Indian Journal of Experimental Biology Vol.52, March 2014, pp. 267-280

Effect of Dactylogyrus catlaius (Jain 1961) infection in Labeo rohita (Hamilton 1822): Innate immune responses and expression profile of some immune related genes

Pujarini Dash, Banya Kar, Arpita Mishra & P K Sahoo* Fish Health Management Division, Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751 002, India

Received 21 March 2013; revised 12 September 2013

The monogenean ectoparasite, Dactylogyrus sp. is a major pathogen in freshwater aquaculture. The immune responses in parasitized fish were analyzed by quantitation of innate immune factors (natural agglutinin level, haemolysin titre, antiprotease, lysozyme and myeloperoxidase activities) in serum and immune-relevant gene expression in gill and anterior kidney. The antiprotease activity and natural agglutinin level were found to be significantly higher and lysozyme activity was significantly lower in parasitized fish. Most of the genes viz., β2-microglobulin (β2M), major histocompatibility complex I (MHCI), MHCII, tumor necrosis factor α (TNFα) and toll-like receptor 22 (TLR22) in gill samples were significantly down-regulated in the experimental group. In the anterior kidney, the expression of superoxide dismutase and interleukin 1β (IL1β) were significantly up-regulated whereas a significant down regulation of MHCII and TNFα was also observed. The down-regulation of most of the genes viz, MHCI, β2M, MHCII, TLR22 and TNFα in infected gills indicated a well evolved mechanism in this parasite to escape the host immune response. The modulation of innate and adaptive immunity by this parasite can be further explored to understand host susceptibility.

Keywords: Dactylogyrus catlaius, Gene expression, Immune response, Labeo rohita

Aquaculture has emerged as one of the most marketability of the product and thereby cause promising food producing sectors due to its role in significant economic losses. improving food security and meeting the nutritional In recent years monogenean parasites have needs of the world population. Since the initiation of emerged as serious pathogens of commercially intensive aquaculture, infectious diseases have important fishes. Among these parasites, the genus emerged as a limiting factor for further expansion of Dactylogyrus is the largest helminth genus consisting the sector. There is now an increasing economic of more than 900 species, parasitizing many fish importance of fish parasitic diseases in aquaculture species3. Members of this genus are ectoparasites and fisheries. Heavy parasitic infections damage the commonly known as gill flukes generally infecting integument of the fish which may develop into the gill of freshwater cyprinids. Gill flukes have wounds facilitating entry of secondary infectious high host specificity and do not cause pathogenic agents1. Parasites interfere with the nutrition, problem in nature due to their narrow host range4. metabolism and secretary function of alimentary However, under favourable aquaculture conditions canal, damage nervous system and also upset the monogeneans may become pathogenic to the host. normal reproduction of the host2. Moreover, parasitic Heavily infected fish of Cyprinidae show swollen and diseases also affect the general perception of people pale gills, accelerated respiration rate, excessive towards aquaculture. Thus, parasitic diseases play a mucous secretion, loss of appetite and less tolerance crucial role in aquaculture by causing mortality or in low oxygen environment which often leads to high retarding growth rate of fish and decreasing mortality5. In severe cases gill hemorrhages and —————— metaplasia occur which become portals for secondary *Correspondent author bacterial and fungal infections5. Among all Telephone: +91-674-2465446 ectoparasitic infections dactylogyral infections alone Mob: 09437015120 Fax: +91-674-2465407 constitute a major portion of about 37% in three 6 E-mail: [email protected] districts of West Bengal, India . Thus, dactylogyral 268 INDIAN J EXP BIOL, MARCH 2014

infections contribute to fish loss in many ways and Paralichthys olivaceus was observed during the the control of this parasite has now become an infection by another monogenean, Neoheterobothrium urgent need in fish farming. The development of hirame13. In another study, cytokines IL-1β economically viable and environmentally sound (spleen and gills) and TGF-β (gills) were up-regulated methods of controlling these parasites would not in Dicentrarchus labrax infected with Diplectanum be possible without adequate knowledge of fish aequans14. Very little information is available on immunology and a complete understanding of antibody responses and acquired protection in the host host-parasite interaction. during monogenean attacks. Specific antibodies With advancement in fish immunology, researchers against D. aequans were detected in sera of sea now have general agreement as to presence of the bass infected by the monogenean using Western 15 main immune mechanisms in teleosts that have been blot analysis . Antibodies also seem to be involved in described for mammals. Teleosts have elements of the partial immunity observed in Oncorhynchus both innate and adaptive immune system. Studies on mykiss immunized with Discocotyle sagittata 16 identification and molecular characterization of fish extracts . immune genes have facilitated their expression However, in particular not much is known about profiling during different infections. It is very much the immune response of fishes in relation to essential to understand which molecules or which Dactylogyrus infection. Moreover, the successful use pathways get activated during a particular infection of immune prophylactic measures in piscine parasitic that would assist in the development of therapeutic infections without doubt requires the basic studies on strategy against the pathogen. Over the past, immune immune responses of host. In absence of any relevant genes for both adaptive and innate immunity information regarding the immune response of the have been identified in various fish species which Indian major carp, Labeo rohita against Dactylogyrus encode different molecules like cytokines, sp., this study has been conducted to reveal the complements, immunoglobulins, lectins and cell immune response in rohu against D. catlaius with surface receptors7. Molecular identifications have respect to the differential expression of some been the focus of fish immunology. Functional important immune genes. Toll like receptor molecules characterization of various immune relevant genes are pattern recognition receptors that induce with respect to various infections has just gained expression of regulatory cytokines like IL 8 and momentum in the recent past8. However, information TNFα through MyD88 pathway thereby assisting in on immune relevant gene expression during initiation of inflammatory response and induction of monogenean parasitic infections is very scarce. adaptive response. To understand, whether the host Oncorhynchus mykiss parasitized by Gyrodactylus immune response against Dactylogyrus is CTL/Th derjavini showed a clear induction of IL1β expression mediated and what role the inflammatory cytokines in the skin, during the initial phases of primary play, few of the genes viz., MHCI, MHCII and infections whereas the type II IL1β receptor was β-2 microglobulin and some cytokines were expressed later9. In addition, there was up-regulation considered for the study. Role of various non-specific of TNFα, iNOS and COX-2 expression, whereas no immune parameters during infection cannot be clear parasite related changes in transcript levels of ignored as they form the first line of defence. In the TCR-β and MHCIIβ could be observed10. Similarly, present study, some of the indicators of innate in Salmo salar infected by , immunity were also evaluated during Dactylogyrus Mc1-1 (related to IL1β) was up-regulated11. Further catlaius infection. studies also indicated increased expression of immune relevant genes like MHCI, Mx, IFN-γ and CD8a in Materials and Methods susceptible responding Baltic S. salar, and slight —Rohu (L. rohita) juveniles (weighing changes in highly susceptible non-responding 70.62±3.05 g) showing no signs of disease (under Danish salmon12. An upregulation in the expression gross and microscopic examination of skin, gill and levels of several immune-related genes [(matrix kidney tissues of representative samples) and no metalloproteinases)-9 and 13, leukotrine-B4 receptor, previous history of parasitic infections were obtained CD20 receptor, MHC class I and class II b chain, Ig H from the farm of CIFA, Bhubaneswar. Fishes and L chains] in peripheral blood leucocytes of were acclimatized in plastic tanks of 500 L capacity DASH et al.: IMMUNE RESPONSE OF LABEO ROHITA TO DACTYLOGYRUS CATALAIUS INFECTION 269

