Transcriptional changes in three immunoglobulin isotypes of rohu, Labeo rohita in response to Argulus siamensis infection

Fish & Shellfish Immunology 47 (2015) 28e33

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Transcriptional changes in three immunoglobulin isotypes of rohu, Labeo rohita in response to Argulus siamensis infection Banya Kar a, Amruta Mohapatra a, Jyotirmaya Mohanty b, Pramoda Kumar Sahoo a, * a b

Fish Health Management Division, ICAR-Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751 002, Odisha, India Fish Genetics and Biotechnology Division, ICAR-Central Institute of Freshwater Aquaculture, Kausalyaganga, Bhubaneswar 751 002, Odisha, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 June 2015 Received in revised form 18 August 2015 Accepted 20 August 2015 Available online 22 August 2015

Immunoglobulin heavy chains of three isotypes viz., IgM, IgD and IgT/IgZ are described in teleosts. In this study, a challenge experiment with an ectoparasite Argulus siamensis was conducted to evaluate the changes in adaptive immune response by quantitation of expression of Ig heavy chains in skin, head kidney and mucus of infected rohu, Labeo rohita. Rohu were challenged with 100 metanauplii of A. siamensis/fish. Head kidney, skin and mucus samples were collected at 0 h, 12 h, 24 h, 3 d, 7 d, 15 d and 30 d by sacrificing four fish each from infected and control groups at each time point. The expression of IgM, IgD and IgZ in these tissues were measured by reverse transcription real time quantitative PCR. IgM level was found to reach its peak significantly 30 d post-infection in head kidney tissue, while IgM transcripts were below detectable range in skin and mucus at all time points. IgZ and IgD levels were significantly up-regulated post-infection in all the three tissue samples. Early up-regulation of IgD was observed in skin and mucus, compared to head kidney. This study showed that parasitic invasion can trigger varied expressions of immunoglobulin types to provide systemic as well as local protection in the host. In particular, the appearance of high level of expression of IgZ and IgD in skin and mucus will pave the way for vaccine development against A. siamensis which feeds on those tissues. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Immunoglobulin Argulus siamensis Labeo rohita Gene expression

1. Introduction Phylogenetically, fish are the most primitive group of vertebrates to possess an adaptive immune system capable of generating an effective antibody response to pathogenic challenges [1]. The immunoglobulin (Ig) genes encode these defense proteins, antibodies which are the major effective molecules of humoral immunity in vertebrates. The Ig molecule is composed of a basic four chain unit in which two heavy (IgH) chains are held together and two light (IgL) chains are associated with the heavy chains. These associations are generally stabilized by disulfide bridges, although some exceptions do exist [2,3]. In mammals, immunoglobulin heavy chains are of five isotypes, namely IgM, IgG, IgA, IgD and IgE [4]. In teleosts, four types of Igs have so far been reported, namely, IgM [5,6], IgD [7e9], IgZ or IgT [10,11], and IgMeIgZ chimera [4]. IgM was the first immunoglobulin to be studied in many species of

* Corresponding author. E-mail addresses: [email protected] (B. Kar), [email protected] (A. Mohapatra), [email protected] (J. Mohanty), [email protected] (P.K. Sahoo). http://dx.doi.org/10.1016/j.fsi.2015.08.023 1050-4648/© 2015 Elsevier Ltd. All rights reserved.

teleosts such as salmon (Salmo salar) [12], zebrafish (Danio rerio) [13] and Atlantic cod (Gadus morhua L.) [14]. The existence of IgD in teleosts was first reported in catfish (Ictalurus punctatus) [7] and Atlantic salmon [8]. Recently, new Ig isotypes IgZ, IgT and a chimera of IgMeIgZ have been identified from zebrafish [10], rainbow trout (Oncorhynchus mykiss) [11], fugu (Takifugu rubripes) [15] and common carp (Cyprinus carpio L.) [4], respectively. The production of humoral antibodies in response to piscine parasites has received some attention in the past [16] though the information of their gene expression in response to parasitoses is still scarce. Argulus siamensis, the fish louse feeds on host mucus, skin tissue, tissue fluid and blood. The parasites usually cause only abrasive wounds on the host skin but nevertheless lead to systemic stress responses and modulations of the immune system and physiology [17e20]. A. siamensis is controlled mainly by pesticides and at present only a few types are in wide use like deltamethrin and avermectins [21]. However, future of this strategy is questionable due to development of pesticide resistance, occurrence of treatment failures, and undesirable environmental impacts. Hence, there is a need to develop new control methods. Since there exists a possibility of immunization against parasites of the genus Argulus

