Effect of chrysophanic acid on immune response and immune genes transcriptomic profile in Catla catla against Aeromonas hydrophila

The effect of chrysophanic acid (CA) (2, 4, and 8 mg kg−1) on the immunity and immune-related gene profile of Catla catla against Aeromonas hydrophila is reported. In both control and treated groups fed with 2 mg kg−1 (2 CA), the phagocytosis, hemolytic, myeloperoxidase content, and superoxide anion production decreased significantly between 6th and 8th weeks, whereas when fed with 4 mg kg−1 CA (4 CA) the H2O2 production and nitric oxide synthase increased significantly between 4th and 8th week. When fed with 2 CA and 4 CA diets, the total protein, bactericidal, and antibody titer increased significantly from the 4th week onwards. When fed with 2 CA, the IL-1β and IL-10 mRNA expression of head kidney leucocytes were significant between weeks 6 and 8. The expressions of toll-like receptors significantly increased when fed with a 4 CA diet from 4th week onwards. The 4 CA group significantly increased in TNF-α, TNF receptor-associated factor 6 (NOD), which influences protein expression, after the 4th week. The mRNA transcription of MHCI, lysozyme-chicken and goose type expressions significantly increased in 4 CA group within the 4th week. In summary, the dietary administration of 4 mg kg−1 of CA (4 CA) provides better immunity and enhances the up-regulation of immune-related genes in Catla against A. hydrophila.


Results
Immunological response. Both healthy and infected groups fed with the 2 CA enriched diet had increased phagocytic and hemolytic activity and SOD generation (P < 0.05) between the 6th and 8th weeks, but not in the 2nd or 4th week. Fish fed with 4 CA diet in both groups exhibited these activities at the 4th, 6th, and 8th weeks. However, the 8 CA administration significantly enhanced these parameters in the 2nd week only; no differences were observed in either the 1st week or after the 6th week (Figs. 1a, 2a, 3a). In both 2 CA groups, enhanced LP index, MPO content, bactericidal action, and antibody levels were observed between the 6th and 8th weeks. A  a a a a a a a a a a a a a  www.nature.com/scientificreports/ similar trend existed in both groups fed with 4 CA from week 2 to 8. The maximum values of the immune parameters were obtained when both groups were fed the 8 CA diets between weeks 2 and 4; however, no increases were observed in the initial study period (week 1) or after prolonged periods (after week 8) (Figs. 4a,b, 5a, 6b). Low respiratory burst (RB) activity was observed in both 2 CA and 4 CA groups in the 2nd and 4th weeks; however, the highest (P < 0.05) values were noted after week 6. In both 8 CA groups, the highest RB activity (P < 0.05) manifested between weeks 4 and 6, but these values were insignificant in the 1st week and after week 8 (Fig. 1b). The nitric oxide (NO) synthase was low in both 2 CA groups (weeks 2-6); increased significantly after the 8th week. Similarly, both 4 CA groups had slightly higher levels (P > 0.05) of NO synthase in the 2nd and 4th weeks, and significantly (P < 0.05) higher levels between weeks 6 and 8. NO synthase levels varied moderately (P > 0.05), except in week 2 (Fig. 2b).  www.nature.com/scientificreports/ Hydrogen peroxide (H 2 O 2 ) production did not differ (P > 0.05) significantly in the 2 CA fed groups from weeks 2 to 6 but attained significant levels (P < 0.05) in the 8th week. The highest H 2 O 2 production was observed in both 4 CA groups between the 4th and 8th weeks, and the lowest production activity (P > 0.05) occurred in the 8 CA groups (Fig. 3b). Lysozyme levels and TP levels decreased slightly (P > 0.05) in the 2nd week in the 2 CA and 4 CA groups. These values rose significantly (P < 0.05) in both 8 CA groups from 4 to 8 weeks, though increases did not vary in either the 1st or after the 8th weeks (Figs. 5b, 6a).