for 15 days before conducting the experiment. with RNASE AWAY (Molecular Bio Products, USA) They were fed with commercial pellet diet at 1.5% of to avoid contamination. their body weight. About 10% of water was removed Infected gill samples were also collected in 10% daily along with the left-over feed and fecal matter. neutral buffered formalin (Fisher, India) for light The basic physico-chemical water parameters were microscopic (squash preparations) examinations to measured systematically at seven-day intervals to detect the parasite species and parasite load following maintain its optimal level throughout the experiment. standard protocol5. The water temperature in the tanks varied from For further confirmation and early detection of 25 to 28 °C during the experiment. Dactylogyrus infection, genomic DNA was extracted Experimental design and co-habitational from gill tissue collected from three infected challenge—The fish were randomly divided into individuals. Before extraction of DNA, tissue samples six tanks with eight fish in each tank. In another tank (50-100 mg) were rinsed with 1 mL of PBS, cut into rohu juveniles infected with Dactylogyrus sp. as fine pieces with sterile scissors and incubated in (examined through light microscopic study of infected 1 mL of TENS buffer (10 mM Tris Cl, 1 mM EDTA, gill samples), which were collected from an infected 0.5% SDS, 0.1 M NaOH) and 25 µL of proteinase K pond of the host institute, were maintained. One (10 mg mL-1) for 3 h at 50 °C followed by general fin-clipped heavily infected rohu (based on phenol-chloroform extraction. Prior to extraction observation of gill fluke load in gill samples taken all the samples were treated with 4 µL of RNase A from the infected fish stock) was introduced into three (10 mg mL-1) and incubated at 37 °C for 30 min. The of the tanks for co-habitational challenge. The other extracted gDNA was dissolved in TE buffer (10 mM three tanks served as uninfected control. The fish Tris Cl, 1 mM EDTA) and stored at -20 °C until were observed daily for development of infection. subsequent PCR analysis. The concentration of the Within 11 days post-challenge, the fish revealed nucleic acid in the sample was quantified by development of infection as observed from gross and measuring absorbance at 260 nm using NanoDrop microscopic examination of gill mucus of randomly ND-1000 spectrophotometer (Thermo Scientific, collected samples. The heavily infected fish started USA). The purity of the samples was also checked dying in the infected group during second week by measuring the ratio of OD260 nm: OD280 nm. post-challenge. A region comprising partial 28S rDNA was Sampling procedure—The fish from infected and amplified using the forward primer, 5′ control groups (12 numbers from each group) on day ACCCGCTGAATTTAAGCAT 3′ and reverse 15 post-challenge were anaesthetized with MS222 primer, 5′ CTCTTCAGAGTACTTTTCAAC 3′. (Argent Chemical, Redmond, WA, USA) and bled The reaction consisted of 1 µL of genomic DNA, non-lethally through the caudal vein using a plastic 19.85 µL of nuclease free water, 2.5 µL of 2 mL syringe (24 gauge needle). Blood samples of Taq buffer, 0.5 µL of dNTP, 0.5 µL of forward and both groups were allowed to clot at room temperature reverse primers each and 0.15 µL (1.5 units) of for 30 min and then kept at 4 °C for 3 h. The clotted Taq DNA polymerase (Bangalore Genei, Merck samples were centrifuged at 5,000 g for 5 min at Specialities Pvt. Ltd., India). The amplification 4 °C to collect sera, which were stored immediately at reaction was carried out initially at 94 °C for 3 min, -70 °C until further analysis for various innate followed by denaturing temperature of 94 °C for immune parameters. The serum samples from each 45 sec for 32 cycles, annealing at 56 °C for 45 sec and experimental fish were processed separately for extension at 72 °C for 1 min 30 sec and final various immune parameters. extension of 10 min at 72 °C. The PCR product was Gill and anterior kidney tissue samples were visualized in 1% agarose gel with ethidium bromide collected from the infected and control group (three staining. The amplified product was purified from fish each) aseptically after anaesthetizing the fish with agarose gel by using GeneiPureTM Gel Extraction MS222 (Argent Chemical, Redmond, USA) and a Kit (GeneiTM, Merck Specialities Pvt. Ltd., India) portion of it was stored in RNAlater (Sigma, USA) at according to manufacturer’s instructions and –20 °C for extraction of total RNA. Throughout the sequenced commercially. The sequence homology procedure, care was taken to use RNAse free tubes was determined by using NCBI BLAST online and dissection tools. Also the workbench was wiped application. DNA sequences of the closely related 270 INDIAN J EXP BIOL, MARCH 2014

