Evaluation of Seeds of Celastrus Paniculatus Willd. – a Potential Medicinal Plant of Pharmacopoeia

Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 1-2

Evaluation of seeds of Celastrus paniculatus Willd. – A potential medicinal plant of Pharmacopoeia

Kaushal Kumar, S. G. Abbas and M.H. Siddiqui Faculty of Forestry, Birsa Agricultural University, Kanke, Ranchi-834006 Email: [email protected]

Abstract : Celastrus paniculatus Willd. (Celastraceae) is a potential medicinal plant extensively used in ayurvedic medicinal systems. The seeds oil of these medicinal plants is used for the treatment of large numbers of diseases. The seeds of this plant were collected from four regions of Jharkhand state and the various constitution and oil were compared with the parameters described in the Ayurvedic pharmacopoeia of Govt. of India. The percentage of oil contents of Celastrus paniculatus Willd. ranges from 43.2 to 44.67 % while the contents of the oil mentioned in pharmacopeia is 45%. The similarities in other contents like foreign matter, total ash, acid insoluble ash, alcohol soluble extract and water soluble extract too is identical. It is, therefore, suggested that the Celastrus paniculatus Willd. should be protected and awareness should be generated amongst public private sector for the production of seed oil at cheap rate for medicinal use. Keywords: Celastrus paniculatus, Seed oil, Pharmacopoea, Quality evaluation

Introduction Methodology of works Celastrus paniculatus Willd. is a large climbing The seeds of Celastrus paniculatus Willd were deciduous shrub, yellowish wood, with lenticelled collected from four different places from the district of branches. It belongs to family Celastraceae. The plant Ranchi (Jonha), Gumla (Bishunpur), Dumka (Baghraidih) mostly occurs in sub Himalayan tract up to 6000 ft. It is also and Pakur (Satia) and in Jharkhand state. They were found in Central India, Western and Eastern Ghats. It is collected with the help of native people in the months of extending from Rajmahal hills and Chhotanagpur plateau December- January. The above seeds sample were brought of Jharkhand and Orissa states. It is one of the potential to laboratory and dried in an oven at 45o C to 50oC. The medicinal plants of India. The plant is known in Sanskrit powdered of the dried seeds was made at 25oC and kept in and ayurveda as 'Jyotishmati'. It is principally a 'Medhya airtight container. The identity, purity and strength of drug'. In ayurvedic medicine it is used as a brain tonic and samples of seeds have been evaluated according to the also used in treatment of some the nervous disorders. The methods upon which the standards of Pharmacopoeia plant is utilized as ethnomedicine among the tribal people depend. and they call it 'Kujri' 'Malkangni', 'Konjri' 'Kusur', 'Rangud' etc. The seeds oil is utilized topically as well as Results internally among the tribal people ((Jain, 1991). It is also The samples were collected from four sites of used for bodyache, rheumatism, leprosy, eczema and skin Ranchi (Jonha), Gumla (Bishunpur), Dumka (Baghraidih) diseases (Asolkar et al. 2005; Atal et al., 1978 ; Handa, and Pakur (Satia) designated as SS-1, SS-2, SS-3 and SS-4 1988; Rekha et al., 2005). According to Bhanumathy et al, respectively. The % of foreign matter, the total ash, acid (2010). the seeds have great potentials as acrid, bitter, soluble ash, alcoholic soluble extract, water-soluble and oil thermogenic, emollient, stimulant, intellect promoting, contents from sample of Ranchi (Jonha, SS-1), Gumla digestive, laxative, emetic, expectorant, appetizer, (Bishunpur, SS-2), Dumka (Baghraidih, SS-3) and Pakur aphrodisiac, cardio tonic, anti-inflammatory, diuretic, (Satia, SS-4) were presented in Table1. The seeds were diaphoretic, febrifuge and tonic, abdominal disorders, separated in the laboratory and the color and identical seeds leprosy, skin diseases, paralysis, cephalalgia, arthralgia, in structure and diameter were recorded (Fig.1). The seeds asthma, leucoderma, cardiac debility, inflammation etc. were processed for the foreign matter, total ash, acid The oil contains fatty acids composition, sesquiterpene insoluble ash, alcohol soluble extract, water soluble extract alkaloid viz. celapanin, celapanigin and celapagin and a and oil contents were studied and presented in Table 1. The number of sesquiterpene esters namely malkanguine- I to table also contains the data available in Ayurvedic VIII and sterol. The present work is based to confirm the Pharmacopoea for comparison to know the purity and contents of quality of seeds and their oil of Celastrus strength of the contents of the seeds obtained from four paniculatus useful for the use of illness as reported by locations of Jharkhand. It has been observed that all Department of AYUSH, Ministry of Health and Family parameters are similar with the reported parameters in the Welfare, Govt. of India, New Delhi. Ayurvedic pharmacopoea.

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Discussion to 45% described in Ayurvedic Phamacopoea, Government of India. The above studies have great significant towards In the present investigation, genetic resources of understanding of seed samples of the plant occurs in Celastrus paniculatus available in Jharkhand has almost Jharkhand state for bioprospection in perspectives to standard in quality for the extraction of oil contents for the pharmaceutical standards. The above data analyzed in the preparation of ayurvedic medicine at a cheaper cost as the present work is most useful for manufactures of herbal contents of oil of this plant is identical with the contents as drugs and other products based on the Celastrus per Ayurvedic Pharmacopoeia. The mean of samples taken paniculatus. from four different places is about 44.92, which is similar

Table 1. : Identity, purity and strength of samples of seeds

Parameters/ Foreign Total Acid- Alcohol soluble Water soluble Oil contents Samples Matter ash insoluble extractive extractive (%) (%) (%) (%) Ash (%) (%) Ayurvedic 2.0 6.0 1.15 20.0 9.0 45.0 Pharmacopoea SS-1 2.1 5.1 1.16 18.3 9.1 44.1 SS-2 1.9 5.2 1.17 19.2 8.95 43.2 SS-3 2.2 5.4 1.10 19.6 8.73 44.4 SS-4 1.8 6.1 1.14 19.5 8.5 44.67 Mean 2.0 5.7 44.92

Acknowledgements The authors are grateful to the native peoples of different districts of Jharkhand who have provided the samples of seeds used in the evaluation of present works.

Fig-1. : Seeds of Celastrus paniculatus

References Journal of Pharmaceutical Sciences and Drug Research 2 (3): 176-181 Asolkar L.V., Kakkar K.K. and Chakre O.J. (2005): Second supplement to Glossary of Indian Medicinal Plants Handa S.S. (1988): Indian Herbal Pharmacopoeia with active principles, NISCARE, New Delhi.188- Celastrus paniculatus, IDMA, Mumbai and RRL, 189. Jammu, 1998, II: 26-34. Atal C.K, Srivastava J.B., Wali B.K., Chakravarthy R., Jain S.K. (1991): Dictionary of Indian Folk Medicine and Dhawan B.N. and Rastogi R.R. (1978): Screening of Ethnobotany, (Deep Publication, New Delhi), 48. medicinal plants for Pharmacological activity Part III Rekha K., Bhan M .K, Balyan S.S. and Dhar A.K. (2005): Indian Journal of Experimental Biology 16: 330-349. Cultivation prospects of endangered species Celastrus Bhanumathy M., S. B. Chandrasekar S.B., Chandur U, paniculatus Willd. Natural Product Radiance Somasundaram T. (2010): Phyto-pharmacology of 4(6):482- 486. Celastrus paniculatus: An Overview. International

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Foraminiferal studies along the littoral zone of the east coast of India and its zoogeographical affinity: an overview

P. K. Kathal and V. K. Singh Centre of Advanced Study in Geology Dr. Harisingh Gour University, Sagar 470003, India

E-mail : [email protected]

Abstract: An overview of the studies, mostly related to the taxonomy, ecology of the littoral Recent foraminifera (Protozoa) and the zoogeographical affinity of the east coast of India has been attempted on the basis of the published literature. The study shows that the Recent foraminifera along the east coast of India show affinity with the Indo-Pacific faunal province. Keywords: Foraminifera, Taxonomy, East coast beaches, India

Introduction coast of India has been given. Foraminifera, the shelled protozoans thrive as the So far as the beaches of 5800 km long coastal most diverse group in the modern oceans. They derived stretch of the east coast of India are concerned, a total of 27 their name from 'foramen' the 'pores' connecting the papers have been published (Table 1) covering taxonomy, chambers in their tests. Varying in size/diameter size from ecology, geographical distribution of the Recent less than a millimetre (micro-foraminifera) to 100 mm foraminifera and establishing the zoo-geographical (mega-foraminifera they also occur as fossils in the marine affinity of the east coast of India based on the Q-mode Phanerozoic rocks (560 Million Years to Recent) and a cluster analysis (Kathal, et al., 2000 and Kathal and Bhalla, wide range of applications in geological, biological, 2001). Besides, paleoenvironment/climate studies (Kathal, environmental and oceanographic studies. 1996 and Kathal and Bhalla, 1996a) show their utility in petroleum exploration. Some of foraminifera are agglutinated that acquire foreign-material to make a test, but most of them Taxonomic and ecological studies form calcareous 'test' (shell) by secreting calcium The taxonomic and ecological studies on the carbonate present in dissolved state in the marine waters. Recent foraminifera in the beach sediments beaches along Being unicellular, highly abundant in sea and very the east coast of India (Fig 1) include the work carried out sensitive to even minute changes in the environmental by Sarojini (1958), Bhatia and Bhalla (1959), Bhalla parameter, their ecological studies help deducing the (1968, 1970), Gosh (1966), Ameer Hamsa (1971, 1973), paleoenvironmental conditions of the sediments/rocks Kathal (1989, 1991, 1999, 2002a, 2002b, 2004), Bhalla besides wide geological applications, like- oil exploration, and Kathal (1998), Kathal and Bhalla (1996b, 1998), higher resolution biostratigraphy, environmental Kathal and Matoba (2001), Kathal and Singh 2010), Singh /paleoenvironmental interpretations. Their 'planktic' forms (2009) and Singh and Kathal (2010). display 'surface water-' while the 'benthic' species help studying the 'bottom water' conditions of the oceans of the Singh and Kathal (2011) compared foraminiferal species present/past. from littoral-zone along east coast of India with that of the east coast of Japan. Although the studies on Recent foraminifera along the east coast of India started quite early indeed Foramgeographical affinity of the east and west (Schwager, 1857; Chapman, 1895; Hofker, 1927 & 1933; coasts of India and Cushman, 1939) yet it received less attention in comparison to the studies carried out, elsewhere. The cluster analysis of the Recent foraminiferal assemblages obtained from the 26 beaches from the two It has been endeavoured here to make a review of coasts was carried out to delineate their foramgeographical the work conducted on the Recent foraminiferal fauna affinities. The study shows that the east coast belongs to the obtained from the littoral-zone along the east coast of India warm water 'Indo-Pacific' realm while the west coast in order to pave way for future studies in this field of shows close affinity to the 'East African' realm with a research. 'mixed-zone' in between (Kathal et al., 2000) and Kathal Observations and Bhalla (2001). Sastry (1963), Shetty (1982) and Khare et al. Morphological aspects of foraminifera (2007) have reviewed the literature but no detailed Importance of various morphological features account on studies made exclusively on the Recent and their response to ambient physico-chemical conditions foraminifera from the exposed littoral-zone of the eastern has been well recognized. Along the east coast of India

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Location map east coast of India

Fig.1 : Beaches, Littoral-Zone and bathymetry, east cost of India

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Kathal and Bhalla (1996b) studied migratory trends and Environmental/climatic inferences from foraminifera morphological variations in Rotorboides granulosum – a Studies have been made to infer/reconstruct paleoclimatic less known Recent foraminifera of tropical region that changes (Kathal, 1996 and Kathal and Bhalla, 1996c). lived in 'low oxygen' conditions between latitudes 25 N° to Accordingly, these micro-organisms have emerged as 25° S in shallow warm water conditions since Middle reliable proxies of paleoclimatic/ paleoenvironmental Miocene. (23.03 million year ago). conditions. However, studies on the role of foraminifera in addressing various issues related to climatic/ environmental changes especially along the littoral regions of east coast of India still has a lot of scope.

Table 1 - Major work carried out on littoral sediments region along the East coast of India.

Sr. Year Author Area Remarks 1. 1958 Sarojini Bay of Bengal Studies on littoral foraminifera 2. 1959 Bhatia & Beach sand of Puri Recorded and illustrated 14 species of Recent Bhalla foraminifera 3. 1966 Ghosh Digha beach Asterorotalia trispinosa (Thalmann) a Southern Bengal spinose rotalid 4. 1968 Bhalla Visakhapatnam Recent foraminifera and its relation to foramogeographical province in Indian Ocean 5. 1970 Bhalla Marina beach Identified 15 species of foraminifera (Madras) 6. 1971 Ameer Palk Bay, Gulf of Reported some foraminifera Hamsa Mannar 7. 1973 Ameer Palk Bay, Gulf of Listed 34 species from the beach sands and Hamsa Mannar illustrated 12 species 8. 1989 Kathal East coast of India Recent foraminifera from the beach sands 9. 1991 Kathal Puri to Recent foraminifera from the beach sands of Vishakhapatnam the east coast of India (Puri to Vishakhapatnam) 10. 1996 Kathal Kakinada Bay Significance of ecophenotypes in paleolatitudinal interpretations 11. 1996a Kathal, East coast of India Distribution of Recent foraminifera from & Bhalla littoral zone 12. 1996b Kathal, East coast of India Intraspecific variation and Palaeolatitudinal & Bhalla significance of Rotorboides granulosum 13. 1996c Kathal & East coast of India Migratory trends and paleolatitudinal Bhalla significance of Rotorboides granulosum 14. 1998 Bhalla & Gulf of Mannar Reported 43 species of Recent foraminifera Kathal discussed the ‘mixed-zone’ of the East African and Indo-Pacific realms

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Sr. Year Author Area Remarks 15. 1998 Kathal & Palk Strait & Presented the taxonomic observations of Bhalla Kakinada Bay Rotorboides granulosum 16. 1999 Kathal Gulf of Mannar Foramgeographical affinity of the Indian Ocean during Quaternary 17. 2000 Kathal et East and West Statistical treatment of 26 beaches, coasts of al. coasts of India India showed ‘Indo -Pacific’ affinity to the east coast of India (up to Vedranniyam) and the east African affin ity to the west coast of India 18. 2001 Kathal & Indian coasts Reported 56 species and discussed the Bhalla foramgeographical affinity of east coast of India with the known foramgeographical provinces 19. 2001 Kathal & East coast of India Reported 85 common species of the two Matoba and Japan distantly located areas within the warm - water Indo-Pacific province 20. 2002a Kathal Sixteen stations Taxonomy and distributional pattern of Recent foraminifera 21. 2002b Kathal Sixteen stations Distribution and ecology of Recent species 22. 2004 Kathal Kakinada bay Biostratigraphic and paleolatitudinal implications of ecophenotypes 23. 2009 Singh East coast of India Morphological comparison 41 common and Japan species out 120 species. 24. 2010 Kathal & Seven beaches First reported of 12 foraminiferal species Singh from the Indian water 25. 2010 Singh & Five beaches First reported of 12 foraminiferal species Kathal from the Indian water and 1 from east coast 26. 2011 Singh & East coast of India Out of 41species, 20 show narrow; 17 show Kathal and Japan medium; and 4 show medium to wide ranges of intraspecific variations

References Ocean. Bull. Nat. Inst. Sci. Ind. 376-392. Ameer Hamsa K. M. S. (1971): Some foraminifera from Bhalla S. N. (1970): Foraminifera from Marina beach Palk Bay and Gulf of Mannar. In: Sym. Ind. Ocean sands of Madras and faunal provinces of the Indian Adjacent Sea, Sec.10. J. Mar. Biol. Assoc., Ind. 28. Ocean, Contri. Cushman Found. Foram. Res. 21: 156-163. Ameer Hamsa K. M. S. (1973): Foraminifera of the Palk Bay and Gulf of Mannar. J. Mar. Bio. Assoc.,14: 418- Bhalla S. N. and Kathal, P. K. (1998): Recent foraminiferal 423. thanatocoenoses from the Gulf of Mannar, India. N. Jb. Geol. Paläont. Abh. 207: 419-431. Bhalla S. N. (1968): Recent foraminifera from Vishakhapatnam beach sands and its relation to the Bhatia S. B. and Bhalla, S. N. (1959): Recent foraminifera known foramgeographical provinces in the Indian from beach sand at Puri, Orissa. J. Pal. Soc. Ind. 4: 78-81. 6 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 3-8

Chapman F. (1895): On some foraminifera obtained by the foraminifera from littoral zone along east coast of Royal Indian Marine Survey's SS Investigator from India. Bull. Ind. Geol. Assoc. 29(1-2): 89-94. the Arabian Sea, near Laccadive Islands. In: Proc. Kathal P. K. and Bhalla S. N. (1996c): Intraspecific Zool, Soc., London, 1-55. variation and Palaeolatitudinal significance of Cushman J. A. (1939): Notes on some Pliocene Rotorboides granulosum- less known recent foraminifera described by Schwager from Car foraminifera of tropical waters. Rev. de Paleobio. Nicobar. J. Geol. Soc., Japan, 46: 149-154. 15(10): 79-85. Ghosh B. K. (1966): 'Asterorotalia trispinosa' Kathal P. K. and Bhalla S. N. (1998): Taxonomy and (Thalamann), A spinose rotaliid foraminifera from paleolongitudinal significance of Rotorboides Digha Beach, southern Bengal. Contri. Cushman granulosum – a less known Recent foraminiferal of Found. Foram. Res. 17: 104-108. tropical region, J. Geol. Soc. Ind. 51: 799-802. Hofker J. (1927): Foraminifera of the Siboga Expedition, Kathal P. K. and Bhalla S. N. (2001): Blending of Recent Part1 Siboga Exped. Monogr. 4: 1-78. foraminiferal biogeoraphical provinces of India Hofker J. (1930): Foramenifera of the Siboga Expedition, waters- a statistical approach. N. Jb. Geol. Paläont., Part 2 Siboga Exped. Monogr. 4(A): 79-170. Abh. 4: 250-256. Kathal P. K. (1989): A study of Recent foraminifera from Kathal P. K. and Matoba Y. (2001): Morphological the sandy beaches of east coast of India. Ph.D. variations in the common elements of Recent 'Indo- Thesis, Agra University. Pacific' and 'Atlanto-Caribbean' foraminifera, off the eastern Japanese and Indian coasts. In: Neogene Kathal P. K. (1991): Studies on Recent foraminifera from Climate, Indian Institute of Technology, Kharagpur, sandy beaches of the east coast of India (Puri to India. (Ed) A.K. Gupta, IIT Kharagpur Pp. 20-21. Vishakhapatnam). Project Completion Report, UGC/8/3[1]88[SR-II], 22p., pls. 6. Kathal P. K. and Singh V. K. (2010): First report of some Recent benthic foraminifera from the east coast of Kathal P. K. (1996): Foraminiferal ecophenotypes in India. J. Geol. Soc. Ind. 76(1): 69-74. paralic environment: significance in paleolatitudinal interpretations. In: Proc. IX Ind. Geol. Congr. Kathal P. K. Bhalla S. N. and Nigam R. (2000): (1993): 120-123. Foramgeographical affinities of the west and east coast of India: An approach through cluster analysis Kathal P. K. (1999): Foramgeographical affinity of Indian and comparison of taxonomic, environmental and Ocean during Quaternary based on the Recent ecological parameters of Recent foraminiferal foraminiferal thanacoenoses, Gulf of Mannar India. thanatotopes, In: Proc. XVI Indian Colloq. Micropal. Tiwari, M. P. & Mohabay, D. M. eds. Gond. Geol. Stratigr., NIO, Goa. R. Nigam and A. K. Bhandari ed. Mag., spl. pub. 4: 281-292. (Bull. ONGC), 37: 65-75. Kathal P. K. (2002a): Taxonomy, distribution pattern and Khare N., Chaturvedi S. K. and Mazumdar A. (2007): An ecology of the Recent littoral foraminifera of the east overview of foraminiferal studies in near shore coast of India. N. Jb. Geol. Paläont. Abh. 224(1): regions off eastern coast of India and Andaman and 115-160. Nicobar Islands. Ind. J. Mar. Sci. 36: 288-300. Kathal P. K. (2002b): Distribution and ecology of Recent Sarojini D. (1958): Studies on littoral foraminiferal from foraminifera from littoral sediments of eastern India. the Bay of Bengal. Ph. D. Thesis, Andhra Univ. J. Geol. Soc. Ind. 60: 429-454. Sastry V. V. (1963): Bibliography of papers published since Kathal P. K. (2004): Ecophenotypes in Recent 1939 on foraminifera from the Indian region. foraminifera, Kakinada bay, east coast of India: Micropal. 9: 107-110. biostratigraphic and paleolatitudinal implications in Tertiaries. In: 4th Inter. Conf. Environ. Micropal. Schwager C. (1866): Fossile foraminiferen von Kar Microbio. Meioben. (EMMM 2004), Turkey, 101- Nikobar, Reise der Osterreichischen Fregatte Novara 102. um Rede inden Jahren 1857, 1858, 1859. Unter den Befehlem des Commodore B. Von Wullerstorf- Kathal P. K. and Bhalla, S. N. (1996a): On the first report Urban Geologische Theil, geologische from the Indo-Pacific region, migratory trends and Beobachtungen, no.2, palaontologische paleolatitudinal significance of Rotorboides Mitheilungen, 2: 187-268. granulosum – a tropical water foraminifer since middle Pliocene (late Miocene?), In: Proc. XV Indian Setty M. G. A. P. (1982): Recent marine microfauna from Colloq., Micropal, Stratigr. Ed. (J. Pandey, R.J. the continental margin, west coast of India. J. Sci, Azmi, A. Bhandari and A. Dave), pp. 317-320. Ind. Res. 6: 674-679. Kathal P. K. and Bhalla, S. N. (1996b): A note on recent Singh V. K. (2009): Study of comparative morphology of common Recent benthic foraminifera from the east

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coast of India and off Choshi Province, east coast of Japan. Ph.D. Thesis, Dr. Haringh Gour Univ. Sagar, India. Singh V. K. and Kathal P. K. (2010): First report of twelve Recent benthic foraminifera from the Indian waters. Gond. Geol. Maz. 25(2): 259-266. Singh V. K. and Kathal P. K. (2011): Morphological variations in common Recent benthic foraminifera from the east coast of India and the southern east coast of Japan. J. Pal. Soc. Ind. 57(1): 67-84. Stubbing H. G. (1933): Stratification of biological remains of marine deposits of the Arabian Sea, British Mus. (Nat. Hist.). John Murray Expd. 3: 159-192.

