J. Comp. Path. 2014, Vol. 150, 316e324 Available online at www.sciencedirect.com ScienceDirect www.elsevier.com/locate/jcpa DISEASE IN WILDLIFE OR EXOTIC SPECIES Comparative Study of Infection with Tetrahymena of Different Ornamental Fish Species G. Sharon*, M. Pimenta Leibowitz†, J. Kumar Chettri‡, N. Isakovx and D. Zilberg* * Aquatic Animal Health Laboratory, French Associates Institute for Dryland Agriculture and Biotechnology, J. Blaustein Institutes for Desert Research, Ben Gurion University of the Negev, Midreshet Ben Gurion 84990, † Colors Aquaculture R&D, Moshav Hatzeva 86815, Israel, ‡ Laboratory of Aquatic Pathobiology, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg C, Denmark and x Shraga Segal Department of Microbiology, Immunology and Genetics, Ben Gurion University of the Negev, Beer Sheva, Israel Summary Tetrahymena is a ciliated protozoan that can infect a wide range of fish species, although it is most commonly reported in guppies (Poecilia reticulata). The aim of this study was to compare the susceptibility to infection with Tetrahymena of five different ornamental fish species from two different super orders. The species examined were platy (Xiphophorus), molly (Poecilia sphenops) and angelfish (Pterophyllum scalare) of the Acanthopterygii super order (which also includes guppies) and goldfish (Carassius auratus auratus) and koi carp (Cyprinus carpio) of the Ostariophysi super order. These two super orders are phylogenetically distant from each other. Infection with Tetrahymena resulted in parasite invasion of internal organs, skin and muscle in all fish species. A relatively strong inflammatory response was observed in infected goldfish and koi, with negligible response in fish species of the Acanthopterygii super order. Guppies were the most susceptible to Tetrahymena infection, exhibiting a mortality rate of 87% and 100% in two separate experiments. A high mortality rate was also observed in platy (77%), while that of molly and angelfish was significantly lower (23% and 33%, respectively). Goldfish and koi carp were less susceptible to infection compared with guppies (24% and 59% mortality, respectively). Immunization studies revealed that the Tetrahymena are immunogenic, since infection of koi carp increased their Tetrahymena immobilization response by approximately three-fold at 3 weeks post infection, while immunization with Tetrahymena plus adjuvant increased their immobilization response by approximately 30-fold. Ó 2013 Elsevier Ltd. All rights reserved. Keywords: fish; histopathology; protozoa; Tetrahymena Introduction Tetrahymena spp. is a ciliated protozoan of the phylum ciliophora (Corliss, 1952). It is a saprozoic ciliate that feeds on organic matter and bacteria in natural habitats (Ponpornpisit et al., 2000). This protozoan is common in nature and appears to have no geographical limits (Hoffman et al., 1975). Tetrahymena spp. is the causative agent of tetrahymeniosis or ‘tet disease’, also known as ‘guppy killer disease’ in tropical aquarium fish, which causes severe economic losses in commercial guppy farms worldwide. Tetrahymena is an Correspondence to: D. Zilberg (e-mail: dzilberg@bgu.ac.il). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2013.08.005 invasive pathogen, which has predilection for guppies for reasons that are not clear, but infections have been reported in other species of ornamental fish, edible fish and even freshwater leeches (Nephelopsis obscura). The latter are themselves parasites of trout (Salmo spp.) and can therefore transfer the protozoa to their host (Saglam and Sarieyyupoglu, 2002). Ornamental fish species reported to be infected with Tetrahymena spp. include zebrafish (Danio rerio; Astrofsky et al., 2002), angelfish (Pterophyllum scalare), platy (Xiphophorus variatus), neon tetra (Paracheirodon innesi) (Ponpornpisit et al., 2000; Pimenta Leibowitz et al., 2005) and the bristle-nosed catfish (Ancistrus spp.; unpublished). Ó 2013 Elsevier Ltd. All rights reserved. Tetrahymena Infection of Ornamental Fish Susceptibility to Tetrahymena increases in fish that are wounded and/or weakened by stress conditions, such as high ammonia level, high organic load, extreme water temperature, non-optimal shipment conditions (e.g. high