species were downloaded and multiple sequence diluted with PBS to make 1% v/v. Two-fold serial alignments were done using Clustal W programme on dilution (up to 8 dilutions) of the unheated sera was Bioedit version 7.0.9.017. The phylogenetic tree was made in PBS, and then 50 µL of 1% rabbit RBC was constructed using MEGA version 5 using neighbor added to each well of the microtitre plate. The plate joining method18. was incubated at 37 °C for 1 h. Haemolysin titer was Innate immune parameters—Total serum defined as the last dilution of serum showing myeloperoxidase (MPO) activity: Total serum complete lysis of RBC22. myeloperoxidase (MPO) activity was measured Natural agglutinin level: To study the serum according to method of Quade and Roth19 with a natural agglutinin level, the serum samples were heat partial modification. Serum (10 µL) was mixed inactivated at 56 °C for 20 min to inhibit the with 90 µL of Hank’s balanced salt solution without complement activity. The heat inactivated sera were Ca2+ or Mg2+ in transparent U-bottom 96-well diluted two-fold serially in PBS (with Ca2+ and Mg2+) microtitre plates (Axygen, USA). Then, 35 µL of in a 96 well microtitre plate. Then 50 µL of formalin- freshly prepared 20 mM 3,3′-5,5′-tetra methyl killed Aeromonas hydrophila (adjusted to OD 2) was benzidine hydrochloride (Genei, Bangalore, India) added to each well. The plate was incubated overnight and 5 mM H2O2 were added. The colour change at 25 °C. The bacterial agglutination titre was defined reaction was terminated after 2 min by adding 35 µL as the last dilution of serum showing maximal 23 of 4 M H2SO4. The OD was taken at 450 nm in a positive agglutinin . TM microplate reader (iMARK , Microplate reader, Expression analysis of immune related genes—RNA BIO-RAD, India). isolation and cDNA synthesis: Skin and anterior kidney Total serum anti-protease level: Total serum tissues (100 mg each) stored in RNAlater of each fish antiprotease in fish serum was determined according were utilized for extraction of total RNA using TRI to Zuo and Woo20 with minor modification. Serum reagent (Sigma) following the manufacturer’s (10 µL) was mixed with 100 µL of trypsin instructions. The resulting RNA was treated with (bovine pancreas type I, Sigma; 200 µg/mL of PBS). DNase I, RNase-free (Fermentas, USA) followed by One positive control was taken containing only inactivation of DNase I according to the trypsin and PBS but no serum. All tubes were manufacturer’s instructions. The absence of DNA incubated at 25 °C for 30 min. After incubation, 1 mL contamination was further confirmed by PCR using of casein dissolved in PBS (2.5 mg/mL) was added to β-actin primers as described later (Table 1). Total RNA all tubes and incubated for 15 min at 25 °C. (1 µg) was reverse-transcribed for preparation of The reaction was stopped by adding 500 µL of 10% complementary DNA (cDNA). In the first step, RNA tricholoroacetic acid (TCA). The sample was was incubated with random hexamer primer centrifuged at 10, 000 g for 5 min. The OD of the (Fermentas, USA, 100 µM) for 5 min at 70 °C and supernatant was measured at 280 nm and the cooled at 25 °C for 10 min for the annealing of the percentage of trypsin inhibition was calculated. primer and RNA. To this reaction mixture, MMLV-RT Serum lysozyme activity: Turbidometric assay was buffer (Sigma), dNTP, RNAse inhibitor (40 U/µL, carried out to study the serum lysozyme activity Bangalore Genei, Merck Specialities Pvt. Ltd., India), according to method of Sankaran and Gurnani21 with and MMLV-RT enzyme (200 U/µL, Sigma) were partial modification. A suspension of 150 µL of added and incubated for 5 min at 25 °C, 1 h at 42 °C Micrococcus lysodeikticus (0.2 mg/mL) in 0.02 M and 2 min at 95 °C subsequently in a thermal cycler sodium acetate buffer (pH 5.5) was added to 15 µL of (Master Gradient Eppendorf, USA). cDNAs thus serum previously taken in 96 well U-bottom synthesized were stored at 0 °C till further use. microtitre plates (Axygen, USA). Initial OD was read PCR conditions: For semi-quantitative expression at 450 nm immediately after adding the bacteria and analysis of few immune-related genes, β-actin, which final OD was read after 1 h incubation at 24 °C. expresses constitutively, was taken for sample Serum lysozyme values were expressed as µg/mL normalization as well as positive control. All the PCR equivalent to hen egg white lysozyme activity. conditions and number of cycles were optimized prior Serum haemolytic activity: To study the haemolytic to the final expression analysis. Certain immune activity of serum, rabbit blood (2 mL) was collected genes such as tumor necrosis factor α (TNFα), major in Alsever’s solution and separated RBC was further histocompatibility complex I (MHCI), major DASH et al.: IMMUNE RESPONSE OF LABEO ROHITA TO DACTYLOGYRUS CATALAIUS INFECTION 271

histocompatibility complex II (MHCII), β2 30 sec and final extension of 10 min at 72 °C . The microglobulin (β2M), toll-like receptor 22 (TLR22), details of primer sequences used for all genes are interleukin 8 (IL8), interleukin 1β (IL1β), manganese given in Table 1. The PCR product of each amplified superoxide dismutase (MnSOD), natural killer cell gene was visualized in 1% agarose gel with ethidium enhancement factor B (NKEF-B), tumor necrosis bromide staining. factor receptor (TNFR2a) and TNF receptor Expression analysis: The relative level of associated factor (TRAF6a) that hold important place expression was analyzed through densitometric in parasitic infections were amplified using either analysis using Alpha Ease_FC Imaging software self-designed or previously published primer (Alpha Innotech Corp., USA). To assess the sequences. The sequence and optimum annealing differences in expression of different genes between temperature for primers used are given in Table 1. control and gill fluke infected fish, the ratios of Each PCR reaction consisted of 0.5 µL of cDNA, immune related genes to β-actin expression product 20.35 µL of nuclease free water, 2.5 µL of Taq buffer, for each gene of interest was calculated. 0.5 µL of dNTPs, 0.5 µL of forward and reverse Statistical analysis—All experimental serum primers each and 0.15 µL (1.5 units) of Taq DNA parameters were carried out taking each serum sample polymerase (Bangalore Genei, Merck Specialities Pvt. in duplicate and the mean ± SE for each parameter Ltd., India). All amplification reactions were carried was calculated. The mean values were compared by out initially at 94 °C for 3 min, followed by Student’s t-test to determine significant difference at denaturing temperature of 94 °C for 45 sec for 28-32 5% (P ≤ 0.05) level. cycles, annealing temperature of respective target Mean values (±SE) of the target gene expression, genes for 45 sec and extension at 72 °C for 1 min relative to β-actin, were derived from triplicates.

Table 1—Primers used with their optimum annealing temperatures and sizes of PCR amplicons Target gene Primer sequence (5’ to 3’) Optimum annealing Size of PCR Accession temperature (°C) amplic-on (bp) number Natural killer cell F-ACCGAGATCATCGCGTTC 49 285 n.s. 65 enhancing factor-B R-CCGGCAGGTCATTGATG Superoxide dismutase F-ACGAGACCTGTAGTGCCCTGC 57.4 178 n.s., s.d. R-CGGAAGCCATCAAGCGTG Interleukin 1β (IL1β)66 F-ATCTTGGAGAATGTGATCGAAGAG 57.4 561 AM932525 R-GATACGTTTTTGATCCTCAAGTGTGAAG Interleukin8 (IL 8) F-GGGTGTAGATCCACGCTGTC R- 53.5 167 n.s., s.d. AGGGTGCAGTAGGGTCCAG MHCI F-CAGTATGGGTATGATGGA R- 44.2 291 n.s., s.d. TCTGCCAGGAGATTGTT MHCII F-AGGAGATGCCGAATGGAG R- 55.3 200 n.s.,s.d. GATGATTCCCAGCACCAG TRAF 6a F-AGATCCGGGAGCTGTGCATCC 59.4 441 n.s., s.d. R-GCCTCTGGAATGCCTGCAAGTC Tumor necrosis F-CCAGGCTTTCACTTCAGG 51.6 181 FN543477 43 factor-α R-GCCATAGGAATCGGAGTAG Toll like receptor 228 F-TCACCCCATTTCGAGGCTAACAT 56.0 520 FN548000 R-GAAGGCGTCGTACTGGAATGTC β2-microglobulin43 F-TCCAGTCCCAAGATTCAGGTG 59.7 175 AM774150 R-TGGTGAGGTGAAACTGCCAG TNFR 2a F-TGGAGGAACATAGTGAAGCC 55.3 227 n.s., s.d. R-AGTTTGAGAACCATCAGACCC β-actin43 F-GACTTCGAGCAGGAGATGG 55.3 138 n.s. R-CAAGAAGGATGGCTGGAACA s.d=self-designed; n.s.=not submitted 272 INDIAN J EXP BIOL, MARCH 2014

