In vitro free radical scavenging activity of hepatic metallothionein induced in an Indian freshwater fish, Channa punctata Bloch

Chemico-Biological Interactions 162 (2006) 172–180

In vitro free radical scavenging activity of hepatic metallothionein induced in an Indian freshwater fish, Channa punctata Bloch Fahim Atif, Manpreet Kaur, Seema Yousuf, Sheikh Raisuddin ∗ Department of Medical Elementology and Toxicology, Jamia Hamdard (Hamdard University), New Delhi 110062, India Received 18 April 2006; received in revised form 17 June 2006; accepted 22 June 2006 Available online 27 June 2006

Abstract Mammalian metallothioneins (MT) have been reported to scavenge free radicals. There is no experimental evidence to show that fish MT has a similar property. In the present study cadmium-induced MT (Cd-MT) from the liver of an Indian freshwater fish Channa punctata Bloch was investigated for its free radical scavenging activity using three different in vitro assays. Exposure to cadmium chloride (0.2 mg/kg body weight; three doses on alternate days) resulted in a marked induction of Cd-MT in liver. Only a single isoform of Cd-MT was found to be induced. Molecular weight of Cd-MT was found to be 14 kDa as deduced by • SDS-PAGE analysis. The purified Cd-MT effectively scavenged the following free radicals: superoxide radical (O2 − ), 2,2 -azinobis •+ • 3-ethylbenzothiazoline-6-sulfonic acid (ABTS ) and 1,1-diphenyl-picrylhydrazyl radical (DPPH ). The radical scavenging effect was found to be concentration-dependent. Also, the purified MT exhibited an inhibitory effect on ferric nitrilotriacetate (Fe-NTA) induced oxidative DNA damage in vitro. The cysteine residues of MT are proposed to be the main candidate for its radical scavenging activity. Findings of the present study strongly suggest a free radical scavenging role for fish MT. Present study adds to the little existing knowledge about fish MT and its possible biological functions. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Fish metallothioneins; Free radicals; Lipid peroxidation; DNA damage

1. Introduction Reactive oxygen species (ROS) are continuously produced in biological system by the action of mitochondrial electron transport system and nicotinamide adenine dinucleotide phosphate (NADP) oxidase [1,2]. These oxygen free radicals are cellular renegades and wreak havoc in biological system by damaging DNA, altering biochemical compounds, corroding cell membranes and killing cells outrightly [3]. Such molecular mayhem ∗

Corresponding author. Tel.: +91 11 26059688x5568; fax: +91 11 26059663. E-mail addresses: [email protected], [email protected] (S. Raisuddin).

plays a major role in the development of ailments like cancer, heart and lung diseases and cataracts [3]. There are various key antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), glutathione (GSH) and ascorbic acid (AA) which mitigate the impact of ROS by different mechanisms. A number of these antioxidants directly scavenge free radicals [4]. Recently, attention has been focused on a role for metallothioneins (MT) as a free radical scavengers. All mammalian species so far examined have multiple MT genes coding for different isoforms. In mammals, MT falls into at least four subgroups, namely MT-1, MT-2, MT-3 and MT-4. The MT-1 and MT-2 isoforms, which differ by only a single negative charge, are the most