fish density) or a disease (Ferguson et al., 1987; Imai et al., 2000; Hatai et al., 2001; Pimenta Leibowitz et al., 2005). In the present work we tested parameters related to Tetrahymena infection of different ornamental fish species. Species selected for this study are popular ornamental fish that are often cultured in close proximity on individual farms. Our aim was to study the threat that this infection, common in guppies, poses on other fish species that are cultured in the same system. Guppy (Poecilia reticulata) is native to the Trinidad Island and the northern part of South America, where it lives in natural water at 24e30 C and exhibits tolerance to a wide range of salt concentrations. There are w300 subspecies of guppies and they are among the most popular ornamental fish species, widely traded across the world. Molly (Poecilia sphenops) is native to Central and South America (Mexico to Colombia) where it lives in tropical water at a temperature range of 18e28 C. Platy is a common name for two related freshwater species of the Xiphophorus genus, the Southern platy (Xiphophorus maculatus) and the Variatus platy (X. variatus), both native to the Eastern coast of Central America and Southern Mexico. Most ornamental platy are hybrids of both species, which have been interbred to the point that they are difficult to distinguish. Platy lives in temperatures ranging between 18 and 25 C. The origin of angelfish is the Amazon, Orinoco and Essequibo rivers in the tropical South America. Its Latin name, Pterophyllum, means ‘winged leaf’ and it is among the most common aquarium species, prized for its unique shape, colour and behaviour. Angelfish live in tropical freshwater at temperatures ranging between 24 and 30 C and reach a maximal length of 7.5 cm. The guppy, molly, platy and angelfish belong to the super order Acanthopterygii (spiny finned fish). Goldfish (Carassius auratus auratus) was one of the first fish species to be domesticated and is the most commonly kept aquarium fish. It is closely related to the less colourful carp, C. auratus, which is native to Eastern Asia and was domesticated in China more than a thousand years ago. The freshwater subtropical goldfish inhabits rivers, lakes, ponds and ditches with stagnant or slow-flowing water. It lives better in coldwater. The koi (meaning ‘carp’ in Japanese), or more specifically nishikigoi, represents ornamental varieties of domesticated common carp (Cyprinus carpio) that are kept for decorative purposes in outdoor ponds or water gardens. Koi carp and 317 goldfish belong to the family Cyprinidae, super order Ostariophysi. The relative susceptibility of the different fish species to infection with Tetrahymena was analyzed and a comparative histopathological analysis carried out. Materials and Methods Fish Naive guppy, platy, molly, angelfish, koi and goldfish were obtained from commercial aquaculture farms in Israel and acclimated for a minimum of 2 weeks before experimentation. Fish were maintained at a water temperature of 25  1 C. Feeding occurred daily at 2% of the body weight (Tropical Orange, Tzemah, Israel, for the tropical ornamental species and Hazorea Food, Ranan Marketing, Israel, for the coldwater species, koi and goldfish). To maintain adequate water quality, submerged biological filters were used and 40% of the water was exchanged every other day. Water quality parameters were monitored weekly and ammonia, nitrite and nitrate were measured by visocolour kits (MachereyeNagel, D€ uren, Germany). Ammonia and nitrite levels were maintained at >0.5 ppm and nitrate levels were maintained at 5e10 ppm. The water pH was kept constant at 7.6 (pH meter, Eutech Instruments, Singapore) and dissolved oxygen was maintained at >80% saturation (YSI 52-dissolved oxygen meter, YSC incorporated, Yellow spring, Ohio, USA). Fish were treated in compliance with the principles for biomedical research involving animals. The experimental protocol was approved by the BenGurion University Committee for the Ethical Care and Use of Animals in Experiments (http://in.bgu. ac.il/fohs/AnimalFacility/Pages/default.aspx), authorization numbers IL-67-11-2002 and IL-51-8-2008. Tetrahymena Maintenance The Tetrahymena spp. (Tet-NI) used in this study was originally