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Journal of Stress Physiology & Biochemistry, Vol. 5, No. 3, 2009, pp. 4-12 ISSN 1997-0838 Original Text Copyright © 2009 by Kumar, Kumar, Bora, Amb
ORIGINAL ARTICLE
Photosynthetic, biochemical and enzymatic investigation of Anabaena fertilissima in response to an insecticide-hexachloro-hexahydro-methano-benzodioxathiepine-oxide.
Kumar, Nirmal J.I*1, Kumar Rita N.2, Bora Anubhuti1 and Amb Manmeet Kaur 1
1P.G. Department of Environmental Science and Technology, Institute of Science and Technology for Advanced Studies and Research (ISTAR), Vallabh Vidyanagar -388 120, Gujarat, India. 2Department of Biosciences and Environmental Science, Natubhai V Patel College of Pure and Applied Sciences (NVPAS), Vallabh Vidyanagar -388 120, Gujarat, India.
E-mail: istares2005@yahoo.com
Received May 7, 2009
A study on the heterocystous, nitrogen fixing cyanobacterium, Anabaena fertilissima was carried out to investigate the effect of an organochlorine insecticide (hexachloro-hexahydro-methano-benzodioxathiepine-oxide, called as endosulfan) at different concentrations of 3, 6 and 12 ^gml-1 on the photosynthetic pigments-Chl-a, Carotenoids and Phycobiliproteins-phycocyanin, allophycocyanin and phycoerythrin, stress metabolites such as carbohydrates, proteins, amino acids, phenols and enzyme activities-nitrate reductase and glutamine synthetase. The insecticide- Endosulfan showed to be deleteriously affecting the activities in the cyanobacterium. As early as the 4th day, chl-a and carotenoids reduced by 38% and 20% respectively. The phycobiliproteins declined by 60%, 64% and 28% with respect to Phycocyanin, Allophycocyanin and Phycoerythrin. Moreover, Endosulfan adversely depleted the cellular activities, leading to a marked decrease in the carbohydrates, proteins, phenols and amino acids and enzymes-nitrate reductase and glutamine synthetase. Despite of deleterious effects of Endosulfan on the cyanobacterium Anabaena fertilissima, a unique regenerating ability in presence of the insecticide was observed by the end of 12 days in the lower doses of insecticide.
key words: Anabaena fertilissima / Endosulfan / Enzymes / Photosynthetic pigments / Stress metabolites.
ORIGINAL ARTICLE
Photosynthetic, biochemical and enzymatic investigation of Anabaena fertilissima in response to an insecticide-hexachloro-hexahydro-methano-benzodioxathiepine-oxide.
Kumar, Nirmal J.I*1, Kumar Rita N.2, Bora Anubhuti1 and Amb Manmeet Kaur 1 1P.G. Department of Environmental Science and Technology, Institute of Science and Technology for Advanced Studies and Research (ISTAR), Vallabh Vidyanagar -388 120, Gujarat, India.
2Department of Biosciences and Environmental Science, Natubhai V Patel College of Pure and Applied Sciences (NVPAS), Vallabh Vidyanagar -388 120, Gujarat, India.
E-mail: istares2005@yahoo.com
Received May 7, 2009
A study on the heterocystous, nitrogen fixing cyanobacterium, Anabaena fertilissima was carried out to investigate the effect of an organochlorine insecticide (hexachloro-hexahydro-methano-benzodioxathiepine-oxide, called as endosulfan) at different concentrations of 3, 6 and 12 ^gml-1 on the photosynthetic pigments-Chl-a, Carotenoids and Phycobiliproteins-phycocyanin, allophycocyanin and phycoerythrin, stress metabolites such as carbohydrates, proteins, amino acids, phenols and enzyme activities-nitrate reductase and glutamine synthetase. The insecticide- Endosulfan showed to be deleteriously affecting the activities in the cyanobacterium. As early as the 4th day, chl-a and carotenoids reduced by З8% and 20% respectively. The phycobiliproteins declined by 60%, 64% and 28% with respect to Phycocyanin, Allophycocyanin and Phycoerythrin. Moreover, Endosulfan adversely depleted the cellular activities, leading to a marked decrease in the carbohydrates, proteins, phenols and amino acids and enzymes-nitrate reductase and glutamine synthetase. Despite of deleterious effects of Endosulfan on the cyanobacterium Anabaena fertilissima, a unique regenerating ability in presence of the insecticide was observed by the end of 12 days in the lower doses of insecticide.
key words: Anabaena fertilissima / Endosulfan / Enzymes / Photosynthetic pigments / Stress metabolites.