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Effect of growth regulatory hormones on the germination stored seeds of Withania somnifera

Monika Christian Tissue culture & Seed Technology laboratory, Botany Department, School of Sciences, Gujarat University, Ahmedabad-380009, Gujarat, India

E-mail : [email protected]

Abstract : In the present study the effect of gibberellic acid (GA3) and kinetin (KIN) on the germination and dormancy on the stored seeds of medical plant, Withania somnifera (Var. WS 20) has been studied. Seeds of Withania somnifera (Var. WS 20) were stored at constant temperature in desiccators and the germination data was taken up to 2 years with interval of 6 months. The parameters include are speed of germination (QI) , average root length (SVI) and average total length (SVI – II). In addition to these parameters, the measurements of average root length, average shoot length, average total length, average fresh weight, and average dry weight, average root length, average shoot length, average total length, average fresh weight, average dry weight were also calculated. The quantity/concentration of both plant growth regulators was used between 10-5M- 10-7 M. The presoaking -5 treatment was given to seeds. The findings of the study show that gibberellic acid (GA3) with 10 M and kinetin (KIN) 10-7M are better for acquiring % germination (%G), the quality index and seedling vigour index (SVI-1 and SVI-11) in fresh and partially aged (1 year and 2 year) seeds . Comparative data suggest that the storage potential of seeds, Withania somnifera (Var. WS 20) can be improved significantly with the help of PGRs. Keywords : Regulatory hormones, Germination, Withania somnifera

Introduction improve the seedling vigour with presoaking treatment of Gibberellic Acid (GA ) and Kinetin (KIN) on germination. The term plant hormone for growth regulating 3 substances was introduced by Fitting (1909). The seed is Methodology one of the astonishing innovations of nature and it is now established that plant hormones affect seed germination by Studies of germination are divided into different acting on different parts of the seed (Srivastava, 2002). steps during 2005 as per the procedure followed by Recent advances in agricultural research has suggested Agrawal and Dadlani (1987). that the improvement in crop productivity and quality can Plant Growth Regulators (PGRs) were procured be further improved by incorporating new technologies from Sigma laboratory. The Seeds were treated with PGRs into traditional breeding programs and influence for experimental purpose. The seeds were dried and soaked harvesting stages on seed vigour (Anonymous, 1985 & in different concentrations of gibberellic acid (GA3) Mhatre and Rao, 1998). Grove et al. (1979) reported that ranging between 10-5M to 10-7M. Similarly the same brassinolide is a plant growth-promoting steroid, which concentration was taken for kinetin (KIN) in distilled they isolated from Brassica napus pollen. Tsai F-Y et al. water. Soaking period of seeds was for 5 hours. Then seeds (1997) made a comparative study of the effects of abscisic were removed from the solution and transferred to the tray, acid and methyl jasmonate, plant growth regulators, on which was covered with filter paper and dried at room seedling growth of rice. They found that growth regulators temperature till than original weight was measured. Dry promote flowering, cellular division, and in seeds growth seeds were put for germination up to the final count. after germination. Gopikumar and Moktan (1994) studies the effects of plant hormones on seed germination and Hormones preparation growth of true seedlings in the nursery. Tsai et al. (1997) –3 Stock solution of GA3 (10 M): Accurately studied the metabolism of gibberellins and suggested that weighed 34.64 mg GA3 was dissolved in 2 ml acetone and gibberellins are important in seed germination affecting final volume was made up to 100 ml DW. Different enzyme production that mobilizes food production used concentrations (10– 5M to 10– 7M) were prepared from stock. for growth of new cells. Saxena (1974) described the –3 presowing hardening treatments improves crop Stock solution of KIN (10 M): Accurately production. Ethylene upregulate auxin biosynthesis in weighed 21.52 mg KIN was dissolved in 2 ml 1N NaOH Arabidopsis seedlings to inhibition of root cell elongation and final volume was made up to 100 ml DW. (Swarup et al, 2007). Nitric oxide signaling in plants and Seeds were germinated in sterilized chamber. help in the synthesis in the chloroplast (Shapiro, 2005 & Intensity of light was maintained at 500 lux from tube light. Roszer, 2012). Keeping in view the aforesaid facts a study e: Constant temperature 25 + 20C was maintained during was taken to observe the seedling vigour of fresh and germination, the relative humidity of the room was kept at partially aged seeds (6 month to 2 year old seeds and to 50-55%. 9 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 9-12

Germination parameters were recorded as suggested by correlation of auxins and cytokinins in plant, known as a ISTA (1985 a,b): A/C= constant. They further held that a gene encoding a Normal seedling count, abnormal seedling count were protein modify by the phytohormones, indoleacetic acid counted. Germination percentage (%G) was calculated as acting by modulating chromosomal transcription. follows on the basis of the number of normal seedlings as Gibberellins include a large range of chemicals that are suggested by Agrawal (1987). produced naturally within plants and are important in seed germination, affecting enzyme production that mobilizes %G = N / T X 100, food production used for growth of new cells (Agrawal and N = Number of normal seedlings, T = Total number of Dadlani,1987). Gopikumar and Moktan (1994) studies on seeds kept for the germination the effects of plant hormones on seed germination and growth of true seedlings in the nursery and found that plant Seedling length was recorded in cm and divided into root hormones are suitable to cover the dormancy due to storing length and shoot length. of seeds and initiate germination. Plant hormones like GA3 Fresh and dry weight of seedlings: Average fresh weight and KIN act upon a responsive plant system by interaction (per seedling) was recorded. Normal seedlings were the molecules and effect the morphological, physiological packed in blotting paper and kept in oven at 80 + 20C till and biochemical responses. Chinoy (1942, 1967) had first constant dry weight was recorded. time tried presowing treatment of PGRs in wheat seeds. The formula quality Index (QI) or Speed of germination The critical studies were made on proper concentration, was calculated as follows (Maguire, 1962); soaking volume and application of PGRs on Indian plants (Saxena, 1974, 1990; Murlikrishna, 1993). In the present Number of seeds germinated investigation the pretreated stored seeds were with 10-5 M QI = ------7 to 10 M of GA3 and KIN kept in distilled water for five Day of inspection hours gave good (Table 2 & Table 3). The results were better in comparison to control (Table 1). The present study Seedling Vigour Index (SVI): SVI-I and SVI-II were is in conformity with the observations of earlier calculated according to method given by Jayraj and investigation in the field (Chinoy, 1942 & 1967; Saxena, Karivartha Raju (1992) and Abdul Baki, James and 1974 & 1990; Murlikrishna, 1993). Out of three -5 -6 -7 Anderson (1973): The formulae are as follows: concentration of GA3 and Kin (10 M, 10 M, 10 M), GA3 -5 SVI-I = %G X Dry weight of seedling (10 M) gave more germination percentage (%G, 60), seedling length (3.3), fresh and dry weight, quality index SVI-II = %G X Total length of seedling (QI, 2.6), Seedling Vigour Index (SVI-1,40.20) and(SV1- Results and Discussion II,330.0) are more as compare to control. On the other hand, KIN (10-7 M) is better results in r fresh and partial (1 Plant hormones are considered effective year and 2 year) aged seeds in % G , Q.I., SVI-I and SVI-II. molecule in development of seeds. The hormones affect The PGRs are known to break seed dormancy in a number seed germination and dormancy by acting on different of plants (Gopikumar and Moktan, 1994). parts of the seed. Walz et al. (2002) stated that there is a

Table 2: GA3 treatment to Fresh and Partially aged seeds of WS 100 variety of Withania somnifera

Treatments No. of Normal Abnormal % G Average Average Average Average Average Q.I. SVI - I SVI - II seeds seedlings seedlings root shoot total fresh dry length length length weight weight (cm) (cm) (cm) (mg) (mg) Treatment to 0 Month Old seeds (Fresh seeds) -5 GA310 M 25 15 0 60 3.3 2.2 5.5 34.60 0.67 2.67 40.20 330.0 -6 GA310 M 25 15 0 60 2.0 1.5 3.5 27.33 0.40 2.67 40.20 210.0 -7 GA310 M 25 12 3 48 1.2 1.2 2.4 25.90 0.36 2.12 17.28 115.2 Treatment to 12 Month Old seeds -5 GA310 M 25 12 0 48 1.4 1.2 2.6 25.00 0.62 1.85 29.76 124.8 -6 GA310 M 25 9 0 36 1.2 1.5 2.7 27.77 0.55 1.28 19.80 97.2 GA 10-7M 25 6 0 24 1.0 1.5 2.5 42.50 0.48 0.86 11.52 60.0 3 Treatment to 24 Month Old seeds -5 GA310 M 25 5 1 20 3.0 1.0 4.0 52.00 0.70 0.20 14.00 80.0 -6 GA310 M 25 6 4 24 1.8 1.2 3.0 40.00 0.42 1.45 10.08 72.0 -7 GA310 M 25 6 0 24 1.0 1.2 2.2 28.33 0.52 0.69 12.48 52.8 10 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 9-12

Table 3: KIN treatment to Fresh and Partially aged seeds of WS 100 variety of Withania somnifera

Treatments No. of Normal Abnormal % G Average Average Average Average Average Q.I. SVI - I SVI - II seeds seedlings seedlings root shoot total fresh dry length length length weight weight (cm) (cm) (cm) (mg) (mg) Treatment to 0 Month Old seeds (Fresh seeds) KIN 10 -5M 25 8 3 32 2.6 1.9 4.5 36.9 0.50 1.01 16.00 144.0 KIN 10 -6M 25 9 3 36 3.3 2.3 5.6 31.7 0.33 1.54 11.88 201.6 -7 KIN 10 M 25 14 1 56 1.2 2.2 3.4 33.6 0.38 2.21 21.28 190.4 Treatment to 12 Month Old seeds KIN 10-5M 25 6 4 24 1.8 1.3 3.1 40.0 0.43 1.40 10.32 74.4 -6 KIN 10 M 25 5 1 20 3.0 1.1 4.1 52.0 0.71 0.23 14.20 82.0 KIN 10-7M 25 12 3 48 1.2 1.0 2.2 25.9 0.36 2.12 17.28 105.6 Treatment to 24 Month Old seeds KIN 10-5M 25 5 0 20 0.8 1.0 1.8 27.2 0.21 0.20 4.20 36.0 KIN 10-6M 25 6 2 24 0.7 1.1 1.8 28.9 0.25 0.38 6.00 43.2 -7 KIN 10 M 25 7 0 28 0.9 1.6 2.5 35.0 0.40 0.52 11.20 70.0

Table1: Seed vigour and germination data of WS 100 variety of Withania somnifera

Storage period No. of Normal Abnormal % G Average Average Average Average Average Q.I. SVI - I SVI - seeds seedlings seedlings root shoot total fresh dry II

length length length weight weight (cm) (cm) (cm) (mg) (mg) 0 Month 25 14 0 56 1.5 1.0 2.5 21.73 0.65 1.35 36.40 148.0 6 Month 25 14 0 56 1.0 1.0 2.0 21.73 0.50 1.83 25.00 112.0 12 Month 25 14 0 56 0.7 1.2 1.9 21.42 0.39 1.54 21.84 106.4

18 Month 25 10 0 40 0.9 1.0 1.9 29.00 0.45 1.23 18.00 76.0 24 Month 25 9 0 36 0.9 1.0 1.9 29.44 0.49 0.93 17.64 68.4

Referance N.; Worley, J. F.; Warthen J. D.; Steffens G. L.; Flippen-Anderson J. L. et al. (1979): Brassinolide, a Abdul Baki A. A., James and Anderson (1973): Vigour plant growth-promoting steroid isolated from estimation in soyabean by multiple criteria, Crop Brassica napus pollen. Nature. 281, 5728 Sci., 13: 630-633 International Seed Testing Association (ISTA) (1985 a): Agrawal P. K. (1987): Germination test under controlled International rules for seed testing, rules 1985, Seed conditions and its evaluation, In: Techniques in seed Sci. & Technol., 13: 299-355 science and technology. (Eds.) Agrawal, P. K. and Dadlani, M. (South Asian Publishers, New Delhi) International Seed Testing Association (ISTA) (1985 b): International rules for seed testing, annexes 1985, Agrawal P. K. and Dadlani, M. (Eds.) (1987): Techniques Seed Sci. & Technol., 13: 358-513 in seed, science and technology, (second edition, South Asian publishers, New Delhi) Jayraj T. and Karivartha Raju T. V. (1992): Influence of harvesting stage on seed vigour in groundnut Anonymous (1985): International rules for seed testing, cultivars, Seed Research, 20: 41-44 Seed Sci. & Technol., 13 (2): 307-520 Maguire J. D. (1962): Speed of germination - a new vigour Chinoy J. J. (1942): Presowing treatment and phasic test for crop seeds, In: Seed production. (Ed.) development, Curr.Sci., 11, 400 Habblethwaite (Butterworth, London). pp, 647-660 Chinoy J. J. (1967): Role of ascorbic acid in crop Mhatre M. and Rao P. S. (1998): Plant tissue culture - production, Poona Agr. College Magaz., 57: 1-6 current trends and future prospects, In: Advances in Fitting H. (1909): Die Bee influssung der orchideenbluten plant physiology, (Ed.) Hemantranjan A. (Sci. durch die bestaubung und durch andere umstande, Publ., Jodhpur, India) pp: 72-101 Z. of Bot., 1: 1 Murli Krishna S. (1993): Physiological studies on castor Gopikumar K. and Moktan M. R. (1994): Studies on the and cotton, Ph.D. Thesis, Gujarat University, effects of plant hormones on seed germination and Ahmedabad growth of true seedlings in the nursery, J. Tropical Roszer T. (2012): Nitric Oxide Synthesis in the Forestry, 10: 45-50 Chloroplast. In: The Biology of Subcellular Nitric Grove M. D.; Spencer G. F.; Rohwedder W. K.; Mandava Oxide (Ed) Roszer T. (Springer New York London)

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Heidelberg). Saxena O. P. (1974): Presowing hardening treatments and crop production – a review, Planta, 4: 1-11 Saxena O. P. (1990): Some aspects of seed germination, In: International Symposium Environmental Mechanism. In Recant Advances in Research and Technology, Jodhpur. Jan. 27-28 Shapiro A. D. (2005): Nitric oxide signaling in plants. Vitam Horm. 72:339-98. Srivastava, L. M. (2002). Plant growth and development: hormones and environment. (Academic Press). p. 140. Swarup R., Perry P., Hagenbeek D. (2007): Ethylene upregulates auxin biosynthesis in Arabidopsis seedlings to enhance inhibition of root cell elongation Tsai F-Y.; Lin C.C.; Kao C.H. (1997): A comparative study of the effects of abscisic acid and methyl jasmonate on seedling growth of rice. Plant Growth Regulation 21 (1): 37–42. Walz A, Park S, Slovin JP, Ludwig-Müller J, Momonoki Y. S, Cohen J. D (2002). "A gene encoding a protein modified by the phytohormone indoleacetic acid". Proc. Natl. Acad. Sci. U.S.A. 99 (3): 1718

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Evaluation of antimicrobial activity of Azadirachta indica, Syzygium aromaticum and Cinnamomum zeyalnicum against oral microflora.

Haripriya Parthasarathy and Smruti Thombare Deptt. of Biological Sciences, Ramniranjan Jhunjhunwala College, Ghatkopar (W) - Mumbai 86

E-mail : [email protected]

Abstract : For several years, varieties of plants have been used throughout India to treat oral care issues and are used till date in rural as well as urban areas. The inhibitory effects of aqueous extracts and essential oil of three plants, Neem (Azadirachta indica), Clove (Syzygium aromaticum) and Cinnamon (Cinnamomum zeylanicum) against clinical isolates identified as Staphylococcus auricularis, Micrococcus species, Acinetobacter lwoffii and Candida albicans, were studied. Cinnamon extract successfully inhibited all four organisms showing largest zones of inhibition. Clove oil inhibited S. auricularis, A. lwoffii and C. albicans but not Micrococcus. Neem oil did not show any significant activity against any organisms. Toothpastes were then formulated using effective plant extract showed the antimicrobial activity. Key words: Aqueous extract, Azadirachta indica, Syzygium aromaticum, Cinnamomum zeylanicum, Staphylococcus auricularis, Micrococcus species, Acinetobacter lwoffii, Candida albicans.

Introduction the growth of Candida hence preventing & curing oral thrush. It has been proven to be active against many Microflora refers to the collective bacteria and pathogens (Suresh et al., 1992). other microorganisms present in a particular ecosystem. The ecosystem can be an animal or human host or a single Materials and Methods part of its body. Our body is a host to billions of bacteria of Collection of plant Material: Azadirachta indica twigs, many kinds. The mouth harbours many microorganisms Syzygium aromaticum buds and Cinnamomum zeylanicum and is an ecosystem of considerable complexity that has bark were obtained from local market. Plant essential oil not been fully investigated yet and the mechanism/ was obtained from commercial outlet of Dr. Urjita Jain chemistry has also not been completely understood. More Herbal ltd, Mumbai. than 30 genera of bacteria have been detected in human mouth. The oral microflora can be beneficial or Collection and maintenance of oral flora: Mouth swabs detrimental. They supply some nutrients and digestive from 6 people were collected and isolated on sterile enzymes such as amylase, lipase and proteases and Nutrient Agar and Sabouraud's Agar plate and incubated at contribute to the hosts defences by protecting from 37ºC and room temperature respectively for 24hrs. The exogenous microbes (Marsh et al., 2006). Any change in colonies obtained were transferred to sterile slants and sent the host's oral environment or immune system can cause a to Metropolis Labs, Worli for identification. number of different oral problems. Use of plants for Preparation of plant Extracts: The plant parts were sun- maintaining oral hygiene is common till date in rural areas dried and ground into a course powder in a blender. 2 g of where toothpastes, mouthwashes, massage gels and other powdered plant material was added to 10 ml of sterile oral care products are not available. These products contain distilled water and heated to 70-80ºC for 2 hours. The synthetic substances such as triclosan, phenol, mixtures were then allowed to cool, filtered through muslin benzydamine hydrochloride, zinc chloride, stannous cloth and centrifuged. The supernatant were then passed fluoride etc. as antimicrobial agents. However, use of through a 0.45µm millipore filter to obtain sterile extracts leaves, stem, fruits, seeds and bark of some plants which were stored at 4ºC (Nazia et al., 2006). (Neem/babool etc.,) for oral hygiene is common. Azadirachta indica has anti-inflammatory, antibacterial, Determination of antimicrobial activity: Kirby Bauer antimalarial, antiulcer, antiparasitic, antifungal, disc diffusion method was used to determine the effect of antiprotozoal and antiviral properties (Biswas et al., 1973; aqueous extracts and plant essential oil on the isolates. The Siddique et al., 1992; Ian et al., 1994). All parts of the tree broth culture of each of the isolate was swabbed on sterile have been used traditionally to cure various disorders as Mueller-Hinton agar. Sterile discs were dipped in plant stated by Subapriya et al., 2005. Buds of Syzygium extracts and placed onto the agar plates. Plates were aromaticum are used as an analgesic, antiseptic and a incubated at 37ºC for 24 hours. The zone of inhibition was carminative. Cinnamomum zeylanicum is known to inhibit observed after 24 hours.

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Formulation of herbal toothpaste: inhibits amino acid decarboxylase activity as suggested by Wendakoon and Sakaguchi (1995). Cinnamon oil contains The herbal toothpaste was prepared as per the benzoic acid, benzaldehyde and cinnamic acid whose formulation given by Jadge, 2008. The ingredients and lipophyllic part is responsible for its antimicrobial quantities were mixed(Table 1). properties (Ramos-Nino et al., 1996). Both clove and Evaluation of toothpaste's activity against the cinnamon contain eugenol, which is known to be isolates bacteriostatic and bactericidal depending on the concentration used (Pelczar et. al., 1988). Essential oil Agar cup Method was used for testing the from cinnamon bark also contains cinnamyl acetate antimicrobial activity of the toothpastes prepared using the (8.7%), which increases the activity of the parent essential oil. The broth culture of the isolates was swabbed compound (Gupta et al., 2008). The mechanisms or modes on Mueller-Hinton agar plates. Wells were bored using of action of the compounds on the bacteria and fungi sterile borer. Toothpaste formulations were added in the include cytoplasmic granulation, cytoplasmic membrane wells and plates were incubated at 37ºC for 24 hours. The rupture and inhibition of intracellular and extracellular zone of inhibition was observed after 24 hours. For each enzymes. The mechanism of oils is generally strain a control was maintained using a formulation hydrophobicity leading to partition in the lipid bilayer of without the active ingredient. the cell membrane, leading to alteration in the permeability Results and discussion and consequent leakage of cell contents. It also inhibits respiration in the cell and causes potassium ion leakage. Asikainen and Chen ( 2000) stated that the oral ecology is different from person-to- The present study suggests that the aqueous extracts were not able to inhibit the isolates. However, person in transmission of Actinobacillus essential oil can inhibit the growth at larger extent. Further actinomycetemcomitans and Porphyromonas gingivalis study of the components responsible for the biological Smith (1993) studied the oral streptococcal colonization of activity is required and possibly a combination of these infants while Pearce (1995) found viridans streptococci in components would yield better results. The tooth pastes the oral cavity of human neonates. In the present study the also were found to be effective in controlling the growth of isolates form the oral cavity were identified as these organisms hence such an herbal product may have a Staphylococcus auricularis, Micrococcus species, potential to replace the standard formulations. Acinetobacter lwoffii and Candida albicans. Aqueous extract of Azadirachta indica and Table 1: Formulation of Toothpaste Syzygium aromaticum could not inhibit any of the culture. Ingredient Quantity in % Essential oil of Azadirachta indica showed the activity only against S. auricularis. The essential oil of Syzygium Calcium Carbonate 35.00 aromaticum was found to be effective against Sodium Lauryl Sulphate 01.50 Staphylococcus auricularis, Acinetobacter lwoffii and Candida albicans. Aqueous extract and the essential oil of Glycerin 30.00 Cinnamomum zeylanicum were found to be effective Sodium Alginate 01.00 against all the four isolates. These results have been compiled in Table 2. Sodium Benzoate 00.12 The toothpastes formulated were found to be effective Sodium saccharine 00.30 against the isolates. The zone sizes observed after 24 hrs Plant Extract 02.50 incubation are tabulated in Table 3.The activity of cinnamon is due to the presence of cinnamaldehyde that Purified water q.s.