Immune gene expression. The mRNA transcripts of IL-1β and 10, TLR-4, as well as MHC-I in head kidney leucocytes, were significantly low between weeks 2 and 4 when fed with 2 CA in both groups, whereas its expression was up-regulated between the 6th and 8th weeks. Feeding with 4 CA could induced IL-1β, IL-10, TLR-4, and MHC-I mRNA expression in both groups significantly from weeks 4 to 8, compared with that of the control. However, the IL-1β, IL-10, TLR-4, and MHC-I mRNA expression were significant in both groups in the 4th and 6th weeks in the 8 CA groups; yet no up-regulation was observed in the first or after the 8th week (Figs. 7a,b, 8b, 10c). The expression levels of TRAF6 and TNF-α were not significantly up-regulated in the 2 CA groups from week 2 to 6, while significant up-regulation was observed in the 8th week. Both groups fed with 4 CA showed down-regulation in the 2nd and 4th weeks, whereas up-regulation was observed in the 6th and 8th weeks. Nevertheless, the TRAF6 and TNF-α expression levels were slightly higher in the groups fed with 8 CA from week 4 to 8 (Fig. 9a,b).
The expression of INF-γ, TLR3, TLR5, Lyz-C, and Lyz-G genes showed no up-regulation in both 2 CA groups from weeks 2 to 6; however, mRNA gene expressions were up-regulated in the 8th week. The 4 CA groups had a significant induction of INF-γ, MyD88, TLR3, TLR5, Lyz-C, and Lyz-G genes expression between weeks 4 and 8, but not in the 1st week (Figs. 8a,c, 10b,d, 11a,b). Interestingly, a slight up-regulation of NF-kB and TLR2 expression was observed in weeks 2 and 4 in the 2 CA group. However, there was a significant up-regulation between weeks 6 and 8. Similar results were found with the 4 CA and 8 CA groups in the 2nd and 4th weeks; however, all the values decreased after the 6th week (Figs. 8d, 10a). The expression of NOD1 and NOD2 were not significantly improved in the 2 CA group (weeks 2-6), yet there was a sudden up-regulation in the 8th week. Significant NOD1 and NOD2 mRNA expressions were also observed in the 4 CA group after the 4th week, as well as in the 8 CA groups (weeks 4 and 6), but not in the 1st week or after the 8th week (Fig. 12a,b). www.nature.com/scientificreports/ RPS. Survival was 100% in group I (H-0 mg). In both post-challenged and un-challenged groups (groups III and IV), treated with 4 CA and 8 CA resulted in higher survival rates between 80.5 and 89%. On the other hand, post-challenged or un-challenged (groups II and VI) treated with 2 CA had low survival between 72.5 and 79%. However, post-challenged group V treated with the control diet had the least survival of 64% (Fig. 13).

Discussion
The ability of phagocytic cells to kill the invading pathogens is an essential innate defense mechanism 33 . In the present study, phagocytic activity significantly improved in both groups (P < 0.05) with doses of 2 mg kg −1 CA (2 CA) between weeks 6 and 8; and in the 4 CA group between weeks 4 and 8. The phagocytic leucocytes harvested from HK of the 2 CA and 4 CA groups revealed high RB activity, attributed as a metabolic function due to CA in the 6th and 8th weeks, resulting in higher amounts of SOD synthesis. The HK leukocytes RB activity is another important innate immune function widely considered a bio-indicator of immune-competence, exclusively triggered by immunostimulants 33,34 . Extracellular O 2 − production was highly significant in the 2 CA www.nature.com/scientificreports/ groups (6 and 8 weeks) and