Parasites from cultures exposed to cortisol enriched medium for 18 h had much stronger green fluorescence signals than those parasites from the control medium; and the green fluorescence (indicating GRs) in the parasites were predominantly confined to the kinetoplast and nucleus in the superimposed images (Fig. 1B). A series of Z-scanned multiview live images (total 26 optical sections, 0.2 lm per section) from GR stained parasites from a cortisol-enriched culture clearly showed GR signals on the circumference of the images (i.e., the parasite’s cell membrane), and the signals were evenly distributed on the first and last images of the parasite indicating that GRs were distributed throughout the entire membrane (Fig. 1C). 3.2. Incubations with RU486 confirm that the numbers of GRs on the haemoflagellate are finite 3.2.1. Experiment 1 Using two different doses of RU486 (100 and 50 ng ml 1) on the same number (14,000 parasites ml 1) of C. salmositica, the inhibition on parasite replication was detectable at 2 weeks PE, was significant (P < 0.01) after 3 weeks PE and continued to 5 weeks PE at both RU486 dosages compared with controls. The inhibition was significantly different (P < 0.05) between the two levels of RU486 at 3 weeks PE. However, a greater inhibition of RU486 on the parasite’s replication was seen for 100 ng ml 1 compared with the 50 ng ml 1 cultures from 4 to 5 weeks PE (Fig. 2). 3.2.2. Experiment 2 When the same concentration of RU486 (50 ng ml 1) was applied to cultures containing 14,000 or 7,000 parasites ml 1, RU486 had, but not significant, inhibitive effects on parasite multiplication until 5 weeks PE in the 14,000 parasites ml 1 group (Fig. 3A), whereas in the 7,000 parasites ml 1 group, the inhibitive effects of RU486 on the replication of the parasite was already significant (P < 0.05) at 3 weeks PE, and highly significant (P < 0.01) at 4 and 5 weeks PE (Fig. 3B). 4. Discussion In an earlier study (Li et al., 2013), we postulated that the stimulatory effect of glucocorticoids on the replication of the unicellular organism, C. salmositica, involved a metazoan GR-like protein. In that study, the in vitro responses of the parasite to cortisol, a cortisol analogue (dexamethasone) and a GR antagonist (RU486) were consistent with the interactions that mediated the parasite’s responses via some form of GR-related cell signalling. The current
study clearly demonstrates that C. salmositica contains metazoan GR-like proteins that are present on both the plasma (cell) membrane (as evidenced by the in vitro agglutination test (Table 1)) and in the cytoplasm (as indicated by the in situ IHF staining; (Fig. 1)). Also, the number of GR on the parasite appears to be fixed as evidenced by the dose (RU486)- and parasite number-dependent responses (Figs. 2 and 3). This is, to our knowledge, the first known report of a functional steroid hormone receptor on and in a protozoan. Steroid hormone receptors such as GR are characteristic of metazoan animals (Sáez et al., 2010; Baker, 2011; Bertrand et al., 2011; Taubert et al., 2011; Wu and LoVerde, 2011; Lecroisey et al., 2012), and functional steroid hormone receptors such as oestrogen receptor (ER) were detected and identified in Taenia crassiceps, a multicellular flatworm (Ibarra-Coronadoa et al., 2011). It should be mentioned, however, that steroid nuclear receptors (NRs) are not limited to metazoans; they are also present in fungi and yeast cells (Phelphs et al., 2006; Fox et al., 2008; Näär and Thakur, 2009). In metazoan animals and yeast, the NRs are present in the cytoplasm of the target cell and when activated by the steroid ligand, they are translocated to the nucleus or mitochondria where they act as transcription factors for the regulation of gene expression (Antebi, 2006; Phelphs et al., 2006; Fox et al., 2008; Oakley and Cidlowski, 2011). In addition, some steroid hormone receptor proteins are inserted in the plasma membrane of steroid-responsive cells; these membrane-associated NRs can be activated by steroids, or their minics located in the extracellular compartment as reserves and are linked to complex intracellular signalling pathways (non-genomic signalling) in metazoans (Zhu et al., 2003; NääR and Thakur, 2009; Pang and Thomas, 2011; Filardo and Thomas, 2012). In metazoans, the NRs play many roles in the immune response, developmental biology and cellular responses to pharmaceuticals (Antebi, 2006; Zanchi et al., 2010; Oakley and Cidlowski, 2011). Antibodies against trout GR agglutinated the parasite (Table 1) within 20 min, with large clumps (up to 20 parasites) forming by 40 min. The parasite was not agglutinated by naïve rabbit serum. The results indicate for the first time that a protozoan animal has functional GRs and they are located on the surface membrane of the parasite (Fig. 1C). GR-like proteins were also detected on the cell membrane and in the cytoplasm of the parasite using IHF staining; intracellular GR was found in parasites cultured in both cortisol-enriched medium and the control medium. This may be because the control medium, which contained 22% FBS, had some endogenous cortisol in FBS. It might also be that the intracellular reserves of the GR protein are in an inactive form; however, the fluorescence signals from the parasites in the control medium were weak compared with the parasites from cortisol-enriched cultures (Fig. 1A), in which stronger GR signals were evident in the nucleus and kinetoplast (Fig. 1B) of the parasites. Taken together, these observations would indicate that the GRs were inducible and activated by cortisol, and that the