Table 2: Evaluation of antimicrobial activity of plant extracts

Plant Average Zone size in mm S.auricularis Micrococcus spp A.lwoffii C.albicans A.E E.O A.E E.O A.E E.O A.E E.O A. indica No 13.30 ± 1.5 No No No No No No Zone Zone Zone Zone Zone Zone Zone S. aromaticum No 26.0 ± 1 No No No 18.0 ± 1 No 26.3 ± 0.5 Zone Zone Zone Zone Zone C. zeylanicum 11.00 ± 1 45.3 ± 3 11.6 ± 0.5 23.6 ± 1.5 11.3 ± 0.5 22.3 ± 2.5 1 1.0 ± 0 4 5.6 ± 1 Key: AE: Aqueous extract, EO: Essential oil

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Table 3: Evaluation of toothpaste's activity

Plant Average Zone size in mm S.auricularis Micrococcus spp A.lwoffii C.albicans A. indica 11.6 ± 0.5 - 10.6 ± 0.5 11 ± 1 S. aromaticum 16 ± 1 - 15 ± 1 11.6 ± 1.5 C. zeylanicum 13.3 ± 0.5 13.6 ± 1.5 13.6 ± 0.5 13 ± 0 Control No Zone No Zone No Zone No Zone

References Ramos-Nino M. E., Cliford M.N., Adams M.R. (1996): Quantitative structure activity relationship for the Asikainen S. and Chen C. (2000): Oral ecology and person- effect of benzoic acid, cinnamic acids and to-person transmission of Actinobacillus benzaldehydes on Listeria monocytogenes. J. Appl. actinomycetemcomitans and Porphyromonas Microbiol. 80: 303-310. gingivalis. Periodontol, 20, 65–81. Siddiqui Salimuzzaman, Shaheen Faizi, Beena Siddiqui, Biswas Kausik, Ishita Chattopadhyay, Ranajit K.Banerjee Ghiasuddin (1992): Constituents of Azadirachta and Uday Bandyopadhyay. (2002): Biological indica: Isolation and Structure Elucidation of a New activities and medicinal properties of Neem Antibacterial Tetranortriterpenoid, Mahmoodin, and (Azadirachta indica). Current Science. 82(11), 1336- a New Protolimonoid, Naheedin. Journal of Natural 1345. Products (55) 3, 303-310. Gupta Charu, Garg Amar, Uniyal ramesh, Archana Smith D.J., Anderson J.M., King W.F., van Houte J, Kumari. (2008): Comparative analysis of the Taubman M.A., (1993): Oral streptococcal antimicrobial activity of cinnamon oil and cinnamon colonization of infants. Oral Microbiol Immunol ; 8: extract on some food-borne microbes. African 1-4. Journal of Microbiology Research. 2(9): 247-251. Subapriya R., Bhuvaneswari V., Nagini S. (2005): Ian W. Jones, Alastair A. Denholm Steven V. Ley, Helen Ethanolic Neem (Azadirachta indica) Leaf Extract Lovell, Anthony Wood, Robert E. Sinden (1994): Induces Apoptosis in the Hamster Buccal Pouch Sexual development of malaria parasites is inhibited Carcinogenesis Model by Modulation of Bcl-2, Bim, in vitro by the Neem extract Azadirachtin, and its Caspase 8 and Caspase 3. Asian Pacific Journal of semi-synthetic analogues. FEMS Microbiology Cancer Prevention. 6, 515-520. Letters. 120(3): 267–273. Suresh P., Ingle V.K. and Vijayalakshima V. (1992): Jadge D. R. et al. (2008): Formulation of toothpaste from Antibacterial activity of eugenol in comparison with various forms and extracts of tender twigs of neem. other antibiotics. J. Food Sci. Technol. 29: 254-256. Journal of Pharmacy Research. 1(2):148-152. Wendakoon C.N., Sakaguchi M. (1995): Inhibition of Marsh P. and Percival R. (2006): The oral microflora — amino acid decarboxylase activity of Enterobacter friend or foe? Can we decide?. International Dental aerogenes by active components of spices. J. Food Journal. 56, 233–239. Prot. 58: 280-283. Nazia Masood Ahmed Chaudhry and Perween Tariq. (2006): Anti-microbial activity of Cinnamomum cassia against diverse microbial flora with its nutritional and medicinal impacts. Pak. J. Bot. 38(1): 169-174. Pearce C., Bowden G.H., Evans M., Fitsimmons S.P., Johnson J,Sheridan M.J. (1995): Identification of pioneer Viridans streptococci in the oral cavity of human neonates. J Med Microbiol.42: 67–72 Pelczar M. J., Chan E.C.S., Krieg N.R. (1988): Control of microorganisms, the control of microorganisms by physical agents In:Microbiology, (New York: McGraw-Hill International), pp. 469 509.

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The Study of Nidification Behavior In Red Wattled Lapwings, Vanellus indicus

V.L. Saxena and A. K. Saxena* Department of Zoology, D.G.College, Kanpur-208001 * Department of Zoology, D.A.V.College, Kanpur-208001

E-mail id. - [email protected]

Abstract : The present study focuses on the nidification and courtship behavior of Red Wattled Lapwings. During breeding season the male bird select the territory. Both male and female take part in building nest. The nest is generally a circular depression encircled by stones or pieces of hard clay. Four eggs are led in each nest. During the mid-March and June the birds exhibit courtship behavior. Male first gives signals of courtship. The female respond by emitting a mating call. The incubated is completed by both parents. Eggs hatch into nidifugous chicks in 28-30 days. Both parents protect territory and protect their own plumage completely (3-5 weeks) tills become good fliers. Key Words- Nidification, Courtship, Clutch size, Nidifugous

Introduction Material and Method The description about Indian birds has been well The study was conducted on 6 pairs of birds in documented by Whistler (1948) and Salim Ali and Ripley Kanpur city, including one nest located at the rooftop of (1980). Birds possess note worthy tendency to build a D.G.College, Kanpur. The period of study was during the home to raise their young ones (Holway,1965 & months of March 2010 to June 2010, which is the time of Collias,1997). The incubation and broody behavior in spring followed by hot summers till the onset of Pheasant (Phasianus colchicus) been studied monsoons. (Breihenback et al., 1965). Rhythm of incubation from The observations were taken with from full egg laying to hatching in mountain white-crowned sparrow precautions without disturbing birds. For the concealment has been reported by Zebra and Morton (1980). The nest of human activities without the knowledge of birds. A 6 and detailed nidification activities of the spoonbill, feet height frame was constructed which was covered by a Platalea leucorodia has been described by Dayanadi and cloth with two openings small enough to observe the Hosetti (2008 & 2009). They stated that casting of nest is an nidification, courtship behavior etc needed for the study. important part of bird's biology as it plays a key role in This 'hide' was left near the nest for 2-3 days to acclimatize shaping the relationships between parents with their offspring. The procedure of site selection for nidification in Savannah sparrow and procedure of nest has been critically described by Wheelwright et al.( 1997). The parental building roles of male and female under different environmental has been studied in Western Gull and several species of birds ( Plerotti and Ridley.,1981; Bryant &Tatner,1990;Conrad & Robertson,1993;Collias,1997; Brawn et al., 2011). The activities and pattern of behavior are successful courtship, acquiring of a nesting area, building a safe and protected nest (Clark and Shulter,1999) laying of the clutch, taking care of the hatchlings, feeding and protecting them (Krebs, 1987) till they become good fliers. This behavioral pattern finds its origin in the direction towards self and racial survival. Nidification behavior is also very important for maintaining the ecological and genetically balances. Saxena et al.(2008) stated that breeding and nidification in Colonbia livia and Stretopelia chinensis undergoes throughout the year, although pigeon breeds preferably during January to May while Dove breeds during January to August. Recently, Vaithianathan problems faced by the species due to changes in environment as a result of pollution. The use of territory has been traced by early as 1941 by Nice Fig.1 : Hide used for observation

17 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 17-21 the birds (Figure 1). The observations were taken on daily basis by naked eye starting in early hours of dawn and ending with dusk. The photographs were taken through a Nikon camera. The eggs that were laid by the birds were measured for size with the help of a thread and a scale and average were calculated. The permission from ethical committee of the college has been obtained for the study of these endangered birds. Observations Courtship Behavior The birds were spotted moving in pairs between 18-22 March, 2010 in Kanpur and its suburbs. The male initiated the courtship at all the station. Male reaches first Fig.2. Eggs of lapwing towards the female with its fanned tail and then stood erect There were deep brownish black markings or spots, which keep the neck in stretched position. The breast of the male are distributed all over the surface of the eggs. The egg size was fully puffed out and to sought attention of the female, varied from 1.2 inches-1.6 inches on an average. (Figure 2) the male bird showing tumbling flights and produce songs. Female responded to these antics of the male by giving Incubation short, quickly repeated calls. Pair formation included Lapwings incubated the eggs by sitting on them. display flights with tail fanned; shallow rapid wing beats Incubation started with the laying of the first egg. Both the and gives a number of calls. sexes shared the duty of sitting on the eggs. Mostly female Number of mates did the duty but male assisted her a lot. Incubation took 3 weeks and 4 days. The season of summer made the birds The male and female were observed to jointly wet their breast feathers regularly to keep the eggs wet. take up the responsibility of rearing the young. (Figure 3, 4) Territory The breeding pair maintained a territory in the surrounding of their nest. It is treated as a special area and intrusion in this area by strangers is protected by parents. Male first acquires a territory and then gives a call note to the female. The male greets the arrival of the female joyfully by wheeling in the air and squawking happily. These birds give alarming calls protesting intrusion; they sing a territory song to get it distinguished and noticed to avoid strangers. Nest construction and nesting material Nest building started on 2nd April 2010. Nest was built during early morning up to noon and then in late afternoon on successive days. Nest building is a joint effort Fig.3. Incubation of eggs of both the parents with almost equal contribution. Clutch Size The clutch size in Lapwing was observed to be of four eggs. The eggs were laid on alternate day starting from 8 April 2010 till 14 April 2010. Eggs were laid during afternoon hours. The eggs were so arranged by the bird that their small ends meet in the center, making for even sitting and easier incubation by the parent. The bird was observed to rearrange the disarranged eggs. Eggs The eggs were of plover type, broad at one end and much pointed towards the other. They were pyriform with color varying from a pale olive green to a reddish buff. Fig.4. Incubation of eggs 18 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 17-21

Hatching pattern Young hatched out one after the other starting on 4 May 2010, at an interval of 46-48 hours, in the order in which they were laid. Hatching was synchronous. (Figure 5)

Fig.7. Physical feature of lapwing hatching Brood care and feeding The brooding mother kept freshly hatched young warm at night. During the day the male and both the female protected them from the hot sun taking turns. Day brooding Fig.5. Hatching pattern of lapwing stops after 5 days of first hatching. Night brooding stopped only after the chicks were partly fledged which took about Physical features of hatchlings 16 days. The hatchlings were covered with brown On the appearance of any danger or an intruder coloured down feathers. They had upper parts grayish the brooding pair slowly creeped away to some distance brown, mottled black. They had a broad white collar, a and began calling to lure the enemy away from the nest. black pectoral band, the chin and rest of the under parts were white tinged whereas the belly and the flanks were If birds of prey flew overhead the brooding bird crouched buff colored. The newly born chickens were nidifugous. silently on the eggs or the young to conceal them. The parents cared for these precocial young. The young left (Figure-8) the nest as soon as they were dry and were able to move about with great ease. Their brown color helped them to blend in the surroundings. They were able to fly within three-five weeks. (Figure 6, 7)

Fig.8. Brood care Behavior of the nestlings The nestlings were nidifugous and precocial. They start running about as soon as they emerged out of egg Fig.6. Physical feature of lapwing hatching shell. They were born feathered and could feed themselves. They needed their parents only for their protection till they could learn to defend themselves. The nestlings spread over an area to feed, ran a few steps, stopped, pecked and then stood up straight. The chicks fed themselves easily but were safely protected by

19 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 17-21 the parents. They ran for cover in some plant or debris (Sordhl, 2001). Monogamy assures better care for the whenever their parents uttered a warning note or an alarm young, these birds practice monogamy to assist in parental call. care (Ali and Ripley, 1980). Parental care has also been Nest sanitation observed in Western Gulls (Collias, 1997; Plerott and Ridly, 1981). Lapwings maintain a well guarded and a Lapwings kept the nest clean and tidy. The well-distinguished territory to aid the welfare of their eggshells after hatching were removed from the nest young ones The present study supports the observation of providing both sanitation and concealment. earlier author for the nest site selection as proposed by Nestlings were nidifugous and precocial. They Clark and Shulter., 1999). Territory also aids in start running about as soon as they emerged out of egg concealment and protection of young ones ( Holway, 1991) shell. They were born feathered and could feed themselves. . Lapwings build their nest in open while selection of site They needed their parents only for their protection till they places or even at the roof of the building keeping the could learn to defend themselves. concealing coloration for protection of eggs. They scoop a The nestlings spread over an area to feed, ran a shallow hallow in the ground and line it with pebbles. few steps, stopped, pecked and then stood up straight. The Vicinity of water is preferred. The male scrapes the ground chicks fed themselves easily but were safely protected by for the nest and the female lines it with local materials the parents. They ran for cover in some plants or debris when she finds it satisfactory. Lapwings construct their whenever their parents uttered a warning note or an alarm nests early in the mornings and late in the afternoon during call. April, similar to nest building of passerine birds. (Collias, 1997). Nest sanitation Nest and Nidification Activities of the Spoonbill Lapwings kept the nest clean and tidy. The Platalea leucorodia in Westerghat Region of Shimoga, eggshells after hatching were removed from the nest Karnataka is also being studied and The nesting activity, providing both sanitation and concealment. nest site selection, nesting habitat, nesting material, nest Discussion size, and nidification of Platalea leucorodia are discussed (Dayananda and Hosetti, 2009). Such studies on According to IUCN (2009 ), Red Wattled nidification is done on different birds including Indian grey Lapwings new zoological name is Vanellus indicus instead horn bill,black headed ibis pig (Charde et al., 2011), pegion of Lobivanellus indicus .The species has been spread over and dove (Saxena et al., 2008), Spot-billed Pelican in a large range. It maintains a well guarded and a well- (Vaithianathan and Jeganathan, 2012), tropical birds distinguished territory which is in the interest for the (Jeffrey et al., 2011) etc. are done in recent years. Lapwings welfare of their young ones. Territories selection has been kept the nest clean and tidy. The eggshells after hatching started to select the site for the construction of nest. The were removed from the nest (Smith, 1993). The hatchlings site selection has been studied in birds (Clark and start running a few steps, then stopped, pecked and then Shulter,1999). Saxena et al.(2008) reported that in pigeons stood up straight as soon as they emerged out of egg shell. and dove the territory is selected and claimed by mutual It is critically noticed that new born bears feathered and consent from both the parent only after pair formation. could feed themselves. They need their parents only for They further stated that individual breeding pairs maintain their protection till they could learn to defend themselves. a territory surrounding the nest. In pigeon the male defends its territory by holding its wings aloft, landing on Clutch size of Lapwings is four in the present its rival at times or by clubbing the rivals head with bent study, however, some workers have also reported three to wings. In dove male as well as female both defend the five eggs (Conrad and Robertson,1993). The present territory by chasing the intruders out of defined space. The investigation the incubated is 25 days in natural conditions present study is in confirmation to above referred authors without using hormone treatment (Smith, 1993) and both that the breeding pair maintained a territory in the the sexes share in the incubation simultaneously. Studies of surrounding of their nest. It is special area protected by incubation behavior have been done as early as 1965 on parents in this area strangers are not permitted by the Pheasants (Breitenbach et al., 1965). The young hatchlings parents. These birds give alarming calls protesting of Lapwings are grayish brown with white on the intrusion; they sing a territory song to get it distinguished underside. Sibling competition and behavior of siblings in and noticed to avoid strangers. Male first acquires a Swiftlets and Bee-eaters has been observed (Bryant and territory and then gives a call note to the female. The male Tatner.1990) while behavior of adults and young of greets the arrival of the female joyfully by wheeling in the Acadian Flycatcher has also been reported in detailed( air and squawking happily. Whitehead and Taylo, 2001; Wesolowski, T. 1994). The eggs are often collected by people and used in traditional These birds show courtship behavior by giving remedies for asthma and typhoid. (Negi,et .al .2007). mating call. The mating call is in the form songs, show flights and mock displays by the male (Singh, 2004). Similar behavior has been reported in American Avocets

20 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 17-21

References Introduction to Behavioural Ecology. 9, 191-220.

Ali S and Ripley S.D. (1980): Handbook of the birds of LaPergola Joshua B., Mortensen Jennifer L., and Curry India and Pakistan. (2 Ed.). (Oxford University Robert L.(2012): Nest, Eggs, and Nesting Behavior Press). 212–215. of the Gray Trembler (Cinclocerthia gutturalis) on St. Lucia, West Indies. The Wilson Journal of Bird Life International. Vanellus indicus. In: IUCN (2009). Ornithology .123(2):390-395. IUCN Red List of Threatened Species. Naik.RM et al (1961): For behavior when incubating. Brawn Jeffrey D., Angehr George, Davros Nicole, JBNHS. 58,222-230. Robinson W. Douglas, Styrsky Jennifer N., Tarwater Corey E.(2011): Sources of variation in the nesting Negi Chandra S : Traditional Uses of Animal and Animal success of understory tropical birds. Journal of Avian Products in Medicine and Rituals by the Shoka Biology. 42 (1), 61–68. Tribes of District Pithoragarh, Uttaranchal, India. Ethno-Med. 1 (1): 47–54. Breitenbach R.P., Nagra C.L. and Meyer R.K.( 1965): Studies of Incubation and Broody behaviorin the Plerotti R. and Ridly (1981): Male and female parental Pheasant (Phasianus colchicus). Anim. Behav., 13: roles in the Western Gull under different 143-148. environmental conditions. Auk. 81:532-549. Bryant D.M. and Tatner P. (1990): Hatching asynchrony, Saxena V.L., Pandey Eshita, Agarwal Sona and Saxena Sibling competition and Siblicide in nestling birds. A.K.(2008): Execution of Breeding and Nidification Studies of Swiftlets and bee-eaters. Anim. Behav, Behaviour in Pigeon (Columba livia) and Dove 39:657-671. (Streptopelia chinensis). Asian J. Exp. Sci. 22(3), 405-410. Charde Pravin, Kasambe Raju and Tarar Jeevan L. (2011): BREEDING BEHAVIOUR OF INDIAN GREY Silverin B. and Goldsmith A. (1983): The effects of HORNBILL IN CENTRAL INDIA.THE RAFFLES modifying incubation on prolactin secretion in free BULLETIN OF ZOOLOGY. Supplement No. 24: living Pied-Flycatchers. Gen.Comp.Endocrinol. 59–64. 55:239-244. Clark R.G. and Shulter D. (1999); Avian habitat selection Singh R. (2004);. Behavior of Silver Pheasant (Lophura pattern from process in nest site use by ducks. nycthmera) Ph.D. Thesis C.S.J.M.Univ. Kanpur. Ecology. 80, 272-287. Smith Staart (1993): The instinctive nature of nest Collias N.E. (1997); On the Origin and Evolution of Nest sanitation. Part II Brit. Birds. 36:186-188. Building by Passerine Birds. Condor, 99: 253-270. Sordhl T.A. (2001) Copulatory behavior of American Conrad K.F. and Robertson, R.J. (1993):Clutch size in Avocets and Black Necked Stills. Auk. 118(4): 1072- eastern Phoebes (Sayornis phoebe). I. The cost of 1076. nest building. Canadian Journal of Zoology. Wesolowski, T. (1994): On the Origins of early evolution 71:1003-7. of male and female parental roles in birds. American Dayananda G.Y. and Hosetti B.B.(2009): Nest and Naturalists. 43:39-58. Nidification Activities of the Spoonbill Platalea Wheelwright, N.T., Lawler J.J. and Weinstein J.H. (1997) : leucorodia in Westerghat Region of Shimoga, Nest site selection in Savannah Sparrows: using Karnataka . Our Nature. 7: 26-31.s Gulls as scarecrows? Anim. Behav.; 53:197-208. Dayananda G. Y.; Hosetti, B. B.(2008): Nesting and Whistler H. (1948): Population Hand Book of Indian nidification activities of the Black-headed Ibis Birds. (Natraj Publication, Dehradoon). 459-461. (Threskiornis melanocephalus) in mid-western ghat Whitehead D.W. and Taylor T. (2001): Acadian Flycatcher region of Karnataka, South India. Current Biotica. 2 (Empidonax virescens). In the birds of North (4), 439-452. America. (Eds). A.Poole and F. Gill, (Birds of North Holway D.A. (1991): Nest site selection and the America Inc. Philadelphia). No.614 importance of nest concealment in the Black throated Zerba E. and Morton, M.L. (1983): The rhythm of Blue Warbler. Condor. 93:575-581. incubation from egg laying to hatching in mountain Hume, Allan O (1889-90) The nests and eggs of Indian White-crowned Sparrows. Ornis Scand, 14:188-197. Birds.:3. Kannan Vaithianathan and Pandiyan Jeganathan (2012): Nesting Ecology of the Spot-Billed Pelican Pelecanus Philippensis in Southern India. World Journal of Zoology. 7 (4): 295-302. Krebs J.R. (1987) Parental care and mating system. An

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Histochemical characterization of protocell-like supramolecular assemblies “Jeewanu”, synthesized in a irradiated sterilized aqueous mixture of some inorganic and organic substances

Vinod Kumar Gupta and Indira Chaturvedi Department of Zoology C.M.D. Post Graduate College, Bilaspur -495001 (Chattisgargh) India

Email: [email protected]

Abstract The present study shows the occurrence of autoreplicative protocell-like model “Jeewanu” for the origin of life in the possible prebiotic atmosphere. The Jeewanu has been prepared in the laboratory in a sunlight exposed sterilized aqueous mixture of some inorganic and organic substances(Bahadur and Ranganyaki;1970). An attempt has been made to study histology of these artificially prepared particles with acidic and basic dyes. The investigation revealed that Jeewanu has orderly structural organization with metabolic characteristics. In prebiotic atmosphere possibly energy transducing systems similar to Jeewanu existed which had an ability to convert solar energy into useful forms. Keywords : Jeewanu, Protocell, Supramolecular assemblies, Abiogenesis, Origin of life , Chemoton model.