the 4 CA groups (4-8 weeks). Similar results were found in rohu, in which a higher level of O 2 − production was reported (Days 4, 6, and 14) post-vaccination with A. hydrophila 35 . The O 2 − production in macrophages exhibits immunity after the initiation of phagocytes in fish that generate reactive oxygen products; such as H 2 O 2 and OH, during a period of robust O 2 intake, termed as RB 36 . This ability to kill microorganisms by phagocytes has an essential role in anti-microbicidal mechanisms in fish 37 . The trout HK leucocytes collected after zymosan exposure showed maximum oxygen consumption or RB activity that produced 12 nM or 13 nM superoxide anion for 10 7 cells 38 . In the present study, both groups fed 2 and 4 mg kg −1 CA diets (2 CA and 4 CA) showed no significant change in RB activity in weeks 4-6. These results were in agreement with a recent study in which a significant observance of RB activity was reported in Atlantic salmon and rainbow trout against Lepeophtheirus salmonis 39 . However, significantly high RB activity was observed in Pangasianodon hypophthalmus against pathogen 40 . Similarly, Munoz, et al. 41 reported that a higher O 2 − level afforded protection to Dicentrarchus labrax against Sphaerospora dicentrarchi. Natural immunostimulants do not significantly affect the innate defense response in fish 42,43 ; numerous studies have reported a higher RB activity in fish through various immunostimulants 44,45 . H 2 O 2 production was significantly high in the 8th week of the 4 CA feeding, whereas the RB activity in the 8 CA group was manifested in the 2nd week. Similar increases in H 2 O 2 production were www.nature.com/scientificreports/ observed in Psetta maxima and Sparus aurata via the administration of glucan 46 . High ROS production due to glucan may be associated with augmented bactericidal and phagocytosis, as reported in Atlantic salmon and rainbow trout 47,48 . The ability to generate H 2 O 2 was reported in neutrophils due to O 2 breakdown during RB in channel catfish 49 . However, in the present study, the H 2 O 2 production was low in both groups fed 2 mg kg −1 CA (2 CA) from week 2 to 6. Similarly, the native or heat-denatured lectin of Abrus precatorius induced low phagocytosis levels, bactericidal, and H 2 O 2 production in mice macrophages 50 . A significantly higher proliferative response was observed in both 2 CA groups after week 6, while in the 4 CA group, the same effect was observed after week 4. Consequently, with the administration of the A. hydrophila vaccine, the proliferative response in fish increased 51 . A lower response was observed in the earlier stage, week 2, in the 2 CA group, suggesting the induction of low memory. However, the potential influence of leukocyte function on enhancing specific immunity related to immunostimulant is yet to be explored. Notably, NO production was directly related to the granulocytes associated with the innate immune system 52 . In this study, NO synthase production was low in both 2 CA groups between weeks 2 and 6, which became significant in the 8th week. However, the 4 CA groups produced a significantly higher level of NO synthase in weeks 6 and 8. Das et al. 35 reported similar effects of NO production in leukocytes in rohu against A. hydrophila. Consequently, the present results suggest that CA potentially influences NO synthesis of ROS and RNS in stimulated leukocytes as an immediate response to pathogens.