Cyanobacteria are photosynthesizing and nitrogen fixing micro-organisms, which contribute significantly towards soil fertility and crop yield (Jha et al. 1999). Most paddy soils have a natural population of cyanobacteria which provides a
potential source of nitrogen fixation at no or low cost (Mishra and Pabby, 2004). A large number of pesticides are used in rice fields to protect the rice seedlings and crops, and selectively destroy the pests, but the indiscriminate use of pesticides causes great
danger to the rice field cyanobacteria and other beneficial micro-organisms (Da Silva et al., 1975). Under water logged conditions as commonly observed in rice fields, pesticides may induce many cellular disorders in cyanobacteria (Singh et al., 1986). Depending on the type and concentration of pesticides and composition of growth media, pesticides have been found to exert stimulatory, inhibitory or no effect on the growth and nitrogen fixing ability by cyanobacteria (Tamilselvam et al. 2002).
The toxicity of BHC, Lindane and Endrin towards nitrogen fixing cyanobacteria- Cylindrospermum sp., Aulosira fertilissima and Plectonema boryanum have been studied (Lakshmi and Annamalai, 2007). Study on the pesticides like Dimecron-100, Ekaluk-EC and Dithane revealed that they were more toxic at higher concentrations but could be noticed lesser toxic at lower concentrations to heterocystous cyanobacteria (Anand and Veerappan, 1980). It has been also studied that insecticide; carbofuran reduced the growth of cyanobacteria Oscillatoria sp., and Westiellopsis prolifica considerably (Ravindran et al., 2000).
A hexa-chlorinated insecticide, Endosulfan has a broad spectrum of activity and is widely used on different crops such as rice (Goebel et al, 1982). Owing to an extensive usage of Endosulfan in various tropical and subtropical countries to control the insect population, it also declines the growth of microflora including cyanobacteria considerably (Shetty et al., 2000). Present study investigates the effect of Endosulfan insecticide on the pigment contents (Chl-a, Carotenoids and Phycobiliproteins-phycocyanin, allophycocyanin and phycoerythrin), metabolites (carbohydrates, proteins, phenols, amino acids) and enzymes (nitrate reductase and glutamine synthetase) of the cyanobacterium Anabaena fertilissima.
Materials and methods:
Organism and culture conditions: for the present study, filamentous, nitrogen-fixing, heterocystous
cyanobacterium Anabaena fertilissima was used and raised to axenic culture. The culture was grown in BG-ii medium (pH 7.5) in the culture room at 25±2°C under 3000 lux light with a photoperiod of 14: 10 h. Exponentially grown cyanobacterial cells were used throughout the experiment. Each experiment was conducted in replicates of three and their ±SE values were calculated.
Endosulfan treatment: Endocel (35% EC,
Endosulfan (6, 7, 8, 9, 10, 10-hexachloro-1, 5, 5a, 6, 9, 9a-hexahydro-6, 9-methano-2, 4, 3-
benzodioxathiepine-3-oxide), an organochlorine insecticide manufactured by Excel Crop Care Ltd, Gujarat, India, was used for the present study. On the basis of a series of experiments for LC50, the effective doses of 3, 6 and 12 ^g ml-1 of Endosulfan were selected. Various concentrations (0, 3, 6 and 12 ^g ml-1) of Endosulfan were prepared from the stock solution (200 ^g ml-1) by dissolving it into the sterilized nutrient medium. Exponentially growing 2 ml of the culture was inoculated to each effective dose and made up to 20.0 ml. For each experiment, the solution of Endosulfan was freshly prepared.