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widely expressed isoforms in different tissues [5]. In case of fish, three isoforms of MT (MT-1, MT-2 and MT-3) have been characterized mainly from liver and brain [6–8]. Amongst these three isoforms, MT-1 and MT-2 are the most prevalent isoforms [9]. Antioxidant role for mammalian MT is well documented. The induction of MT under radical generating circumstances has led to the speculation that MT might be involved in free radical scavenging activity [10,11]. Electron spin resonance (ESR) spectroscopic studies have demonstrated that MT could scavenge free radicals including superoxide and hydroxyl radicals [12]. MT have also been found to inhibit nephrotoxicity of cisplastin by scavenging free radicals in MT-null mice [13]. MT is reported to mitigate cardiotoxicity of doxorubicin both in vivo and in vitro mainly due to its free radical scavenging property [14]. Protective effect of MT against ROS-induced DNA damage and apoptosis has also been reported [15–17]. Moreover, mouse MT protects rasDNA strand scission induced by ferric nitrilotriloacetic acid (Fe-NTA) [18]. There are some structural differences between mammalian and fish MT. Fish MT display the displacement of a cysteine residue located in the carboxyterminal half of the molecule and a lower number of lysine residues juxtaposed to cysteines [19]. It is also reported that fish MT is less hydrophobic than its mammalian counterpart [19]. NMR spectroscopy showed a selective broadening of the heteronuclear spectra of fish MT, thus suggesting a higher flexibility of the molecule [20]. In vivo studies have reported antioxidant property of MT [13,14]. As regards free radical scavenging activity of fish MT, no direct evidence has so far been offered. Recently, we reported protective effect of cadmiuminduced MT (Cd-MT) on deltamethrin- and endosulfaninduced oxidative stress in different tissues of freshwater fish Channa punctata Bloch [21,22]. It was observed that Cd-MT was responsible for such an effect and suggested that fish MT might be having antioxidant property [21]. In the present study, we report findings on in vitro antioxidant and free radical scavenging activity of CdMT isolated from the liver of C. punctata using a battery of standardized tests.

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DEAE-Sephadex A 25, Sephadex G 75, xanthine oxidase were purchased from Sigma–Aldrich Co. (St. Louis, USA). Acrylamide, agarose, ammonium persulphate (APS), bromophenol blue, cadmium chloride (CdCl2 ), ethidium bromide (EB), N,N methylene-bisacrylamide, N,N,N N -tetramethyl ethylenediamine (TEMED), ␣-naphthylamine, orthophosphoric acid (OPA), sulfanilic acid, Tris–hydrochloride (Tris–HCl), thiobarbituric acid (TBA), xanthine were procured from HiMedia (Mumbai, India). Coomassie brilliant blue R-250 and 2-mercaptoethanol were purchased from E-Merck (Germany). For evaluating modulatory effect of MT on DNA damage Fe-NTA was used as DNA damaging agent. Fe-NTA solution was prepared afresh using the procedure of Awai et al. [23]. Ferric-nitrate nonahydrate and nitrilotriacetic acid disodium salt (Sigma–Aldrich) were dissolved in deionized water to form solutions of 300 and 600 mM, respectively. The two solutions were mixed in a ratio of 1:2 under continuous stirring at room temperature and the pH was adjusted to 7.4 with sodium bicarbonate. 2.2. Fish C. punctata Bloch (Spotted snake-head murrel) weighing 50–75 g were commercially procured and maintained in 60-l aquaria with oxygen saturated water prior to experimentation. Fish were acclimatized for 2 weeks. The temperature of water was maintained at ambient laboratory temperature (25 ± 2 ◦ C) with constant illumination. Water was changed every other day to minimize contamination from metabolic wastes. Fish were fed standard Spirulina based white fish feed pellets five times a week. Physico-chemical characteristics of water such as dissolved oxygen (DO), biological oxygen demand (BOD), chemical oxygen demand (COD), temperature and pH were monitored on alternate days. The ranges of these parameters were as follows: DO 7.2 ± 0.6 mg/l, BOD 3.5 ± 0.2 mg/l, COD 6.8 ± 0.6 mg/l, temperature 22–25 ◦ C, pH 7.6 ± 0.08. The cadmium concentration in aquarium water was below the maximum permissible limit of 3 ␮g/l. 2.3. Cadmium exposure

2. Materials and methods 2.1. Chemicals 2,2 -Azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), butylated hydroxyl toluene (BHT), 1,1-diphenyl-2-picrylhydrazyl (DPPH),

Cadmium chloride was dissolved in normal saline. A group of acclimatized fish (n = 15) was exposed to CdCl2 at a dose of 0.2 mg/kg body weight for 7 days (three i.p. doses on alternate days). This dose of cadmium has been reported to induce MT in tilapia and C. punctata [24,21].