Neutrophil granules release MPO enzymes during oxidative RB that produce toxic hypochlorous acids that react with pathogenic microorganisms 53 . MPO content was highly significant (P < 0.05) in both 2 CA groups at 6 and 8 weeks, similar to that of the 4 CA groups from weeks 2 to 8. A significantly elevated level of MPO content was reported in P. hypophthalmus against monogenean infection 40 . Conversely, a recent study in rohu found that MPO activity was not significantly different between healthy and infected fish 54 . The present results indicate that a relatively lower concentration (2 mg kg −1 CA) stimulates MPO after 6 weeks, whereas a medium concentration (4 mg kg −1 CA) stimulated MPO earlier, at week 4. Hemolytic activity was statistically influenced (P < 0.05) in the www.nature.com/scientificreports/ 2 CA groups in weeks 6 and 8, whereas the 4 CA groups induced MPO between weeks 4 and 8. Among various immune mechanisms, only the complement pathway has the potential to avert microbial infection. Therefore, the hemolytic exertion in fish serum is recognized as an intersperse complement pathway, which plays a vital role against infectious pathogens 55 . In this study, a significant hemolysin titer was obtained with the 4 mg kg −1 CA, suggesting the influence of complement pathways against pathogens 56 . However, the groups fed with a low dose (2 mg kg −1 CA) produced a high hemolysin titre in the later stages, whereas a high dose (8 mg kg −1 CA) produced similar results in the earlier stages. Additionally, a more recent study reported less hemolysin titre in rohu against dactylogyrid monogenean 54 . However, these changes may be dependent upon fish species, the pathogenicity of the microbes, or the blocking of certain microbe epitopes, capable of influencing the alternate complement cascade. Apart from the RB mechanism, lysozyme is a lysis enzyme produced by granulocytes during non-specific oxygen-independent pathways that play an essential role in innate immunity [57][58][59] . In vertebrates, lysozyme is an imperative humoral component in the systemic and mucosal immune systems; it further acts as a defensive factor against pathogenic microorganisms 57 ; lysozyme secreted by human granulocytes attach to the hyphae of Candida albicans 60 . These studies strongly suggest the existence of a lysozyme-conflict defence mechanism in fish against pathogens. TP is also an important compound involved in the immune system. Both lysozyme activity and TP concentration reached significant levels (P < 0.05) in both groups fed with 2 CA and 4 CA diets between the 4th and 8th weeks. Elevated serum lysozyme activity was also reported in the immunostimulant administration in rainbow trout and rohu 61,62 .
Bactericidal and antibody levels were statistically improved (P < 0.05) in both 2 CA groups after week 6 and in the 4 CA groups from weeks 4 to 8. The relationship between antibody production and protection against pathogens has been demonstrated in Catla against furunculosis 51 . However, the significance of protection or resistance to pathogen within antibodies further suggests cellular immunity following immunostimulants. Antibody synthesis depends primarily on effector-cell proliferation and its specific antibody secretion or memory cell differentiation, which representing the complex progression needed for cytokine secretion and cell cooperation 63 .
In the present study, immune defense system changes when infected with A. hydrophila led to both up-and down-regulation of selected immune genes. The immune-related gene expression patterns in fish have been investigated only recently; and, consequently, the understanding of immunity to pathogen is limited. IL-1β is an important inflammatory mediator in microbial infections 64 . In the 2 mg kg −1 CA diets, the IL-1β and IL-10 expression levels in HK were significantly low between weeks 2 and 4, and relatively high in weeks 6 and 8. The gene's significant up-regulation was also present in the 4 CA groups in the 6th and 8th weeks. IL-1β was up-regulated in the skin of trout against Gyrodactylus derjavini 65 . However, in the present study, IL-1β showed a significant down-regulation in Catla infected with A. hydtophila; suggesting the release of pathogen toxins in different phases of pathogen attachment, which play a significant role in arbitrating inflammation, thereby leading to down-regulation 62 ; an increase in IL-1β and IL-10 expression. The exact actions of CA involvement in fish against A. hydtophila may be contained within the immune system. IL-10 is involved in the synthesis of the multifunctional cytokine inhibitory factor, which promotes immune-suppressive function 66 , and plays a major role in undermining the proinflammatory cytokines responses; such as IL-1β, TNFα, and IFN-γ; as well as preventing tissue damage 67,68 . Increased IL-10 mRNA expression levels have been reported in fish due to different immunostimulants related to the transcription arising in innate immune cells, such as macrophages 69,70 .
TLRs facilitate inflammatory immune responses when binding to molecules with PAMPs, and trigger an inflammation-related genes expression through signal transduction 71,72 , also referred to as endogenous TLR ligands, which acts as an ominous early alarm to innate and adaptive immunities 73 . The TLR2, TLR3, TLR4, and TLR5 expressions were significantly up-regulated in the 4 CA groups after 4 weeks and in the very early and late stages in the 2 CA and 8 CA groups. Modulation of negating innate immune gene receptors, like TLRs and NLRs, plays a fundamental role in forming an Ig repository 74 . A recent study reported that similar functions were demonstrated in the embryo stages in different organs or tissues of L. rohita TLR2 and TLR5 genes 75 . The TLR4 has been recently demonstrated with the ability to recognize viral or bacterial motifs 76 .