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Fig. 1. Confocal immunohistofluorescence images of trout glucocorticoid receptors on/in the parasite, Cryptobia salmositica, detected using secondary antibody conjugated with FITC; the immunostaining was repeated in six separate culture samples. (A) Parasites isolated from control culture minimum essential medium supplement with foetal bovine serum (MEM + FBS), fixed in 2% paraformaldehyde and stained either by naïve rabbit serum ( , negative staining) or anti-trout glucocorticoid receptor rabbit antiserum (+, positive staining). There were no fluorescence signals in parasites with naïve rabbit serum ( ); evenly distributed fluorescence signals in the parasites were visible when the trout glucocorticoid receptor antibody was the primary antibody (+). (B) Parasites isolated after 18 h in cortisol-enriched MEM (MEM + FBS + 25 ng ml 1 cortisol) cultures, fixed in 2% paraformaldehyde and either stained with naïve rabbit serum ( ) or trout glucocorticoid receptor antisera (+). There were no fluorescence signals in parasites when the naïve rabbit serum was the primary antibody ( ); fluorescence signals were visible when the trout glucocorticoid receptor antibody was the primary antibody (+), and the signals were stronger around the nucleus and kinetoplast in the merged images. The blue signals (DAPI stain) indicate DNA in both the nucleus and kinetoplast; the green signals (FITC stain) indicate the presence of trout glucocorticoid receptors in the parasite’s cytoplasm; the merged images are after DAPI and FITC staining images were superimposed. Bar = 10 lm. (C) Merged glucocorticoid receptor confocal images of the parasite (0.2 lm optical sections) from a typical glucocorticoid receptor antiserum stained parasite’s image after 18 h in cortisol-enriched MEM culture. Fluorescence signals were present over the entire surface of the cell membrane as indicated by images from the first (a) and last (f) optical sections, and the circumferences of the parasite sections (b–e). Bar = 10 lm.
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Fig. 2. Effects on Cryptobia salmositica multiplication (initial number 14,000 parasites ml 1) with two different concentrations of mifepristone (RU486). Compared with controls (no RU486), parasite multiplication at both concentrations of RU486 was significantly (P < 0.01) suppressed at 3 weeks post-exposure; 100 ng ml 1 of RU486 was more suppressive than 50 ng ml 1 throughout the 5 week period; however the suppression of between 100 ng ml 1 and 50 ng ml 1 cultures on parasite replication was significantly different (P < 0.05) only at 3 weeks postexposure. The graphs show the parasite number per ml in means ± SEM, n = 8, and the different lower case letters indicate significant differences in parasite number per ml (P < 0.05) between the two RU486 concentration cultures.
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activated GRs were relocated to the nucleus and kinetoplast of the haemoflagellate. RU486 has been widely used as a GR antagonist in animal stress physiology (Spiga et al., 2007; Alderman et al., 2012), in steroid hormone research, and in reproductive animal clinics including humans, due to its efficacy as an anti-pregnancy drug. However, the antagonistic properties of RU486 on progesterone receptors (PRs) and GRs are complex (Leonahart and Edwards, 2002). The response of the parasite to RU486 indicates that the GR-like protein on/in the parasite is less specific than that in metazoans. In the current in vitro study with RU486 on parasite replication, the same number of parasites responded differently to different dosages of RU486 (Fig. 2); the 100 ng ml 1 of RU486 significantly inhibited (P < 0.05) parasite replication more than that of 50 ng ml 1 of RU486 at 3 weeks PE, even though both RU486 dosages significantly inhibited (P < 0.01) parasite multiplication compared with controls. Furthermore, different numbers of parasites (14,000 and 7,000 ml 1) responded differently to the same RU486 dosage (50 ng ml 1) (Fig. 3), indicating that the inhibitive action of RU486 was dose-related. The inhibition of RU486 (50 ng ml 1) on 14,000 parasites ml 1 was not significant at 3 and 4 weeks PE, even though the inhibitory effects were evident, but not significant (only 7% and 13% lower compared with controls at 3 and 4 weeks PE, respectively). The less inhibitive effects at low level of RU486 (50 ng ml 1) on the high inocula (14,000 parasites ml 1) may indicate that concentrations of RU486 were too low and did not occupy all the hormone receptors. However, the inhibition of RU486 (50 ng ml 1) on 7,000 parasites ml 1 was significant at 3 (P < 0.05), 4, and 5 (P < 0.01) weeks PE. Collectively, these results further support the presence of a functional GR-like receptor (or NR) on/in C. salmositica and that the parasite has a limited number of receptors. It has yet to be established whether the GR-like protein that responds to cortisol in C. salmositica is specific for glucocorticoids or that it also responds to other steroids. Previous studies suggested that there was no replication stimulating effect of oestrogen when 17b-oestrodial was added to in vitro parasite cultures (Currie and Woo, 2008), and preliminary studies using polyclonal antibodies to mammalian ER (a polyclonal IgG antiserum produced in rabbit against mouse oestrogen receptor a: sc-542; Santa Cruz Biotechnology Inc., CA, USA) only weakly agglutinated the parasite (M. Li and P.T.K. Woo, unpublished data). These preliminary findings suggest that the GR-like protein on C. salmositica may be glucocorticoid-specific. Acknowledgements The research and ML were supported by a Grant from the Natural Sciences and Engineering Research Council of Canada to P.T.K.W. We sincerely thank Dr. Matt Vijayan of University of Calgary, Canada for the trout GR polyclonal antibody, and Dr. John Lumsden and Dr. Keith Betteridge, University of Guelph, Canada for their kind donation of the naïve rabbit serum and ERa polyclonal antibody for the study. We are also indebted to Dr. John Leatherland for the use of some equipment and reagents, and reviewing early drafts of the manuscript.