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2.4. Purification of MT 2.4.1. Sephadex G 75 chromatography MT was purified from liver of cadmium-exposed fish following the gel filtration chromatography procedure of Wu et al. [24]. Liver of exposed fish was dissected out and homogenized in 4 volumes of homogenizing buffer (10 mM Tris–HCl buffer, 5 mM 2-mercaptoethanol, pH 8.6). Homogenate was centrifuged at 15,000 × g for 40 min and supernatant was heated at 65 ◦ C for 10 min. Cooled supernatant was ultracentrifuged at 40,000 × g for 1 h. One millilitre cytosolic fraction was loaded onto a Sephadex G 75 column equilibrated with elution buffer (10 mM Tris–HCl, 0.02% NaN3 , pH 8.6) at the rate of 0.7 ml/min. MT containing fractions were monitored with UV detector at 254 nm and collected for atomic absorption spectrophotometer (AAS) analysis of cadmium content. 2.4.2. Metal detection in protein fractions Protein fractions eluted from Sephadex G 75 column were analyzed for metal content by graphite furnace atomic absorption spectrometer (AAS ZEEnit 65, AnalytikJena AG, Germany) using the procedure of Ahmad et al. [25]. Briefly, 1 ml of sample was acid digested with 5 volumes of 70% nitric acid at 120 ◦ C for 1 h. The samples were then cooled and 0.75 volume of H2 O2 was added. The digested samples were evaporated to dryness at 120 ◦ C. Finally, 5 volumes of 1% nitric acid was added to digestion vials and 20 ␮l sample was loaded on AAS for cadmium content analysis. The concentration of protein in loaded sample was 1.2 mg/ml. Thus, the loaded protein was 24 ␮g in 20 ␮l sample. 2.4.3. DEAE-Sephadex A 25 anion exchange chromatography All cadmium-containing fractions were pooled and loaded onto a DEAE-Sephadex A 25 anion exchange column (25 cm × 2 cm column). The column was eluted with a Tris–HCl linear gradient (from 250 to 600 mM Tris–HCl; flow rate 0.7 ml/min). The elutions were collected and dialyzed with double distilled water in 3500 MW dialysis bag for 36 h (changed every 12 h for two times). After lyophilization, the pellet was dissolved in 1 ml double distilled water. 2.4.4. Western blotting A low molecular weight SDS-PAGE system (12.5% acrylamide gel) was used for the molecular weight determination and western blot analysis of purified protein. Two gels (6 cm × 8 cm × 0.75 mm) were run at con-

stant voltage (100 mA at start and 60 mA at the end of electrophoresis). One gel was stained with Coomassie brilliant blue and destained. The other gel was used for immunoblotting using Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad, USA) following the instructions as provided by the manufacturers. Protein was transferred to PVDF membrane (BioRad) at 30 V, 4 ◦ C for overnight. The transferred membrane was cut into small pieces and proceeded for the detection of MT using antibodies. The membrane was coated with a blocking agent, 3% solution of bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 1 h at room temperature. Membrane was washed thrice with TBS and placed in primary antibody solution (rabbit anti-cod polyclonal MT, Cayman Chemicals Co., USA, 1:200) for 1 h. After washing with TBS, membrane was placed in secondary antibody solution (goat anti-rabbit IgG conjugated with HRP, Banglore Genei Pvt. Ltd., India, 1:1000) and incubated for 1 h at room temperature. After incubation, membrane was washed thrice and incubated with developing reagent (chloronaphthol solution; 30% H2 O2 ) and left for 5–30 min. Colour development was stopped by washing membrane with distilled water for 30 min with three changes. 2.5. Free radical scavenging activity •