TRAF6 and TNF-α were up-regulation in the HK in both 4 CA groups after the 6th week and after the 8th week in the 2 CA groups. A similar expression pattern of TNF-α was recorded in zebrafish embryos and adults against Edwardsiella tarda infection 77 and in carp HK, due to LPS 78 . However, TRAF6 and TNF-α expression presented low in the HK in both 4 CA groups between the 2nd and 4th weeks and between the 4th and 6th weeks in the 8 CA groups. Similarly, a low TNF-α expression was reported in rohu HK against E. tarda 79 . The TRAF6 response to different PAMPs treatments in Epinephelus tauvina revealed its contribution in influencing immune responses 80 . MHC receptors are associated with antigen-presenting cells (APCs), which assist T-cells in initiating the immune system 81 . In HK, leucocyte MHC-I expression was up-regulated in the 2 CA groups in the 6th and 8th weeks and the 4 CA groups on or after the 4th week. The high level of MHC expression in Catla, due to A. hydrophila, suggests that MHC contains cells (macrophages) within the HK that mediate the inflammation. However, no such change in MHC-I expression was observed in the 2 CA groups between weeks 2 and 4. Gharbi et al. 82 reported the MHC receptors susceptibility in Atlantic salmon to L. salmonis.
The up-regulation of NF-kB occurred in both 2 mg kg −1 CA groups in weeks 6 and 8, whereas MyD88 expression significantly was up-regulated in the same period, in the 4 CA groups in weeks 4-8, and the 8 CA in weeks 4-6. The IL-10 mechanism was described in Catla by blocking the NF-kB-signals in the HK. Association of PAMP-TLR induced oligomerization, triggered intracellular signaling cascade via recruitment of the myeloid differentiation factor 88 (MyD88)-dependent or -independent pathways 83 . Combined stimulation of intracellular MAP kinase pathway and Iκβ deprivation irritates the triggering of transcription factors, like AP-1 and NF-κB; which triggers proinflammatory cytokine secretion in the B and T cells leading to B cell proliferation 84 . In zebrafish, the TLR4-induced MyD88 dependent signals triggered the regulation of anti-inflammation 85  www.nature.com/scientificreports/ to regulate the activation of catla lymphocytes and lymphoid organs. NF-κB is capable of binding to Ig kappa light-chain of pathogens encountered within the B cells 86 . Therefore, the tight NF-κB phosphorylation and adhesion regulation are necessary to inhibit dysfunction of immune function 87 . Thus, the sudden increase in NF-κB levels observed in this study indicates their vital role in the pathophysiology linked to TLR signaling. When fed with 4 mg kg −1 CA, both NOD1 and NOD2 demonstrated significant expression on or after week 4. In addition to TLRs, NLRs is also responded to microbial components 88 and endogenous ligands, as a result of tissue or cellular injuries 89 . The TLR2 and TLR5, NOD1, and NOD2 genes were up-regulated carp's embryonic stages 90,91 . The TLR and NOD constitutive expression suggest the possibility of innate immune receptors in the early stage of CA treatment in Catla.
The chicken (Lyz-C) and goose (Lyz-G) gene expressions were not up-regulated in the 2 CA groups in the 8th week and in the 4 CA groups between weeks 4 and 8. Similar Lyz-C and Lyz-G up-regulation was recorded in Japanese flounder kidney, intestine, heart, whole blood, spleen, and ovary against E. tarda 92,93 . Hence, the Lyz gene up-regulation may be responsible for the rise in serum lysozyme concentration in Catla fed with CA diets against A. hydrophila infection.