isolated at our laboratory in 2005 from guppies imported to Israel by a commercial fish farm. The fish were found to be infected with Tetrahymena sp. during the quarantine stage, brought to our laboratory and stocked in 10 litre aquaria. Comparative DNA barcode analysis indicated that the parasite Tet-NI, was a new species of Tetrahymena (Chantangsi et al., 2007). The disease-causing parasite was maintained in vivo and in vitro, as described by Pimenta Leibowitz and Zilberg (2009). Briefly, in-vivo infection was maintained in two separate containers of 100 litres each by the regular addition of na€ıve fish to replace 318 G. Sharon et al. mortalities. For in-vitro culture, Tet-NI was isolated aseptically from the internal organs (excluding the gastrointestinal tract), skin lesions, gills or tail of infected guppies and cultured in RM-9 medium (consisting of protease peptone, tryptone, glucose, liver extract and di-potassium hydrogen phosphate) in a Petri dish incubated at 25 C. Penicillin G (3 mg/l) and streptomycin sulphate (3 mg/l) were added to prevent bacterial growth. Subculturing was conducted weekly under sterile conditions without antibiotic in a sterile hood (ADS Laminar, Paris, France). As Tet-NI appears to lose pathogenicity under prolonged culture conditions (Pimenta Leibowitz and Zilberg, 2009), it was regularly passaged through guppies. Infection of Different Fish Species The infection method was based on the protocol previously described by Chettri et al. (2009) for guppies. Tetrahymena, from a 3- to 4-day-old culture, was harvested by centrifugation (300 g for 5 min at 10 C; Heraeus Labofuge 400R, Langenselbold, Germany), the supernatant was discarded and the residing Tetrahymena washed three times in phosphate buffered saline (PBS; pH 6.2, 0.07 M). Tetrahymena was then resuspended in PBS or RM-9 and its concentration was determined by direct counting using a haemocytometer. Thirty fish were used from each of the following species: guppy, platy, angelfish and molly. Fish were injected intraperitoneally (IP) with Tetrahymena in PBS, at a dose of w20,000 Tetrahymena/g of fish body weight (see Table 1), as described by Chettri et al. (2009). In a second experiment, 20 koi carp, 41 goldfish and 36 guppies were similarly infected by IP injection of Tetrahymena in RM-9 medium (Table 1). Fish of each species were placed in a separate 30 litre aquarium equipped with submerged biological filters. Tetrahymena-infected fish were sampled for histological examination at the indicated days post infection (dpi). Fish mortality was monitored daily and dead fish underwent post-mortem examination and were tested for the presence of Tetrahymena in the skin, gills and internal organs. Histological Analysis Histological analyses were performed on tissues from the different infected fish. Fish exhibiting signs of disease (lethargy or showing characteristic white skin lesions) were sampled for histology on the following days: molly at 7 and 13 dpi, angelfish at 7 and 14 dpi, koi carp at 3 and 12 dpi, goldfish at 5 dpi and guppies at 7 dpi. Whole fish were fixed in formalin for 48 h and kept in 70% alcohol until processed. Fixed fish were sliced to 0.5 cm wide slices, decalcified (in formic acid 44% and sodium citrate 12.5% for 12e24 h) and then transferred to 70% alcohol. Processing was conducted in a microwave histoprocessor (RHS-1, Milestone, Italy). Samples were embedded in paraffin wax blocks and sectioned (5 mm). Tissue sections were stained with haematoxylin and eosin (HE). Infection, inflammatory response and other pathologies observed in different organs were documented and photographed (Axioskop microscope and AxioCam MRc5; Carl Zeiss, Oberkochen, West Germany). Anti-Tetrahymena Antibody Production Serological analysis was performed in koi carp, from which serum could be easily drawn (Table 1). Twenty fish averaging 23 g body weight were placed in 100 litre plastic containers equipped with biological filters. The fish were infected with Tetrahymena by IP injection of 460,000 Tetrahymena/fish in RM-9 medium. This same group was used for both histology Table 1 Experimental infection of different ornamental fish species with Tetrahymena Experiment number 1 2 3 Species (number infected) Body weight (g) Age Infection dose (Tetrahymena/fish) Analyses performed Guppy (30) Platy (30) Molly (30) Angelfish (30) Guppy (36) Koi carp (29) Goldfish (41) Guppy (10) Koi carp (20) Goldfish (10) 0.67  0.17 1.02  0.21 2.14  0.33 2.12  0.69 0.50  0.13 0.45  0.14 0.51  0.25 0.58  0.16 23  6.23 0.96  0.21 2.3 months 3 months 3.3 months 3.5 months 2 months 1.7 months 1.5 months 2 months 6 months 1.7 months 10,000 20,000 40,000 40,000 10,000 10,000 10,000 10,000 460,000 17,455 Mortality Mortality and histology Mortality and histology Mortality and histology Mortality Mortality Mortality Histology Histology and serology Histology Koi carp were stocked in 100 litre plastic containers. All other groups were stocked in 30 litre aquaria. 319 Tetrahymena Infection of Ornamental Fish Cumulative mortality (%) a 100 Guppy 80 Molly a Platy a Angelfish 60 40 b b 20 0 0 2 4 6 8 10 12 14 Days post infection b Cumulative mortality (%) and serology (Table 1). Tetrahymena were prepared as described above. Fish were bled 3 weeks prior to the infection and then 3 weeks post infection (total of two bleeds). Bleeding was performed on anaesthetized fish (clove oil, 250 ml/l) and blood was drawn from the caudal vein using a 1 ml syringe with a 24 gauge needle. Tubes were marked and centrifuged at 9,350 g for 5 min at room temperature (Lumitron Eppendorf centrifuge 5424, Hamburg, Germany) and serum was stored at 80 C. At 3 and 12 dpi, fish were sampled for histology as described above. Five additional koi carp were immunized with Tetrahymena in adjuvant according to Chettri et al. (2009). A suspension of Tetrahymena (750,000 in 1 ml PBS) was sonicated twice for 10 sec (Branson Digital Sonifier, Danbury, Connecticut, USA) on ice and emulsified with adjuvant (by 12e15 min vortexing) at a ratio of 2:1 (Tetrahymena lysates:adjuvant). Freshly prepared emulsion was used for IP injection at a volume of 200 ml per fish. Freund’s complete adjuvant (FCA) was used at the initial injection and Freund’s incomplete adjuvant (FIA) in the booster (4 weeks after the initial injection). Blood was drawn 4 weeks after the booster. An immobilization assay was conducted according to Clark et al. (1987) and Chettri et al. (2009), with minor modifications. Cultured Tetrahymena (Tet-NI, invitro passage 6) was washed in RM-9 (300 g at 10 C for 5 min) and the concentration adjusted to 4,000 Tetrahymena/ml. The assay was conducted in 1.5 ml tubes. Fifty microlitres of the koi carp serum tested were added to the tube and two-fold serial dilutions in double distilled water (DDW) were prepared. Two hundred Tetrahymena were added to each tube in 50 ml DDW. Tubes were then incubated for 1 h at 25 C and samples were examined on a glass slide for immobilization using an Axiovert 40 CFL inverted microscope (Zeiss). The percentage of immobilized Tetrahymena was calculated out of the total number of Tetrahymena viewed. Values in Table 2 represent serum dilutions that led to >50% immobilization. 100 a Guppy 80 Koi Goldfish 60 b 40 c 20 0 1 3 2 4 5 6 7 8 9 10 Days post infection Fig. 1. Mortality rate of different ornamental fish species infected with Tetrahymena. The upper and lower panels show results obtained in two separate experiments. Statistical differences between (a) versus (b) versus (c) are significant (P <0.05). Statistical Analysis Statistical analyses were carried out with Sigma Stat 3.1 (Systat Software Inc., Chicago, Illinois, USA). Mortality rates were compared using the Kaplan Meier survival analysis and the log rank test. Differences were considered significant at P <0.05. Table 2 Immobilization of Tetrahymena by sera from koi carp Fish sampled Non-infected Sick Survivors Vaccinated Number of fish tested 8 1 10 5 Number of positive fish 2 1 8 5 Serum dilution resulting in 50% immobilization Range Average 5 10 5e20 80e320 5.00 e 14.37 144.00 Values are expressed as the reciprocal of the dilution at which 50% immobilization occurred; results are presented for those fish that showed immobilizing activity. 