Fig. 3. Effects on Cryptobia salmositica, multiplication using different parasite numbers at a concentration of mifepristone (RU486, 50 ng ml 1). (A) Initial number of parasites was 14,000 parasites ml 1. Compared with the controls (no RU486), 50 ng ml 1 of RU486 showed weak suppression of parasite replication with suppression only significant (P < 0.01) at 5 weeks post-exposure. (B) Initial number of parasites was 7,000 parasites ml 1, Compared with the controls (no RU486), 50 ng ml 1 of RU486 significantly suppressed parasite replication at 3 (P < 0.05), 4 (P < 0.01) and 5 (P < 0.01) weeks post-exposure. The graphs show parasite number as means ± SEM, n = 8.
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Pang, Y., Thomas, P., 2011. Progesterone signals through membrane progesterone receptors (mPRs) in MDA-MB-468 and mPR-transfected MDA-MB-231 breast cancer cells which lack full-length and N-terminally truncated isoforms of the nuclear progesterone receptor. Steroids 76, 921–928. Phelphs, C., Gburcik, V., Suslova, E., Dudek, P., Forafonov, F., Bot, N., MacLean, M., Fagan, R.J., Picard, D., 2006. Fungi and animal may share a common ancestor to nuclear receptors. Proc. Natl. Acad. Sci. U.S.A. 103, 7077–7081. Sáez, P.J., Lange, S., Pérez-Acle, T., Owen, G.I., 2010. Nuclear receptor genes: evolution. In: Encyclopedia of Life Sciences (ELS), John Wiley & Sons Ltd, Chichester. http://dx.doi.org/10.1002/9780470015902.a0006145.pub3. Sathiyaa, R., Vijayan, M.M., 2003. Autoregulation of glucocorticoid receptor by cortisol in rainbow trout hepatocytes. Am. J. Physiol. Cell Physiol. 284, C1508– C1515. Schreck, C.B., Contreras-Sanchez, W., Fitzpatrick, M.S., 2001. Effects of stress on fish reproduction, gamete quality, and progeny. Aquaculture 197, 3–24. Spiga, F., Harrison, L.R., Wood, S.A., Atkinson, H.C., MacSweeney, C.P., Thomson, F., Craighead, M., Grassie, M., Ligthman, S.L., 2007. Effect of the glucocorticoid receptor antagonist org 34850 on basal and stress-induced corticosterone secretion. J. Neuroendocrinol. 19, 891–900. Taubert, S., Ward, J.D., Yamamoto, K.R., 2011. Nuclear hormone receptor in nematodes: evolution and function. Mol. Cell. Endocrinol. 334, 49–55. Woo, P.T.K., Leatherland, J.F., Lee, M.S., 1987. Cryptobia salmonsitica: cortisol increases the susceptibility of Salmo gairdneri Richardson to experimental cryptobiosis. J. Fish Dis. 10, 75–83. Woo, P.T.K., 1978. The division process of Cryptobia salmonsitica in experimentally infected rainbow trout (Salmo gairdneri). Can. J. Zool. 56, 1514–1518. Woo, P.T.K., 2003. Cryptobia (Trypanoplasma) salmositica and salmonid cryptobiosis. J. Fish Dis. 26, 627–646. Woo, P.T.K., 2010. Immunological and therapeutic strategies against salmond cryptobiosis (special issue: immunology and cell biology of parasite diseases), J. Biomed. Biotechnol. Article ID341783. http://dx.doi.org/10.1155/2010/341783. Woo, P.T.K., 2012. Cryptobia (Trypanoplasma) salmositica. In: Woo, P.T.K., Buchmann, K. (Eds.), Fish Parasites: Pathobiology and Protection. CABI Publishing, Wallingford, Oxfordshire, UK, pp. 30–54. Wu, W., LoVerde, P.T., 2011. Nuclear hormone receptors in parasitic helminthes. Mol. Cell. Endocrinol. 334, 56–66. Zanchi, N.E., Filho, M.A., Felitti, V., Nicastro, H., Lorenzeti, F.M., Lancha Jr., A.H., 2010. Glucocorticoids: extensive physiological actions modulated through multiple mechanism of gene expression. J. Cell. Physiol. 224, 311–315. Zhu, Y., Rice, C.D., Pang, Y., Pace, M., Thomas, P., 2003. Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes. Proc. Natl. Acad. Sci. U.S.A. 100, 2231–2236.