Introduction formation of vesicles by various scenarios of self- organization (Hanczyc Szostak, 2004; Orgel,2004; One of the most fundamental problems of origin 2006;Eschenmoser, 1999). The synthesis of amino acids of life is to know of functional cells in prebiotic times might be initiated by spark discharge in a mixture of (Maynard 1986 &, 1987; Dawkins 1976). The origin of life reduced gases (Miller, 1953). The abiogenesis of organic presumably occurred by self-assembly of organic compounds on the early earth became central point in the compounds on the prebiotic earth into encapsulated postulation for the origin of life (Willis and Bada, 2000). molecular systems capable of catalyzed polymer synthesis. Life has been defined as a chemical system capable of Such a self-assembly might possess a membrane bound Darwinian evolution (Joyce and Orgel, 1998). Prior to self reproducing molecular cell systems (Deamer, 2002). evolution of biochemical machinery the growth and simple The self assembly of molecular systems within a variety of primitive cells (protocells) must have been driven by cell-sized has been extensively studied by workers in the environmental factors ( Zhu et al., 2012). field (Deamer 1997, Segre et al., 2001). It is suggested that mineral surfaces have an important role into pre-cellular Laboratory model of protocell systems should be evolution. Life began as a series of reactions resembling helpful in modeling various hypotheses of origin of life. metabolism. The autocatalytic pathways were established Assuming if protocell reproduction can be achieved and perhaps on mineral surfaces in aqueous phases as the adaptive innovations are possible in the spontaneous suggested by Bernal (1967). Pinto et al. (1980) was of the evolution it will then be simple chemical system. The opinion that endogenous synthetic stages need to be nature of such adaptations may provide clues as how investigated in detail. Overtime the systems became modern cells evolved from their earliest ancestors (Zhu et increasingly complex to the point that self reproducing al., 2012 ). polymers may be synthesized with cellular compartments Chemical evolution would have produced in course of time primitive self maintaining chemical systems with (Wachtershäuser,1988, Cody 2000). Prior to the rudimentary mechanisms of energy transduction but evolution of biochemical machinery the growth and without (replication and mutable) records. We call such division of simple primitive cells ( protocells) must have systems as “Infrabiological Systems” (Szarthmáry, 2005). been driven by environmental factors (Zhu et al., 2012). Several concepts of models of cells have been Self assembly processes can produce supramolecular produced to test various theories for the origin of cellular complex structures with certain properties of living state. life (Cavalier-Smith, 1987; Luisi, 1998; Szostak et Such structures are able to capture energy available in the al.,2001; Pohorille and Deamer, 2002). environment and initiate primitive reactions associated with metabolism, growth and replication (Deamer,2007). “Chemoton model” of living system was given by The self assembly and replication of membrane, the nature Gánti (1971). He emphasized combination of a metabolic of potential polymers and the nonenzymatic template cycle and a membrane was also proposed called a self directed copying nucleic acid sequences postulated reproducing microsphere (Gánti, 2003). In contrast, a

23 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 23-28 protocell-like entity with a boundary and template Solution (i) : replication but no metabolic system was conceived earlier Ammonium molybdate 4.00 gm and (Szostak, 2001). Based on Chemoton concept protocells diammonium hydrogen phosphate 12.00 gm were have been visualized as the functional integration of dissolved in 100 ml of distilled water. metabolism, containment and information processing (Rasmussen et al., 2003). The photochemical, formation of Solution (ii) : protocell-like microstructures “Jeewanu” in a laboratory Mineral solution : It was prepared by dissolving simulated prebiotic atmosphere capable of showing the following salts in 100 ml of distilled water. Each salt multiplication by budding, growth from within by actual was added when one salt was dissolved completely. synthesis of material and various metabolic activities has been reported (Bahadur et al., 1963;1964; 1966;1967; NaCl 3.00 gm

1970; 1975;1980). Jeewanu have been analysed to contain Ca(CH3COO)2 0.30 gm a number of compounds of biological interest viz. amino K SO 0.30 gm acids in free as well as in peptide combination (Bahadur et 2 4 al., 1954; 1961;1965; 1970; Briggs;1965; Maurya;1977), MgSO4 0.50 gm sugars as ribose as well as deoxyribose (Bahadur et al. FeSO 0.50 gm 1963; 1964; 1966; 1967; 1970 ), nucleic acid, bases as 4 purines as well as pyrimidines (Bahadur et al., 1970, 1972, Solution (i) and (ii) were mixed, as a result of Ranganayaki et al.,1976), phospholipids ( Bahadur et al., which, a white precipitate was formed, which was digested 1970; Singh, 1975) and ferredoxin -like material ( Rao et in the least quantity of HCl by boiling. al., 1978; Bahadur et al., 1980) in them. The presence of After cooling, the volume of the solution was various enzyme like activities viz., phosphatase, ATP-ase, made up to 300 ml by distilled water. Mixture was cotton esterase ( Briggs, 1965; Bahadur et al., 1970; Singh, 1973; plugged and then sterilized in an autoclave at 15 lb pressure Gupta 1980 ), nitrogenase ( Smith et al., 1975; Bahadur et for 30 minutes. al., 1980 ) have been also been detected in Jeewanu mixture. It was found that under certain specific conditions After cooling, 3 volume of above mixture and 1 Jeewanu can catalyse photolytic decomposition of water volume of 36% formaldehyde were aseptically added in a utilizing sunlight as a source of energy. Further it was conical flask. observed that H2 thus released is utilized in photochemical A part of mixture was kept in dark as control. fixation of nitrogen, and carbon dioxide. These findings Another part was taken in a separate conical flask, cotton 14 15 were confirmed using C , N and D2O ( Smith et al ; plugged and covered with black cloth and was kept as Bahadur et al., 1980). Gáinti (2003) discussed that control. Jeewanu posseses a promising configuration similar to The mixtures were exposed to sunlight for protocell-like model. Therefore, an attempt has been made varying periods of exposure as per requirement of the made to investigate histological characteristics of experiment. The control mixtures covered with black cloth Jeewanu to characterize their structural organization. The were examined for the formation of photoproducts at present investigation is aimed to acertain the possible regular intervals. nature of earliest living system on the earth. Observations Materials and Methods The Jeewanu mixture kept in dark as control The high mineral Jeewanu were prepared by the showed negative results. It is colourless and did not show method as described by Bahadur and Ranganyaki (1970) any initiation of photochemical reaction. and Verma (1980). The histochemical localization of acidic material in the Jeewanu was studied by Eosin The mixture after exposure to sunlight (Baker, 1969) while the basic materials were stained with immediately became bluish in colour. The intensity by Gentian Violet (Baker, 1969) and Methyl Green (basic (bluish) of colour increases on further increasing the dye) (Brachet, 1944; Pearse,1961). The histochemical exposure time of sun light. The photochemical formation localization of RNA-like activity was studied by Pyronin Y of Jeewanu becomes visible under high power of staining technique (Brachet, 1944; Pearse,1961). The microscope even in few minutes (2-5 minutes) of exposure staining procedure employed in the present study is routine to sunlight. procedure. Morphologically, Jeewanu are spherical in shape The High Mineral Jeewanu mixture was exposed and exhibit bluish colour. The microscopic (1500x to sunlight and the photochemical formation of Jeewanu magnification) observation of Jeewanu showed that they was studied at regular intervals under high power and oil became yellowish in colour with a definite boundary wall immersion (1500x) of optical microscope. and possesses intricate internal structures. The number of Jeewanu formed increases in number on further exposure The ingredients of the solution for the preparation to sunlight. The size of Jeewanu forms varies from 0.5µ to of Jeewanu are as follows :

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3.5µ in diameter (Fig. 1 & 2) With Methyl Green (basic dye) the central structure ofl With Gentian Violet staining, the central structure Jeewanu take intense green stained and diffused greenish of high mineral Jeewanu presence of basic material-like colouration was also seen in the peripheral region showing substance. In some particles bluish colour was seen the presence of a basic material-like substance in the diffused in the peripheral region (Figs 3 & 4). Jeewanu mixture (Figs 5 and 6). The microscopic examination of slides under The bright red colour of Pyronin Y was diffused throughout high power and oil immersion (1500 X) showed that extra the High Mineral Jeewanu showed the presence of RNA central region of High Mineral Jeewanu was stained pink like activity in the mixture (Figs 7 &8). showing the localization of acidic structure in the extra central region.

Fig 1 & 2 : Micrograph showing photochemical formation of high mineral Jeewanu showing multiplication by budding and growth from within under optical microscope (1500 X)

Fig 3 & 4 : High mineral Jeewanu stained by Gentian Violet Ó showing histochemical localization of acidic in the central region

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Fig 5 & 6 : High mineral Jeewanu stained by Eosin (micrograph c, d) showing histochemical localization of basic material in the extracentral region

Fig 7 & 8 : High mineral Jeewanu stained by Pyronin Y (micrograph c,d,) showing histochemical localization of RNA-Like activity diffused throughout the micro structure

Discussion forms of metabolism arose inside closed compartments and became more complex later. At certain concentration One of the major challenges is to artificially additional molecules no longer dissolve but instead begin synthesize protocell which can ensemble organic and to associate into small aggregates called “micelles” inorganic substances into self-reproducing system of life ( (Bahadur, 1967). The abiogenesis of biogenic materials Szostak et al., 2001; Hanczyc et al.,2004). Eigen and viz. amino acids, nucleic acid bases, sugars, phospholipids Shuster (1979) described certain organic molecules which have been discussed for the origin of life. He has formed can spontaneously organize into larger structures. Haldane photochemically self- sustained protocells- like (1928) and Oparin (1957& 1968) gave theory of suprarmolecular assemblies called Jeewanu responsible coacervation of colloidal material as the beginning of for the origin of life. The present study is in conformity to growth material characterization of life. Molecular the observations of Bahadur (1964 & 1966) that sunlight evolution refers to the ultimate formation of molecules by exposed sterilized aqueous high mineral mixture of chemical transformation of substances with the condition ammonium molybdate, diammonium hydrogen that earliest living systems were madeup of the same phosphate, biological minerals and formaldehyde material of which the present day living system is madeup confirms photochemical formation of protocell-like of (Bahadur, 1964 & 1966). Bahadur postulated that first molecular associations.They have a definite boundary wall

26 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 23-28 and intricate internal structure. Their size varies from 0.5 µ Bahadur K., Ranganayaki S., Verma H.C., Srivastava R.B., to 3.5 µ in diameter. It is also held that formation of Agrawal K.M.L., Pandey R.S., Saexna I., Malviya Jeewanu is strictly an outcome of a photochemical A.N., Kumar V., Perti O.N. and Pathak H.D. (1964): reactionin the possible prebiotic atmosphere. Synthesis of Jeewanu – Part II Photochemical The present cytochemical studies of Jeewanu preparation of growing, multiplying units with with basic dyes Gential Violet, Methyl Green and Pyronin metabolic acitivities. Zbl. Bakt. Abt II, 117, 575 – 584. Y showed the presence of acidic material-like substance in Bahadur K. Saxena, I. (1964): Conversion of lifeless the central region. Staining with Pyronin Y suggested that matter into living systems. Zbl. Bakt., 118, 671. RNA-like activity is possibly diffused throughout High Bahadur K., Pandey R.S., Perti O.N. and Pathak H.D. Mineral Jeewanu. The histochemical staining of High (1965): Origin of living systems. Agra Univ. J. Res. Mineral Bahadur et al Jeewanu with acidic and basic stains (Sci.), 13 (II), 197 -224. showed that Jeewanu possesses an ordered structural organization. The present investigation confirms the Bahadur K.(1966) In: Synthesis of Jeewanu, The Protocell observations of Bhadur et al. (1970, 1980, 1972,1973, (Ramnarain Lal Beniprasad, Allahabad U.P., India), 1976) that RNA monomers are more readily 20. photochemically synthesized in Jeewanu mixtureas The Bahadur K. (1967): Synthesis of Jeewanu : The Protocell, also supported the view that primitivity of RNA in the Zbl. Bakt,121(2), 291-319. possible primitive atmosphere (Joyce and Orgel, 2006). The microscopic examination of Jeewanu, the Bahadur K. and Ranganayaki S. (1970): Photochemical autoreplicative protocell-like abiogenic microstructure formation of self-sustaining coacervates. J. Brit. shows that it possesses a definite ordered structure Interplanetary Soc., 23 (12), 813 – 29. (Bahadur,1975; Bahadur and Gupta, 1972; Gupta,1980; Bahadur K. (1975): Photochemical formation of self- Gupta,2002). sustaining coacervates capable of growth, All cellular life today incorporates two processes multiplication and metablolic activity. Zbl. Bakt., 130 – (a) self-assembly; and (b) directed assembly. The (II), 211 -218. directed assembly involves the formation of covalent Bahadur K., Randanayaki S., Folsome, C. and Smith, A. bonds by energy dependent synthetic reactions and require (1980): A Functional Approach to the Origin of Life a coded sequence; while spontaneous self-assembly Problem, National Academy of Sciences, India, occurs when certain compounds interact through non- Golden Jubilee Commemoration Volume, 1-18. covalent, hydrogen bonds, electrostatic forces and non- Bernal, J.D. (1967): The Origin of Life (Weidenfeld & polar interactions to form closed membrane bounded Nicholson : London). micro-environment (Pohorille and Deamer, 2002). It is also suggested that it is possible that in the primitive Briggs M.H. (1965): Experiments on origin of cells. Space atmosphere there may nonlinear collaboration of photo- flight, 7(4), 129. redox transformations at mesoscopic level possibly led to Cavalier-Smith T.(1987): The origin of cells a symbiosis emergence of supramolecular assemblies similar to between genes, catalysts and membranes. Cold Harb Jeewanu, showing properties of biological order, viz. Symp. Quant. Biol., 2, 805 - 824. multiplication by budding, growth from within by actual synthesis of material and metabolic activities. It may be Cody G.D., Boctor N.Z., Filley T.R., Hazen R.M., Scott postulated that earliest energy transducing systems were I.H., Sharma A. and Yoder H.S. Jr.(2000): Primordial possibly a photoautotroph similar to Jeewanu which had an carbonylated iron-sulphur compounds and the ability to convert solar energy into useful forms. synthesis of pyruvate. Science, 289,1337 -1340. Acknowledgement Dawkins R. (1976) In: The selfish gene (Oxford University Press, Oxford, UK). V.K. Gupta gratefully acknowledges University Grant Deamer D.W. (2002): Workshop papers on Signs of Life Commission, Central Regional Office, Bhopal– M.P.,India (National Research Council, Washington D.C., USA, for financial assistance under a minor research project. National Academic Press), 68. References Deamer D.W. (1997): The first living systems : a Bahadur K. (1954): Photosynthesis of amino acids from bioenergetic perspective. Microbiol. Mol.Biol. Rev., paraformaldehyde and potassium nitrate. Nature, 61, 230 – 261. 173,1141. Deamer D.W. (2007) : Emergent phenomenon in biology : Bahadur K. and Srivastava R.B. (1963): Preparation of the origin of cellular life: In Planetary systems and Jeewanu units capable of growth, multiplication and origin of life (Eds. Pudritz, R.E., Higgs Paul g., Stone, metabolic activity. Vijnana Parishad Anusandhan J.R., Cambridge University Press). Patrika, 6, 63-117. Eigen M. Shuster P. (1979) In: Hypercycle: A principle of

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Barriers to Breast Cancer Screening In Young Indian Women: A Tale of Two Cities

A.Vidyarthi*, A.Soumya**, S.Choudhary!, B.K.Sinha# *Onco-Surgeon, Guru Nanak Hospital and Research Center **Fashion Designer, Shri Arvind Mills, Banglore !

Department of Psychology, RLSY College, Ranchi #Department of Zoology,SSM College, Ranchi

Abstract : Screening for breast cancer reduces mortality by 30 – 40% but there are many psychological barriers to screening. A comparative case study was undertaken in young well educated Indian women to map the barriers to screening and their relation to the socio-cultural milieu. Volunteers from two premier educational institutes of Ranchi and Chennai were required to respond to a self completion questionnaire recording their attitudes to breast cancer screening. The responses were marked on a scale of 1-5. The two cohorts were from similar social and economic backgrounds and had equally fair knowledge of Breast cancer but differed in their access to healthcare and their choice of dress. The significant differences in outcome variables of embarrassment, fear and barrier in the two cohorts were found to correlate to the centre of study only. The authors postulate that this may be the due the influence of their present social environment i.e. 'neighbourhood'. Key Words: Breast cancer, Screening, Barriers, Socio-cultural parameters, Knowledge of breast cancer

Introduction environment of the subject on the barriers to screening. Regular screening for breast cancer reduces mortality by about 30 – 40 % (Vaino et al., 2002; Tabar et Materials and Methods al., 2003; Swedish Organized Service Evaluation Group, A comparative case study (Hantrais, 1996) was 2006). But there are many psychological, social and designed, selecting women from two premier educational economic barriers to screening e.g. embarrassment, lack of institutes of Chennai and Ranchi with the following access to health-care, or lack of physician referral. inclusion criteria: (McGarvey et al., 2005; Secginli et al., 2006; Ansnik et al., 2008) 1. Women less than 30 years with a graduate's degree. Patients undergoing mastectomy have a worse 2. Women with no breast diseases “Quality-of-Life” (The WHOQOL Group, 1994; Fleck et 38 females from Chennai and 40 from Ranchi, al., 1999; Kluthcovsky et al., 2007;) compared to those from two premier institutes, volunteered for the study. undergoing Breast conserving surgery, especially the Embarrassment felt to activities relating to breast physical and social components (Ganz et al., 2004; Pandey examination, fear of loss of breast and knowledge of breast et al., 2006;), with a poorer body image (Falk Dahl et al., cancer were tested through a self-completion 2010). questionnaire. They were also tested for their knowledge of Screening uptakes in US and UK are 75 – 80 % breast cancer through ten questions answered in true/false (Cole and Bryant, 1997; Patnick, 2008 ;), but it is almost nil format. A pilot study was carried out to establish the in India. This study attempts to define the barriers that internal reliability of the questionnaire and Cronbach alpha prevent Indian women from volunteering for breast cancer scores of the various sections measured 0.919, 0.816 and screening and compare the magnitude of those barriers in 0.765 respectively (Cronbach, 1951). young women from a metropolitan city and a state capital. Independent variables included age, annual The authors have interviewed several educated women family income, family history of breast cancer, and access with a fair knowledge of Breast cancer who have been to healthcare (access to family physician, history of advised breast self-examination but refuse to practice even previous gynecological consultation and access to Health that. What prevents them from undergoing screening? insurance). The socio-cultural parameters included the In this backdrop the present study aims to: preferred style of dress and the place of residence, now and as a child. 1. Map the barriers to screening in young, educated, well-off Indian women with good access to The pilot survey showed that some subjects who healthcare facilities and, had consented to the study had not answered one or more questions. The possible reasons may be: 2. Define the influence of the socio-cultural

