Moirangthem Kameshwor Singh JaiGopal Sharma Rina Chakrabarti

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1 Agri Res (June 2013) 2(2): DOI /s z FULL-LENGTH RESEARCH ARTICLE Impat of UV-B Radiation on the Physiology of Freshwater Carp Laeo rohita Larvae and Evaluation of UV-B Protetive Properties of Seeds of Ahyranthes aspera and Vitamin C Moirangthem Kameshwor Singh JaiGopal Sharma Rina Chakraarti Reeived: 11 August 2012 / Aepted: 2 April 2013 / Pulished online: 28 May 2013 Ó NAAS (National Aademy of Agriultural Sienes) 2013 Astrat Effet of harmful UV-B radiation and their remedial measures have een studied in Laeo rohita (rohu) larvae. Larvae were fed with four different diets: D1 and D2 ontained 0.1 and 0.5 % Ahyranthes aspera seeds, respetively; D3 ontained 800 mg vitamin C/kg diet, and D4 ontained ontrol diet. After 50 days, all groups were exposed to UV-B radiation (80 lw/m 2 ). One ath of ontrol diet-fed fish was exposed to UV-B (D4Ex), and the other group remained unexposed (D4UEx). Survival rate of rohu was signifiantly (P \ 0.05) higher in D2 when ompared to others. Among the exposed groups, highest average weight was reorded in D2 (103 ± 4 mg). Total tissue protein onentration was higher in exposed groups ompared to the unexposed one. Glutami oxaloaeti transaminase (GOT) and glutamate pyruvate transminase (GPT) levels were minimum in unexposed ontrol diet-fed fish. Vitamin C provided protetion against lipid peroxidation whih was evident from the lowest level of thioarituri aid reative sustane in exposed fish (D3). Signifiantly (P \ 0.05) higher nitri oxide synthase level in fish fed with Ahyranthes aspera seeds (0.5 %) supplemented diet showed the immunomodulatory property of the seeds. A omination of vitamin C and seed may e more UV-B protetive for arp larvae. Keywords Rohu UV-B radiation Survival Immunomodulation Ahyranthes aspera Laeo rohita Introdution Ultraviolet (UV) radiation is a potent environmental stressor to aquati organisms that are exposed to it. In freshwater eosystem, UV radiation an easily penetrate as the water is transparent and onsequently the effet of radiation is more pronouned [26]. The harmful effet of UV-B inludes damage that ompromises the physiology, iohemistry, reprodution, and growth of the exposed animals [1, 19, 25, 37]. The early developmental stage is more prone to UV-B damage as the less pigmented skin is very transparent, and therefore, vulnerale to the harmful radiation. The emryos M. K. Singh R. Chakraarti (&) Aqua Researh La, Department of Zoology, University of Delhi, Delhi , India aquaresearhla@yahoo.o.in J. Sharma Department of Biotehnology, Delhi Tehnologial University, New Delhi, India and larvae of many speies are more sensitive to environmental stressors like UV-B ompared to their later life stages [2, 7]. Sharma et al. [33] reported that exposure of UV-B affeted the survival rate of atla, Catla atla larvae under laoratory onditions. UV-B radiation affeted the skin and eyes of ayu, Pleoglossus altivelius larvae, and gills of atla larvae [31, 32]. In reent years, aquaulture industry is suffering from aute disease prolem. In India, arp seed is produed mainly in nursery with optimum water depth, and sunlight shows inreasing trend during reeding season (espeially in May June). Therefore, the larvae are more prone to the exposure of harmful UV-B radiation. UV-B exposure indues pronouned immunomodulation in yprinids [22]. The digestive physiology and immune system of atla were affeted y UV-B radiation [34]. Biologial protetion from UV-B may e ahieved y speifi responses, like the intensifiation of melanin pigmentation and antioxidant defenses, and y improvement of ell-repair mehanisms [4, 7, 11, 38]. However, these responses give only inomplete protetion and UV-B