Photosynthetic, biochemical and enzymatic response of Anabaena fertilissima to Endosulfan was studied at an interval of every four days up to sixteen days. At the end of every four days, the treated as well as untreated cultures were assayed for various characters including photosynthetic pigments-chlorophyll-a, carotenoids and phycobiliproteins-phycocyanin, allophycocyanin and phycoerythrin, metabolites like carbohydrates, proteins, amino acids, phenols and also enzyme-nitrate reductase and glutamine synthetase. Analytical grade (Merck Ltd, and Himedia Ltd, India) chemicals were used throughout the study.
Photosynthetic pigments measurement: Chlorophyll a and carotenoids were extracted in 80% acetone and determined spectrophotometrically by measuring the absorbance at 663, 630nm and 480 nm, respectively (Jeffrey and Humphrey, 1975; Parsons and Strickland, 1963). Phycobilin content was
extracted in 50mM potassium phosphate buffer (pH measured at 562, 615 and 652 nm, respectively
7.0) after repeated freezing and thawing and (Bennett and Bogorad, 1973).
(a) Chl-a, (b) phycocyanin, (c) allophycocyanin,(d) phycoerythrin, (e) carotenoids,
Fig: 2 shows the metabolic response of Anabaena fertilissima to the insecticide Endosulfan: (a) carbohydrates, (b) proteins, (c) amino acids, (d) phenols
t І
-0 dav
concentration of endosulfan (ug ml'1)
-4 dav -*-Sdav -X-12 dav -4-16 dav
0.1 -і 0.09 ■ ; 0.0S
І s 0 07 s f 0.06 ■ ;1 0.05 ■ I q 0.04 -I = 0.03 ■
I 0.02 ■ 0.01 ■ 0
control з б 12
concentration of endosulfan (ug ml1)
-0 dav
-4 dav
-S dav
-12 dav -t-16 dav
Fig: 3 shows the enzymatic response of Anabaena fertilissima to the insecticide Endosulfan: (a) nitrate reductase, (b) glutamine synthetase
Biochemical estimation: The total carbohydrates, proteins, amino acids and phenols were extracted in 80% ethanol. Carbohydrates were estimated by the method of Hedge and Hofreitte (1991) using sucrose as a standard. Protein estimation was done by Lowry et al (1951) with Bovine Serum Albumin (BSA) as the standard. The total amino acids were measured using ninhydrin reagent (Lee and Takahanshin, 1966). Phenols estimation has been carried out as per Malick and Singh (1980).
Enzyme assays: Nitrate reductase enzyme was extracted using cysteine buffer (pH 8.8). The enzyme activity was assayed spectrophotometrically following the protocol given by Sempruch et al, (2008). Glutamine synthetase enzyme was extracted in Tris HCl buffer (pH 7.5). The enzyme assay was determined by the Mn2+ y-glutamyl transferase activity and estimated by a slight modification of the method described by Yuan et al, (2001). The end product y-glutamyl hydroxamate formed was measured spectrophotometrically at 540 nm and represented in optical density readings.
Results and discussion:
Pigments:
The cyanobacterium Anabaena fertilissima showed inhibitory growth response against the insecticide endosulfan. The inhibitory effect of endosulfan on photosynthetic pigments of A. fertilissima was found to be dose dependent (Figures 1a-e). Within 4 days of treatment a low concentration (3 ^g ml-1) of insecticide reduced Chl a and carotenoid contents by 38% and 20% respectively. Moreover, the phcobiliproteins also were found to decrease significantly with respect to increasing endosulfan concentrations as well as an increase in days of exposure The lowest concentration of 3 ^g ml-1 reduced the Phycocyanin, Allocphycocyanin and Phycoerythrin content by 16, 18 and 14 % respectively. The declining trend in the pigment
contents continued with the rising concentration of
JOURNAL OF STRESS PHYSIO
insecticide as 12 ^g ml-1 of endosulfan sharply lowered chlorophyll a and carotenoid contents by 58% and 50% respectively. Phycocyanin, Allocphycocyanin and Phycoerythrin also reduced significantly by 60%, 64% and 28% respectively. Such decrease in chlorophyll a, carotenoid and phycobilin contents may be ascribed to the inhibition of pigment synthesis directly by the insecticide or accelerated degradation of pigments. However, by the end of the 12th day the cultures began to show signs of a change in the colour turning from yellow to green. Moreover, the observation was further supported by pigment analysis where 12 ^g ml-1 of insecticide after 12 days of exposure exhibited less reduction in Chl-a and carotenoid content (32% and 36 % respectively), thus showing a significant recovery in the levels of chlorophyll a and carotenoids. Prasad et al (2005) also quoted similar observations while studying on the growth, photosynthesis, active oxygen species and antioxidants responses of paddy field cyanobacterium Plectonema boryanum to Endosulfan stress.