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[22,23]; a better understanding of acquired immune responses becomes imperative for vaccine development. It is essential to identify protective antibody responses following infection and challenge. Until recently, studies have addressed relatively few immune parameters and immune related genes with respect to Argulus-host system [17,18,23,24]. Knowledge of immune responses of the host to the parasite, Argulus and their role in protection against the parasite is still limited and this host-parasite system is yet to evolve as a research domain. In this study, we report the modulation in the transcriptional changes in genes encoding three immunoglobulins, namely IgM, IgZ and IgD in response to A. siamensis infection over a period of 30 days post-infection. Gene expression profiling was carried out in skin, mucus and head kidney tissues using real-time RT-qPCR analyses. 2. Material and methods 2.1. Animals Rohu (Labeo rohita) juveniles (55.89 ± 0.99 g) showing no signs of disease (under gross and microscopic examination of skin, gill, intestine and kidney tissues of representative samples) and no previous history of parasitic infections were obtained from the farm of the Central Institute of Freshwater Aquaculture, Bhubaneswar, India. Fishes were acclimatized in plastic tanks of 1000 L capacity with tap water, for 15 days before conducting the experiment. They were fed with commercial pellet diet at 2.5% of body weight. About 10% of water was removed daily along with the left-over feed and fecal matter. The basic physico-chemical water parameters were measured systematically at seven-day intervals to maintain at optimal level throughout the experiment. The water temperature in the tanks varied from 25 to 28
C during the experiment. 2.2. Parasite A population of A. siamensis was maintained on stock rohu (approximately 500 g) in tanks of wet laboratory. The eggs deposited on the sides of the tank were collected in beakers containing tap water and incubated at 28
C with daily refreshment of water for hatching in a previously standardized way [25]. Upon hatching, the metanauplii were counted and maintained in similar conditions as the eggs till used in the challenge test. Care was taken to use the metanauplii within 6e8 h of hatching. 2.3. Experimental design and sampling procedure For expression analysis of various immunoglobulin genes, twenty-eight fishes were divided into seven groups (seven time points 0 h, 12 h, 24 h, 3 d, 7 d, 15 d and 30 d) of four fish in each and maintained in separate tanks for the experiment. Another lot of twenty eight fish were also maintained in similar conditions to serve as control for each time point. The rohu juveniles were challenged with 100 numbers of metanauplii each as per earlier standardized protocol [20]. For sample collection, at each time point four fish were collected from the tanks. The mucus (approximately 500 ml) was collected aseptically from the integument of the fish using a glass slide carefully after anaesthetizing the fish with MS222 (Argent Chemical, Redmond, USA) and transferred to TRI reagent for storing at 70
C until RNA extraction. Skin from the site of parasite attachment (generally around pelvic fin base or ventral surface) and head kidney tissue samples were subsequently collected at 0, 12, 24 h, 3 d, 7 d, 15 d and 30 d post-infection (p.i.) aseptically after sacrificing the fish with overdose of anaesthesia and stored in RNAlater (Sigma, USA) at 70
C until extraction of