Contents lists available at ScienceDirect
International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara
Glucocorticoid receptors on and in a unicellular organism, Cryptobia salmositica Mao Li ⇑, Patrick T.K. Woo Department of Integrative Biology, University of Guelph, Guelph, ON N1G 2W1, Canada
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Article history: Received 15 August 2013 Received in revised form 26 October 2013 Accepted 28 October 2013 Available online 11 December 2013 Keywords: Glucocorticoid receptor (GR) Protozoan Cryptobia salmositica Cortisol RU486 In vitro culture Host–parasite interaction
a b s t r a c t This is the first report to our knowledge that demonstrates a functional steroid hormone receptor in a protozoon. The study used Cryptobia salmositica, a pathogenic haemoflagellate found in salmonid fishes. It has been previously shown that cortisol and dexamethasone (a synthetic glucocorticoid) enhanced the multiplication of C. salmositica under in vitro conditions indicating the presence of glucocorticoid receptors on/in the parasite. Also, the glucocorticoid receptor antagonist, mifepristone (RU486), inhibited the stimulatory effect of the two glucocorticoids on parasite multiplication. In the present study, we used an antibody (produced in a rabbit against glucocorticoid receptor protein) agglutination test and confocal microscopy with immunohistofluorescence staining to demonstrate cortisol-glucocorticoid receptor-like protein receptors on the plasma membrane and in the cytoplasm of the parasite. In two in vitro studies, the addition of 50 ng ml 1 of RU486 was more effective in inhibiting parasite replication in cultures with 7,000 parasites ml 1 than in cultures with 14,000 parasites ml 1. Also, 100 ng ml 1 of RU486/ml was more effective than 50 ng ml 1 in inhibiting parasite multiplication in the 14,000 parasites ml-1 cultures. These in vitro studies indicate that the number of binding sites on/in the parasite is finite. The findings may be important in future studies especially on steroid receptor signalling pathways and dissection of ligand–receptor interactions, and for evaluating the adaptations that develop in pathogens as part of the host–parasite interaction. Crown Copyright Ó 2013 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc. All rights reserved.
1. Introduction Cryptobia salmositica is a pathogenic unicellular haemoflagellate present in salmon (Oncorhynchus spp.) collected from streams on the Pacific Coast of North America; the parasite is transmitted by the freshwater leech, Piscicola salmositica. Briefly, the pathogen has been cloned and attenuated using serial culture in minimum essential medium (MEM) supplemented with foetal bovine serum (FBS). A single dose of the attenuated strain protects 100% of several juvenile and adult salmonid species from cryptobiosis and mortality. Also, the vaccine has no detectable bioenergetic cost to the fish and does not affect growth of juvenile fish. The vaccine and the pathogenic strains have been used extensively to study the biology of C. salmositica and its host–parasite relationships (Woo, 2003, 2012), resulting in the development of protective strategies against the pathogen and disease (Woo, 2010). Outbreaks of cryptobiosis with high mortalities (in pre- and postspawning salmonids) have occurred in hatcheries and sea cages, and approximately 50% of gravid salmon from some streams brought into hatcheries die from the disease (Woo, 2003, 2012). ⇑ Corresponding author. Tel.: +1 519 824 4120; fax: +1 519 767 1656. E-mail addresses: [email protected], [email protected] (M. Li).