2 Agri Res (June 2013) 2(2): may ause moleular damage to lipids, proteins, and nulei aids, esides induing oxidative stress on living organisms [18, 19]. Photoprotetive pigments (MAAs, arotenoids), antioxidant defense ompounds (flavonoids, vitamins C and E), and ell yle development genes are some moleules involved in UV radiation defense [10]. We have oserved the damaging effet of UV-B on freshwater arp larvae in our earlier studies [31, 33, 34]. Dietary supplementation of antioxidant vitamin C and plant ingredient may e enefiial for fish larvae to fae the hallenge of exposure of harmful UV-B irradiation. Ahyranthes aspera, a her elonging to the family Amaranthaeae has showed immunostimulatory effet in arps [5, 28]. Laeo rohita (Family: Cyprinidae), ommonly known as rohu, is an omnivorous speies, ontriuting onsideraly in Indian aquaulture prodution. Outreak of disease is reported in this speies. The present investigation was arried out to study the effet of dietary supplementation of seeds of Ahyranthes aspera and vitamin C(L-asorate-2-triphosphate alium salt) separately on the physiology of UV-B-irradiated rohu larvae. The aility of larvae to fae the hallenge of UV-B radiation was evaluated. Materials and Methods Experimental Diets Four different diets were prepared for the experimental trial (Tale 1). Two diets D1 and D2 ontained 0.1 and 0.5 % Ahyranthes aspera seeds, respetively; diet 3 (D3) ontained 800 mg vitamin C (L-asorate-2-triphosphate alium salt, Himedia Laoratory Pvt. Ltd., Mumai, India)/ kg of diet, and diet 4 (D4) was ontrol diet without seeds and vitamin C. Food was given at the rate of 5 % of ody weight throughout the study period. Culture of Fish and Exposure to UV-B Radiation Larvae of Laeo rohita, rohu were otained from Chatterrjee Brothers fish farm, Mogra, West Bengal having indued reeding faility. Larvae (1.0 ± 0.01 mg) were alimated for 10 days, and then randomly distriuted in glass aquarium (15 l eah) kept under indoor ondition. The stoking density was 50 larvae/aquarium. Feeding with experimental diets started from day 11. After 50 days of ulture, fish were hallenged with UV-B radiation. The dose was 80 lw/m 2 [31]. All fish fed with vitamin C and seed-inorporated diets were exposed to the UV-B radiation, whereas, ontrol diet-fed fish were sudivided into two athes one ath was exposed to UV-B (D4Ex) and the other remained unexposed (D4UEx). Four repliates (aquaria) were used for eah treatment. Fish were exposed to UV-B radiation every day at p.m. for 10 min, and this was ontinued for 40 days. Total duration of experiment was 90 days. Then the experiment was terminated, survival rate was reorded, and weight of individual fish was measured. Dehlorinated, transparent water was used and the depth of water in the aquarium was 20 m. During experiment, different physio-hemial parameters like temperature, ph, and dissolved oxygen level were monitored regularly. Water temperature and ph ranged from 30 to 31 C and 7.5 to 8.1, respetively, throughout the study period. Dissolved oxygen level was always maintained aove 5 mg/l with the help of aerators. The soure of UV-B ( nm) was a Philips tue light (TL 20/12 RS, Holland) fixed aove eah aquarium. All tues were preurned for 100 h to give a stale output. The spetral output of the tues, as defined y the manufaturer has the maximum emission at 313 nm, with negligile energy aove 320 [3]. UV-B tues were overed with ellulose aetate whih asors wavelength of \280 nm. Floresent lamp (Philips 20 W) was used as amient light soure for oth treated and ontrol fish. The UV-B radiation was measured y Mirotops II, Sunphotometer, Solar Light Company, Philadelphia, USA. Biohemial Assay Fish were euthanized, tissue was olleted without sales, head, tail, and ones from individual fish. Tissue sample Tale 1 Composition of experimental and ontrol diets Ingredients (g/kg) Experimental diet Control diet D1 D2 D3 D4 Dry fish powder Wheat flour Cod liver oil Vitamin mineral premix Ahyranthes aspera seeds Vitamin C D1 diet ontained 0.1 % seeds, D2 diet ontained 0.5 % seeds, D3 diet ontained vitamin C, D4 ontrol diet

3 168 Agri Res (June 2013) 2(2): otained from eah aquarium was pooled. Then, the samples were preserved at -80 C. Tissue (100 mg) was homogenized with 1 ml of hilled phosphate uffer; then spun down at 10,0009g for 10 min, and supernatant was immediately used for assay. Four repliates were used for eah assay. Total tissue protein was measured aording to the method of Lowry et al. [21]. The asorane was reorded at 750 nm using Miroplate Reader (BioTek, Synergy HT, New York, USA). Tissue glutami oxaloaeti transaminase (GOT) and glutamate pyruvate transminase (GPT) were assayed using diagnosti kits (Siemens Heathare Diagnostis Ltd., Baroda, India). The asorane was reorded at 340 nm. GOT and GPT levels were expressed as IU/l. Thioarituri aid reative sustane (TBARS) was measured aording to the method of Ohkawa et al. [27]. The asorane was measured at 532 nm. The standard was prepared using 1,1,3,3-tetramethoxy propane, and the result was