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RNA. 2.4. RNA isolation and quantification One hundred milligrams of skin and head kidney tissues of each fish stored in RNAlater were utilized for extraction of total RNA using TRI reagent (Sigma, USA) following the manufacturer's instructions. RNA was also extracted from the mucous samples stored directly in TRI reagent. The resulting RNA was treated with RNasefree DNase I (Fermentas, USA) followed by inactivation of DNase I according to the manufacturer's instructions. The concentration of the total RNA in the sample was quantified by measuring absorbance at 260 nm. The purity of the samples was also checked by measuring the ratio at OD260 nm and OD280 nm using NanoDrop ND1000 (Thermo Scientific, USA) with expected values between 1.9 and 2.0. The integrity of RNA was checked by electrophoresis on 1% agarose gel containing 0.5 mg/ml ethidium bromide at 100 V and also screening through RT-PCR using b-actin expression. Out of the four RNA samples collected for each time point, three best samples were used for further expression study. 2.5. cDNA synthesis One microgram of total RNA was used for generation of firststrand complementary DNA by reverse transcription using Thermo Scientific RevertAid First Strand cDNA synthesis kit (Thermo Fischer Scientific Inc., USA) following the manufacturer's instructions. Briefly, template RNA (1 mg) was added to random hexamer primer (1 ml) and nuclease free water to make the volume to 12 ml. Subsequently, 5 reaction buffer (4 ml), Ribolock RNase inhibitor (1 ml), 10 mM dNTP mix (2 ml) and RevertAid M-MuLV RT (200 U/ml) (1 ml) were added to make the final volume of the reaction mixture to 20 ml. The mixture was incubated at 25
C for 5 min followed by 60 min at 42
C. The reaction was terminated by heating at 70
C for 5 min. The reverse transcription reaction product was stored at 20
C for further use in the expression studies. 2.6. Real time quantitative PCR The primer pairs for IgM (F-50 ACGCTTCACCATCTCCA30 and R50 AGCCACCGTAGCCTCTT30 ), IgZ (F-50 CAGCCCTAAACTCGG30 and R50 GGTTGTGCCCTCTATGT30 ) and IgD (F-50 GGGACTCAAAGCAAAGAA30 and R-50 TAACCTCACAGGCAAAGAC30 ) were self-designed using Primer Premier 5 (version 5.0, Premier Biosoft International, Palo Alto, CA) from transcriptome data of rohu [26] and homologous sequences of related species available in NCBI database (http://www.ncbi.nlm.nih.gov). b-actin was used as the reference gene based on our earlier observations (20). Real time quantitative PCR was carried out using cDNA samples of mucus, skin and head kidney of rohu for IgM, IgZ and IgD expression analysis in Light Cycler 96 SW 1.1 (Roche, Germany) using FastStart Essential DNA GreenMaster (Roche, Germany) according to the manufacturer's instructions after standardization. Briefly, 1 ml of cDNA synthesized was used as a template in a total reaction mixture of 10 ml containing 5 ml of 2 Light cycler SYBR green I mix, 0.5 ml each of primer pairs (5 pmole) and 3 ml of H2O provided in the kit. The qPCR program consisted of pre-denaturation at 95
C for 10 min and 45 cycles of amplification at 95
C for 10 s, annealing temperature Ta (58
C for IgZ and 59
C for IgM and IgD) for 10 s, and 72
C for 20 s. All reactions were performed simultaneously for each gene with bactin, in the same plate in triplicate. qPCR specificity was verified by melt curve analysis at a temperature of 95
C for 10 s, 65
C for 1 min and 95
C for 1 min. No-template controls were also included in each run. Tm analysis was done to check primer specificity.

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2.7. Relative quantification

3.2. IgZ expression

The quantification cycle (Cq) values were calculated using Light Cycler 96 SW 1.1 and the data were exported. N-fold differential expression was calculated using the comparative Cq method [27] by calculating the average of each Cq for the triplicate samples. The Cq value of the gene for each cDNA was subtracted from its respective Cq value of b-actin to get the DCq value. Since the samples for each time period were taken in triplicate, an average of the DCq values was obtained. Further, the DDCq was calculated by subtracting the DCq of the samples from the DCq value of the calibrator. Fold difference was calculated as 2DDCq. Mean fold difference was calculated and represented as ± standard error. The control group at each time point was taken as calibrator for the infected samples of the respective time point.