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1. They did not know the answer 0.22).There was no correlation between the knowledge 2. Perceptual Defense (Blum , 1955; Minard, 1965; scores of the respondents and their embarrassment, fear Loiselle and Williamson, 1966;) caused them to and barrier scores. The difference in the knowledge scores ignore questions that evoked strong negative between the respondents from Chennai and Ranchi did not emotions reach statistical significance (Chennai = 4.79 Ranchi = 4.35, p = 0.36). Therefore, “barrier”, a third dependent variable was added to account for the unanswered questions. All Measures of Embarrassment variables were marked on a scale of 1-5 and final scores Five questions measured embarrassment and the were obtained by taking the section mean. responses are shown in figure 1.Briefly, 35 per cent would The results were analyzed using PAST 2.17b refuse breast examination by a male doctor, 46 per cent will (Hammer, Harper, and Ryan 2001) software. The statistical hide a breast lump from their sons, 40 per cent would refuse tests included Chi-square test, ANOVA, and Kruskal – a Mammogram because it is too embarrassing, and 76 per Wallis test. cent may ignore any advice regarding breast examination due to embarrassment. The mean Embarrassment score in Results: Chennai was 2.24 and in Ranchi was 3.01(p < 0.01).This The demographic characteristics of the sample score did not vary in relation to any of the independent have been presented in table 1.The mean age of the variables except the center of study. respondents from Chennai and Ranchi were 20 years and 22 years respectively.78 per cent were graduates and 22 per Measures of Fear of mastectomy cent post-graduates. The two cohorts were from The absence of fear of loss of breast (mastectomy) comparable economic backgrounds (Family income, p = was used as a surrogate measure of resistance to screening 0.051;).They were significantly different in their access to and the responses are shown in table 2 and figure 2. Briefly, a family physician (p < 0.001) and access to health 49 per cent would not demand breast prosthesis, 62 per cent insurance (p < 0.0001) but similar in the number of would not feel any restriction in their choice of dress, 77 volunteers who had visited a gynecologist (p = 0.16). 12/38 per cent would not feel deformed, and 71 per cent fear the volunteers from Chennai had seen breast cancer in their cancer more than the deformity after mastectomy. The family compared to 5/40 from Ranchi (p = 0.041). mean Fear scores in Chennai were 2.50 and those in Ranchi were 3.14 (p < 0.001). None of the recorded independent Socio-cultural background variables correlated with the fear score except the center of 63 per cent respondents from Chennai were residents of a study. non – metropolitan city and 60 per cent had spent their It is implicit from figure 3 that a significantly childhood in one. In comparison, 95 per cent respondents greater number of respondents from Ranchi than Chennai from Ranchi had spent their childhood in a non – avoided consenting to mammograms (Chi^2 = 16.56; p < metropolitan city and 92 per cent were now staying in one. 0.001).Only 50 per cent respondents from both centers The difference did not reach statistical significance (Place chose regular mammograms! The mean barrier scores in of residence as a child, p = 0.80; present place of residence, Chennai were 1.81 and Ranchi were 2.31 (p < 0.001). p = 0.86). 87 per cent Chennai respondents preferred western dresses compared to 53 per cent from Ranchi (p < Discussion 0.001). Respondents from Ranchi also displayed a This survey attempted to define the emotional and markedly heightened 'perceptual defense' response to psychological barriers against breast cancer screening in uncomfortable questions – either due to embarrassment or Indian women. Therefore, the respondents were chosen lack of knowledge. In fact, 38 per cent Ranchi volunteers from a population which would face minimum practical or didn't know the size of their brassier cup compared to 0 per logistic problems in accessing healthcare services. The cent Chennai volunteers! questions, too, were designed to strike deep emotional That, two cohorts of women of similar age chords in educated women and were based on situations groups, educational and economic backgrounds who have commonly encountered by the first author in his clinical spent their lives in similar urban social environments practice. should display such varied response to embarrassing The results revealed deep emotional distress to questions points to the effect of their social environment situations commonly encountered by breast cancer i.e. the effect of the 'neighborhood'. patients. For example, a woman often discovers a lump in Knowledge of Breast cancer her breast when she is a widow and dependent on her son. Our survey shows that in such situations 46 per cent The average score was 4.57/10 ranging from 0 – respondents would hide the lump from their son and allow 9.The mean score of respondents with a family history of the disease to progress. When asked to react to the slogan 'if breast cancer was 5.12 and those without a family history only women paid as much attention to their breasts as men was 4.41; the difference was not significant (p = do'; one out of four respondents ignored the question and a

30 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 29-35 similar number opined that it is a 'necessary evil' - men will women was surprising. When specifically questioned if ogle at female breasts! Very few respondents really they would suffer the embarrassment of mammograms to understood that the slogan refers to breast-self examination avoid the deformity due to loss of breast 70 per cent as a means of cancer screening. respondents chose to 'get the tumor out anyhow'! This These deep –seated emotional barriers have been indicates that fear of a diagnosis of cancer overrides the found in highly educated women. The conventional fear of deformity. Therefore, fear of being detected with wisdom is to overcome them through education about cancer would prove a barrier to screening rather than fear cancer. Therefore, the respondents were also tested for of deformity motivating them to undergo screening. A their knowledge of breast cancer through ten questions similar result was obtained by Tejeda et al. (2009) and answered in true/false format. The median score was five Watts et al. (2009) when surveying women to find barriers with most scoring between 3 and 6.Thus the respondents to mammography and pap smears respectively. displayed a high level of emotional avoidance combined The second aim of the study was to define the with fair knowledge of the disease and minimal practical or influence of socio-cultural differences on screening logistic barriers. Forbes et al. (2011) surveyed the health attitudes. Therefore, two groups of volunteers of seeking attitudes of women from various ethnic comparable educational, economic and cultural communities in East London. They found that women of backgrounds were chosen from a metropolitan city and a Indian origin reported emotional rather than logistic or state capital. In spite of their comparable backgrounds practical barriers to seeking medical help. 59 per cent more respondents from Ranchi preferred ethnic dresses reported embarrassment as a barrier, 46 per cent worried than those from Chennai. Given that dress choices are about what the doctor might find, and 53 per cent reported vulnerable to peer pressures this change in style reflects the not feeling confident talking about their symptoms. influence of 'neighborhood'. In the authors' opinion the Another survey amongst South Asian women living in differences in their embarrassment, fear and barrier scores Canada (Bottorff et al., 1998) found that many respondents are also due the effect of 'neighborhood', since we could not would not visit a doctor unless accompanied by a friend or detect any correlation to either family history of breast relative. cancer, ease of access to health care or knowledge of breast These studies and the present study show that cancer. barriers to health – seeking behavior, in Indian women, in Many authors have linked perceptions of general and cancer screening in particular are neighborhood to general health status (Ross and predominantly emotional and may not be influenced by the Mirowsky, 2001; Hill, Ross and Angel, 2005; Wen, subjects' health – related knowledge. Embarrassment, Fear Hawkley and Cacoppo, 2006) .Schempf, Strobino and and Barrier scores of the respondents were not associated O'Campo (2009) were able to show that neighborhood with either their having a patient in the family or their structures and processes shaped maternal behavioral risks knowledge of cancer (p values; 0.15, 0.79 and 0.14 thus impacting infant birth weight. In the authors' opinion respectively). Forbes et a.l (2011) and Scanlon and Woods the social environment of a metropolitan city helps reduce (2005) both found that South Asian women have better the barriers to screening thus influencing health-risk knowledge of age-related risks of breast cancer but are less behavior of respondents. At the same time, as more and likely to examine their breasts than their western more urban centers develop there is hope that tier two cities counterparts, same as the findings of our survey. like Ranchi will also develop social environments like The volunteers for this survey were young Chennai in the future to positively impact the health women given to wearing western dresses and those from behavior of its residents. Chennai were students at a national fashion designing Conclusions institute. It is expected that such women would pay more attention to their body image than the general Indian The study seems to indicate that barriers to cancer female. Cash, Melnyk and Hrabosky (2004) have screening are an emotional response of the subject and that postulated that body image includes an attitude of 'neighborhood' i.e. place of residence may have an impact satisfaction or dissatisfaction in one's body that varies with on the risk behavior of the residents. two factors-self-evaluation and investment in appearance Limitations of the study: or the respondent's view of the importance of her Being a comparative study, the results of this appearance. Assuming that younger women are likely to study can't be generalized. But the first author has come invest more in their appearance we hypothesized that fear across many instances in his clinical practice when women of disfigurement by loss of breast may prompt them to of this socio-economic group avoided screening. The choose regular screening, but the results failed to support question; why are they avoiding screening, seemed to beg our hypothesis. Surveys in Indian women from low socio – an answer! economic strata (Khan et al., 2010) did find that such patients did not pay much importance to their appearance, The study asks young women to respond to but finding the same result in a group of young educated situations they have not faced. But a majority has rightly said that mastectomy would result in a poorer body image

31 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 29-35 and dress restriction, similar to the results of Falk Dahl et Acknowledgments: al. (2010) and Ohsumi et al. (2009) Therefore the other responses are unlikely to differ significantly. The authors wish to express their gratitude to the students and faculty of National Institute of Fashion The study also suffers from small sample size but Technology, Chennai and Xavier Institute of Social in a conservative society such as India it is very difficult to Sciences, Ranchi for their cooperation. persuade women to answer intimate questions about their breasts.

Table 1.Comparison of the demographic characteristics of the respondents from Chennai and Ranchi

Demographic characteristic Chennai Ranchi Total number of respondents 38 40 Mean age (range) 20(17 – 23) 22(20 – 27) Education Graduate (%) 37(97.4) 22(56.4) Post – Graduate (%) 1(2.6) 17(43.6) Annual Family Income (%) < INR 15000(%) 0 1(2.7) INR 15000 – 500,000(%) 23(60.5) 27(73.0) INR 500,000 – 1,000,000(%) 4(10.5) 9(24.3) > INR 1,000,000(%) 10(26.3) 0 Positive history of Breast Cancer in Family (%) 12(31.6) 5(12.5) Respondents having Family Physician (%) 27(71.1) 16(40) Respondents who have seen Gynecologist (%) 13(34.2) 20(50) Respondents with Health Insurance (%) 33(86.8) 9(22.5) Dress Style Ethnic +/- dupatta (%) 5(13.2) 17(42.5) Western +/- dupatta (%) 33(86.8) 21(52.5) No response (%) 0 2(5.0) Present place of residence Tier 1 city (%) 14(36.8) 3(7.5) Tier 2 city (%) 9(23.7) 23(57.5) Tier 3 town ((%) 15(39.5) 14(35.0) Residence during childhood Tier 1 city (%) 14(36.8) 2(5.0) Tier 2 city (%) 6(15.8) 19(47.5) Tier 3 town (%) 17(44.7) 19(47.5) Mean knowledge of cancer score(CI)* 4.79(4.13 – 5.42) 4.35(3.7 – 5.0)

* F (1, 73) = 0.8399p (same) = 0.3624Not Significant

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Table 2: A Comparison of Mean Embarrassment, Fear and Barrier scores of respondents from Chennai and Ranchi.* Chennai Ranchi P value Embarrassment 2.237 3.096 <0.01 Fear 2.504 3.143 <0.001 Barrier 1.81 2.31 <0.001

*The respondents from Ranchi have significantly higher scores in spite of having similar scores when tested for knowledge of Breast cancer (ref: Table 1)

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Improved Maturation of Wild and Pond-reared Black Tiger Shrimp Penaeus monodon ( Fabricius) using different Combinations of Live and Wet Feeds

K. Ramesh Babu Department of Marine Living Resources, College of Science and Technology Andhra University, Visakhapatnam- 530 003. A.P., India.

Corresponding author Email Id: [email protected].

Abstract : The impact of various combinations of live feeds and frozen feeds such as polychaete, squid meat, crab meat and beef liver was tested on the maturity and spawning of the shrimp Penaeus monodon. Females with weight ranges 86g to 96g were selected for the present experimentation. The shrimps were fed with five different combinations at the rate of 15% of their body weight. Feed combinations selected for current study was crab and polychaete, oyster and polychaete, squid and clam, beef liver and squid, and oyster and beef liver. We applied different ablated methods to evaluate the reproductive ability of wild and pond reared Penaeus monodon The great reproductive performance was achieved when shrimps fed with Feed-I, the reproductive performance based on fecundity was identified by the number of eggs in gravid females. Future research efforts focused on the artificial diets which contain both live and frozen feed combinations will be essential for the improved egg quality in tank- reared Penaeus monodon. Thereby increasing the consistency and reliability of maturation diets for better quality nauplii production. Key Words: Penaeus momodon, Spawning, Fecundity, Hatching, nauplii, Survival rate

Introduction development as spawners. The initial weight of females In addition to eyestalk ablation, various naturally and males ranged from 86.18 to 96.52 g and from 50.61 to occurring and formulated feeds are proved fertile in 65.21 g respectively. The shrimps were fed with five inducing maturation in pond reared and naturally collected different feeds combinations as crab and polychaete, oyster penaeid shrimps. Fish meal, Vitamin-C, and fish oil play and polychaete, squid and clam, beef liver and squid, and important role in growth and reproduction in aquatic oyster and beef liver. Shrimp were fed at the rate of 15% of animals (Jia-Yuan Xu, et al., 2010). In general the fresh or their body weight. It took about 60 days for the conversion frozen diets currently used for maturation of shrimps under of pre-vitellogenic female into the vitellogenic stage. The captivity have (1) rich animal protein (approximately 60% developed spawners were transferred to a hatchery to on dry weight basis) with amino acid profiles similar to that assess the efficiency of spawners in terms of spawning, of shrimps (e.g. shrimp and some bivalves such as Tapes hatching and survival of larvae. sp.) (2) High cholesterol and (3) Phospholipids, with The gravid females were transferred to hatchery, Phosphatidylcholine (Lecithin) fraction which is probably where they were gradually acclimatized to the hatchery of major importance. Other than these generalities, little is conditions and were disinfected with 10 ppm of known of maturation diets. Nevertheless, the inhibition of formaldehyde and 50 ppm of Treflon (Trifluralin) ovarian development under captive conditions was respectively for 20 minutes. Spawners were fed at the rate overcome by eye stalk ablation (Beard and Wickin, 1980). of 15-20% of their bio-mass at regular interval of time both Unilateral eye stalk ablation is simple techniques for seed during the day and night. Water temperature ranged from production (Villaluz et al., 1972). 28-31C and sea water salinity 29-32.5 ppt in the hatchery Food is an important factor for sexual maturation and also in the laboratory. Prior to initiation of the trail, each spawner was weighed and the weight was recorded. (Browday, 1992) and male reproductive performance 2 (Meunpol et al., 2005) Many advances were made in the Spawners were stocked at the rate of 2-3 m in female to development of technology for maturation and male ratio of 2:1. Females were checked for the presence of reproduction of penaeid shrimp (Bray and Lawrence, 1992 spermatophores in the thelycum. Unilateral and bilateral and Browday, 1992). The present study was undertaken to eyestalk ablation was carried out in the early morning demonstrate the larval survival of ablated pond – reared P. hours during the intermoult stage by cutting the eyestalk monodon by using five different practical diets. with sterilized scissors. After eyestalk ablation and a period of five to seven days, females were checked by with under Materials and methods water torchlight (sourcing light) for ovarian maturation by For studies on the impact of fresh feeds on external observation. Based on the relative size of the fecundity & hatching rates, the brooders , raised in shrimp ovary gravid females in maturation stage IV were farms were transferred to 10 ton capacity epoxy resin transferred to round fibre tanks having 0.5 ton of filtered coated rectangular RCC tanks of 52.51 m for further sea water of 30 ppt for spawning. After spawning, the

37 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 37-42 water was drained using fine meshed net (100 µ mesh) to daily growth (ADG) of 0.257 g in female shrimps was retain the eggs. The spawned eggs were washed with recorded when the shrimps were fed with oyster and beef treated seawater containing Treflon as anti-fungal reagent, liver. The lowest average daily growth (ADG) of 0.183 g concentration ranging from 0.02 to 0.05 ppm. After 2 to 3 was recorded in male shrimps fed with oyster and beef liver washings, the eggs were stirred and three one ml aliquots (Fig.1 & 2). were drawn from the beaker to count the number of eggs.

Then the eggs were transferred to 500 litre capacity black Crab+Polychaete Oyster+Polychaete Squid+Clam paint coated hatching tanks. After 40-45 minutes of Beaf Liver + Oyster Squid + Beaf Liver spawning and upon the appearance of hatching envelop, the number of nauplii in 50 ml aliquots drawn from the hatching tanks was enumerated. The hatching rate was calculated by the following formula. Total nauplii Hatching rate (%) = ------X 100 Total eggs spawned Morbid larvae and Dead larvae were removed from larval rearing tanks by siphoning. A water temperature of 29- 31C, pH 7.8, and salinity of 29-32.5 ppt were maintained at the optimum levels throughout out the study period. For studies on larval diet and larval development, the treated seawater as said above was used in the laboratory and Fig. 1. ADG of Female shrimps (P. monodon) hatchery. The larval rearing tanks (LRT) have capacities of fed with different feed combinations 14-16 tons, which are parabolic in their structure and design. Crab+Polychaete Oyster+Polychaete Squid+Clam

The larvae were maintained in 1.2 m 7.5 m 1.8 m size Beaf Liver + Oyster Squid + Beaf Liver tanks. The water level in each tank was maintained at 16 tones. Tanks with a water holding capacity of 14 tonnes were used for rearing the post larvae. A haemocytometer was used for algal cell counting in the medium. A salinity refractrometer was used for regular checking of the water salinity. Algal culture was done in 250 ml, 500 ml, 1000 ml and 2000 ml conical flasks (Borosil glass) and 20 litre carboys were used for indoor algal culture. 100 litres plastic cans, 1000 litre to 1200 litre FRP tanks, and 4 tonnes capacity rectangular concrete outdoor tanks were used for outdoor algal culture. Round, clylindro-conical fibre glass tanks of 500 litres capacity were used for artemia culture. The various prophylactic measures taken with treatment groups are presented in Table I. In all the treatments, the water quality parameters such as temperature and salinity Fig. 2. ADG of Male shrimps (P. Monodon) were in optimal range of 29-31C, 28-32 ppt respectively. fed with different feed combinations

Results A notable difference in body weight gain was An important aspect of the present study is on the recorded among the shrimps fed with different influence of feeding condition on the growth and combinations of feed. The maximum weight gain of 24.06 maturation of shrimps under captivity. This study is aimed g was noticed in females fed with crab and polychaete in 60 in filling up the gap in aquaculture industry. Considerable days of time, whereas in males the weight gain obtained variations were noticed when the shrimps were fed with was 18.12 g in the same duration of feeding condition and combined fresh feeds. The feeds have remarkable with the same feed. The results with respect to the average influence on the growth body weight, and also on fecundity daily growth (ADG) and net body weight are summarized rates. as follows. The maximum Average daily growth (ADG) of 0.401 g Females was recorded when the female shrimps were fed with crab and polychaete. In males the highest average daily growth Average Daily Growth (ADG): Crab and polychaete, (ADG) of 0.301 g, was observed when the shrimps were 0.401 g; oyster and polychaete, 0.364 g; squid & clam, fed with crab and polychaete, while the lowest average 0.340 g: beef liver and squid, 0.298 g; oyster and beef

38 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 37-42 liver 0.257 g. (Fig.1) Maturation Net weight gain in 60 days: Crab & polychaete, 24.06 g; Ovarian maturation in shrimps fed with different oyster and polychaete, 21.84 g; squid & clam, 20.40 g; beef combinations of feeds was found to vary significantly liver and squid, 17.88 g; beef liver & oyster, 15.42 g; between the treatments. The minimum elapsed period (Fig.3). recorded among wild unilaterally ablated was 6-9 days in ADG of Males: shrimp fed with crab and polychaete followed by 5-10 days in shrimp fed with oyster and polychaete and the maximum Crab and polychaete, 0.302 > oyster and polychaete 0.273 of 10-16 days was recorded in the case of shrimp fed with > squid and clam 0.234 > beef liver and squid 0.191 g > oyster and beef liver (Table 1) oyster and beef liver, 0.183 g. (Fig. 2). The minimum elapsed period recorded among Net weight gain: Crab and polychaete 18.12 > oyster and captive unilateral ablated was 9-15 days in shrimp fed with polychaete 16.38 > squid and clam 14.04 > beef liver and crab and polychaete, and the maximum of 19-27 days was squid 11.46 g > oyster and beef liver 10.98 g. (Fig. 4) recorded in case of shrimp fed with oyster and beef liver. The minimum elapsed period in recorded among wild Crab+Polychaete Oyster+Polychaete Squid+Clam bilateral was 10-13 days in shrimp fed with crab and Beaf Liver + Oyster Squid + Beaf Liver polychaete, and the maximum of 20-24 days was recorded in case of shrimp fed with oyster and beef liver (Table 1) Table: 1. Efficiency of different fresh feed combinations on maturation of shrimps (elapsed time) from first stage to spawning (days).

Feed Wild Captive Wild combinations unilaterally unilaterally Bilaterally ablated ablated ablated Crab and polychaete 6 to 9 9 to 15 10 to 13

Oyster and polychaete 5 to 10 11 to 16 12 to 18

Fig. 3. ABW of Female shrimps (P. monodon) Squid and clam 6 to 13 13 to 20 15 to 20 fed with different feed combinations Beef liver and squid 8 to 15 16 to 24 18 to 22 Oyster and beef liver 10 to 16 19 to 27 20 to 24 Crab+Polychaete Oyster+Polychaete Squid+Clam

Beaf Liver + Oyster Squid + Beaf Liver Wild unilaterally ablated Experiments on eyestalk ablation were conducted on shrimps collected from the wild (from the Sea) and the following observation ware made. Crab and polychaete 6- 9 days > oyster and polychaete 5-10 days > squid and clam 6-13 days > beef liver and squid 8-15 days > oyster and beef liver 10-16 days (Table 1). Captive unilateral ablated: Eyestalk ablation was conducted on shrimps initially reared in aquaculture ponds and the results are like this. Crab and polychaete 9-15 days > oyster and polychaete 11-16 days > squid and clam 13-20 days > beef liver and squid 16-24 days > oyster and beef liver 19-27 days (Table 1). Fig.4. ABW of Male shrimps (P. Monodon) Wild bilateral ablated: fed with different feed combinations Experiments on eyestalk ablation were conducted on shrimps collected from the wild (from the Sea) and following observation were made. Crab and polychaete 10-13 days > oyster and polychaete 12-18 days > squid and clam 15-20 days > beef liver and squid 18-22 days > oyster and beef liver 20-24 days (Table 1).