Statistical differences in gene expression between gill partial 28S rDNA generated in this study was 366 bp fluke infected and uninfected control samples were size (GenBank Accession no. KC687091). The assessed using Mann-Whitney U test (P-value of phylogenetic analysis by neighbor joining method less than or equal to 0.05 was considered statistically revealed this species to be most closely related to significant). members of the genus Dactylogyrus, while the reference sequence of genus Dactylogyroides formed Results a different clade (Fig. 2). All the healthy fish exposed to infected fish during In the present study, total serum antiprotease was co-habitation got infected within 4-5 days of found to be significantly higher in gill fluke infested challenge (as observed from gill samples collected fish in comparison to control (Fig. 3a). Parasite from experimental group and their microscopic infected fish showed a higher agglutination titre examination) showing the first pathological signs of (Fig. 3b) and a lower natural haemolysin titre was swollen and pale gills. The number of parasites observed in the parasitized fish (Fig. 3c). No significant attached and feeding on each individual fish varied difference was found in serum myeloperoxidase during experimental period. However, in general level between control and fluke infected group the level of infection was too high (Fig.1) after (Fig. 3d). The lysozyme activity of serum was found co-habitational challenge on the day of sample to be significantly lower in parasitized fish collection. Due to heavy infection, hemorrhages in comparison to control (Fig. 3e). The results appeared after infection on gills and mortality of the immune parameters studied are illustrated in was also started in the experimental group during Fig. 3. the study period. The infection was regarded as In the present study, the expression of some acute with high degree of severity. At this stage, the immune relevant genes was investigated in the gill samples from infected fish were collected. The and anterior kidney tissues of D. catlaius infected fish control fish did not show any infection either in gill or relative to the control group. Densitometric kidney squash examination. quantification analysis of in-vivo expression of the The parasite was identified as Dactylogyrus immune-related genes is shown in Figs 4 (gill) and catlaius through morphological characterization. The 5 (anterior kidney). Most of the genes were found to be significantly down-regulated (P≤0.05) in infected fish as compared to control fish in gill samples except antioxidant genes viz., NKEF-B and MnSOD, and receptors like tumor necrosis factor receptor 2a (TNFR2a) and TNF receptor associated factor 6a (TRAF6a) (Fig. 4a, b, c and d). The expression of MHCI, MHCII, TNFα, TLR 22 along with β2M was significantly down-regulated (P≤0.05) in the infected fish gills relative to control fish gills (Fig. 4e, f, g, h and j). There was no significant change in the expression of IL8 in the infected fish relative to the control ( Fig. 4 i) while expression of IL1β was not detected in gill samples (Fig. 4). On the other hand, the expression of MnSOD and IL1β were significantly up-regulated (P≤0.05) in infected fish anterior kidney samples as compared to control anterior kidney tissue (Fig. 5 b and k). At the same time significant down regulation (P≤0.05) of MHCII and TNFα was observed in infected anterior

kidney samples (Fig. 5 f and g). There was no significant Fig. 1—Light microscopy (magnification 10X) of gill lamellae of L. rohita showing the parasite D. catlaius (arrow) change in expression level for other genes studied in attachment. anterior kidney samples of infected fish (Fig. 5). DASH et al.: IMMUNE RESPONSE OF LABEO ROHITA TO DACTYLOGYRUS CATALAIUS INFECTION 273

Fig. 2—A phylogenetic tree constructed by neighbor-joining method (1000 bootstraps) for the 28S region of different species of Dactylogyrus including the isolate used (D. catlaius) in the present study.

Discussion This study was carried out with the objective of examining the effect of Dactylogyrus infection on the immune system of L. rohita, which is still unexplored. Any type of pathogen attack triggers the innate immunity of the host which bears the responsibility of activating the whole immune system by producing an array of immune molecules to combat the infection. In the present study an attempt was made to analyze the modulation of the immune system of L. rohita, with reference to innate immune responses and differential expression of few immune-related genes during experimental challenge with D. catlaius as a reflection of changes in both innate and specific immunity. Various indicators of disease and stress response that are used for general immunological defense mechanism are cells of immune system (leucocytes, non-specific cytotoxic cells, eosinophilic granular cells, macrophages and other cells) and their products [myeloperoxidases (MPO), superoxides, acute-phase proteins, lysozyme, interferon, complement, Fig. 3—Effect of Dactylogyrus catlaius infection on various non- properdin, lysins and agglutinins, etc.]24-26. The specific immune parameters (a-antiprotease activity, b-natural agglutination titre, c-haemolysin titre, d-myeloperoxidase activity superoxide production was estimated by and e-lysozyme activity) in Labeo rohita. Data are presented as myeloperoxidase activity which is produced by mean values (±SE). Significant difference between control and neutrophils and acts against pathogens by forming infected group is indicated by *, (P ≤ 0.05). 274 INDIAN J EXP BIOL, MARCH 2014

reactive oxygen molecules27. It acts upon hydrogen immune system to react positively. C type lectin peroxide during respiratory burst and produce receptors, which are considered as the sensitive hypochlorous acid which is fatal to foreign invaders28. receptors of innate immune system are found to act as In the present study, no significant difference was binding ligands for some glycan moieties of parasites. found in the myeloperoxidase activity between Carbohydrates expressed on the surface of parasitic control and gill fluke infested fish. The result falls in helminthes are reported to be potential targets for parallel with the study on Sparus aurata where no these lectin molecules38. An increase in lectin acitivity difference in myeloperoxidase level was observed in D. catlaius infected fish serum suggests that this during Enteromyxum leei (Myxozoa) infection parasite might possess some carbohydrate receptors between parasitized and non-parasitized fish29. Also a study on L. rohita infected with Argulus showed no difference in myeloperoxidase content between uninfested control and parasite infested fish30. These observations suggested that myeloperoxidase activity may not be playing major role in the control of parasites especially ectoparasites of fish; even they do cause severe tissue damage. Host antiproteases are considered as an important branch of host defense system as proteases are known to play major role in pathogenesis of parasitic diseases. In the present study, the total antiprotease activity was found to be significantly higher in gill fluke infested fish in comparison to control that resulted as a reflex to counter parasitic release of proteases. In other parasitic infections like E. scophthalmi exposed turbot31 and E. leei exposed Diplodus puntazzo, total antiproteases levels were reported to be generally higher than control levels32. Similarly, serum antiprotease level in L. rohita infested with an ectoparasite A. siamensis also showed a significant increase30. This higher level of antiproteases in serum can be attributed to the fact that most of the antiproteases like α2 macroglobulin are responsible for clearance of the active proteases from body fluids which might be secretions of the parasites. Lectins are important innate immune molecules which agglutinate foreign cells by adhering to their surface carbohydrate and promote phagocytosis by leucocytes. Lectins are well studied in mammals and vertebrates33,34. In fish, a number of lectins have been discovered35 and most of the experiments have been Fig. 4—Expression of immune relevant genes relative to β-actin carried out on their agglutination activity and gene in gill samples of Dactylogyus catlaius infected and control carbohydrate specificity36. These molecules not only rohu. Bars represent mean values (±SE) of three samples. Statistically important down-regulation (P≤0.05) are denoted by * initiate the immune response but also get involved in mark. (a-NKEF-B, natural killer-cell enhancing factor B; downstream effector functions like complement b-MnSOD, manganese superoxide dismutase; c-TNFR2a, tumor activation and initiation of adaptive immune necrosis factor receptor 2a; d-TRAF6a, TNF receptor associated response37. In the present study, a significantly higher factor 6a; e and f-MHC-I and II, major histocompatibility complex I and II; g-TNFα, tumor necrosis factor α; h-TLR22, level of serum agglutinating activity was found in toll-like receptor 22; i-IL-8, interleukin 8; j-β2-M, parasitized fish signifying the stimulation of the host β2- microglobulin). DASH et al.: IMMUNE RESPONSE OF LABEO ROHITA TO DACTYLOGYRUS CATALAIUS INFECTION 275