There was 100% survival in H-0 mg, but only 64% of survival rate was observed in the infected group fed with the control diet. Both post-challenged and un-challenged groups treated with median dose (4 mg kg −1 ) and high doses (8 mg kg −1 ) of CA had high survival (between 80.5 and 89%), but the low survival rate was observed in both groups treated with low dose (2 mg kg −1 ) of CA resulting in low survival (between 72.5 and 79%). Similar reports in different fishes when fed with diets enriched with various active constituents indicate the same trends [94][95][96] .
In conclusion, to the best of our knowledge, this is the first report on the positive influence of CA in C. catla on innate and adaptive immune responses. The optimum level of CA is 4 mg kg −1 , which manifested during the 2nd week of treatment. The pathway related to the inflammation and immunomodulation of genes also triggered a similar response. The 8 mg kg −1 of CA resulted in such response much later, after the 6th week. Further comprehensive investigations are necessary to explain immune gene expressions' ability to elucidate the mechanism of action in other fish species, with different chrysophanol doses, against different pathogens.

Materials and methods
Formulation of experimental diet. The basal/control diet contained maize grain, fish meal, finger millet, and pearl millet as protein sources; rice-bran and wheat-flour as carbohydrate sources; and groundnut oil cake and vegetable oil as lipid sources (Table 1). Each ingredient was finely pounded and mixed with the required water volume to make a soft bread. The prepared feed was kept in an aluminum vessel and steamed in a pressure cooker for 15 min (at 15 psi). The formulated blend was cooled at room temperature (RT); then incorporated with the pre-mix of vitamins and minerals. Four experimental diets were prepared, by mixing chrysophanic acid (CA) in four different concentrations: (1) 0 mg kg −1 , (2) 2 mg kg −1 (2CA), (3) 4 mg kg −1 (4CA), and (4) 8 mg kg −1 (8CA). Pellet feeds were prepared using a manual pelletizer (2 mm). The prepared diets were immediately ovendried at 60 °C for 12 h. and tightly packed, stored in appropriate containers, and labeled. The experimental pellet feed constitutions were analyzed using regular procedures. www.nature.com/scientificreports/ Aeromonas hydrophila. Aeromonas hydrophila (MTCC 1739) was acquired at Himedia (India), and subcultured at 37 °C for 24 h. in a nutrient broth. The bacterial suspension obtained was centrifuged at 3000 × g for 10 min, and the supernatant was then discarded. The remaining pellet bacteria was re-dissolved in phosphate buffered saline (PBS, pH 7.4); until an optical density (OD) of the suspension [0.5 at 456 nm at 1 × 10 6 colony forming unit (CFU)] was achieved using a microplate reader and stored in a deep freezer for further use.
Fish. Healthy, Catla catla (36.7 ± 2.1 g, 20.3 ± 2.9 cm) were procured in a local farm and kept in 500 L fiberreinforced plastic (FRP) containers, sufficiently aerated, and filtered with de-chlorinated freshwater. The fish were immediately immersion in a KMnO 4 solution for 2 min to evade any dermal infection and then kept for 2 weeks under standard laboratory conditions within natural photoperiod. During the acclimation period, fish were provided the control diet (Table 1). Fecal and unfed materials were siphoned off daily to avoid the accumulation of ammonia content in the tanks. The following measurements were recorded: pH 7.2 ± 0.05, dissolved oxygen 8.8 ± 0.02 mg l −1 , temperature 24 ± 1 °C, and CaCO 3 190 ± 0.2 ppm. Blood and tissues sampling procedure. Six fish from each group were arbitrarily chosen at the end of weeks 2, 4, 6, and 8 post-challenged with A. hydrophila. Post-anesthetized (MS-222, Sigma, USA), blood samples were drawn separately from the cardinal vein via a 1 mL plastic syringe. Each blood sample was equally divided into two separate sterile tubes with and without heparin, respectively. The heparinized blood was immediately analysed for hemato-biochemical analysis and immunological study, whereas the non-heparinized blood samples were permitted to clot at RT, then preserved at 4 °C for 4 h. All samples were centrifuged (2300 × g) for 5 min at 4 °C. The resulting serum was collected and stored in individual sterile tubes (− 80 °C) for further analyses. Lastly, the anterior kidney was dissected out aseptically from each fish, and RNAlater was added (Ambion, USA) and then preserved at − 80 °C awaiting RNS extraction.