2.5.1. Superoxide radical (O2 − ) Superoxide radicals were produced by the standard xanthine/xanthine oxidase (X/XO) reaction [26,27]. Different concentrations of Cd-MT (viz., 0.5, 1, 2, 5, 10, 20, 50, 100 ␮M) purified from fish liver were incubated with 1.49 ml of 65 mM potassium phosphate buffer (65 mM, pH 7.8), and 0.1 ml of 100 mM hydroxyl ammonium chloride. The reaction was initiated by the addition of 0.25 ml of xanthine oxidase (30 ␮g) and incubated at 25 ◦ C for 20 min (total volume 2 ml). After incubation, 0.5 ml of sulfanilic acid (0.03 mM) was added to 0.5 ml of incubation mixture which was again incubated at room temperature for 5 min followed by addition of 0.5 ml of ␣-naphthylamine. The absorbance was recorded at 530 nm. 2.5.2. ABTS radical assay The ABTS decolourisation assay was performed • using procedure of Re et al. [28]. ABTS + was generated by oxidation of ABTS (7 mM) with potassium persulfate (2.45 mM). Both ABTS and potassium persulfate were mixed in ratio of 2:1 and kept in dark at room temperature for 12–16 h. Three millilitres of ABTS cation solution were mixed with

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30 ␮l MT of various concentrations (100, 200, 300, 400, 500 ␮M) in 1 cm path length cuvette and the decrease of absorption was measured for 6 min at 734 nm. 2.5.3. DPPH radical assay • DPPH radical scavenging assay was carried out using method of Miliauskas et al. [29]. The solution of • DPPH in phosphate buffer (6 × 10−5 M) was prepared freshly, before UV measurements. Three ml of this solution were mixed with 77 ␮l MT of various concentrations (100, 200, 300, 400, 500 ␮M) in 1 cm path length cuvette. The samples were kept in dark for 15 min at room temperature and decrease in absorption was measured at 515 nm. Blank sample containing the same amount of • methanol and DPPH solution was prepared. Radical scavenging activity was calculated by using the following formula: Percentage inhibition =

AB − AA × 100 AB

where AB is the absorption of blank sample (t = 0 min) and AA is the absorption of test solution (t = 15 min). 2.6. Fe-NTA-induced DNA damage DNA damage was evaluated by agarose gel electrophoresis method of Cai et al. [30]. Fish genomic DNA was isolated from liver using QIAmp DNA isolation mini kit (QIAGEN® catalogue no. 51104, lot no. 11550244). Briefly, standard reaction mixture consisted of 20 mM chelex-treated phosphate buffer (pH 7.0), 66.4 ␮g/ml DNA, 200 mM Fe-NTA, 2 mM H2 O2 , and 2 mM sodium ascorbate. To measure the effect of MT on DNA damage, 50, 100, 150 mM fish liver CdMT was added to the reaction mixture after addition of Fe-NTA and before addition of both H2 O2 and ascorbic acid. Reaction was carried out at 30 ◦ C for 30 min. A total of 30 ␮l including 10 ␮g of DNA plus 3 ␮l loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol FF, 30% glycerol in water), were loaded onto agarose gel (1.0%) in TAE buffer (40 mM Tris–acetate, 1 mM EDTA) and subjected to electrophoresis in presence of ethidium bromide (0.5 mg/ml in TAE) for 2–3 h.

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Table 1 Cadmium content in different tissues of C. punctata Bloch Sample

Liver Kidney Muscles

Cadmium (␮g/gm tissue) Control

Cadmium-exposed

44.39 63.98 14.13

58.36 69.97 14.33

Percent increase (%) 31.47 9.36 1.41

Values are means of three samples.

3. Results 3.1. Cd-MT AAS analysis of liver, kidney and muscles of C. punctata showed highest cadmium concentration in liver (31.47% over control) followed by kidney (9.36% over control) and muscles (1.41% over control) (Table 1). Fig. 1 shows a single peak of protein fractions having maximum absorbance at 254 nm. Samples with maximum absorbance were analyzed for cadmium content by AAS and the observed peak is shown in Fig. 2. Cadmium containing samples were pooled and subjected to DEAE-Sephadex A 25 anion exchange chromatography. One major peak of protein containing fractions was eluted with Tris–HCl gradient (Fig. 3), that was subsequently analyzed for cadmium content (Fig. 4). The elution profile along with cadmium estimation of protein fractions by AAS showed that there was induction of single isoform of MT. Purified hepatic MT was resolved on SDS-PAGE to determine the molecular weight in denatured conditions. Samples showed presence of a single band of molecular weight 14 kDa as its migra-

2.7. Statistical analysis Data were expressed as means ± S.E.M. of 3–4 experiments.

Fig. 1. Gel filtration profile of hepatic cytosol fraction in fish exposed to cadmium chloride (0.2 mg/kg body weight, three doses on alternate days) purified over Sephadex G 75 packed column.