which are being recognized by the lectin molecules boosting the host immune system to neutralize the infection. The increased lectin activity may be a result of the host – parasite recognition as well as the host response to the parasite as lectins are reported to play dual role in both the cases39. Lysozyme is an active molecule of fish defense system produced by neutrophils/phagocytes which restricts pathogen attack by disrupting the glycosidic bond of the peptidoglycan layer. In the present study, the lysozyme level showed a decrease in parasite infested fish. Reduction in levels of lysozyme was also earlier observed in gilthead sea bream parasitized by Polysporoplasma spari40 and in D. puntazzo infected by E. leei32. Lysozyme activity is dependent on level of stress and it is well recorded that serum as well plasma lysozyme activity is significantly depleted in stressed fish41 and that may be a reason for reduced lysozyme levels in infected fish. Complement is one of the main mechanisms to ward off any infection as it is involved in initiation of innate immune response as well as mounting of an adaptive response. The haemolytic activity of fish serum is attributed to the alternate complement pathway. Monogenean parasites like Gyrodactylus derjavini and G. salaris can be killed by alternate complement pathway32. However, in the present study a lower haemolysin titer was observed in parasitized fish, suggesting their increase in susceptibility to this infection as found here. In dactylogyral infections, considerable hemorrhaging with bleeding in gill lamellae occurs and it is suggested that the bleeding is promoted by secretions of the parasite42. The decrease in the heamolysin titre may be attributed to this loss of blood and tissue damage. During tissue injury or exposure to pathogens there are changes in the levels of many plasma proteins. In the present study the levels of antiprotease and lectin, both of which are acute phase proteins (APP) significantly increased during infection. This increase Fig. 5—Expression of immune relevant genes relative to of APPs during inflammation probably aids the host β-actin gene in anterior kidney tissues of Dactylogyus catlaius in regaining homeostasis43. infected and control rohu. Bars represent mean values (±SE) of The expression study of immune-related genes at three samples. Statistically important up-regulation /down- regulation (P≤0.05) are denoted by * marks. ( a-NKEF-B, transcription level gives a real picture of the immune natural killer-cell enhancing factor B; b-MnSOD, manganese status of the organism and it helps in analyzing superoxide dismutase; c-TNFR2a, tumor necrosis factor receptor differential expression of target genes up to a very 2a; d-TRAF6a, TNF receptor associated factor 6a; e and f- convenient level. Due to scarcity of genomic MHC-I and II, major histocompatibility complex I and II; g-TNFα, tumor necrosis factor α; TLR22, h-toll-like receptor 22; information on Indian carps, expression studies of i-IL-8, interleukin 8; j- β2-M, β2- microglobulin and k- IL-1β, immune relevant genes have been initiated interleukin 1β ) recently8,43,44. Two organs, gill, the primary site of 276 INDIAN J EXP BIOL, MARCH 2014

attachment of gill fluke and anterior kidney, the major infection is not explored. The expression of TLR 22 immunocompetent organ in fish were taken in the was found to be significantly down-regulated in gills present study for the transcriptional analysis of of parasitized fish but did not differ significantly in various immune related genes to study the changes anterior kidney. There is a possibility of migration of during D. catlaius infection. The gill assumes greater receptor bearing cells from the site of infection after a importance as an organ of study in case of gill fluke certain period post-infection which could be the infections, as it is the organ first damaged by reason for the down regulation of TLR 22 in gill. The attachment of the fluke and later shows extensive up-regulation of TLR 22 has been reported in skin of tissue damage due to feeding. Hyperplasia of the A. siamensis infested rohu8. However, changes in the epithelial cells and subsequent lamellar fusion, goblet expression pattern of a single TLR may not state the cell proliferation as well as the migration of status of the whole TLR pathway. The cause and the eosinophilic granular cells (EGCs) to gills of fishes mechanism of down-regulation of TLR 22 need to be infected with these parasites have been recorded45. explored further. TNFα is an inflammation associated gene, which is IL1β is a pro-inflammatory cytokine that stimulates produced by macrophages, monocytes, natural killer T and B cells production, helps in phagocytosis and cells and T cells46. It stimulates the migration of induces expression of several genes like COX2, MHC dendritic cells into lymph node and thereby and IL1β itself52. In the skin of Oncorynchus mykiss encourages its interaction with CD4+ T cells. TNFα parasitized by Gyrodactylus derjavini, a clear induces adhesion molecules on endothelial cells and induction of IL1β expression was observed during the helps in bringing the leucocytes closer. In the present initial phases of primary infection4. It was also experiment, a down regulation was observed in the reported to be up-regulated in spleen and gills of expression of TNFα and its receptors like TNFR2a Diplectanum aequans-infected Dicentrarchus and TRAF6a in both anterior kidney and gill of labrax53. In the present study, IL1β expression was parasitized fish. Expression level of TNFα was found to be up-regulated in anterior kidney tissue of found to be down regulated in the head kidney of parasite infected fish, whereas it was not detected in E. leei infected gilthead sea bream at different time gill. The very purpose of this cytokine to remain points post-exposure47. There is a little information unchanged at the site of infection suggests that host available on down regulation of cytokine expression inflammatory response is silent or slow to render during fish-parasite interaction but is well reported in protection to this infection, particularly through some other models48. Several mechanisms could be induction of specific immunity. The present work also suggested for this down regulation of cytokines showed no significant difference in IL8 expression during parasite infection. At some level of infection, level in both anterior kidney and gill tissues of the host might adopt a mechanism to reduce the parasitized and non-parasitized fish. Hence, the down- inflammation in order to prevent unnecessary tissue regulation or no major change of the cytokines injury. A decreased level of TNFα was also observed (TNFα, IL8 and IL1β) in infected fish clearly revealed in the intestine of Sparus aurata upon infection with that the parasite suppresses the action of few E. leei49. inflammatory cells (particularly phagocytes or Toll-like receptors (TLRs) are the best leucocytes) at the site of attachment to establish its characterized class of pathogen recognizing receptors pathogenicity. (PRRs) which are capable of inducing production of Major histocompatibility complex (MHC) plays a cytokines, reactive nitrogen and oxidative radicals and key role in the recognition of self and non-self, differentiation of cells of immune system50. They also thereby regulating T cell activation. Properly trigger maturation of dendritic cells and inflammatory processed antigens are expressed in the membrane gene expression. Some TLR molecules like TLR 9 bound to either class I or class II MHC proteins and and TLR 21 have shown to play a role during then recognized by lymphocytes through the cell- parasitic infection by changing their expression surface T-lymphocyte receptors (TCR). However, pattern at different levels of infection51. TLR 22 is contact with the CD8 (in cytotoxic T lymphocytes) or unique to teleost and is known to be modulated by CD4 (in helper T lymphocytes) co-receptors are ligands such as peptidoglycans and poly I: C50. necessary for triggering the adaptive immune However, the role of this molecule in parasitic response leading to pathogen elimination54. In the DASH et al.: IMMUNE RESPONSE OF LABEO ROHITA TO DACTYLOGYRUS CATALAIUS INFECTION 277