Immunological assays. Non-specific immune parameter. Phagocytosis was analysed via Aeromonas hydrophila, whereas the respiratory bursts were determined by the reduction of nitroblue tetrazolium (NBT) assay (Sigma, MO, USA) to measure neutrophils reactive oxygen radical production 97 . Bactericidal activity was determined using a 96-well microtiter plate with 3-(4, 5 dimethyl-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), according to the method of Kampen et al. 98 . Serum lysozyme levels were measured via turbidimetric assay Ellis 57 . Nitric oxide synthase (NOS) was analysed as prescribed by Lee et al. 99 . The production of hydrogen peroxide (H 2 O 2 ) was measured by phenol red oxidation method using horseradish peroxidase 33 . The HK leukocytes proliferative reactions were measured via MTT assay 100,101 . The synthesis of superoxide anion (SOD) was estimated following 51 . Respiratory burst (RB) in terms of oxygen radical generation was measured by phagocytes using NBT reduction assay 97 . Total serum myeloperoxidase (MPO) and total protein contents were measured by the technique employed by Mohanty and Sahoo 79 and Gornall et al. 102 . Serum hemolytic activity was estimated by microtitre plate 54 .
Specific immune parameters. The serum antibody levels of fish infected with A. hydrophila was determined using an indirect enzyme linked immunosorbant assay (ELISA) 103 with some modifications 104 .
Immune-related gene expression study. Total RNA extraction. Roughly 50-100 mg of HK tissues was dissected in each fish for isolation of total RNA using TRI reagent (Sigma), per manufacturer's instruction. The total RNA concentration was determined using spectrophotometer (Nanodrop ND-1000, Thermo Scientific, USA). The sample's purity was achieved by quantifying the ratio of OD 260 nm/OD 280 nm (1.8-2.0). The purified RNA was then used for cDNA synthesis. www.nature.com/scientificreports/ Accu-Taq la), 0.2 µL of 10 pmol of both primers (forward and reverse), and 2 µL cDNA. The extension parameters of the PCR as followed: 95 °C for 3 min; followed by denaturation for 30 cycles for 45 s (94 °C), and 45 s for appropriate annealing temperature (Table 2), followed by additional 45 s extension at 72 °C, and a 10-min for final extension (72 °C). The synthesized PCR products were confirmed by 1.0% agarose gel electrophoresis. The relative expression profile of β-actin and immune-relevant genes were evaluated by densitometry (Gel DOC, BIO RAD Laboratories, India).

Relative percentage survival (RPS).
Analysis of RPS was studied in all the experimental groups as mentioned in "Experimental setup". Each experimental group was maintained triplicate, and 20 fish were used in each group. The bacterial culture and concentration of bacterial density were the same as mentioned in "Aeromonas hydrophila". The bacterial challenge and administration of pathogen were the same as mentioned in "Experimental setup". The survival rate was determined at the end of the experiment. Relative percentage survival (RPS) was calculated following standard formula: RPS = 1 − % mortality in treatment group / % mortality in control group × 100. Table 2. List of gene primers used for tissue specific expression pattern of selected immune related genes in C. catla. IL-1β/10 interleukins1β/10, IFN-γ interferon-gamma, TNF-α tumor necrosis factor-alpha, TRAF6 TNF receptor-associated factor 6, TLR2/3/4/5 toll-like receptors 2/3/4/5, LYZ-C/G lysozyme-C/G, NOD1/2 nucleotide binding oligomerization domain containing 1/2, NF-kB nuclear factor kappa-light-chain-enhancer of activated B cells, MyD88 myeloid differentiation primary response 88, MHC-I major histocompatibility complex-class I.