The results of IgZ expression analysis are illustrated in Fig. 2. In head kidney, the expression of IgZ transiently raised at early time point of 12 h (3.08-fold) as compared to control which subsequently decreased to control levels at 24 h and 3 d p.i. At later time points of 7 d and 30 d p.i., the expression showed significant increase of 1.37 and 1.85 folds, respectively, as compared to control. In skin, the expression reached its peak at 3 d p.i. (2.77-fold) and statistically significant increase was also evident at 24 h p.i. (1.3fold). In mucus the IgZ expression reached its peak at 30 d p.i. (3.43-fold). However, statistically significant increase in the expression was also observed at other time points viz., 12 h (2.3fold), 24 h (1.7-fold) and 15 d p.i. (2.8-fold) in infected mucus samples.

2.8. Statistical analysis The difference between the mean values of the gene at a certain time point was compared to its respective control using Student's ttest. The expression levels of the gene in infected tissue samples at different time-points were analyzed using one-way ANOVA followed by Duncan's multiple range tests, with values p < 0.05 as significantly different. All values of n-fold differential expression were plotted in a graph.

3.3. IgD expression For IgD, variable expression pattern was detected in different tissues used in the study (Fig. 3). In head kidney, the expression

3. Results 3.1. IgM expression An increase in expression of IgM transcripts at almost all the time points (except at 3 d as compared to control) was noticed in the head kidney tissue of rohu infected with A. siamensis (Fig. 1). The expression level peaked a week after infection showing significant increase in levels as compared to the time point controls as well as 0 h control, being higher at 30 d post-infection (3.57-fold). IgM expression was not in detectable range in skin and mucus tissue samples collected from infected rohu at all time points.

Fig. 1. Real time quantitative gene expression profile of IgM in the head kidney of L. rohita at different time points (0, 12, 24 h, 3, 7, 15 and 30 d) following infection with A. siamensis (black bars). Control samples (grey bars) have been included at each time point. Bars represent as the mean ± SEM (n ¼ 3) calibrated normalized relative quantities (CNRQs). Statistical analysis was made by one way ANOVA among expression levels of infected samples of the different time points to analyze if the different time point groups varied from each other. The significant differences (P < 0.05) amongst the seven groups are represented by alphabets (a, b, c) over the bars. Statistical analysis between expression levels of control and infected sample at each time point was done by Students't-test and significant statistical difference is represented by *.

Fig. 2. Real time quantitative gene expression profile of IgZ in the head kidney (A), skin (B) and mucus (C) of L. rohita at different time points (0, 12, 24 h, 3, 7, 15 and 30 d) following infection with A. siamensis (black bars). Control samples (grey bars) have been included at each time point. Bars represent the mean ± SEM (n ¼ 3) calibrated normalized relative quantities (CNRQs). Statistical analysis was made by one way ANOVA among expression levels of infected samples of the different time points to analyze if the different time point groups varied from each other. The significant differences (P < 0.05) amongst the seven groups are represented by alphabets (a, b, c) over the bars. Statistical analysis between expression levels of control and infected samples at each time point was done by Students't-test and significant statistical difference is represented by *.

B. Kar et al. / Fish & Shellfish Immunology 47 (2015) 28e33

Fig. 3. Real time quantitative gene expression profile of IgD in the head kidney (A), skin (B) and mucus (C) of L. rohita at different time points (0, 12, 24 h, 3, 7, 15 and 30 d) following infection with A. siamensis (black bars). Control samples (grey bars) have been included at each time point. Bars represent the mean ± SEM (n ¼ 3) calibrated normalized relative quantities (CNRQs). Statistical analysis was made by one way ANOVA among expression levels of infected samples of the different time points to analyze if the different time point groups varied from each other. The significant differences (P < 0.05) amongst the seven groups are represented by alphabets (a, b, c, d) over the bars. Statistical analysis between expression levels of control and infected sample at each time point was done by Students't-test and significant statistical difference is represented by *.