Reproductive migration in salmon and reproduction itself are stressors that result in elevated cortisol levels in fish (Schreck et al., 2001; Norris and Hobbs, 2006) and increased susceptibility to infectious pathogens. It was shown that in vivo implantation of cortisol into rainbow trout (Oncorhynchus mykiss) significantly increased Cryptobia parasitaemias in infected fish by decreasing the host’s immune response (Woo et al., 1987). More recently, Li et al. (2013) showed that both cortisol and the glucocorticoid analogue, dexamethasone, significantly enhanced in vitro multiplication of C. salmositica, while the glucocorticoid receptor (GR) antagonist, mifepristone (RU486), neutralised the enhancing effects of glucocorticoids. These observations suggest that C. salmositica may contain GR-like proteins that are involved in the interaction of the parasite with glucocorticoid ligands. The present study used a GR antibody agglutination test and confocal microscope immunohistofluorescence (IHF) methodology to determine whether GR-like proteins are present in the unicellular flagellate and if so, where they are located. 2. Materials and methods The C. salmositica used for the study were originally isolated from its leech vector found on spawning coho salmon
0020-7519/$36.00 Crown Copyright Ó 2013 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc. All rights reserved. http://dx.doi.org/10.1016/j.ijpara.2013.10.006
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(Oncorhynchus kisutch) on Vancouver Island, Canada (Woo, 1978). The parasite was cloned, maintained by serial culture in MEM + FBS, subpassaged in rainbow trout (O. mykiss), and cryopreserved at 90 °C (Woo, 2003). 2.1. Experiments to demonstrate GRs on C. salmositica 2.1.1. Agglutination test Parasites cultured in MEM + FBS were centrifuged at 12,000g for 20 min at 4 °C. The parasite pellet was resuspended in Cold-Blooded Vertebrate Ringer’s solution (CBVR) and washed three times in CBVR. After the last wash, the pellet was vortexed in 0.5 ml of CBVR and resuspended to approximately 1 107 parasites ml 1. Three microlitres of parasite suspension (1 107 parasites ml 1) were mixed with 3 ll of polyclonal IgG antibody (produced in rabbit and heat inactivated at 37 °C) against rainbow trout GR (Sathiyaa and Vijayan, 2003) on a clean chilled glass slide, covered with a wax-sealed coverslip and placed on ice. The control was 3 ll of heat inactivated naïve rabbit serum and 3 ll of parasite suspension (1 107 parasites ml 1). Various dilutions (undiluted, 1/2 and 1/4 in CBVR) of the trout GR antibody and naïve rabbit control serum were mixed with the parasite suspension to give overall dilutions of 1/2, 1/4 and 1/8 respectively. The slides were examined for parasite agglutination every 20 min under a light microscope. 2.1.2. In situ IHF staining Cryptobia salmositica does not survive and multiply in MEM unless FBS is present to satisfy the nutrient requirements of the parasite (Li and Woo, 1991). Since the FBS contained some cortisol, it was anticipated that the parasites from the control medium (without additional cortisol) would also stain weakly for GRs. 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 (1 mg ml 1 in ethanol) and added to MEM + FBS as described previously (Li et al., 2013). Briefly, parasites from cultures were exposed to cortisol-enriched medium (MEM + FBS + 25 ng ml 1 of cortisol) for 18 h at 10 °C. Parasites from cortisol-enriched medium and control medium (MEM + FBS) were isolated by centrifugation at 12,000g for 20 min at 4 °C, then washed three times in CBVR and resuspended in 0.5 ml of CBVR. After the last wash, smears of the parasite were made on chilled clean slides, fixed for 10 min at 22 °C in 2% buffered paraformaldehyde, rinsed three times in PBS and used for IHF or kept moist at 4 °C until needed. The in situ IHF staining protocol of Madan et al. (2007), and modified by Li et al. (2012) was used. The test was repeated on at least six separate occasions using rabbit polyclonal IgG antiserum (against trout GRs). The antibody has been used in at least three previous studies (Sathiyaa and Vijayan, 2003; Alderman et al., 2012; Li et al., 2012). Fluorescence images were taken using a Zeiss LSM 410 confocal microscope (Olympus America, New York, USA); objective (100) at 405 nm wavelength for DAPI, and at 488 nm for FITC. The fluorescent dye was conjugated to the secondary antibody of Alex Fluor⁄ 488 donkey anti-rabbit IgG (H + L) (Invitrogen, Burlington, ON, Canada). The laser confocal microscope was set at the optimal setting for imaging by adjusting the laser intensity for the background. All images were captured and stored as TIFF files. Z-stack images by Z-scan were saved as multiview live files and analysed using the Zeis LSM software package (ImagePro, NY, USA). 2.2. The GR antagonist, RU486, was used to confirm the apparent finite numbers of GRs that were present on/in C. salmositica RU486 (Sigma–Aldrich, St. Louis, USA) was dissolved in absolute ethanol to produce a stock solution (1 mg ml 1 in ethanol) and