present study, a clear down-regulation of both MHCI attachment in response to the parasites and their and MHCII was seen in gill tissues of infected fish. subsequent elimination by antioxidant molecules like Also in the anterior kidney, MHCII was significantly superoxide dismutase. down-regulated. This suggested that the parasite It is evident from the present study that gill fluke probably inhibits the development of adaptive infection modulates the innate as well as adaptive immunity through some mechanism which needs to immune system of rohu in different aspects, resulting be further explored. In fact, a down-regulation of in up-regulation of few antioxidants and pro- MHCII was reported in Atlantic salmon in response to inflammatory cytokines on one hand in tissue located Neoparamoeba perurans which explained high far away from the site of infection (i.e. anterior susceptibility of Atlantic salmon to amoebic gill kidney) and down-regulation of few of the genes like disease (AGD)55. In addition to MHCI and MHCII, TLR 22 and other genes associated with adaptive β2M was also significantly down-regulated in gill of immunity on the other hand in both gills and anterior infected fish suggesting the inability of infected fishes kidney. The down-regulation of genes associated with to mount a specific response against gill fluke. These antigen presentation i.e. MHCI and β2M along with might be one of the probable reasons of making this MHCII and TLR22 in infected gills clearly indicated fish more susceptible to this parasite infection that this parasite evolves strong mechanism of repeatedly in a culture environment. escaping out of host defense machinery to establish Natural killer cell enhancing factor (NKEF) them. This may be the possible reason for no/poor belongs to peroxiredoxin (Prx) family. Natural killer- production of antibody in the host in a natural like cells in fish are involved in both kinds of immune environment to protect the fish from subsequent responses, acting as important effectors of innate infections that needs to be confirmed. Similarly, the immune response and also priming the adaptive parasite could also down-regulate one of the most 56,57 important cytokine i.e. TNFα in both anterior kidney response against pathogens . NKEF is also known and gill tissues, thus indicating the insignificant role to act as an antioxidant preventing DNA and protein of inflammatory cells in rendering protection. As a from being damaged by oxidative stress, whole it indicated a weak immunity of the host to the chemotherapy agents and inflammation induced parasite infection and this may be one of the major monocyte adhesion58. This factor enhances the causes how the parasite was able to progress very fast cytotoxic effect of natural killer cells and strengthens to produce acute infection and heavy tolls of mortality the immune response. In the present study there was in carp culture. The present study also showed that the no major change in NKEF in both gill and anterior host innate immunity, which is considered as the first kidney tissues of infected fish in comparison to the line of defense is not that much effective to combat control. This indicates that this parasite infection does this parasite. No such protective mechanisms of not influence the host defense to stimulate NK-like innate immunity were observed, by the measurement cell activity to get rid of this infection. of certain innate immune parameters during gill fluke Manganese superoxide dismutase is another infection which might be due to the strong antioxidant enzyme which comes to use when a cell is pathogenicity of this parasite. As a result of which, under oxidative stress. Mn-SODs are involved in the Dactylogyrus succeeds in infecting fishes so severely innate immune responses of animals and its worldwide leading to high mortality. transcription is modulated in response to challenges In conclusion, the infection of D. catlaius induces 59,60 61,62 63,64 with endotoxins , bacteria or viruses . or modulates the expression of several inflammatory However, role of Mn-SOD in defense against and other immune relevant genes in different tissues parasitic infestations is not explored much yet. In the of rohu. The data available for monogenean present study a significant up-regulation of parasitoses is scarce. Most of the work in this group transcription was noticed in anterior kidney tissues of of parasites is related to Gyrodactylus sp., infected fish. Also its transcription was up-regulated Diplectanum aequans, Neoheterobothrium hirame though not significantly in gill tissues of experimental etc., and gill flukes being ignored though they are group in comparison to control. The up-regulation of capable of causing heavy economic losses. This Mn-SOD in anterior kidney and gill tissues of appears to be the first report that looks into the infected rohu suggests the production of ROS by the changes in immune system of a fish in case of host to protect its own damaged tissue at the site of D. catlaius infection. 278 INDIAN J EXP BIOL, MARCH 2014