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Spawning rate: critical habitat for Penaeus monodon in Andhra Pradesh, A comparison is made on the number of eggs laid India. per each brood in the shrimps raised under captivity and In present studies argue that the mangrove also shrimps caught in the sea. For this care is taken to ecosystem is crucial prerequisition for the recruitment include the same weight groups of animals and it is clearly success of many penaeid shrimp species, particularly evident that the number of eggs laid by the shrimps caught P.monodon. In the state of Andhra Pradesh, the Godavari in the sea are significantly higher when compared to those river delta should be a key nursery ground, since this delta that were raised under captivity Shrimp fed with crab ad alone harbours 56% of the total mangrove cover in the polychaete and oyster and polychaete showed interesting state. This is also supported by the fact that the coastal results of higher spawning. areas off the Godavari delta constitute the main trawling Hatching rate: grounds for both fin and shell fish in Andhra Pradesh. Hatching rate was more or less the same for successive Numerous investigations made on shrimp spawning in ablated females. This could be related to batch reproduction pointed out that various factors as availability and variability of sperm in the thelycum of the photoperiod, light intensity, physico - chemical parameters female. Hatching rate was more in shrimps collected from of waters and color of maturation tanks play a vital role the Sea. Among wild ones the maximum hatching rate of (Chamberlain and Lawrence, 1981 and Wyban et al., 1987) 90.56 was recorded in unilateral ablated wild shrimp fed and eyestalk ablation (Chamberlain and Lawrence, 1981) with crab and polychaete and the minimum of 69.85 in the was reported to be of much help in inducing maturation. unilateral ablated wild shrimp fed with oyster beef liver. When all the requirements are provided, eyestalk ablation The maximum number of nauplii hatched was 71.05 in the is of immense use in inducing gonadal maturation. In the unilateral ablated captive shrimp fed with crab and present study, brood stock developed from wild grown polychaete and minimum of 32.25 captive shrimp fed with shrimps was converted as spawners making use of various oyster and beef liver. In the wild bilateral ablated shrimps feed combinations. Their reproductive performance was fed with crab and polycheate maximum 72.75 and tested in a commercial hatchery. Detailed comparison is minimum of 27.45 shrimp fed with beef liver and oyster made with naturally caught shrimps with respect to respectively (Table 2). survival rate, maturation, spawning rate and hatching rate to drive home the advantage of developing brood stock and Discussion: spawners from farm grown ones due to the inherent merits. Table: 2. Percentage of hatching rate in In the feeding experiments, importance has been wild and captive spawners given to live polychaetes in the present study towards maturation and spawning as suggested by (Brown et al., Feed Wild Captive W ild 1979 and Middleditch et al., 1979), Next to polychaetes, combinations unilaterally unilaterally bilaterally ablated ablated ablated squid was found to be the best single - food diet, as it produced higher levels (although not significantly higher Crab and polychaete 90.46 ±1.34 71.05 ±1.72 72.75 ±1.20 in every case) of growth, molting, maturation and spawning than the other single food diets, the growth Oyster and polychaete 84.25 ±2.44 53.13 ±1.05 65.20 ±2.83 promoting characteristics of squid have earlier been Squid and clam 72.66 ±1.95 42.83 ±1.42 51.99 ±1.56 reported (Deshimaru & Shigeno, 1972 and Fenucci et al., Beef liver and squid 78.65 ±1.93 40.88 ±1.27 45.82 ±1.68 1980). The high maturation performance may be related to the high percentage (98%) of sterol in the form of Oyster and beef liver 69.85 ±1.57 32.25 ±1.16 27.45 ±1.17 cholesterol (Lytle and Lytle, 1989) have pointed out that *Mean values standard deviation crustaceans such as Artemia and Mactra chinensis (Teshima et al., 1988) are considered as good diets with As far as hatchery production of shrimp seed is essential feed ingredients for maturation of captive shrimp. concerned, continuous production of high quality nauplii Nevertheless, it would appear that mussels are a much has certainly remained an elusive goal. In this backdrop, more productive in inducing maturation than clams. the greater and better ovarian maturation and spawning (Brown et al., 1979) found that Penaeus setiferus accepted, success in captive spawners developed by feeding crab and mussel more readily than oysters (Crassotrea sp.). polychaete was better, this argues in general with the suggested that food organisms for maturation should have findings of (Brown et al., 1979, Primavera et al., 1979, ripe gonads for ingestion by brood stock and spawner. This Beard and Wickins, 1980, Chamberlain & Lawrence 1981, would agree well with the present study where the various Menasveta et al., 1994 and Sangpradub et al., 1994). In food items were not reproductively active when they were order to estimate the ecosystem support area that is needed offered as food and therefore, were less effective than to supply hatcheries with shrimp spawners, the supporting polychaetes which were reproductively active and rich in C ecosystem or habitat type must first be assigned. Present 20: C 20 6, C 20:5 3 and C 22:6 3 compounds. Combined studies will argue that the mangrove ecosystem is the diets were proved to be more effective than a single diet.

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The relationship between reproduction and growth is The highest rate, 90% hatching was recorded in dependent up on the division of energy resources. Due to wild unilateral eyes talk ablated shrimps fed with crab and the division of energy towards reproduction a gradual polychaete and lowest hatching rate, 69% was obtained in decline in weight of females occurred. wild unilateral ablated shrimps fed with oyster and beef Presently shrimp fed with crab and polychaete liver. The hatching rate in captive raised spawners, 71% as showed higher growth rates when compared to others. The higher and unilateral ablated shrimps fed with crab and next highest growth rate was recorded in shrimps fed with polychaete, and minimum lowest of 32% was obtained in crab and polychaete and oyster and polychaete. The same shrimps fed with oyster and beef liver. Hatching rate was trend was found in males also, that maximum growth rate more or less similar for successive spawning of wild and in shrimps fed with crab and polychaete and in shrimps fed captive shrimps. Effective fertilization is possible only with oyster and beef liver. Santiago (1977) reported that P. with males, weighing > 40 g showing signs of full maturity. monodon gained an ABW of 16 g. after four months of (Primavera et al., 1979, Beard and Wickins, 1980 and pond culture. Eyestalk ablation induces maturation in Alfaro, 1993), recorded less hatching rate due to shrimps, whether they are caught in the sea or they are insufficient quantity of sperms. Captive and wild shrimp reared in the pond. Elapsed time from eyestalk ablation up males weighing > 60 g were used in the treatment groups to first maturation will be less initially compared to and there were good spawning rates from captive reared subsequent maturation; more elapsed time also leads to spawners as of wild ones. However the hatching rate was failure of hatching because of low fertilization. (Beard and low in captive spawner and the reason might be long Wickins, 1982,) found that 22 days of elapsed time in pond- elapsed time, poor sperm quality and over stocking during reared females, but with less fertilization rate. Presently, spawner development. These should be overcome through highest elapsed time was recorded in captive animals fed further study. In the present work combination of crab and with oyster and cow liver which showed the lowest polychaete, and oyster and polyhchaete showed better percentage of hatching. The lowest elapsed time was result in terms of lower elapsed time, high rate of spawning recorded in both wild and captive spawners fed with crab and hatching than other feeds. Improvement of quality of and polychaete which showed higher hatching rates. water used in maturation tanks, spawning and hatching Unilateral ablated shrimps showed better hatching rates tanks through UV radiation found to reduce the levels of with low elapsed time compared to bilateral ablated specific pathogenic micro organisms in the incoming sea captive shrimps. (Emmerson and Andrews,1983; Hiller, water may be of help in the overall improvement of the 1984) recorded a higher spawning of 2,57,650 to 5,50,330 reproductive performance. Efforts should also be made to eggs per spawn from wild ablated P. monodon and 60,000 investigate the male shrimps, which will be of help in to 7,47,500 eggs from non-ablated females. The wide enhancing the fertilization and hatching rates. Poor water range in egg numbers obtained could be due to variations in parameters also affect reproductive quality (Perez- weight of females (50 - 200g) and inclusion of eggs in Velazquez et al., 2001). Nevertheless, the water quality for counts from both partial and complete spawning (Alikunhi all treatments was considered ideal for the species (Peixoto et al., 1975; Muthu & Laxminarayana, 1977). However, et al., 2005), with temperature between 24.5 °C to 29 °C (Sangpradub et al., 1994) found that the average number of and salinity of 33 to 35 ppt. Thus, food probably was eggs per spawner fed with a fresh diet was 1,47,000 important factor for aggrieved the spermatophore contrarily to shrimps with an average mean weight of 136.5 degeneration reported in the results of the present study. gr. fed with a fresh and pelleted diet yielded 1,39,000 eggs. Conclusion (Menasveta et al., 1994) stated that the total egg production ranged from all 9, 11,460 in smaller (86.68.20 g) to 5, Our present findings gave satisfactory results on ovarian 73,086 in larger ones (135.715.989) reared in the pond. maturation and spawning in captive spawners, among five different feed combinations crab and polychaete The result obtained in the present findings combinations was better which elicit a greater success in indicated highest spawning rate of 4, 45,440 per shrimp in the captive and wild caught spawners , further research wild unilateral shrimps fed with crab and polychaete. The should continue to establish other good combinations of next highest egg production of 3, 56,487 per spawner was feeds which will helpful for the production of high quality obtained in wild bilateral ablated shrimps fed with the same of nauplii, as hatchery production of shrimp seed is feed combination. Lowest egg production of 1, 52,945.6 concerned, this will definitely fulfil the gaps in was recorded in shrimp fed with oyster and beef liver of aquaculture. captive unilateral. In pond reared shrimps fecundity and hatching rate were 2, 04,000 eggs. In the present study the Acknowledgement hatching rate was found to be low in captive spawners The author thankful to “Pavan Aqua Pvt Ltd” located at compared to wild ones. The differences in hatching rates Timmapuram, Visakhapatnam. may be due to nutrition, sex ratio, water depth and other bio-physical factors. It is also important to maintain certain male: female ration in the maturation tanks.

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References Decapoda), Aquaculture 41, e 252-e 259. Alfaro J.J., Palacios A., Tito M.A. and Angulo R.A. (1993): Lytle J.S. and Lytle T.F. (1989): Fatty acid composition and Reproduction of the shrimp Penaeus occidentalis variation in individual blood worms. Gulf coast (Decapoda: Penaeidae) in the gulf of Nicoya, Costa research laboratory, P.O. Box. 7000, Ocean springs, Rica. Revistta de Bilogia Tropical. 41(3 part A): 563- Mississippi 39564, USA 572. Menasveta P., Sangpradub S., Piyatiratitivorakul S. and Alikunhi K.H., Poernomo A., Adisukresno S., Budiono M. Fast A.W. (1994): Effects of broodstock size and and Busman S. (1975): Preliminary observations on source on ovarian maturation and spawning of induction of maturity and spawning in Penaeus Penaeus monodon (Fabricius) from the gulf to monodon ( Fabricius) and Penaeus merguiensis de Thialand. J. World Aquaculture Soc. 25(1):41-49. Man by eyestalk extirpation. Bull. Shrimp. Cult. Res. Meunpol O., Meejing P. and Piyatiratitivorakul S. (2005): Cont. Jepera. 1(1): 1-11 Maturation diet based on fatty acid content for male Beard T.W., and Wickins J.F. (1980): Breeding of Penaeus Penaeus monodon (Fabricius) broodstock. Aquac. monodon in a laboratory recirculation system. Res. 36, 1216–1225 Acquaculture. 20: 79-89 Middleditch B.S., Missler S.R., Ward P.G., Mc Vey J.B., Bray W.A. and Lawrence A.L. (1992): Reproduction of Broun A. and Lawrence A.L. (1979): Maturation of Penaeus species in capacity. In: A. Fast and L.J. Penaeid shrimp: dietary fatty acids. Proc: World Lester (eds.) marine shrimp culture: Principles and Maricult. Soc: 10: 472-476 practices, (Elsevier Science Publishers), B.V. Muthu M.S., and Laxminarayana A. (1977): Induced Bredene. The Netherlands. maturation and spawning Indian Penaeid prawns. Browday C.L. (1992): A review of the reproductive Indian J. Fish. 24:172-180. biology of Penaeus species: perspectives on Peixoto S., Cavalli R.O. and Wasielesky W. (2005). Recent controlled shrimp maturation system for high quality developments on broodstock maturation and nauplii production. In J. Wyban (Ed.) Proceedings of reproduction of Farfantepenaeus paulensis. Braz. the special session on shrimp farming, World Arch. Biol. Technol. 48 (6), 997–1006. Acquaculture Society, Baton Rouge, Louisiana, Perez-Velazquez M., Bray W.A., Lawrence A.L., Gatlin U.S.A. D.M. and González-Félix M.L. (2001): Effect of Brown A., Mc Vey J.P., Middleditch B.S. and. Lawrence temperature on sperm quality of captive Litopenaeus A.L. (1979): Maturation of white shrimp Penaeus vannamei broodstock. Aquaculture 198, 209–218. setiferus in capacity. Proc. World Maricult. Soc.11: Primavera J.H., Lim C. and Bolongan E. (1979): Feeding 488-499. regimes in relation to reproduction and survival of Chamberlain G.W. and Lawrence A.L. (1981): Maturation, ablated Penaeus monodon, Klikasan. J. Biol. 8:227- reproduction and growth of Penaeus vannamei and 235. P.stylirostris fed different diets. J. World Maricult. Sangpradub S., Fast A.W., Piyatiratitivorakul S. and Soc. 12(1): 209-224. Menasveta P. (1994): Effect of different feeding Deshimaru O. And Shigeno K. (1972): Introduction to the regimes on ovarian maturation and spawning reared artificial diet for Penaeus japonicus. Aquaculture. 1- gaint tiger prawn in Thialand. Acquaculture. Int. 12: 115. 2(1):49-58. Emmerson W.D. and Andrews B. (1983): The effect of Santigo A.C.J.R. (1977): Successful spawning of cultured stocking density on the growth and development and Penaeus monodon (Fabricius) after eyestalk survival of Penaeus indicus (Milne Edwards) larvae. ablation, Aquaculture, 11: 195-196 Acquaculture. 23: 45-47 Teshima S., Kanazawa A. and Kakuta Y. (1988): Role of Fenucci J.L., Zein Eldin Z.P. and Lawrence A.L. (1980): Dietary phospholipids in the transport of 14C The nutritional response of two Penaeid species to tripalmitia in the prawn, Bull. Jpn. Soc. Sci. Fish. 52: various levels of squid meal in a prepared feed. Proc. 519-524. World Maricult. Soc.11: 403-409 Villaluz D.K., Villaluz A., Ladrera B., Sheik M. and Hiller D.D. (1984): Artificial conditions influencing the Gonzaga A. (1972): Production, larval development maturation and spawning of sub adult Penaeus and cultivation of sugpo (Penaeus monodon monodon (Fabricius). Aquaculture, 36: 179-184 Fabricius). Phillip. J. Sci. 98: 205-236. Jia-Yuan Xu, Ting-Ting Wang. Yu-Feng Wang. Yu Peng. Wyban J.A., Lee C.S., Sweeney J.N. and Richards W.K Jr. (2010): Effect of combined fish meal; soyabean (1987). Observations on development of a meal ratio, Vitamin-C, and Fish oil supplementations maturation system for P. vannamei. J. World in diet on the growth and reproduction of red shrimp. Aquaculture. Soc. 18(3) 198-200. Cray fish, Procambarus clarkii (Crustacea-

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Inhibitory effects of aqueous leaf extracts of Lantana camara on the growth of P.hysterophorus in fruiting stage.

Arpana Mishra Department of Botany, Govt. Chhatrasal P. G. College, Panna (M.P.), India

Corresponding author, e-mail: [email protected]

Abstract : Lantana camara is notorious weed and a popular ornamental garden plant belonging to the family verbenaceae. L.camara is native to tropical and subtropical America. An experiment was conducted to understand inhibitory effects of aqueous leaf extracts derived from L.camara on growth of P.hysterophorus in fruiting stage. Bioassay also indicated that the inhibitory effect was proportional to the concentrations of the extracts and higher concentration had the stronger inhibitory effect. Keywords : Allelochemicals, Leaf extract, Fruiting stage

Introduction camara leaf aqueous extract is as. 100gm under of leaf The term allelopathy is derived from two Greek chopped in small pieces and crushed in the mixture grinder words Allelon means each other and Pathos means to after grinding the material of leaf paste were soaked in 200 suffer, the term refers to effects that are both detrimental ml of distilled water for 24 hrs then prepare the following and beneficial among the interacting organisms (Rice, concentrations 100%, 50%, 33%, 25% and water as a 1984). These effects have been observed in all classes of control treatment. The extract of each specimen was plants and also extend to microorganisms. Plants produce filtered with muslin cloth. The concentration volume of chemicals that directly of indirectly influence the each specimen was maintained by adding distilled water. environment. These chemicals are called allelochemicals. Foliar treatment of fruiting Parthenium hysterophorus The origin of Parthenium weed dates back to 4th century with different concentration aqueous leaf extract of BC in the Greek city of Parthenia. This weed has been Lantana camara on alternate days but control quadrates named so according to the name of Greek city. It was sprayed only distil water . distributed throughout the world along with wheat and Plants samples were analyzed for shoot and root belonging to the family Asteraceae. Parthenium is not only length, leaf area. Leaf area was measured with the help of a harmful to crop but also causes several diseases to man e.g. leaf area meter (Model No. 211 Systronice). asthama, contact dermatitis and hay fever. Lantana camara Results and Discussion is a most common weed belonging to the family Verbenaceae. L.camara has an allelopathic potential 1. Effect of Lantana camara aqueous leaf extract on because it contains a number of phenolic compounds shoot and root length of Parthenium hysterophorus in (Narwal, 1994) . The weed is aggressively growing in fruiting stage forest, agriculture, tea garden and wastelands of all over the The different concentration of aqueous leaf country (Ahmed, 1997) . Allelochemicals of L.camara has extract of Lantana camara had significant effect on shoot potential in the development of green herbicides. and root length of Parthenium hysterophorus fruiting The present work was undertaken to study the plant. The plant growth inhibit after aqueous leaf extract effect of L.camara on growth and metabolism of spray on Parthenium hysterophorus. Plant shoot and root Parthenium and to evaluate the allelopathic potential of the length were decreased over control with the increasing former. concentration of extract. Plant shoot and root length were control after 5th spray, 4th spray, 3rd spray and 1st spray of Materials and Methods 25%, 33%, 50% and 100% concentration of aqueous leaf The study area Shakti nagar lies in the Banda extract of Lantana camara respectively. Finally plant were district of Uttar Pradesh in between Latitude 24º 53' and dead after 7th, 5th, 4th and 3rd spray of 25%, 33%, 50% and 25º 55' N, Longitude 80º 07' and 81º 34' E, the geographical 100% concentration of aqueous leaf extract of Lantana area of the district is 4114.20 sq. km. Leaves of Lantana camara respectively. camara were collected from Chitrakoot region of Madhya Maximum growth of shoot and root were Pradesh. Collection of raw material and preparation of observed 54.13% and 54.4% increased respectively in extract in two days advance for each spray. control. Plant shoot and root length were decreased over The preparation process undertaken for Lantana control with the increasing concentration of extract. The

43 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 43-45 plant growth inhibit after aqueous leaf extract spray on Parthenium hysterophorus fruiting plant. In 25% extract the plant growth were observed 8.27% increased in shoot and 6.4% increased in root over control. In 33% extract the plant growth were observed 4.78% increased in shoot and 33% increased in root over control.

Table-1: Effect of Lantana camara leaf extract on growth (cm) of Parthenium hysterophorus in fruiting stage .

Shoot and root length (cm) at the time of spray Number of spray at the alternate days % % Plant Total Concentration Increase Decrease growth Days in % 1 2 3 4 5 6 7 24 days 24 days (cm) (24) Over BT Over BT Shoot 29.0 30.9 32.7 34.7 36.9 39.3 41.9 44.7 54.13 Control Root 25.0 26.7 28.2 29.9 31.8 33.9 36.2 38.6 54.4 Shoot 29.0 29.9 30.6 31.1 31.4 31.4 31.4 - 8.27 45.86 25 % Root 25.0 25.7 26.2 26.5 26.6 26.6 26.6 - 6.4 48 Shoot 23.0 23.6 24.0 24.2 24.2 - - - 4.78 49.35 33 % Root 21.0 21.4 21.6 21.7 21.7 - - - 3.33 51.07 Shoot 28.0 28.4 28.6 28.6 - - - - 2.14 51.99 50 % Root 24.0 24.2 24.3 24.3 - - - - 1.25 53.15 Shoot 29.4 29.4 29.4 - - - - - 0 0 100 % Root 25.3 25.3 25.3 - - - - - 0 0 BT = Before treatment; - = Dead the Parthenium weed.