320 G. Sharon et al. Results Susceptibility of Different Fish Species to Infection with Tetrahymena Mortality rates of different ornamental fish species infected with Tetrahymena (Tet-NI) were recorded daily and results are presented in Figs. 1a and b. Guppies exhibited the highest rate of mortality in two independent experiments, with mortality of 87% and 100% at 14 and 10 dpi, respectively (Figs. 1a and b, respectively). The mortality rate of platy (77% at 14 dpi) was only slightly lower than that of guppy. In contrast, molly and angelfish were significantly more resistant to Tetrahymena infection, exhibiting a mortality rate of only 23% and 33%, respectively, at 14 dpi. Koi carp and goldfish exhibited mortality rates of 59% and 24%, respectively, at 10 dpi. Postmortem examination of freshly dead fish confirmed that the cause of death in all cases was due to Tetrahymena infection. Comparative Histological Analysis of Tetrahymena-infected Fish Comparative histological analysis was conducted on two to four Tetrahymena-infected fish from each species. Presence of Tetrahymena was observed in the dermis and subdermal fat of all infected species (representative data are shown for goldfish, Fig. 2c). In addition, Tetrahymena was observed in the intramuscular space of guppy, goldfish and koi carp (Figs. 2a and b). Tetrahymena was also observed in the gills of most fish species, except for koi carp (not shown). In molly gills, Tetrahymena was seen blocking a blood vessel, resulting in localized hyperaemia and congestion, evident by vascular distension (not Fig. 2. Histological analysis of muscle and associated skin from fish infected with Tetrahymena. (a) Muscle tissue from a guppy showing Tetrahymena between muscle fibres (arrows) with no evident inflammatory response. (b) Muscle tissue from a koi carp with infiltrating leucocytes (primarily mononuclear cells; white arrows) around the parasite (black arrows). (c) Goldfish muscle and associated skin in which Tetrahymena is seen in the epithelium (long arrow), reaching subdermal fat (F) and the musculature (M). Leucocytes infiltrating the muscle and subdermis are indicated by short arrows. HE. Tetrahymena Infection of Ornamental Fish 321 Fig. 3. Histological analysis of liver from fish infected with Tetrahymena. (a) Molly liver showing Tetrahymena (arrow) adjacent to a blood vessel, with little or no associated tissue damage. (b) Molly liver (L) and pancreatic tissue (P) showing Tetrahymena (arrows) penetrating and surrounding the liver and pancreas, with associated, localized tissue destruction and void formation. (c) Koi carp liver, infected with Tetrahymena (black arrows), showing detached hepatocytes (H). White arrows mark areas rich in infiltrating leucocytes (b). HE. shown). Tetrahymena in the gill blood vessels of guppy and molly contained red blood cells in their cytoplasm (not shown). In general, Tetrahymena were seen in the vicinity and within several internal organs. They often aggregated around blood vessels, such as in the liver of molly (Fig. 3a) and guppy (not shown), suggesting a specific attraction mechanism to these sites. Tetrahymena were also observed in the heart tissue of guppy and koi carp (not shown). In all fish species, except for koi carp, Tetrahymena were evident in the kidney (not shown). In addition, they were found in the liver, pancreatic tissue (Fig. 3) and intestinal mucosa and submucosa (Fig. 4) of all the fish species analyzed. Tetrahymena were not seen within the gut lining epithelium or gut lumen in any of the species examined. In angelfish, a single Tetrahymena was observed inside the swim bladder, with no associated tissue damage (not shown). A single parasite was also identified in a nasal cavity of goldfish (not shown). In all fish species, excluding the koi carp, Tetrahymena were present around and within the gonads (Fig. 5) and infected embryos were present in live bearers (Fig. 5a). Focal cell destruction and void formation around the parasites, presumably following its route of migration in the tissue, were evident (Fig. 3b). In the liver, kidney and pancreatic tissue, large numbers of parasites resulted in compression and/or detachment of the tissue cells (Fig. 3b). Varying levels of leucocytes, including mononuclear and polymorphonuclear cells, were associated with the invading parasite in koi carp and goldfish. Leucocytes in koi carp were associated with tissue breakdown in the muscle, liver and heart (Figs. 2b and 3c). High levels of leucocytes were associated 