Acknowledgement leucocytes during monogenean parasite Neoheterobothrium Authors are thankful to Dr P. Jayasankar, Director, hirame infection, Dis Aquat Org, 75 (2007) 79. 14 Faliex E, Da Silva C, Simon G & Sasal P, Dynamic CIFA, Bhubaneswar, India for facilities and to expression of immune response genes in the sea bass, Prof. U. Shameem, Andhra University, Visakhapatnam, Dicentrarchus labrax, experimentally infected with the India for help in identification of the species of monogenean Diplectanum aequans, Fish Shellfish Immunol, parasite. 24 ( 2008) 759. 15 Monni G & Cognetti-Varriale A M, Antigenic activity of Diplectanum aequans () in sea bass References (Dicentrarchus labrax L.) held under different oxygenation 1 Hoffman G L, Lesions due to internal helminths of conditions, Bull Eur Assoc Fish Pathol, 21 ( 2001) 241. freshwater fishes, in The pathology of fishes, edited by W E 16 Rubio-Godoy M, Sigh J, Buchmann K & Tinsley R C, Ribelin and G Higaki (The University of Wisconsin Press, Immunization of rainbow trout Oncorhynchus mykiss against Madison, Wisconsin, USA) 1967, 151. Discocotyle sagittata (Monogenea), Dis Aquat Org, 2 Rahman M R, Prvez M, Jahan M S & Sarker M M, 55 (2003) 23. Histopathology of Beffamya bengalensis (Lamarck) by larval 17 Hall T A, BioEdit: A user friendly biological sequence helminth, Univ J Zool Rajshahi Univ , 17 (1998) 19. alignment editor and analysis program for Windows 3 Dove D M A & Ernst I, Concurrent invades-four exotic 95/98/NT, Nucleic Acids Symp Ser, 1999 (41) 95. species of monogenea now established on exotic freshwater 18 Tamura K, Peterson D, Peterson N, Stecher G, Nei M & fishes in Australia, Int J Parasitol, 28 (1998) 1755. Kumar S, MEGA5: Molecular evolutionary genetics analysis 4 Neary E T, Develi N, Özgül G, Occurrence of Dactylogyrus using likelihood, distance and parsimony methods, Mol Biol species (Platyhelminths, Monogenean) on Cyprinids in Evol, 28 ( 2011) 2731. Almus Dam Lake, Turkey, Turk J Fish Aquat Sci, 19 Quade M J & Roth J A, A rapid, direct assay to measure 12 (2012) 15. degranulation of bovine neutrophil primary granules, 5 Shameem U, Monogenoidean parasites of fish: Vet Immunol Immunopathol, 58 (1997) 239. Dactylogyrosis and Gyrodactylosis infection in fish culture 20 Zuo X & Woo P T K, Natural anti-proteases in rainbow ponds, in Hands-on training programme on techniques for trout, Oncorhynchus mykiss and brook charr, Salvelinus identification, pathology and control of fish parasites fontinalis and the in vitro neutralization of fish α2- (Department of Zoology, Andhra University, macroglobulin by the metalloprotease from the pathogenic Vishakhapatnam, India) 2013, 63. haemoflagellate, Cryptobia salmositica, Parasitology, 6 Chanda M, Paul M, Maity J, Dash G, Gupta S S & Patra B C, 114 (1997) 375. Ornamental fish goldfish, Carassius auratus and related 21 Sankaran K & Gurnani S, On the variation in the catalytic parasites in three districts of West Bengal, India, Chron activity of lysozyme in fishes, Indian J Biochem Biophy, Young Sci, 2 (2011) 51. 9 (1972) 162. 7 Zhu L, Nie L, Zhu G, Xiang L, Shao J, Advances in research 22 Kumari J & Sahoo P K, Effects of cyclophosphamide on the of fish immune-relevant genes: A comparative overview of immune system and disease resistance of Asian catfish, innate and adaptive immunity in teleosts, Dev Comp Clarias batrachus, Fish Shellfish Immunol, 19 (2005) 307. Immunol, 39 (2013) 39. 23 Sahoo P K & Mukherjee S C, Effect of dietary β-1,3 glucan 8 Saurabh S, Mohanty B R & Sahoo P K, Expression of on immune responses and disease resistance of healthy and immune–related genes in rohu Labeo rohita (Hamilton) by aflatoxin B1 induced immunocompromised rohu (Labeo experimental freshwater lice Argulus siamensis (Wilson) rohita Hamilton), Fish Shellfish Immunol, 11 (2001) 683. infection, Vet Parasitol, 175 (2011) 119. 24 Anderson D P & Siwicki A K, Basic haematology and 9 Lindenstrom T, Buchmann K & Secombes C J, Gyrodactylus serology for fish health programs, in Diseases in Asian derjavini infection elicits IL-1b expression in rainbow trout aquaculture II, edited by M Shariff, J R Arthur & R P skin, Fish Shellfish Immunol, 15 (2003) 107. Subasinghe (Fish Health Section, Asian Fisheries Society, 10 Lindenstrom T, Secombes C J & Buchmann K, Expression Manila) 1995, 185. of immune response genes in rainbow trout skin induced by 25 Marsden M J, Freeman L C, Cox D & Secombes C J, Non- Gyrodactylus derjavini infections, Vet Immunol specific immune responses in families of Atlantic salmon, Immunopathol, 97 (2004) 137. Salmo salar, exhibiting differential resistance to 11 Matejusova I, Felix B, Sorsa-Leslie T, Gilbey J, Noble L R, furunculosis, Aquaculture, 146 (1996) 1. Jones C S & Cunningham C O, Gene expression profiles of 26 Sahoo P K & Mukherjee S C, The effect of dietary some immune relevant genes from skin of susceptible and immunomodulation upon Edwardsiella tarda vaccination in responding Atlantic salmon (Salmo salar L.) infected with healthy and immunocompromised Indian major carp (Labeo Gyrodactylus salaris (Monogenea) revealed by suppressive rohita), Fish Shellfish Immunol, 12 (2002) 1. subtractive hybridization, Int J Parasitol, 36 (2006) 1175. 27 Ellis A E, Immunity to bacteria in fish, Fish Shellfish 12 Kania P W, Larsen T B, Ingerslev H C & Buchmann K, Immunol, 9 (1999) 291. Baltic salmon activates immune relevant genes in fin tissue 28 Dalmo R A, Ingebrightsen K & Bogwald J, Nonspecific when responding to Gyrodactylus salaris infection, defense mechanisms in fish with particular reference to the Dis Aquat Org, 76 ( 2007) 81. reticuloendothelial system (RES), J Fish Dis, 20 (1997) 214. 13 Matsuyama T, Fujiwara A, Nakayasu C, Kamaishi T, Oseko 29 Cuesta A, Munoz P, Rodriguez A, Salinas I, Sitja-Bobadilla N, Tsutsumi N, Hirono I & Aoki T, Microarray analyses of A, Alvarez-Pellitero P, Esteban M A & Meseguer J, Gilthead gene expression in Japanese flounder Paralichthys olivaceus seabream (Sparus aurata L.) innate defence against the DASH et al.: IMMUNE RESPONSE OF LABEO ROHITA TO DACTYLOGYRUS CATALAIUS INFECTION 279