Minimum percentage increased 2.14% in shoot length

a 45 and 1.25% in root length were recorded in 50% e 40.74 r concentration, but in 100% extract concentration the plant a 40 f

a 35 growth was completely suppressed after single spray. e l 30 n

Detail result showed in Table 1. i 25 e

s Leaf 2. Effect of Lantana camara aqueous leaf extract on leaf a 20 e r 15 area of Parthenium hysterophorus in fruiting stage c n The different concentration of aqueous leaf I 10 6.42 % 5 3.43 extract of Lantana camara had inhibitory effect on leaf 1.7 0 0 area of Parthenium hysterophorus fruiting plant. Plant leaf Control 25% 33% 50% 100% area was decreased over control with the increasing Concentration concentration of extract. The leaf area was decreased after aqueous leaf extract spray on plant. Maximum leaf area of Fig.1: Effect of different concentration of Parthenium hysterophorus was observed 40.74% Lantana camara leaf extract on leaf area of increased in control. In 25% concentration aqueous leaf Parthenium hysterophorus in fruiting stage. extract the leaf area were observed 6.42% increased and in 33% concentration leaf area were observed 3.43% presence of allelochemicals. The L. camara leaves contain increased over control. Minimum percentage 1.70% allelochemicals like phenolic compounds, mono- and increased was recorded in 50% concentration, but in 100% sesquiterpenes, triterpenes, triterpenoids, quinines, extract concentration, the leaf area was found constant as essential oils, flavonoids, biocides etc (Raghavan, 1976). first spray. observation showed in fig1. The water soluble allelochemicals of Lantana The effect of different concentration of Lantana camara inhibited the initial growth of both the agricultural camara leaf aqueous extracts were recorded and compared ( Oryza sativa, Triticum aestivum, Vigna sinensis, with control (i.e., distil water). Result showed, different Cucurbita pepo, Abelmoschus esculentus, Amaranthus concentration of aqueous leaf caused inhibitory effect on tricolor and forest crops (Acacia auriculiformis, shoot and root elongation, leaf area of Parthenium Paraserianthes falcataria, Albizia procera) in the hysterophorus fruiting plant. Bioassays also indicated that laboratory conditions (Hossain & Alam, 2010). The the inhibitory effect was proportional to the concentration phenolic compounds extracted from the leaves of Lantana of the extracts and higher concentration had the stronger camara were found to be phytotoxic to rice, wheat and inhibitory effect. The reason of inhibition may be the three grass seedlings. The extracts of leaf, stem, flower and

44 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 43-45 fruit of Lantana camara inhibited the seed germination of Parthenium hysterophorus (Mishra & Singh, 2010). The extracts of Lantana camara leaves and their fractions reduced the biomass of Eichhornia crassipes and Microcystis aeruginosa within 7 days under laboratory conditions (Kong et al., 2006). Leaf extract showed pronounced inhibition of shoot length, root length, leaf area, fresh and dry weight of the seedling Parthenium (Mishra & Singh, 2012). Thus, the phytotoxicity of L.camara can be considered as a source of potent green herbicide to control P. hysterophorus fruiting stage. References Ahmed N. (1997) : Wild Flowers of Bangladesh. Dhaka, Bangladesh. (The University Press Ltd.) p.142. Hossain M K and Alam, NMD. (2010): Allelopathic effects of Lantana camara leaf extract on germination and growth behavior of some agricultural and forest crops in Bangladesh. Pak.J.Weed Sci .Res. 16(2), 217-226. Mishra A. and Singh R. (2010): Comparative study of effect of Lantana camara extract of different parts on seed germination of Parthenium hysterophorus L. International Journal of Plant Sciences 5 (1), 74-75. Mishra A. (2012): Allelopathic interaction of L. camara leaf of extract on growth of P. hysterophorus in seedling stage. International journal of plant sciences. 7 (2), 259- 262. Kong C.H: Wang P., Zhang C.X., Zhang M.X. and HU F. (2006): Herbicidal potential of allelochemicals from Lantana camara against Eichhornia crassipes and the alga Microcystis aeruginosa. Weed Research 46(4), 290-295. Narwal S. S. (1994) : Allelopathy in crop production. (Scientific publishers, Jodhpur India) p.288. Raghavan V. (1976): In Recent Advances in Botany. (Ed.) Kachroo P., Bishan Singh and Mahendra Pal Singh, (Dehradun) p.264 Rice E. L. (1984): Allelopathy. II edition. (Academic Press, New York) 422.

Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 47-56

Inhibitory efficacy of Chlorpyriphos and Datura stramonium on Acetylcholinesterase activity, Kinetics and Histology of Brain of Catla catla

Namdeo, A., Tembhre, M.,* Banerjee, S., Gour, S. and Ahirwar, S. MLB Govt Girls (Autonomous) PG College, Bhopal, India M K Ponda College, Bhopal, India. *

Email: [email protected]

Abstract : Influence of ethanol leaf extract of Datura stramonium, an indigenous plant used in ayurvedic medicine in India and commonly used organophosphorus pesticide; chlorpyriphos on acetylcholinesterase (AChE) activity, kinetics and histology were investigated in the brain of Catla catla. The recovery of inhibited AChE by both compounds was also determined. It was observed that 96 hrs exposure of Datura stramonium leaf extract (100 mg/L) produces 47.2% AChE inhibition and chlorpyriphos (0.00073 mg/L) elicits 33% AChE inhibition. A considerable synergistic inhibitory effect was found in the brain AChE with pre-treatment of Datura stramonium leaf extract followed by chlorpyriphos exposure. This treatment could yield 60% inhibition. The recovery of Datura stramonium extract-induced AChE inhibition was ranged from 1.6% (48 hrs) to 11.7% (120 hrs) while chlorpyriphos-induced AChE inhibition was recovered to 8.1% (48 hrs) and 23.8% (120 hrs). Kinetic study of AChE also showed inhibitory potential of these compounds. The Km of control group was 0.41 x 10-3 M. This was increased to 0.9 x 10-3 M (Datura stramonium extract), 0.76 x 10-3 M (chlorpyriphos), and 1.1 x 10-3 M (pre-treated with Datura stramonium extract followed by chlorpyriphos exposure). The Vmax values were constant i.e. 0.11 activity/min/mg protein in control and treated groups showing competitive AChE inhibition. The cyto-architectural profile of treated brain shows histological alterations indicated by varying degree of necrosis and vacuolation in the molecular and granular layers. Keywords: Acetylcholinesterase, Brain, Catla catla, Chlorpyriphos, Datura stramonium Inhibition, Kinetics, Histology.

Introduction inhibition of AChE causing impairment of hydrolysis of The acetylcholinesterase enzyme (AChE; E.C neurotransmitter acetylcholine (Tembhre et al., 2006; 3.1.1.7) is most acceptable target for the assessment of Somnuek, 2007). Chlorpyriphos have been reported to inhibitory action of various organophosphate (OP) inhibit acetylcholinesterase activity in the brain of fish pesticides. Hence, determination of inhibition of AChE is (Rao et al., 2005; Halappa and David 2009; Wang et al., widely employed in bio-monitoring studies and considered 2010). as most reliable biomarker of pesticide pollution (Carr et Study of recovery of pesticide-induced al., 1995; Somnuek et al., 2007, Kumar and Tembhre, 2010 acetylcholinesterase inhibition is important as it reveals and Tembhre et al., 2012). The primary mode of action of status of pesticide-AChE complex. Carr et al. (1995) organophosphorus pesticide is inhibition of AChE activity, reported that after 60 days, the brain AChE of mosquito the enzyme that degrades the neurotransmitter fish was almost fully recovered from inhibition by acetylcholine in cholinergic synapses in the vertebrate chlorpyriphos. Tembhre et al. (2006) observed recovery of nervous system (Silver, 1974). The use of large number of chlorpyriphos-induced AChE inhibition in Cyprinus new generation pesticides is still in progress, which leads carpio. They reported 83%, 88% and 90% recovery in fore- to adverse synergistic implications on non-target species , hind- and mid brain on seventh day after chlorpyriphos (Susan et al., 2010). Many of these pesticides may linger exposure. Complete recovery of inhibited brain AChE was on in the environment and lead to detrimental effects. observed in mosquito fish after 60 days exposure of Inhibitory effects of dimethoate on AChE activity of fish chlorpyriphos (Russel et al., 1997) have been reported by Satyadevan et al. (1993), Tembhre The kinetic study on the brain AChE inhibited by and Kumar (1994), Singh and Kumar (2000). Methylamine malathion in Tilapia mossambica reveals that malathion is also known to produce significant inhibition in fish competitively inhibits AChE (Sahib et al., 1980). Dembele AChE. (Tembhre and Kumar, 1995 & 1997). The chronic et al. (1999) investigated AChE kinetics in the brain of exposure to sub lethal concentration of Diazinon shows Cyprinus carpio exposed to chlorfenvinphos and significant inhibitory effect on AChE in the brain of carbofuran. They reported that both the pesticide induced Clarius gariepinus (Adedeji, 2011). Chlorpyriphos (CPF), competitive inhibition in the brain AChE of the fish. an organophosphate pesticide is widely used for pest control on cotton, corn, almond and fruit trees including Scientists have investigated a large number of oranges and apples. CPF binds with acetylcholinesterase in medicinal plants for screening their pesticides properties cholinergic nerves at synaptic vesicles. This results in (Tiwari and Singh, 2004; Crowch and Okello, 2009). Some

47 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 47-56 medicinal plant extract contain various phyto-chemical solvent. The extract was kept at room temperature for known as alkaloids, tannin, saponin, nicotine etc. These evaporation of ethanol till semi solid mass left. This was active ingredients are toxic to fish (Murthy et al., 2010; kept stored at 4oC for further use. Kumar and Sikarwar, 2003). Since, synthetic pesticides Experimental Animal create various environmental problems because of slow rate of their degradation. The plant based insecticides are Fingerlings of Catla catla collected from the considered to be eco friendly because of their easy fishpond near Kolua village, Raisen road, Bhopal were degradability. Datura stramonium has been reported to be used in this experiment. They were acclimatized for 15 used as medicine in reducing pain and as a narcotic and days prior to the experiment. Fishes were fed daily with local anaesthetic drug in many places (Abena et al., 2003). commercial dry feed pellets (Tokyu, Spirulina, Japan). The Some findings of investigation revealed that Datura fishes had median weight 100 ± 10 gm. The fishes were stramonium extract predominately contained alkaloids viz. stocked in glass aquaria of 60 liters supplied with tap water 0 atropine, hyoscyamine, and scopolamine (Adekomi et al., (temperature 22.7 + 0.61 C, hardness as CaCO3 212 + 4.8 2011). In all species of genus Datura the concentration of ppm, pH 7.3 + 0.05, chlorides 87.62 + 2.39, total alkalinity alkaloid varies depending on species and on the part of the as CaCO3 165 + 1.15 ppm). A Physico-chemical property plant (Duez et al., 1985). Friedman and Levin, (1989) of water was constantly checked according to reported Datura stramonium seeds as neuroinhibitor. APHA/AWWA/WEF (2005). Oxygen content was Several investigators have discussed the effect of ethanol maintained with the help of aerator. The feeding was leaf extract of Datura stramonium on animals (Gidado et stopped before 24 hrs prior to and during the exposure al., 2000 & 2007). period, which extended 96 hrs. Organophosphate pesticides are well known to Fishes were divided into four groups of thirty adversely affect the histological components of various each. Group-I: served as Control; Group-II: exposure to organs including brain of fish (Gupta and Guha, 2006; sub lethal concentration 0.00073 mg/ L was based on the 96

Butchiram et al., 2009). Exposure of sub lethal h LC50 value (0.0034 mg/ L) of CPF for Catla catla was concentrations of malathion and dimethoate to Catla catla selected for test. The fishes were exposed to this showed vacuolation in brain with eccentric nuclei, necrosis concentration daily for 96 hrs with replenishment of water in molecular and granular layers (Singh M, 1998). Sarma et at every 24 hrs. Group-III: Fishes were exposed to 100 al. (2010) also reported mild necrosis in the apical lobe of mg/L of Datura stramonium leaf extract for 96 hrs. ; cerebrum of brain of Channa punctatus intoxicated with Group- IV: The fishes were pre-treated with 100 mg/L of endosulfan. Datura stramonium extract for 96 hrs followed by the The present study was undertaken to investigate that exposure to 0.00073 mg/L chlorpyriphos for 96 hrs. At the whether the use of synthetic pesticides can have a botanical end of the experiment, five fish were removed from each alternative which may proved to be more effective and less group to study the effect of CPF and D. s extract. However, persistent in the environment due to its rapid rate of remaining fishes were transferred to toxicant free water to degradation. Nevertheless, Datura stramonium (D.s) has study recovery of AChE. Water was changed after every 24 never been tried for anticholinesterase activity in fish. hrs. Five fishes from this stock were removed and dissected Therefore, in the present study, we compared the effects of at the end of 24 hrs, 48 hrs, 72 hrs, 96 hrs and 120 hrs. efficacy of an organophosphorus pesticide, chlorpyriphos Sample Preparation and a medicinal herb Datura stramonium leaf extract on Treated fish were euthanized, dissected and the AChE activity, kinetics, and recovery of inhibited AChE brains were removed quickly and washed in 0.9% saline. A and histology of brain of Catla catla. 10% (w/v) tissue homogenate was prepared in Elvehjem- Materials and Methods potter homogenizer and centrifuged at 5000 rpm for 20 min 0 Chemicals in cooling centrifuge (Remi) at 4 C. The supernatants were kept in deep freeze for AChE assay, kinetics and Protein Chlorpyriphos (O,O,Diethyl-O-(3,5,6 trichloro- content. 2-pyridyl) phosphorothioate) 94% purity technical grade, Acetylthiocholine Iodide (ATChI) and DTNB (5- Enzyme Assay 5'dithiobis-2-nitrobenzoic acid) (Himedia, India), Bovine AChE activity was measured spectrophoto- Serum Albumin (BSA) (Loba Ceremic), Folin's reagent metrically according to the method of Ellman's et al. (Merck). (1961) in the Brain of Catla catla. Samples of homogenate Plant Material were diluted with 2.6 ml 0.1 M Sodium phosphate buffer (pH 7.4) to which 100 mM DTNB and 75 mM ATChI was Fresh leaves of Datura stramonium were added. The rate of color production was measured at 412 collected from botanical garden, authenticated, thoroughly nm in SL 164 UV-VIS spectrophotometer. All washed in water and shade dried. Leaves were powdered measurements were done in duplicate. Specific activity and extracted in Soxhlet apparatus with 90% ethanol as was expressed in nmol/min/mg protein.

48 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 47-56

Protein estimation activity in the brain of Clarius gariepinus was Protein was estimated by the method of Lowry et demonstrated up to 85% (Adedeji, 2011). The decrease in al. (1951) using BSA as the standard. Samples of brain AChE activity in Cyprinus carpio exposed to sub homogenate were diluted with reagents then 0.5 ml Folin's lethal concentration 7.5 µg/L of quinalphos was recorded reagent was added and after 20 min read at 620 nm against a with 75.2% AChE inhibition (Chebbi and David, 2009). reagent blank. Measurements were done in duplicate. The present study revealed that CPF produced significant brain AChE inhibition in Catla catla. The neurotoxic effect AChE Kinetics of CPF is potentiated by its biotransformation to a more Kinetic parameters (Km & Vmax) were potent oxon metabolite that inhibits AChE substantially determined by using four different concentrations of (Fukuto, 1990). substrate i.e 0.66 mM, 0.44 mM, 0.33 mM, 0.26 mM. It has been reported that various parts of Datura Lineweaver-Burk plot was made by plotting the stramonium being medicinal plant, were observed to be reciprocals of velocity and substrate concentration. poisonous (Devi et al., 2012). There is a little information Histology in the literature regarding its anticholinergic property to the animals. The present investigation is a first attempt Brain was removed from three fishes from each towards the understanding of the neurotoxic effect of group after treatment. Tissues were fixed in freshly Datura stramonium leaf extract by observing brain AChE prepared aqueous Bouin's fluid in glass vials for 24 hrs, inhibition in Catla catla. The exposure of plant extract at washed in running tap water, dehydrated in graded series of 100mg/L concentration was found to induce significant alcohol, cleared in xylene, infiltrated and embedded in 47.2% AChE inhibition. paraffin wax. Multiple sections were cut at 5-6 micron thickness and stained with Haematoxylin and Eosin. Slides Suganthy et al. (2009) reported 50% AChE were examined using binocular microscope (Olympus) inhibition by methanolic leaf extract of Rhizophora and the selected fields were microphotographed at 100X lamarkii, Suaeda monica, Avicennia officinalis and and 400X with computer-aided microscope (Leica). Sesuvium portulacastrum. An active compound mahanimbine, a carbazole alkaloid isolated from Murraya Statistical analysis koengii displayed a dose-dependent AChE inhibition Graphs of results were prepared by applying (Kumar et al., 2010). Ethanol extract of Bacopa monnieri Excel 2007 software. For the data of statistical comparison and Ginkgo biloba possess AChE inhibitory power as between different treatments and control, data were suggested by Das et al. (2002). They reported that 100 analyzed by Student's t-test to determine the effect of the mg/kg and 300 mg/kg extract of Bacopa monnieri and treatment. The level for the accepted statistical Ginkgo biloba respectively showed dose-dependent in significance was p>0.05. vitro AChE inhibition in brain. Results and Discussion Recently it has been revealed that ethanol extract The effect of sub lethal concentration of CPF of Bacopa monnieri noticeably inhibit AChE activity in (0.0034 mg/ L) for 96 hrs demonstrated significant (p > various regions of the brain (Ahirwar et al., 2012). They 0.01) reduction in acetylcholinesterase activity in the brain reported 72% AChE inhibition in hippocampus, 58% in of Catla catla. The brain AChE activity was declined to brain stem, 50% in pons, 46.5% in cerebellum, 44% in 9.45 + 2.69 nmole/ min/ mg protein against the control thalamus, 40% in Cerebral cortex and 33.3% straitum. The fishes in which AChE activity was 14.14 + 8.41 nmole/ preliminary phytochemical study showed the presence of min/ mg protein. CPF produced -33% inhibition (Table-1: alkaloids including atropine, hyoscyamine and Fig-1). The treatment of ethanol leaf extract of Datura scopolamine that can all elicit anticholinergic poisoning stramonium (100 mg/L) also caused depletion in AChE (Ertekin et al., 2005). The analysis also showed that these activity to 7.46 + 3.68 nmole/ min/ mg protein showing anticholinergic alkaloids are responsible for blocking of 47.2% brain AChE inhibition. However, 96 hrs AChE activity in cholinergic nerves (Friedman, 2004). We pretreatment of fish to D.s extract (100 mg/L) followed by observed that pre-treatment of D.s leaf extract followed by (0.0034 mg/ L) CPF exposure for 96 hrs produced CPF exposure substantiate AChE inhibition in brain significant (p > 0.01) inhibition in AChE activity to 5.65 + further to 60%. It might be causing synergistic 2.11 nmole/ min/ mg protein indicating 60% inhibition physiological effect when used in continuous exposures. (Table-1: Fig-1). Our result showed that AChE activity was Our previous study reported that the AChE recovered gradually to 8.1% at 48 hrs; 17.7% at 72 hrs; activity of Cyprinus carpio intoxicated with CPF displayed 21.6% at 96 hrs & 23.8 % at 120 hrs after exposure to differential inhibition i.e. 66.6% in fore- , 40% in mid- and chlorpyriphos. Recovery of inhibited AChE after exposure 50% in hind brain (Tembhre et al., 2006). Balint et al. to leaf extract of Datura stramonium was found to be 1.6% (1995) reported that the exposure of 2 mg/L methidathion at 48 hrs; 2.4% at 72 hrs; 5.2% at 96 hrs & 11.7% at 120 hrs. for 5 days caused 90-92% decrease in AChE activity in Our findings revealed that there is considerable difference Cyprinus carpio. A dose dependent reduction in AChE in the extent of recovery of chlorpyriphos and Datura

49 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 47-56 stramonium treated fish (Table-2: Fig-2). Brain AChE niloticus and Rhamnus prinoides showed mixed inhibitory inhibition in Cyprinus carpio exposed to sub lethal effects viz. non competitive-uncompetitive type (Crowch concentration of quinalphos was recovered up to 35% after and Okello, 2009). th 14 day (Chebbi and David, 2009). Parathion-induced The histological observations of the control brain brain AChE inhibition in Gasterosteus aculeatus was (Fig-4, 5) showed the molecular layer, granular layer and significantly recovered after 48 hrs of exposure (Wogram purkinje cell layer. The components of these layers were et al., 2000). It is evident from observation of various architecturally normal. CPF treatment mildly affected investigations that recovery period vary with tenure after molecular and granular layers causing necrosis, which is exposure, degradability of inhibitor and degeneration of evidenced by the appearance of narrow spaces and acetylcholinesterase (Russel et al., 1997). Tembhre et al., vacuolation (Fig-6, 7). These changes were moderately (2006) reported 83%, 88% and 90% recovery of AChE in increased in the brain treated with leaf extract of Datura fore-, hind- and mid brain respectively after 168 hrs of stramonium (Fig-8, 9). However, it was slightly greater in exposure of chlorpyriphos in Cyprinus carpio. Our the brain of fish pre-treated with Datura stramonium leaf findings indicate that leaching of D. s extract is slow than extract followed by CPF exposure (Fig-10, 11). CPF in the brain. Intoxication of 0.35 ppm hexachlorocyclohexane to Labeo We have compared AChE Kinetics also to study rohita produced mild vacuolar changes with small spaces the nature of inhibition. The Km value of control brain was in brain, whereas 1.73 ppm exposure showed severe 0.41 x 10 -3 M. Our study showed that Km increased in necrosis (Das and Mukherjee, 2000). Chronic exposure of chlorpyriphos treated Brain to 0.76 x 10 -3 M and in D. s 0.6 and 1.3 µg/L endosulfan to rainbow trout did not cause extract treated to 0.9 x 10 -3 M. However, in group IV it was lesions in brain (Altinok and Capkin, 2007). Severe further enhanced to 1.1 x 10 -3 M. However, Vmax damage in brain cells and neural cells with broken neural remained constant i.e. 0.11 activity/min/mg protein in bundles were observed in 100 ppm malathion treated control and treated fishes (Table-1). Therefore, increasing Ophiocephalus punctatus (Pugazhvendan et al., 2009). pattern of Km and constant Vmax showed that both the Recently, above mentioned alterations were found in CPF and D.s extract inhibit the Brain AChE in a common carp exposed to atrazin and chlorpyriphos competitive manner (Fig-3). Similar findings were (Houjuan et al., 2012). reported with malathion in Catla catla (Singh and Kumar, Thus, present investigation leads us to conclude 2000), with methyl paraoxon in Prochilodus lineatus that leaf extract of Datura stramonium induced more (Silva Filho et al., 2004) and with monocrotophos in neurotoxicity in the brain as compared to chlorpyriphos. Channa punctatus (Rahman et al., 2004). There is lack of The study revealed that Catla catla brain AChE is more information on kinetics of enzyme inhibition in fish by sensitive to herbal extract. Thus, leaf extract of Datura herbal extracts. Our study has demonstrated for the first stramonium may be considered as potential herbal time that Datura stramonium extract is able to display pesticide for the control of predatory fishes. However, brain AChE inhibition kinetics similar to those of OP further isolation and purification of phytochemicals are pesticides. Therefore, we reported for the first time that it necessary to investigate the presence of active ingredient. has competitive inhibitory property of AChE. Acacia

Table-1: Effect of 96 hrs exposure of chlorpyriphos and Datura stramonium on acetylcholinesterase activity, inhibition, Km and Vmax of the Brain of Catla catla. The Specific activity is expressed in nmole/ min/ mg protein. Each value is mean ± SD of five individual observations. Significant: P > 0.05*; Highly significant: P > 0.01**

Parameters Specifi c Vmax % inhibition of -3 activity of Km x 10 M (activity/min/mg AChE Groups AChE protein)

-3 Control 14.14 ± 8.41 - 0.41 x 10 M 0.1 1 -3 CPF 9.45 ± 2.69** -33% 0.76 x 10 M 0.1 1 -3 D. s 7.46 ± 3.68* -47.2% 0.9 x 10 M 0.1 1

Pre -treatment of D. s followed by -3 5.65 ± 2.11** 1.1 x 10 M 0.1 1 exposure of -60% CPF

50 Asian J. Exp. Sci., Vol. 27, No. 2, 2013; 47-56

Table-2: Recovery of inhibited AChE due to chlorpyriphos and Datura stramonium exposure in the Brain of Catla catla. Each value is mean ± SD of five individual observations. Significant: P > 0.05*; Highly significant: P > 0.01**; Very highly significant: P > 0.001***

Group CPF D.s

Control (0.00073 Recovery of inhibited AChE (100 Recovery of inhibited AChE

mg/ L) mg/L) Parameter 72 96 120 48 96 120 48 hrs 72 hrs hrs hrs hrs hrs hrs hrs

AChE activity 14.14± 9.45± 10.2± 11.1±2 11.5± 11.7± 7.46± 7.58± 7.6± 7.85± 8.34± (nmole/min/ 8.41 2.69 ** 4.0** .3 0.68 0.86 3.68* 0.50 0.81 0.48 0.26 mg protein) *** *** *** *** *** *** ***

% inhibition of - 33% 27.7 21.2 18.6 17.2 47.2% 46.3 45.9 44.4 41 AChE

% recovery - - 8.1 17.7 21.6 23.8 1.6 2.4 5.2 11.7 of AChE

BRAIN

70 60 E

h 60 47.2 C 50 A

f o 40 33 n o

i 30 t i

b 20 i h

n 10 i 0

0 % Control Chlorpyriphos Datura Datura stramonium stramonium and chlorpyriphos

Fig-1: Percentage inhibition of AChE activity in the Brain of Catla catla of control group, CPF group (0.00073 mg/ L), D.s group (100 mg/L) and pre-treatment of D.s leaf extract followed by CPF.