322 G. Sharon et al. Fig. 4. Histological analysis of intestine from fish infected with Tetrahymena (black arrows). (a) Angelfish intestine showing Tetrahymena in the intestinal mucosa and no associated inflammation. (b) Goldfish intestine showing Tetrahymena in the intestinal mucosa and submucosa and associated infiltrating leucocytes (white arrows). HE. with the parasite in goldfish, as evident in infection of and around the intestine (Fig. 4b), gonads (Fig. 5b), skin and muscle (Fig. 2c). However, inflammatory responses were not evident in guppy, molly or angelfish, as no parasite-associated leucocytes were observed in any of the infected tissues analyzed. Immunization of Koi Carp with Tetrahymena and Serological Analysis Analysis of sera from healthy fish revealed that two out of eight fish exhibited low levels of Tetrahymena immobilization activity (50% immobilization activity at a dilution of 1 in 5, Table 2). Eight out of 10 serum samples from koi that survived Tetrahymena infection exhibited Tetrahymena immobilization activity at an average dilution of 1 in 144 (Table 2). The surviving fish were kept in the laboratory and remained healthy and vital for at least 3 months post infection, suggesting that they had completely recovered from the disease. Serum from a moribund fish that was sampled at 12 dpi also had Tetrahymena immobilizing activity (at a dilution of 1 in 10, Table 2). This fish exhibited classical clinical signs, including white patches on the lateral skin, scale loss and a high level of Tetrahymena on the skin (not shown). Sera from vaccinated koi carp had the highest anti-Tetrahymena immobilizing activity at an average dilution of 1 in 144, about 10-fold higher than the activity observed in surviving fish (Table 2). Fig. 5. Histological analysis of the reproductive system from fish infected with Tetrahymena (black arrows). (a) Tetrahymena around the muscle tissue of a molly embryo. (b) Tetrahymena adjacent to and invading the ovaries of a goldfish with associated infiltrating leucocytes (white arrows). HE. Tetrahymena Infection of Ornamental Fish Discussion All five fish species tested in the present study were found to be susceptible to infection by Tetrahymena. The fish used in the experiment were immunecompetent, based on estimated timing of immune system maturation in each species. Direct comparison between parameters related to development of the immune response in different fish species is difficult, thus information for a range of fish species was considered. According to studies in zebrafish (D. rerio), trout and carp, antibody production, which is indicative of immune response maturation, occurs before the age of 4 weeks (Zapata et al., 2006). The immune system of freshwater species is known to develop much earlier than that of marine fish, which become immunocompetent at 8e10 weeks of age. In carp (C. carpio), immune development starts as early as 16 days post hatch and immunoglobulin (Ig) M-positive cells appear at 2 weeks of age (Botham and Manning, 1981; Magnadottir et al., 2005). Cell-mediated immunity develops earlier than the humoral immune response (Zapata et al., 2006). There is no direct information on immune system development of guppy, platy, molly, angelfish and goldfish, but based on the above literature, it would be safe to assume that by 1 month of age the immune system of each of these species would be active and so at the age selected for analysis (>1 month) the fish should have been capable of mounting an immune response to Tetrahymena. Tetrahymena was reported previously to affect a large range of fish species, although the most significant impact has been reported thus far on guppy (Ferguson et al., 1987; Astrofsky et al., 2002; Pimenta Leibowitz et al., 2005). The present work confirms that guppies are more susceptible to the parasite than the other species tested. Histological analysis of infected fish revealed high levels of infection with Tetrahymena in the skin, underlying muscle and fat and in the internal organs of all species tested. Parasites surrounded and invaded internal organs. Affected organs included the gills, heart, kidney, liver, pancreatic tissue, gut wall, gonads and peritoneal cavity. The parasite was not seen within