level reached its peak at 30 d p.i. (11.89-fold). Also, the expression was detected to be statistically higher at all time points after 12 h onwards (1.3-fold at 24 h, 1.91-fold at 3 d, 4.62-fold at 7 d and 6.14fold at 15 d). In skin, the expression of IgD was higher at 3 d p.i. in infected tissue (3.04-fold). Statistically significant increase in expression was also detected at 12 h (1.79-fold) and 7 d p.i. (2.65fold). However, a slight reduction in expression level of IgD was noticed at 15 d p.i. (0.10-fold) in comparison to time point control sample. In mucus the expression of IgD was at its peak at 24 h p.i. (8.39-fold). Statistically significant increase in expression was also observed at later time at 7 d (2.97-fold) and 30 d p.i. (3.78-fold). 4. Discussion There is a growing demand for new approaches involving immunological studies to improve the health status of aquatic organisms and design control strategies against diseases. The immunization of fish against A. siamensis may be facilitated by an improved understanding of the adaptive immune system in the host and molecules involved therein. In order to understand how

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the host responds to parasites, the specific response of rohu infected with the ectoparasite A. siamensis was displayed by measuring the transcriptional changes in various isotypes of Igs (IgM, IgD and IgZ) for the first time in head kidney, skin and mucous tissues. Although the repertoire of antibodies is limited in teleosts than mammals; four classes of antibodies do exist in the teleost group, namely IgM, IgZ or IgT, IgMeIgZ chimera and IgD [28]. Head kidney is an important immune competent organ for antibody producing B cells distribution and proliferation, as demonstrated by in situ hybridization studies carried out in numerous fish species [14,29e31]. Skin is considered as the largest immunologically active organ but not much is known about the molecular mechanisms associated with it [32]. Also, in case of ectoparasites like A. siamensis, skin is the primary target organ and therefore it is important to check if a protective immune response is mounted in this tissue. Mucosal immunity is rarely studied in teleosts though mucosal epithelial cells are involved in adaptive immunity by Ag presentation and production of Igs along with complement, lectins, CRP, lysozymes, proteolytic enzymes and other effectors [33e35]. Since A. siamensis colonises on the cutaneous mucosa of host fish, a study of the immune responses mounted in the mucus is important for targeting vaccine development. The predominant immunoglobulin in fish is a tetramer of IgM class [30,36] and its monomer is found in serum of few fish species [37]. In this study, rapid (12 h) up-regulation of IgM was observed in head kidney with a decrease at 3 d p.i. and subsequent upregulation post one week of infection. This suggests the recruitment of B cells in response to the parasite infection. A similar pattern of IgM expression was reported in Atlantic salmon in response to sea lice, Lepeophtheirus salmonis [38]. In this model, IgM transcripts were below detectable level in skin and mucus which might be due to pronounced role of IgM in systemic humoral immunity and not in local adaptive immunity [39]. Furthermore, in some groups of teleosts, IgM produced in mucus and skin are similar but non-identical to the systemic IgM present in serum [40,41] and the IgM levels in fish skin mucous varies greatly among individuals. Therefore, it is essential to look if IgM is produced as a cutaneous antibody in L. rohita or not. IgZ forms the third immunoglobulin class in teleosts that has been shown to behave as the most predominant immunoglobulin in mucosal immune responses [42] like the mammalian class of immunoglobulin, IgA. IgZ and its rainbow trout ortholog IgT have already been reported in many teleosts including carps [4,10,11,43,44]. However, in L. rohita, the transcripts of IgZ along with IgD have been recently reported by RNA-seq [26]. IgZ is primarily expressed in head kidney and spleen [10,45] with weak expression in other tissues like thymus, intestine and skin. However, this distribution varies abundantly among the teleosts species. IgZ, like other two classes of immunoglobulins showed early upregulation (12 h and 24 h) post A. siamensis infection in all three tissue types studied. Similar up-regulation in expression of IgZ transcripts have been earlier noted in skin and head kidney of L. rohita in response to Edwardsiella tarda and Dactylogyrus infection [46]. IgZ expression has also been shown to be highly upregulated in the gill and intestinal mucosal layer in response to Flavobacterium psychrophilum in rainbow trout [47]. These studies indicate the potential role of IgZ in mounting an adaptive immune response (both systemic and local mucosal) in response to both bacterial and parasitic invasions. IgD is a primordial antibody, reported from almost all species examined in teleosts [47] but with little understanding on its function. In human, IgD mediates mucosal immunity and triggers innate antimicrobial response. Earlier workers are of view that IgD is as important as IgM in the adaptive immune response and helps in enhancing the humoral response [48,49]. Studies indicate that