parasite Enteromyxum leei (Myxozoa), Parasitology, 132 47 Cuesta A, Munoz P, Rodriguez A, Salinas I, Sitja-Bobadilla (2006) 95. A, Alvarez-pellitero P, Estaben M A & Mesequer J, Gilthead 30 Saurabh S & Sahoo P K, Non-specific immune responses of sea bream (Sparus aurata L.) innate defence against the Indian major carp Labeo rohita Hamilton to infestation by parasite Enteromyxum leei (Myxozoa), Parasitology, the freshwater louse Argulus siamensis (Wilson), Indian 132 (2006) 1. J Fish, 57 ( 2010) 45. 48 Evering T &Weiss L M, The immunology of parasite 31 Sitja-Bobadilla A, Redondo M J, Bermudez R, Palenzuela O, infections in immunocompromised hosts, Parasite Immunol, Ferreiro I, Riaza A, Quiroga I, Nieto J M & Alvarez-Pellitero 28 (2006) 549. P, Innate and adaptive immune responses of turbot, 49 Sitja-Bobadilla A, Caiduch-Giner J, Saera-Vila A, Scophthalmus maximus (L.), following experimental Palenzuela O, Alvarez-Pellitero P & Perez-Sanchez J, infection with Enteromyxum scophthalmi (: Chronic exposure to the parasite Enteromyxum leei Myxozoa), Fish Shellfish Immunol, 21 ( 2006) 485. (Myxozoa: Myxosporea) modulates the immune response 32 Alvarez-Pellitero P, Fish immunity and parasite infections: and the expression of growth, redox and immune relevant from innate immunity to immunoprophylactic prospects, genes in gilthead sea bream, Sparus aurata L., Fish Shellfish Vet Immunol Immunopathol, 126 (2008) 171. Immunol, 24 (2008) 610. 33 Epstein J, Eichbaum Q, Sheriff S & Ezekowitz-R A B, 50 Aoki T & Hirono I, Immune relevant genes of Japanese The collectins in innate immunity, Curr Biol, 8 (1996) 29. flounder Paralichthys olivaceus, Comp Biochem Physiol, 34 Sumyia M & Summerfield J A, The role of collectins in host Part D 1 (2006) 115. defence, Semin Liver Dis, 17 (1997) 311. 51 Li Y, Luo X, Dan X, Qiao W, Huang X & Li A, Molecular 35 Yano T, The non-specific immune system, in The fish cloning of orange-spotted grouper (Epinephelus coioides) immune system: Organism, pathogen and environment, TLR21 and expression analysis post irritans edited by G Iwama, T Nakanishi (Academic Press, San infection, Fish Shellfish Immunol, 32 (2012) 476. Diego, USA) 1996, 105. 52 Li Y, Luo X, Dan X, Huang X, Qiao W, Zhong Z & Li A, 36 Ewart K V, Johnson S C & Ross N W, Lectins of the innate Orange-spotted grouper (Epinephelus coipides) TLR2, immune system and their relevance to fish health, ICES J MyD88 and IL-1β involved in anti-Cryptocaryon irritans Mar Sci, 58 (2001) 380. response, Fish Shellfish Immunol, 30 (2011) 1230. 37 Vasta G R, Nita-Lazar M, Giomarelli B, Ahmed H, Du S, Cammarata M, Parrinello N, Bianchet M A & Amzel L M, 53 Faliex E, Da Silva C, Simon G & Sasal P, Dynamic Structural and functional diversity of the lectin repertoire in expression of immune response genes in the sea bass, teleost fish: Relevance to innate and adaptive immunity, Dicentrarchus labrax experimentally infected with the Dev Comp Immunol, 35 (2011) 1388. monogenean Diplectanum aequans, Fish Shellfish Immunol, 24 ( 2008) 759. 38 Vázquez-Mendoza A, Carrero J C & Rodriguez-Sosa M, Parasitic Infections: A Role for C-Type Lectins Receptors, 54 Klein J & Figueroa F, Evolution of the major BioMed Res Int, 2013 (2013) (in press). histocompatibility complex, Crit Rev Immunol, 6 (1986) 295. 39 Buchmann K, Lectins in fish skin: do they play a role in 55 Young N D, Crosbie-P B B, Adams M B, Nowak B F & host-monogenean interactions, J Helminthol, 75 (2001) 227. Morrison R N, Neoparamoeba perurans n. sp., an agent of 40 Karagouni E, Athanassopoulou F, Tsagozis P, Ralli E, of Atlantic salmon (Salmo salar), Moustakareas T, Lytra K & Dostika E, The impact of a Int J Parasitol, 37 (2007) 1469. successful anti-myxosporean treatment on the phagocyte 56 Kuznetsov A V, Clark J F, Winkler K & Kunz W S, Increase functions of juvenile and adult Sparus aurata L., Int of flux control of cytochrome C oxidase in copper-deficient J Immunopathol Pharmacol, 18 (2005) 121. mottled brindled mice, J Bio Chem, 271 (1996) 283. 41 Saurabh S & Sahoo P K, Lysozyme: an important defense 57 Li R W & Waldbieser G C, Genomic organisation and molecule of fish innate immune system, Aqua Res, 39 (2008) expression of the natural killer cell enhancing factor (NKEF) 223. gene in channel catfish Ictalurus punctatus (Rafinesque), 42 Schaperclaus W, Helminthiases, in Fish Diseases Volume II, Fish Shellfish Immunol, 20 (2006) 72. edited by W Schaperclaus, H Kulow, K Schreckenbach) 58 Chen Y M, Su Y L, Lin J H, Yang H L & Chen T Y, Cloning 1992, 750. of an orange-spotted grouper (Epinephelus coioides) Mx 43 Mohanty B R & Sahoo P K, Immune responses and DNA and characterization of its expression in response to expression profile of some immune-related genes in Indian nodavirus, Fish Shellfish Immunol, 20 (2006) 58. major carp Labeo rohita to Edwardsiella tarda infection, 59 Abe R, Shimosegawa T, Moriizumi S, Kikuchi Y, Kimura K, Fish Shellfish Immunol, 28 (2010) 613. Satoh A, Koizumi M & Toyota T, Lipopolysaccharide 44 Mishra J, Sahoo P K, Mohanty B R & Das A, Sequence induces manganese superoxide dismutase in the rat pancreas: information, ontogeny and tissue-specific expression of its role in caerulein pancreatitis, Biochem Biophys Res complement C3 in Indian major carp, Labeo rohita Commun, 217 (1995) 1216. (Hamilton), Indian J Exp Biol, 47 ( 2009) 672. 60 Li Q, Kumar A, Gui J F & Yu S X, Staphylococcus aureus 45 Raissy M & Ansari M, Histopathological changes in the gills lipoproteins trigger human corneal epithelial innate response of naturally infected Capoeta aculeata (Cuvier and through toll-like receptor-2, Microb Pathog, 44 (2008) 426. Valenciennes, 1884) with parasites, Afr J Biotechnol, 61 Bao Y, Li L & Zhang G, The manganese superoxide 68 (2011) 15422. dismutase gene in bay scallop Argopecten irradians: cloning, 46 Whyte S K, The innate immune response of finfish, Fish 3D modeling and mRNA expression, Fish Shellfish Immunol, Shellfish Immunol, 23 (2007) 1127. 25 (2008) 425. 280 INDIAN J EXP BIOL, MARCH 2014

62 Wang G X, Zhou Z, Cheng C, Yao J Y & Yang Z W, Osthol Chinese shrimp Fenneropenaeus chinensis: cloning, distribution and isopimpinellin from Fructus cnidii for the control of and expression, Dev Comp Immunol, 31 (2007) 429. Dactylogyrus intermedius in Carassius auratus, Vet 65 Chen J, Wu H Q, Niu H, Shi Y H & Li M Y, Increased Parasitol, 158 (2008) 144. liver protein and mRNA expression of natural killer cell- 63 Anduro G A, Barillas-Mury C V, Peregrino-Uriarte A B, enhancing factor B (NKEFB) in ayu (Plecoglossus altivelis) Gupta L, Gollas- Galva´n T, Hernandez-Lo´pez J & Yepiz- after Aeromonas hydrophila infection, Fish Shellfish Plascencia G, The cytosolic manganese superoxide dismutase Immunol, 6 (2009) 567. from the shrimp Litopenaeus vannamei: Molecular cloning and 66 Engelsma M Y, Stet R J, Saeij J P & Verburg-van Kemenade expression, Dev Comp Immunol, 30 (2006) 893-900. B M, Differential expression and haplotypic variation 64 Zhang Q, Li F, Wang B, Zhang J, Liu Y, Zhou Q & xiang J, of two interleukin- 1beta genes in the common carp The mitochondrial manganese superoxide dismutase gene in (Cyprinus carpio L.), Cytokine, 22 (2003) 21.