expressed as lmol MDA/mg protein. Nitri oxide synthase (NOS) was measured aording to the method of Lee et al. [17]. The asorane was reorded at 540 nm. The nitrite onentration was determined from the nitrite standard urve and was expressed as mol/mg tissue. Speifi Growth Rate (SGR) The speifi growth rate was alulated using the formula: SGR ¼ ðinw t InW i Þ=t 100 where W i and W t were the initial and final ody weight and t, the time in days. Statistial Analysis Results are presented as mean ± SE. Data were analyzed using one way analysis of variane (ANOVA) and Dunan s multiple range (DMR) test [24]. The signifiane level was aepted at P \ Results Survival rate of rohu was signifiantly (P \ 0.05) higher in 0.5 % Ahyranthes aspera seed-supplemented diet fed (D2) exposed group ompared to the other exposed and unexposed groups (Tale 2). This group was followed y 0.1 % seed-supplemented diet fed one. Among the exposed groups, the highest average weight was reorded in D2 diet-fed fish (103 ± 4 mg), ut there was no signifiant (P [ 0.05) differene in the average weight etween D2 and D3 diet-fed rohu. The average weight of ontrol diet-fed unexposed fish (D4UEx) was % higher ompared to the 0.5 % seed-inorporated diet (D2) fed exposed fish. But the average weight of the latter group was 12 % higher ompared to the ontrol dietfed exposed fish (D4 Ex). SGR value was maximum in D4 UEx and D2 treatments (Tale 2). Total tissue protein onentration was higher in all exposed fish (regardless of diets supplied) ompared to the unexposed ontrol diet-fed group (Tale 2). Tissue protein was maximum in 0.5 % seed-inorporated diet (D2) fed group (0.111 ± mg/ mg tissue). Among the exposed fish, there was no signifiant (P [ 0.05) differene in the onentration of tissue protein etween 0.5 % seed- and vitamin C-supplemented diet-fed groups, and etween 0.1 % seed-supplemented and ontrol diet-fed exposed groups. Supplementation of vitamin C and seed of Ahyranthes aspera (0.5 %) in diets enhaned the tissue protein level. GOT and GPT levels were minimum in unexposed ontrol diet-fed fish. There was no signifiant (P [ 0.05) differene in the GOT level among the UV-B exposed treated and ontrol diet-fed rohu (Fig. 1). Among the exposed fish, GPT level was maximum and minimum in vitamin C- and 0.5 % seed-supplemented diet-fed groups, respetively. TBARS level was signifiantly (P \ 0.05) lower in vitamin C-supplemented diet-fed fish ompared to the other groups. Among the UV-B-irradiated fish, two to sevenfold higher TBARS level was found in ontrol dietfed fish ompared to others (Fig. 2a). NOS level was signifiantly (P \ 0.05) higher in 0.5 % seed-inorporated diet (D2)- fed fish ompared to other groups (Fig. 2). This was to 2.8-fold higher in D2 diet-fed fish ompared to the other groups. Disussion The environmental stressor UV-B radiation disturs the physiology of fish. Sustanes having UV-B protetive properties may help to overome the harmful effet of irradiation. In the present study, the UV-B protetive role of seeds of Ahyranthes aspera is very lear. The survival rate was higher in two plant-inorporated diet-fed UV-B hallenged rohu ompared to the other treatments, even higher than that of the unexposed ontrol diet-fed group. Similar result was found y Rao et al. [29] in rohu hallenged with Aeromonas hydrophila. Supplementation of seeds of Ahyranthes aspera inreased survival rate and growth of rohu. The presene of a numer of oleoni aids, edysterone, and various amino aids [14] onfirmed the nutritional quality of seeds. Ahyranthes aspera seeds ontained protein with alorifi value of 3.92/g. The amount of leuine, isoleuine, phenylalanine, and valine were similar to the Bengal gram, while tryptophane, methionine, and ystine ontents were

4 Agri Res (June 2013) 2(2): Tale 2 Effets of seeds of Ahyranthes aspera and vitamin C on the survival, average weight, SGR and total tissue protein level of UV-B irradiated Laeo rohita larvae Diets Parameters D1 D2 D3 D4Ex D4UEx Survival rate (%) 79.5 ± ± 1.29a ± 4.50e 70 ± 3.74d 74.5 ± 2.06 Average weight (mg) 92.0 ± ± ± ± ± 18.6a SGR (%) 5.02 ± ± 0.05a 5.09 ± ± ± 0.15a Total protein (mg/mg tissue) ± ± 0.009a ± 0.008a ± ± Mean (n = 4) sharing different letters in the same olumn are signifiantly (P \ 0.05) different D1 diet ontained 0.1 % seeds, D2 diet ontained 0.5 % seeds, D3 diet ontained vitamin C, D4 ontrol diet (Ex UV-B exposed, UEx UV-B unexposed groups) Conentration (IU L -1 ) GOT GPT a a a a a D1 D2 D3 D4Ex D4UEx Treatments Fig. 1 GOT and GPT levels found in UV-B-irradiated rohu larvae fed with seeds of Ahyranthes aspera and vitamin C-supplemented diets. Vertial ars with different supersripts are signifiantly (P \ 0.05) different higher than the most pulses [15]. Chakraarti et al. [6] reported that the presene of edysterone in the raw seeds of Ahyranthes aspera enhaned the growth rate of Cyprinus arpio ommon arp. Similarly, in the present study, edysterone served as the growth-stimulating omponent for rohu larvae. In this study, vitamin C also enhaned the growth of rohu. Vitamin C is an essential nutrient for optimum growth and maintenane [9, 13, 16, 20]. Total tissue protein onentration was higher in exposed fish fed with seeds (0.5 %) and vitamin C. Asori aid plays important role in ertain aspets of protein metaolism [8, 35]. It ats as a ofator in the enzymati hydroxylation of proline residues of ollagen and onnetive tissue of verterates. It enhaned the growth of parrot fish Oplegnathus fasiatus [39]. Higher levels of GOT and GTP aount for the damage level of tissue. Signifiantly higher levels of GOT and GPT were reorded in UV-B-exposed atla larvae [34]. Similar result was found with rohu in the present investigation. Low GPT level found in 0.5 % seed-inorporated diet-fed fish indiated that seeds helped the fish to fight against the TBARS (µmol MDA mg -1 protein) Nitri oxide synthase (mol mg -1 tissue) 350 (a) () a d irradiation. Vitamin C was not found to e effetive in reduing GOT and GPT levels in UV-B-irradiated rohu. Higher level of TBARS is an indiator of lipid peroxidation. In the present investigation, among the UV-Birradiated groups, signifiantly (P \ 0.05) higher level of TBARS was reorded in ontrol diet-fed fish ompared to others. Vitamin C provided highest protetion against lipid peroxidation to the UV-B-irradiated rohu. Seeds of Ahyranthes aspera (0.5 %) also provided protetion to the fish. Fish tissue ontains large quantities of polyunsaturated fatty aids (PUFAs) essential for memrane funtion [23]. Lipid peroxidation is the proess of oxidative degradation e D1 D2 D3 D4Ex D4UEx Treatments D1 D2 D3 D4Ex D4UEx Treatments Fig. 2 (a) Effet of UV-B radiation on thioarituri aid reative sustane (TBARS) and () nitri oxide synthase (NOS) levels of rohu larvae fed with seeds of Ahyranthes aspera and vitamin C-supplemented diets. Vertial ars with different supersripts are signifiantly (P \ 0.05) different d a

5 170 Agri Res (June 2013) 2(2): of PUFA, and its ourrene in iologial memranes auses impaired memrane funtion, strutural integrity and inativation of several memrane-ound enzymes [12]. Vitamin C is a potent antioxidant and protets tissues from oxidative stress. Loading ells with vitamin C redue oxidative ell death, inhiits FAS-indued apoptosis and onfers genomi protetion through the quenhing of intraellular reative oxygen speies [30]. High level of NOS in fish fed with 0.5 % Ahyranthes aspera seed-supplemented diet showed the stronger defense system of rohu hallenged with Aeromonas hydrophila [36]. The present investigation with UV-Birradiated rohu onfirmed the earlier study. NOS are a family of enzymes that atalyze the prodution of ellular signaling moleule nitri oxide. This nitri oxide plays vital role in many iologial proesses. The induile isoform inos produes a large amount of nitri oxide as a defense mehanism. The presene of long-hain polyunsaturated fatty aids suh as linoleni and olei aids have een reported in the seeds of Ahyranthes aspera. The immunostimulating effet of seeds may e due to the presene of essential fatty aids [6]. Conlusions The results showed that feeding of seeds of Ahyranthes aspera at 0.5 % level helped rohu larvae to fae the hallenge of UV-B irradiation. Higher survival and growth of larvae are positive indiators of the health status of the larvae. Vitamin C is ale to protet the ells from oxidative stress during UV-B irradiation. A omination of vitamin C and seeds may e more UV-B protetive for arp larvae. Aknowledgments This study was supported y a grant from the Department of Biotehnology, DBT, New Delhi to RC. KMS is thankful to the University of Delhi for giving him the UGC Researh Fellowship as Siene Meritorious Students. Conflit of interest of interest Referenes The authors delared that they have no onflits 1. 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