added to MEM + FBS as described previously (Li et al., 2013). Two in vitro experiments were carried out. 2.2.1. Effects of two levels of RU486 on parasite multiplication Nine millilitres of RU486 enriched medium (MEM + FBS) were dispensed into individual 25 ml flasks with four replicates for each RU486 dosage (100 or 50 ng ml 1); an additional four flasks contained 9 ml of medium (MEM + FBS) as controls. One millilitre of parasites (140,000 parasites ml 1 in MEM + FBS) was inoculated aseptically into each flask to produced 14,000 parasites ml 1 initial inoculum cultures. 2.2.2. Effects of a single RU486 dosage on two different parasite inocula Briefly, 9 ml of RU486 enriched medium (50 ng ml 1 of RU486) were dispensed into individual 25 ml culture flasks in two groups (I and II). In Group I, each of four flasks with RU486 enriched medium (MEM + FBS) was inoculated with 1 ml of parasites (140,000) in the flask; in Group II, each of the four flasks was inoculated with 1 ml of parasites (70,000) in the medium. Nine millilitres of MEM + FBS in four replicates in groups I and II was inoculated with 1 ml of parasites as controls (140,000 and 70,000, respectively) to produce 14,000 and 7,000 parasites ml 1 initial inocula as controls, respectively. Parasite inoculations and samplings were conducted aseptically in a biological safety cabinet and parasite numbers were enumerated weekly from 2 weeks post-exposure (PE) using a haemocytometer (Archer, 1965; Li et al., 2013). 2.3. Statistical analysis Data on the effects of RU486 on parasite numbers were analysed using one-way ANOVA followed by the Holm-Sidak method for multiple comparisons; F values indicated statistical significance. If the test for normality or equal variance failed, Kruskal–Wallis’ one-way ANOVA on ranks was used, followed by Dunn’s (multiple comparisons versus controls) or the Student–Newman–Keuls pair-wise multiple comparison method. Student’s t-test was used to compare parasite numbers between the RU486 dosage experiments. For all tests, P < 0.05 was considered significant. 3. Results 3.1. GRs on and in C. salmositica 3.1.1. Demonstration of GR on the plasma membrane of the parasite using an agglutination test Parasites agglutinated rapidly (within 20 min) after the polyclonal rabbit anti-trout GR serum was added to the parasite suspension at a final dilution of 1/2 at 10 °C. At higher final dilutions of the antibody (1/4 and 1/8), agglutinations were also detected but the clumps were smaller and took longer to form (Table 1). There was no agglutination using all dilutions of the naïve rabbit serum (Table 1). In the GR antisera at 1/2 dilution, agglutinations were detected within approximately 20 min and the sizes of the clumps of parasites increased with time (Table 1). 3.1.2. In situ staining of C. salmositica GRs using an IHF test When exposed to DAPI, the parasite nuclei and kinetoplasts stained blue because they contain DNA. In immunostaining with rabbit antiserum (against rainbow trout GR), the primary trout GR antiserum was conjugated by secondary antibody (donkey anti-rabbit labelled by FITC), and produced green fluorescence under UV light in the confocal microscope. This indicates the presence of GRs, which were seen throughout the parasite’s cytoplasm.
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Table 1 Agglutination of live parasites, Cryptobia salmositica, by trout glucocorticoid receptor antibody. Live parasites were agglutinated by trout glucocorticoid receptor antiserum (final dilution, 1/2) within 20 min. At this dilution, the parasite clusters increased in size over time. The agglutination was less pronounced with increasing final dilutions of glucocorticoid receptor antiserum at 1/4 and 1/8 dilutions. There was no agglutination observed with naïve rabbit serum in any dilutions. The experiment was repeated on six separate occasions. Time (min)
20 40 60 120
Agglutination of live parasites with glucocorticoid receptor antiseruma
Agglutination of live parasites with naïve rabbit seruma
1/2
1/4
1/8
1/2
1/4
1/8
++ +++ +++ +++
– + + +
– – + +
– – – –
– – – –
– – – –
+ Indicates at least four to five parasites clustered together; ++ and +++ indicate twice and three times, respectively, of ‘‘+’’ parasites clustered together. a Final dilution when mixed with parasites.
However, in the controls, there were no such signals if the parasite was immunostained with naïve rabbit serum (Fig. 1). GR fluorescence signals from parasites in the control medium (MEM + FBS) were mostly distributed in the cytoplasm around the nucleus and kinetoplast when DAPI and GR stained images were superimposed (Fig. 1A). Parasites from cultures exposed to cortisol enriched medium for 18 h had much stronger green fluorescence signals than those parasites from the control medium; and the green fluorescence (indicating GRs) in the parasites were predominantly confined to the kinetoplast and nucleus in the superimposed images (Fig. 1B). A series of Z-scanned multiview live images (total 26 optical sections, 0.2 lm per section) from GR stained parasites from a cortisol-enriched culture clearly showed GR signals on the circumference of the images (i.e., the parasite’s cell membrane), and the signals were evenly distributed on the first and last images of the parasite indicating that GRs were distributed throughout the entire membrane (Fig. 1C). 3.2. Incubations with RU486 confirm that the numbers of GRs on the haemoflagellate are finite 3.2.1. Experiment 1 Using two different doses of RU486 (100 and 50 ng ml 1) on the same number (14,000 parasites ml 1) of C. salmositica, the inhibition on parasite replication was detectable at 2 weeks PE, was significant (P < 0.01) after 3 weeks PE and continued to 5 weeks PE at both RU486 dosages compared with controls. The inhibition was significantly different (P < 0.05) between the two levels of RU486 at 3 weeks PE. However, a greater inhibition of RU486 on the parasite’s replication was seen for 100 ng ml 1 compared with the 50 ng ml 1 cultures from 4 to 5 weeks PE (Fig. 2). 3.2.2. Experiment 2 When the same concentration of RU486 (50 ng ml 1) was applied to cultures containing 14,000 or 7,000 parasites ml 1, RU486 had, but not significant, inhibitive effects on parasite multiplication until 5 weeks PE in the 14,000 parasites ml 1 group (Fig. 3A), whereas in the 7,000 parasites ml 1 group, the inhibitive effects of RU486 on the replication of the parasite was already significant (P < 0.05) at 3 weeks PE, and highly significant (P < 0.01) at 4 and 5 weeks PE (Fig. 3B). 4. Discussion In an earlier study (Li et al., 2013), we postulated that the stimulatory effect of glucocorticoids on the replication of the unicellular organism, C. salmositica, involved a metazoan GR-like protein. In that study, the in vitro responses of the parasite to cortisol, a cortisol analogue (dexamethasone) and a GR antagonist (RU486) were consistent with the interactions that mediated the parasite’s responses via some form of GR-related cell signalling. The current