the gut lining epithelium or in the gut lumen, presumably due to the unsuitable conditions in those compartments. There was no evidence of spinal cord or brain infection. Red blood cells observed in the cytoplasm of the parasites raised the possibility that the parasite feeds on these cells (Shenberg, 2003; Pimenta Leibowitz and Zilberg, 2009). Studies of immunity have shown that the host genetic background determines the quality and intensity of different types of immune responses. Thus, genetic 323 traits determine the immune responses and disease susceptibility of different fish spices (Magnadottir, 2006) and can explain the differences in resistance to Tetrahymena infection between guppy, platy, molly and angelfish, which belong to the super order Acanthopterygii, and the koi carp and goldfish, which belong to the super order, Ostariophysi. Genetic divergence that exists throughout the jawed vertebrates reflects evolutionary processes and generation of diversity. The variability in the genes that determine the antibody and T-cell antigen receptor (TCR) structure and specificity offer new information as to the complexity of the diversified processes that control antigen recognition in different species and receptor specificity and affinity (Litman et al., 1999). Chemokine receptors are involved in the recruitment of various cells in inflammatory and physiological conditions. The genes encoding these receptors continued to evolve during vertebrate evolution, increasing their diversity predominantly in teleosts (Nomiyama et al., 2011). The genome of Medaka (Oryzias latipes), which belongs to the order Beloniformes, is well conserved and is phylogenetically close to the Cyprinodontiformes (the order to which guppy, platy and molly belong). In contrast, the zebrafish, which is closely related to koi and goldfish (family Cyprinidae), is known to have the highest gene duplication rate in vertebrates (Blomme et al., 2006). There are a total of 41 chemokine receptor-encoding genes in zebrafish compared with 31 genes in Medaka (Nomiyama et al., 2011). The common carp, which is considered the same species as koi (C. carpio), is assumed to have had an extra round of genome duplication resulting in enriched immune-related genes, compared with the zebrafish (Wang et al., 2012). Forty five percent of the genes of the common carp encode immune-related proteins, including components of the complement system, coagulation cascade proteins and molecules involved in antigen processing and presentation. Some of these genes may also be involved in pathogen resistance (Wang et al., 2012). Therefore, genetic variation between fish species may explain the differences in response to Tetrahymena and the ability to mount an inflammatory response in Cypriniformes and not in the phylogenetically distant Cyprinodontiformes and Perciformes (order to which angelfish belong) of the super order Acanthopterygii. Previously we have found that body homogenates of immunized guppies immobilized Tetrahymena in vitro, while homogenates of non-immunized fish exhibited almost no immobilization activity (Chettri et al., 2009). In the present study we tested the ability of na€ıve and immune koi carp sera to immobilize Tetrahymena. Immunization resulted in a relatively strong immobilization response, reminiscent of the 324 G. Sharon et al. results obtained in immunized guppies (Chettri et al., 2009). The Tetrahymena immobilization response in guppies following immunization with Tetrahymena and adjuvant was associated with protection from infection (Chettri et al., 2009). The extent of the inflammatory response observed in some fish species correlated with increased resistance to Tetrahymena infection, although the actual effects of the infiltrating cells on the parasites require further investigation. References Astrofsky KM, Schech JM, Sheppard BJ, Obenschain CA, Chin AM et al. (2002) High mortality due to Tetrahymena sp. infection in laboratory maintained zebrafish (Brachydanio rerio). Comparative Medicine, 52, 363e367. Blomme T, Vandepoele K, DeBodt S, Simillion C, Maere S et al. (2006) The gain and loss of genes during 600 million years of vertebrate evolution. Genome Biology, 7, R43. 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