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Fig. 2. Cadmium content analysis of protein fractions by AAS collected from Sephadex G 75 packed column.

Fig. 5. SDS-PAGE analysis of Cd-MT. Lane A: molecular weight marker in kDa, lane B: protein fraction after gel filtration Sephadex G 75 and lane C is MT purified from anion exchange chromatography.

Fig. 3. The elution of MT fraction obtained from gel filtration on DEAE-Sephadex A 25. The separation was conducted with a linear gradient (250–600 mM) of Tris–HCl buffer (pH 8.6).

tion was closer to the low molecular protein marker band of 14.4 kDa and confirmed by “molecular weight determination programme” in gel documentation system (Fig. 5). Western blot analysis of MT fraction obtained from DEAE-Sephadex A 25 column and characterized on SDS-PAGE analysis showed a single isoform of MT in the liver of C. punctata as evidenced by the presence of a single band on PVDF blotting membrane (Fig. 6). 3.2. Free radicals scavenging activity of fish Cd-MT Fig. 7 shows the inhibition of nitrite formation by different concentrations of Cd-MT. The results show concentration dependent inhibition of nitrite formation. Cd-MT showed highest inhibition of nitrite formation by 83.1% at a concentration of 100 ␮M. While at 0.5 ␮M concentration, Cd-MT caused only 30.01% inhibition over control values. Hepatic Cd-MT also scavenged

Fig. 4. AAS analysis for cadmium content in protein fractions collected from DEAE A 25 anion exchange column.

Fig. 6. Western blot analysis of MT fraction obtained from DEAESephadex A 25 column and separated on SDS-PAGE analysis (lane A).

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Fig. 7. Cd-MT inhibits nitrite formation by scavenging superoxide radical in vitro at different concentrations. Values in parenthesis show the percentage values of nitrite inhibition. Values represent means ± S.E.M. of 3–4 experiments.

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Fig. 9. Percentage scavenging of DPPH radicals by Cd-MT. Values in parenthesis show the percentage values of nitrite inhibition. Values represent means ± S.E.M. of 3–4 experiments.

ABTS radicals as evidenced by a marked decrease in absorbance of reaction mixture at 734 nm. Cd-MT caused a pronounced decrease in absorbance demonstrating thereby free radical scavenging activity of Cd• MT (Fig. 8). It also efficiently scavenged DPPH radicals as indirectly measured by the decrease in absorbance of reaction mixture (Fig. 9). Cd-MT at a concentration of 500 ␮M caused decrease of 94.2% in the absorbance over control values. 3.3. Effect of Cd-MT on Fe-NTA-induced DNA damage Cd-MT caused inhibition of Fe-NTA-induced DNA damage (Fig. 10). The inhibition of DNA damage was highest at 100 and 150 ␮M concentrations of Cd-MT as evidenced by the electrophoretic migration of DNA on agarose gel.

Fig. 10. DNA damage. Lane A is control, lane B is DNA + Fe-NTA complex, lanes C–E is DNA + Fe-NTA with different concentrations of MT (50, 100, 150 ␮M, respectively).

4. Discussion

Fig. 8. ABTS radical inhibition by Cd-MT. Values represent means of 3–4 experiments.

Cadmium-specific metallothionein (Cd-MT) purified from liver of C. punctata scavenged three in vitro gener• • ated free radicals (superoxide, ABTS + and DPPH ) and