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transcripts of IgD are primarily distributed in the head kidney, spleen and to some extent in peripheral blood leukocytes [9,45,50]. In the present study, in head kidney the increase in IgD transcript level was gradual, with expression peaking at 30 d p.i. A panel of Ig transcripts including IgM showed similar cycle of rapid upregulation followed by decrease and subsequent up-regulation in head kidney of Atlantic salmon in response to sea lice [38]. The IgD responses in the skin have not been investigated in the past, thus generating an unclear picture of its role in skin secretory immune system. Here, we show a very rapid up-regulation in IgD transcripts in both mucus and skin with expressions peaking as early as 24 h and 3 d p.i., respectively. This indicates that IgD might have a role in mucosal immunity in addition to enhancement of systemic Ig response along with IgM. Makesh et al. [47] have postulated the involvement of IgD in mucosal immunity in teleost fish following an up-regulation of IgD expression observed in skin, gill and intestinal mucosa of F. psychrophilum infected rainbow trout. The understanding of the functional differences between the three classes of immunoglobulins in teleosts is still limited. IgM is the first Ig to appear in phylogeny, ontogeny and as antibody in an immune response and until recently it was the general belief that only IgM is functional in teleosts [42]. Earlier workers have shown IgM to have a heightened functional role in many teleost species. Zimmerman et al. [51] showed the same by demonstrating excessive expression of IgM in comparison to IgD and IgZ in zebrafish throughout its development. However, recent breakthroughs have added IgD [52] and IgZ/IgT [10,11] as active players to the scene. The findings of Zhang et al. [53] showed that IgT/IgZ class of immunoglobulins is specialized in mucosal immunity being immensely significant for teleost as majority of the fish pathogens initiate disease in the mucus layers. A number of workers have demonstrated the importance of IgZ/IgT and IgD along with IgM in providing protection by mounting immune responses in various disease models [45,47,54,55]. This study also highlights the role of both immunoglobulin classes along with IgM in mounting a response rapidly after parasitic assault. What is worthwhile to note is the significant up-regulation of IgZ and IgD, components of mucosal immunity, as presence of antibodies in mucus being essential for development of a vaccine regime against A. siamensis. In conclusion, this study showed that parasitic invasion can be a triggering factor for varied immunoglobulin type expressions to provide systemic as well as local protection in the host. This appears to be the first report to evaluate the response of all three classes of teleost immunoglobulins in L. rohita in response to an ectoparasitic infection. Furthermore, appearance of highly upregulated expression of IgZ and IgD in skin and mucus could pave the way for development of vaccination regimes against ectoparasites, particularly A. siamensis in rohu. Acknowledgments The authors wish to thank the Indian Council of Agricultural Research, New Delhi for providing the National Fellow Scheme to Dr. P. K. Sahoo. The authors also wish to thank the Director, CIFA, Bhubaneswar for providing necessary facilities during this study. References [1] W.B. VanMuiswinkel, The piscine immune system: innate and acquired immunity, in: P.T.K. Woo (Ed.), Fish Diseases and Disorders, CAB International, Wallingford, 1995, pp. 729e750. [2] H.M. Grey, C.A. Abel, W.J. Yount, H.G. Kunkel, A subclass of human gamma-Aglobulins (gamma A-2) which lacks the disulfide bonds linking heavy and light chains, J. Exp. Med. 128 (1968) 1223e1236. [3] C.A. Mikoryak, L.A. Steiner, Noncovalent association of heavy and light chains in Rana catesbeiana immunoglobulins, J. Immunol. 133 (1984) 376e383. [4] R. Savan, A. Aman, M. Nakao, H. Watanuki, M. Sakai, Discovery of a novel

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