Metabolic changes:
Total carbohydrate content showed drastic reduction with increasing concentrations of Endosulfan (fig. 2a). The reduced carbohydrate levels ranged from 3% to 39% by the end of the fourth day when exposed to 3, 6 and 12 ^g ml-1 of the insecticide. Towards the end of 16 days of endosulfan exposure, carbohydrate levels dropped down to 41% when treated with 12 ^g ml-1of the insecticide. These results have been well corroborated with those of Kumar et al, (2008). The retardation in the carbohydrate content might be due to interference of chemicals with photosynthesis process (Padhy, 1980).
Protein content also exhibited decreasing trend
with increasing exposure days and Endosulfan
concentration (Fig. 2b). The protein content reduced
as low as 85% by the end of 16 days when treated
with 12 ^g ml-1of endosulfan. The results were & BIOCHEMISTRY Vol. 5 No. 3 2009
further confirmed by Shehata et al (2001). Thiel (1990) emphasized that decrease in protein content in Anabaena variabilis was observed in starved cells.
Similarly, Amino acid content reflected a significant increase in the percent reduction with respect to increasing concentrations and days of exposure. Amino acid percent reduction increased consistently ranging from 10% to 83% with increasing endosulfan concentration and exposure time (days) (Fig. 2c). Soliman et al. (1993) reported that the synthesis of some major amino acids depended on the provision of carbon skeleton from TCA cycle, which can be indirectly affected by the herbicide.
In contrast to other metabolites, phenol content in respective treated cells as compared to the control showed a relative decrease in the percentage reduction until the 8th day (Fig. 2d). However, from the 12th day onwards, phenol reduction level expressed an increase with respect to higher concentrations of the insecticide as well as exposure periods. The percentage reduction in case of phenols increased up to 92% by the end of the 16th day when treated to 12 ^g ml-1. Mallick and Rai (1994) also substantiated the findings earlier that phenol could be used as protectants to the organisms during stress or drought conditions.
Enzyme assays:
Nitrate reductase (NR) enzyme declined radically from 32% - 55% by the 4th day (Fig. 3a). A 76% reduction in the nitrate reductase activity was estimated for an increased insecticide concentration (12 ^g ml-1) when incubated for a period of 16 days. The decrease in the nitrate reductase enzyme also simultaneously indicated a fall in the nitrogen fixing ability of Anabaena spp. Adhikary et al, (1984) studied the effect of carbamate insecticide Sevin on the growth, survival and nitrogen fixation of Anabaena spp. and Westiellopsis prolifica stated that higher concentrations of the insecticide showed an inhibitory effect on the NR.
Glutamine synthetase (Fig. 3b) determined a consistent decrease with raise in insecticide concentrations and exposure (days). The percentage reduction ranged from 3% - 21% and 61% - 76% for respective concentrations by the 4th day and 16th day respectively. The depletion in glutamine synthetase activity in response to pesticides has also been reported by Rajendran et al, (2007).
Conclusion:
The present study revealed that Endosulfan-6, 7, 8, 9, 10, 10-hexachloro-1, 5, 5a, 6, 9, 9a-hexahydro-6, 9-methano-2, 4, 3-benzodioxathiepine-3-oxide treatments adversely affected the photosynthetic pigments (Chl-a, Carotenoids and Phycobiliproteins-phycocyanin, allophycocyanin and phycoerythrin), stress metabolites (carbohydrates, proteins, amino acids and phenols) and enzyme activities (nitrate reductase and glutamine synthetase) in the isolate, Anabaena fertilissima even at concentrations as low as 3 ^g ml-1. However, the cells also displayed a unique regenerating ability against the insecticide Endosulfan towards the end of the 12th day in the lower doses.
Acknowledgements: Authors thank the University Grants Commission (UGC), New Delhi for financial assistance in favour of the study.
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