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prevented DNA damage induced by Fe-NTA complex in presence of H2 O2 . This demonstrates that fish MT like mammalian MT has a free radical scavenging activity. Free radical scavenging activity of mammalian MT is reported in several assay systems [12–14]. Mammalian MT have been reported to prevent Fe-NTA-induced DNA damage in vitro. Min et al. [18] have observed that mouse MT prevented Fe-NTA-induced rasDNA strand scission in vitro. Research of fish metallothioneins has centered mainly on its utility as biomarker of exposure. In a number of biomonitoring studies of aquatic polluted habitats, increased level of MT induction in fish and other aquatic organisms has been shown to correlate with level of heavy metal pollution [31–33]. However, except the notion that MT are stress proteins and are the products of stress response, biological significance and its chemo-biological interactions in fish have not been worked out. Cadmium and zinc are two most important metals capable of inducing MT in fish [34]. Of these, MT-inducing effects of cadmium have been extensively studied in fish [35–37]. We observed that three i.p. injections of cadmium chloride (0.2 mg/kg body weight) resulted in a significant induction of MT in liver in C. punctata. Although induction of MT is reported in other organs (brain and kidney in mammals and brain, gills and kidney in fish), studies concerning free radical scavenging activity of MT have primarily been conducted on hepatic MT [12]. In mammals it has been observed that over-expression of MT due to chemical exposure or gene transfer affords resistance to several forms of oxidative injury [38,39]. Moreover, animals with decreased MT levels, due to gene deletion, experience enhanced sensitivity to oxidative injury [38–41]. We have previously observed that fish pre-treated with cadmium prevented deltamethrin and endosulfan-induced peroxidative damage as measured by a number of robust biomarkers of oxidative stress [21,22]. Cd-MT induction was suggested to be one of the factors responsible for protection against peroxidative damage of the prooxidant toxicants as they have been reported to induce oxidative stress in fish [42,43]. MT isolated from liver of C. punctata showed free • radical scavenging efficacy in scavenging ABTS + , • DPPH and superoxide radicals in vitro. All the three radicals are produced by different pathways. There is some resemblance between MT gene of fish and mammals. Both the MT have triplicate structure of the gene and conservation of cysteine residues along with a TATAAA signal and two copies of metal-responsive elements (MREs) [44]. MT of fish and mammals are also identical to

a great extent in the number and position of cysteinyl residues [44]. MT contains many cys-x-cys and cys-cys sequences, and the position of these cysteinyl residues along the polypeptide chain is highly conserved [11]. The free radical scavenging and antioxidant properties of MT are attributed to these cysteine residues of MT [11,12,45]. As low as 50 ␮M of purified MT from the liver of C. punctata showed DNA damage reducing effect in Fe-NTA/H2 O2 assay in vitro suggesting its hydroxyl radical scavenging activity. The DNA was isolated from liver of C. punctata. The high rate of hydroxyl radical generation makes the Fe-NTA/H2 O2 system an excellent model for the study of DNA damage by hydroxyl radicals. Recently, Min et al. [46] have also shown that MT-enriched rat hepatocytes are resistant to Fe-NTA toxicity under GSH depletion. It has also been observed that MT in nuclei is capable of preventing deoxyribose degradation, DNA strand scission, and base modification induced by Fe-NTA/H2 O2 incubation [18]. In another in vitro study it was demonstrated that purified MT protects biological molecules, such as DNA from oxidation [12]. These results suggest that MT may act in the cell as a secondary antioxidant. Involvement of differential MT expression in free radical sensitivity of rainbow trout gonadal cell line (RTG-2) and the Chinook salmon embryonic cell line (CHSE-214) have been reported by Kling and Olsson [47]. They reported that cadmium and zinc pretreatment offered hydrogen peroxide resistance in RTG-2 and CHSE-214 cells via the induction of MT. Present study demonstrates the ability of fish hepatic MT in scavenging hydroxyl radicals in vitro. Findings of the present investigation demonstrated that fish MTs have a definite antioxidant and free radical scavenging activity and, like mammals, cysteine residue is the major contributing domain to this activity. Unlike mammals, fish are exposed to mixtures of pollutants including MT-inducing heavy metals and therefore there is a need to carry out systematic studies to evaluate outcome of synergistic and antagonistic biological and chemical interactions in fish from polluted habitats. The present study offers some insight into such a scenario. Detailed studies are needed to fully elucidate molecular interactions of free radicals with peptide residues of MT of C. punctata. Acknowledgement Financial support of Indian Council of Agricultural Research (ICAR), Government of India is acknowledged.

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