study clearly demonstrates that C. salmositica contains metazoan GR-like proteins that are present on both the plasma (cell) membrane (as evidenced by the in vitro agglutination test (Table 1)) and in the cytoplasm (as indicated by the in situ IHF staining; (Fig. 1)). Also, the number of GR on the parasite appears to be fixed as evidenced by the dose (RU486)- and parasite number-dependent responses (Figs. 2 and 3). This is, to our knowledge, the first known report of a functional steroid hormone receptor on and in a protozoan. Steroid hormone receptors such as GR are characteristic of metazoan animals (Sáez et al., 2010; Baker, 2011; Bertrand et al., 2011; Taubert et al., 2011; Wu and LoVerde, 2011; Lecroisey et al., 2012), and functional steroid hormone receptors such as oestrogen receptor (ER) were detected and identified in Taenia crassiceps, a multicellular flatworm (Ibarra-Coronadoa et al., 2011). It should be mentioned, however, that steroid nuclear receptors (NRs) are not limited to metazoans; they are also present in fungi and yeast cells (Phelphs et al., 2006; Fox et al., 2008; Näär and Thakur, 2009). In metazoan animals and yeast, the NRs are present in the cytoplasm of the target cell and when activated by the steroid ligand, they are translocated to the nucleus or mitochondria where they act as transcription factors for the regulation of gene expression (Antebi, 2006; Phelphs et al., 2006; Fox et al., 2008; Oakley and Cidlowski, 2011). In addition, some steroid hormone receptor proteins are inserted in the plasma membrane of steroid-responsive cells; these membrane-associated NRs can be activated by steroids, or their minics located in the extracellular compartment as reserves and are linked to complex intracellular signalling pathways (non-genomic signalling) in metazoans (Zhu et al., 2003; NääR and Thakur, 2009; Pang and Thomas, 2011; Filardo and Thomas, 2012). In metazoans, the NRs play many roles in the immune response, developmental biology and cellular responses to pharmaceuticals (Antebi, 2006; Zanchi et al., 2010; Oakley and Cidlowski, 2011). Antibodies against trout GR agglutinated the parasite (Table 1) within 20 min, with large clumps (up to 20 parasites) forming by 40 min. The parasite was not agglutinated by naïve rabbit serum. The results indicate for the first time that a protozoan animal has functional GRs and they are located on the surface membrane of the parasite (Fig. 1C). GR-like proteins were also detected on the cell membrane and in the cytoplasm of the parasite using IHF staining; intracellular GR was found in parasites cultured in both cortisol-enriched medium and the control medium. This may be because the control medium, which contained 22% FBS, had some endogenous cortisol in FBS. It might also be that the intracellular reserves of the GR protein are in an inactive form; however, the fluorescence signals from the parasites in the control medium were weak compared with the parasites from cortisol-enriched cultures (Fig. 1A), in which stronger GR signals were evident in the nucleus and kinetoplast (Fig. 1B) of the parasites. Taken together, these observations would indicate that the GRs were inducible and activated by cortisol, and that the
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Fig. 1. Confocal immunohistofluorescence images of trout glucocorticoid receptors on/in the parasite, Cryptobia salmositica, detected using secondary antibody conjugated with FITC; the immunostaining was repeated in six separate culture samples. (A) Parasites isolated from control culture minimum essential medium supplement with foetal bovine serum (MEM + FBS), fixed in 2% paraformaldehyde and stained either by naïve rabbit serum ( , negative staining) or anti-trout glucocorticoid receptor rabbit antiserum (+, positive staining). There were no fluorescence signals in parasites with naïve rabbit serum ( ); evenly distributed fluorescence signals in the parasites were visible when the trout glucocorticoid receptor antibody was the primary antibody (+). (B) Parasites isolated after 18 h in cortisol-enriched MEM (MEM + FBS + 25 ng ml 1 cortisol) cultures, fixed in 2% paraformaldehyde and either stained with naïve rabbit serum ( ) or trout glucocorticoid receptor antisera (+). There were no fluorescence signals in parasites when the naïve rabbit serum was the primary antibody ( ); fluorescence signals were visible when the trout glucocorticoid receptor antibody was the primary antibody (+), and the signals were stronger around the nucleus and kinetoplast in the merged images. The blue signals (DAPI stain) indicate DNA in both the nucleus and kinetoplast; the green signals (FITC stain) indicate the presence of trout glucocorticoid receptors in the parasite’s cytoplasm; the merged images are after DAPI and FITC staining images were superimposed. Bar = 10 lm. (C) Merged glucocorticoid receptor confocal images of the parasite (0.2 lm optical sections) from a typical glucocorticoid receptor antiserum stained parasite’s image after 18 h in cortisol-enriched MEM culture. Fluorescence signals were present over the entire surface of the cell membrane as indicated by images from the first (a) and last (f) optical sections, and the circumferences of the parasite sections (b–e). Bar = 10 lm.
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Fig. 2. Effects on Cryptobia salmositica multiplication (initial number 14,000 parasites ml 1) with two different concentrations of mifepristone (RU486). Compared with controls (no RU486), parasite multiplication at both concentrations of RU486 was significantly (P < 0.01) suppressed at 3 weeks post-exposure; 100 ng ml 1 of RU486 was more suppressive than 50 ng ml 1 throughout the 5 week period; however the suppression of between 100 ng ml 1 and 50 ng ml 1 cultures on parasite replication was significantly different (P < 0.05) only at 3 weeks postexposure. The graphs show the parasite number per ml in means ± SEM, n = 8, and the different lower case letters indicate significant differences in parasite number per ml (P < 0.05) between the two RU486 concentration cultures.
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activated GRs were relocated to the nucleus and kinetoplast of the haemoflagellate. RU486 has been widely used as a GR antagonist in animal stress physiology (Spiga et al., 2007; Alderman et al., 2012), in steroid hormone research, and in reproductive animal clinics including humans, due to its efficacy as an anti-pregnancy drug. However, the antagonistic properties of RU486 on progesterone receptors (PRs) and GRs are complex (Leonahart and Edwards, 2002). The response of the parasite to RU486 indicates that the GR-like protein on/in the parasite is less specific than that in metazoans. In the current in vitro study with RU486 on parasite replication, the same number of parasites responded differently to different dosages of RU486 (Fig. 2); the 100 ng ml 1 of RU486 significantly inhibited (P < 0.05) parasite replication more than that of 50 ng ml 1 of RU486 at 3 weeks PE, even though both RU486 dosages significantly inhibited (P < 0.01) parasite multiplication compared with controls. Furthermore, different numbers of parasites (14,000 and 7,000 ml 1) responded differently to the same RU486 dosage (50 ng ml 1) (Fig. 3), indicating that the inhibitive action of RU486 was dose-related. The inhibition of RU486 (50 ng ml 1) on 14,000 parasites ml 1 was not significant at 3 and 4 weeks PE, even though the inhibitory effects were evident, but not significant (only 7% and 13% lower compared with controls at 3 and 4 weeks PE, respectively). The less inhibitive effects at low level of RU486 (50 ng ml 1) on the high inocula (14,000 parasites ml 1) may indicate that concentrations of RU486 were too low and did not occupy all the hormone receptors. However, the inhibition of RU486 (50 ng ml 1) on 7,000 parasites ml 1 was significant at 3 (P < 0.05), 4, and 5 (P < 0.01) weeks PE. Collectively, these results further support the presence of a functional GR-like receptor (or NR) on/in C. salmositica and that the parasite has a limited number of receptors. It has yet to be established whether the GR-like protein that responds to cortisol in C. salmositica is specific for glucocorticoids or that it also responds to other steroids. Previous studies suggested that there was no replication stimulating effect of oestrogen when 17b-oestrodial was added to in vitro parasite cultures (Currie and Woo, 2008), and preliminary studies using polyclonal antibodies to mammalian ER (a polyclonal IgG antiserum produced in rabbit against mouse oestrogen receptor a: sc-542; Santa Cruz Biotechnology Inc., CA, USA) only weakly agglutinated the parasite (M. Li and P.T.K. Woo, unpublished data). These preliminary findings suggest that the GR-like protein on C. salmositica may be glucocorticoid-specific. Acknowledgements The research and ML were supported by a Grant from the Natural Sciences and Engineering Research Council of Canada to P.T.K.W. We sincerely thank Dr. Matt Vijayan of University of Calgary, Canada for the trout GR polyclonal antibody, and Dr. John Lumsden and Dr. Keith Betteridge, University of Guelph, Canada for their kind donation of the naïve rabbit serum and ERa polyclonal antibody for the study. We are also indebted to Dr. John Leatherland for the use of some equipment and reagents, and reviewing early drafts of the manuscript.
Fig. 3. Effects on Cryptobia salmositica, multiplication using different parasite numbers at a concentration of mifepristone (RU486, 50 ng ml 1). (A) Initial number of parasites was 14,000 parasites ml 1. Compared with the controls (no RU486), 50 ng ml 1 of RU486 showed weak suppression of parasite replication with suppression only significant (P < 0.01) at 5 weeks post-exposure. (B) Initial number of parasites was 7,000 parasites ml 1, Compared with the controls (no RU486), 50 ng ml 1 of RU486 significantly suppressed parasite replication at 3 (P < 0.05), 4 (P < 0.01) and 5 (P < 0.01) weeks post-exposure. The graphs show parasite number as means ± SEM, n = 8.
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