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BRAIN Chlorpyriphos 25 23.8 Datura stramonium 21.6

20 17.7 y r

e 15 v 11.7 o c e 8.1 R 10

% 5.2 5 1.6 2.4 0 48 hrs 72 hrs 96 hrs 120 hrs Duration (in hours)

Fig: 2 - Recovery of inhibited AChE after 48,72, 96 and 120 hours in the Brain of Catla catla exposed to chlorpyriphos (0.00073 mg/ L) and Datura stramonium (100 mg/L) for 96 hours.

Fig-3: Lineweaver-Burk plot showing competitive inhibition in the Brain AChE of Catla catla exposed to CPF (0.00073 mg/ L), D. s leaf extract (100 mg/L) and pre-treatment of D.s leaf extract followed by CPF for 96 hrs.

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Fig-4: Microphotograph of T. S. of the Brain of Catla catla Fig-5: Microphotograph of T. S. of the Brain of Catla catla (Control group) showing molecular layer (ML); granular (Control group) showing molecular layer (ML); granular layer (GL); purkinje cell layer (PCL) (H & E; 100 X) layer (GL) (H & E; 400 X)

Fig-6: Microphotograph of T. S. of the Brain of Catla catla Fig-7: Microphotograph of T. S. of the Brain of Catla catla exposed to CPF (0.00073 mg/L) for 96 hrs showing exposed to CPF (0.00073 mg/L) for 96 hrs showing necrosis in molecular layer (NML); necrosis in granular necrosis in molecular layer (NML); necrosis in granular layer (NGL) (H & E; 100 X) layer (NGL) (H & E; 400 X)

Fig-8: Microphotograph of T. S. of the Brain of Catla catla Fig-9: Microphotograph of T. S. of the Brain of Catla catla exposed to 100 mg/L D.s extract for 96 hrs showing exposed to 100 mg/L D.s extract for 96 hrs showing necrosis in molecular layer (NML); necrosis in granular necrosis in molecular layer (NML); necrosis in granular layer (NGL) (H & E; 40 X) layer (NGL); vacuole formation (VF) (H & E; 100 X)

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Figure-10: Microphotograph of T. S. of the Brain of Catla Figure: 11 - Microphotograph of T. S. of the Brain of Catla catla pretreated with 100 mg/L ethanolic leaf extract of D.s catla pretreated with 100 mg/L D.s extract followed by followed by CPF (0.00073 mg/L) for 96 hrs showing CPF (0.00073 mg/L) for 96 hrs showing necrosis in necrosis in molecular layer (NML); necrosis in granular molecular layer (NML); necrosis in granular layer (NGL); layer (NGL); vacuole formation (VF) (H & E; 100 X) vacuole formation (VF) (H & E; 400 X)

Acknowledgement The authors are thankful to Principal, MLB Govt Girls Nemcsok J. (1995): Biochemical and subcellular College, Bhopal, India for the research facility and to Head changes in carp exposed to the organophosphorus of the Department of Biotechnology, Barkatullah methidathion and the pyrethroid deltamethrin. University, Bhopal, India for providing facility for Aquat. Toxicol. 33, 279. microphotography. Butchiram M.S., Tilak K.S. and Raju P.W. (2009): Studies References on histopathological changes in the gills, liver and kidney of Channa punctatus (Bloch) exposed to Abena A.A., Miguel L.M., Mouanga A. and Hondi A.T. alachlor. J. Environ. Biol. 30(2), 303-306. (2003): Evaluation of analgesic effect of Datura Carr R.L., Straus D.L. and Chambers J.E. (1995): fastuosa leaves and seed extracts. Fitoterapia, 486- Inhibition and aging of channel catfish brain 488. acetylcholinesterase following exposure to two Adedeji O.B. (2011): Response of acetylcholinesterase phosphorothionate insecticides and their active activity in the brain of Clarias gariepinus to sub- metabolites. Journal of Toxicology and lethal concentration of diazinon. Journal of Applied Environmental Health, Part A, 45(3), 325-336. Sciences in Environmental Sanitation, 6(2),13-14 Dembele K., Haubruge E. and Gaspar Ch. (1999): Adekomi D.A., Musa A.A.,Tijani A.A., Adeniyi T.D. and Recovery of acetylcholinesterase activity in the Usman, B. (2011): Exposure to smoke extract of common carp, Cyprinus carpio L. after inhibition Datura stramonium leaf: Some of its effect s on the by organophosphate and carbamate compounds. heart, liver, lungs, kidneys and testes of male Bull. Environ. Contam. Toxicol. 62: 731- 742. Sprague Dawley rats. Journal of Pharmacognosy Chebbi S.G. and David M. (2009) : Neurobehavioural and phytotherapy, 3(5),67-75. responses of the freshwater teleost, Cyprinus carpio Ahirwar S., Tembhre M., Gour S. and Namdeo A. (2012): (Linn) under quinalphos intoxication. Anticholinesterase efficacy of Bacopa monnieri Biotechnology in Animal Husbandary 25(3-4), against the brain regions of rat-a novel approach to 241-249. therapy for Alzheimer's disease. Asian J. Exp. Sci., Crowch C.M. and Okello E.J. (2009): Kinetics of 26(1),65-70. acetylcholinesterase inhibitory activities by Altinok I. and Capkin E. (2007): Histopathology of aqueous extract of Acacia nilotica (L.) and rainbow trout exposed to sub-lethal concentrations Rhamnus prinoides (L' Her). African Journal of of methiocarb or endosulfan. Toxicologic Pharmacy and Pharmacology. Vol. 3(10), 469-475, pathology, 35 , 405-410. October. APHA/AWWA/WEF (2005) – Standard methods for the Das B.K. and Mukherjee S.C. (2000): A histopathological examination of water and waste water. 21st edition, study of carp, Labeo rohita exposed to Andrew D. Eaton, Lenore S. Clesceri, Eugene, W. hexachlorocyclohexane. Veterinarski Arhiv 70(4), Rice and Arnold E. Greenberg editors. 169-180. Balint, T., Szegletes, T., Szegletes, Zs., Halasy, K. and Das A., Shanker G., Nath C., Pal R., Singh S. and Singh

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Russel A. Hardin J., Grand L. and Fraser A. (1997): alcoholic extract of Nerium indicum leaf and their Poisonous plants of North Carolina. Charlotte: biochemical stress response on fish metabolism. North Carolina State University. African Journal Traditional, Complementary and Sahib, K.A., Sailatha, D. and Ramana Rao, K.V. (1980) : Alternative medicines, Vol. 1, Num. 1, 15-29. Impact of malathion on acetylcholinesterase in the Tembhre, M. and Kumar, S. (1994) – Effect of acute and tissues of the fish Tilapia mossambica (Peters) – chronic exposure to sub-lethal dose of dimethoate time course study. J. Biosci. Vol. 2, No.1, 37-41. in the gut of Cyprinus carpio. J. Ecotoxicol. Sarma, K., Pal, A.K., Sahu, N.P., Mukherjee, S.C. and Environ. Monit. 4(3) 205-210. Baruah, K. (2010) – Biochemical and histological Tembhre, M. and Kumar, S. (1995) – Effect of sub-lethal changes in the brain tissue of spotted murrel, concentration of methylamine on AChE activity Channa punctatus (Bloch) exposed to endosulfan. and enzyme kinetics of alimentary canal of Fish Physiol. Biochem. 36 (3): 597- 603. Cyprinus carpio. Ind. J.Z. Spect., Vol. 6, No.2, (39- Satyadevan, S., Kumar, S. and Tembhre, M. (1993) – 42). Acetylcholinesterase activity and enzyme kinectics Tembhre, M. and Kumar, S. (1997) – Acetylcholinesterase in the brain of common carp, Cyprinus carpio inhibition in the fish gut as an indicator of subjected to sub lethal exposure to dimethoate. environmental poisoning by methylamine. Proc. Biosci. Biotech. Biochem. 57 (9): 1566-1567. Acad. Environ. Biol. 6 (1), 29-33. Silver, A. (1974) - The biology of cholinesterases. North- Tembhre, M., Ahirwar, S., Gour, S. and Gaur, M. (2006) – Holland Publishing Company Amsterdam. The Chlorpyriphos induced inhibition of AChE in Netherlands. Cyprinus carpio and recovery during drug leaching. Silva Filho, M.V., Oliveira, M.M., Salles, J.B., Bastos, Journal of cell and tissue research. Vol. 6 (2) 793- V.L., Cassano, V.P. and Bastos, J.C. (2004) – 796. Methyl-paraoxon comparative inhibition kinetics Tembhre, M., Gour, G., Ahirwar, S. and Namdeo, A. (2012) for acetylcholinesterases from brain of neotropical – Diacetylemonoxime reactivation of fishes. Toxicol Lett., Nov 2; 153(2): 247-54. Acetylecholinestrase and butyrylcholinesterase Singh, M. and Kumar, S. (2000) - Effects of sublethal inhibited by dichlorovos in central and peripheral concentrations of dimethoate and malathion on nervous system of Rat. IPCBB Vol. 48, IACIT press AChE activity and enzyme kinetics of liver of Catla Singapore, 126-133. catla (Ham.). Uttar Pradesh Journal of Zoology Wang, C., Lu, G. and Cui, J. (2010) – Response of 2000 Vol. 20 No. 2, 131-135 acetylcholinesterase and GST activities to Singh, M. (1998) – The histopathological study of Brain insecticide co-exposure in Carassius auratus. Wiley and Liver of Catla catla due to effect of some Periodicals, Inc. Environmental Toxicology p e s t i c i d e s w i t h s p e c i a l r e f e r e n c e t o Wogram, J., Sturm, A., Segner, H. and Liess, M. (2000) - acetylcholinesterase kinetics. (Ph.D thesis, Effects of parathion on acetylcholinesterase, Barkatullah University). butyrlcholinesterase and carboxylesterase in three- Somnuek, C., Cheevaporn, V., Saengkul, C. and Beamish, spined stickleback, Gasterosteus aculeatus F.W.H. (2007) – Variability in acetylcholinesterase following short-term exposure. Environmental upon exposure to chlorpyriphos and carbaryl in Toxicology and Chemistry, 1528-1531. hybrid catfish. Science Asia 33: 301-305. Suganthy, N., Pandian, S.K. and Devi, K.P. (2009) – Cholinesterase inhibitory effects of Rhizophora lamarckii, Avicennia officinalis, Sesuvium portulacastrum and Suaeda monica; Mangroves inhabiting an Indian coastal area (Vellar Estuary). Journal of Enzyme inhibition and Medicinal Chemistry, 24 (3): 702-7. Susan, T.A., Sobha, K. and Tilak, K.S. (2010) - A study on acute toxicity, oxygen consumption and behavioural changes in the three major carps, Labeo rohita (Ham.), Catla catla (Ham.) and Cirrhinus mrigala (Ham.) exposed to fenvalerate. Bioresearch Bulletin 1: 33-40. Tiwari, S. and Singh, A. (2004) – Piscicidal activity of

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An Advance Technique for Solving Assignment Problems

P.R. Sharma*, S. Shukla** *Department of Mathematics, University of Rajasthan, Jaipur (India) **S. S. Jain Subodh P.G. College, Jaipur (India) Email Id.: [email protected] , [email protected]

Abstract: The assignment problem is a typical combinatorial optimization problem. Classical Assignment Problem (AP) is a well-known topic world-wide. In this problem cij denotes the cost for assigning the jth job to the ith person. This cost is usually deterministic in nature. But in realistic situations, it may not be practicable to know the precise values of these costs. We consider many classical problems from location theory which may serve as theoretical models for several logistic problems such that some linear or quadratic function attains its minimum. It turns out that linear objective function yields a linear assignment problem, which can be solved easily by several primal- dual methods like Hungarian method, Shortest augmenting path method etc. Aim of the paper is to investigate a new approach to solve assignment problems of different types. This method proposes momentous advantages over similar methods.

Keywords: Assignment Problem, Branch, Bound Technique.

Introduction: The assignment problem is one of the earliest applications of linear integer programming problem. A diversity of practical problems turn out to be a special illustration of the assigning problem, i.e. a problem, where one looks for an assignment of members of set A to members of set B such that some function attains its optimum. We may assume that the numbers of elements in sets A and B are equal and that we want to assign exactly one element from A to each element from B. Solving such problem often means that we have to evaluate some function for each assignment. Since there are n! Possible assignments, where n is the number of elements in A, this may lead to a very hard problem. Different methods have been presented to solve assignment problem by Goel and Mittal (1982), Bazarra et al. (2005) and Hamdy (2007). A considerable number of methods have been so far presented for assignment problem in which, the best known, most widely used, and most written about method for solving the assignment problem is the “Hungarian Method”, originally suggested by Kuhn in 1955. Ford and Fulkerson (1957) provided vital ideas for the untimely approaches used in solving network flow problems, extended to solve the transportation problem [Munkres (1957)] and generalized to solve the linear programming problem [Dantzig et al. (1956)]. It is a dual method with a feasible assignment being obtained only at the last computational step. Aim of the paper is to apply Branch and Bound technique for all type of assignment problems. The proposed method is a methodical process, easy to apply and can be exploited for all types of assignment problems with maximize or minimize objective functions.

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Mathematical Formulation of Assignment Problem: Let there be n persons and n jobs. Each job must be done by exactly one person and one person can do, at

th th most, one job. If the cost of doing j job byi person is cij , then the cost matrix is given by the following table:

jobs 1 2 3 ………. J ………..n Person 1 c11 c12 c13 ………. c1 j ……….. c1n 2 c21 c22 c23 ………. c2 j ……….. c2n 3 c31 c32 c33 ………. c3 j ……….. c3n ……….. …… ….. ….. ………… ………… ……….. …… ….. ….. ………… ………… i ci1 ci2 ci3 ……….. cij ………… cin

……….. ….. ….. …. ……….. …………. ……….. ….. ….. …. ……….. …………. n cn1 cn2 cn3 ……….. cnj …………. cnn Table 1

The problem is to assign the persons to the jobs so that the total cost of completing all jobs become minimum.

We introduce xij , where ⎧1, if the person i is assigned the job j; i,j = 1, 2, ....., n xij = ⎨ ………. (1) ⎩0, otherwise

th Corresponding to the ()ij event of assigning person i to job j, the constraint n i.e. each job must be done by exactly one person, and the constraint ∑ xij ==1,jn 1,2,...., i=1 n ∑ xij ==1,i 1,2,....,n means each person must be assigned at most one job. j=1 Thus the model for crisp Assignment Problem (AP) is given by

Model 1:

nn Min z= ∑∑ cij x ij ………. (2) ij==11

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⎧ n ⎪∑ xij ==1, jn 1,2,...., ⎪ i=1 ⎪ n ...... (3) subject to⎨∑ xij == 1, i 1,2,....,n ⎪ j=1 ⎪x ==0 or 1, ij , 1,2,..., n ⎪ ij ⎩

This cost cij is usually deterministic in nature. But in real situations, it may not be practicable to know the precise values of these costs. In such an uncertain situation, instead of exact values of costs, if the preferences

th th for assigning the j job to the i person is known in the form of composite relative degree ( d ij ) of similarity to ideal solution (maximum degree indicates most preferable combination), then cij can be replaced by d ij in the classical assignment problem in the maximization form which can be solved by any standard procedure ( e.g. Hungarian method or by any software) to get the optimal assignment. In that case the model for the preference AP becomes Model 2:

nn Max z= ∑∑ dij x ij ………. (4) ij==11

⎧ n ⎪∑ xij ==1, jn 1,2,...., ⎪ i=1 ⎪ n ……… (5) subject to⎨∑ xij == 1, i 1,2,....,n ⎪ j=1 ⎪x ==0 or 1, ij , 1,2,..., n ⎪ ij ⎩

Method of Solution: In this paper, the assignment problem is solved by Branch and Bound Algorithm. Using curtailed enumeration technique. The terminologies of the branch and bound technique applied to the assignment problems are presented below: Let k be the level number in the branching tree (for root node, it is 0), σ be an assignment made is the

k current node of a branching tree. pσ be an assignment at level k of the branching tree, A be the set of

k assigned cells (fractional assignment) up to the node pσ from the root node (set of i and j values with respect

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k to the assignment cells up to the node pσ from the root node), and vσ be the lower bound of the fractional

k assignment A up to pσ , such that ⎛⎞ vcσ =+∑∑∑ij⎜⎟ Minc ij ………. (6) ij, ∈∈∈ A i X⎝⎠ jY

th th where cij is the cell entry of the cost matrix with respect to the i row and j column, X is the set of rows

k which are not removed up to the node pσ from the root node in the branching tree, and Y is the set of

k column which are not removed up to the node pσ from the root node in the branching tree. Branching guidelines 1. At level k, the row marked as k of the assignment problem will be assigned with the best column of the assignment problem. 2. If there is a tie on the lower bound, then the terminal node at the lower most level is to be considered for further branching. 3. Stopping rule: - If the minimum lower bound happens to be at any one of the terminal nodes at

the (n− 1)th level, the optimality is reduced, and then the assignment on the path from the root node to that node along with the missing pair of row column arrangement will from the optimum solution. Types of Assignment Problems: There are three types of Assignment Problems. Type-I: In maximization problems jobs effectiveness is frequently measured by profit instead of cost. When a worker is assigned to different jobs, the profit contribution often differs from jobs to jobs. This difference arises from workers capability and experience on a particular jobs, as well as the different nature of jobs to be assigned. Therefore job effectiveness of the worker is expressed in terms of profit matrix. Except for one transformation an assignment problem in which the objective is maximize total payoff measures can be solved by the Branch and Bound Algorithm. The transformation involves subtracting all the entries of the original payoff table from the maximum entry of that table. The transformed entries give us the relative costs and the problem then becomes a minimization problem. Once the optimal assignment for the transformed problem is obtained. The total measure of the original payoff matrix can be found by those cells to which the assignment has been made. In these types of problem the number of persons to be assigned and number of jobs were assumed to be the same. Such as assignment problem is known as balanced assignment problem. Type-II: If the number of persons is different from number of jobs, the assignment problem is said to be unbalanced problem. If the number of jobs is less than the number of persons, some of the persons cannot be assignment any job. In such problems one or more dummy jobs of zero duration are introduced to make the assignment problem balanced. On the other hand if the number of persons is less than the number of jobs

Asian J. Exp. Sci., Vol.27, No. 2, 2013; 57-71 than we add one or more dummy persons with duration time zero to balance the assignment problem. The balanced problem then can be solved by using the Branch and Bound Techniques. Type-III: In some cases, a certain worker cannot be assigned a particular job. The reasons for impossible assignments are numerous. Back of required skills, deficiency in technical know-how, improper training and physical inability are only a few many reasons. For solving such type of assignment problems, infinite cost is put in the cell where no assignment is possible. The remaining procedure is exactly the same as the ordinary assignment problems. Numerical Examples: Example–1. The jobs 1, 2, 3 are to be assigned three machines 1, 2,3 the processing cost (Rs/-) are as given in the matrix shown below, find the allocation which will minimize the overall processing cost Machine 1 2 3 1 19 28 31 Jobs 2 11 17 16 312 15 13

Solution: Initially, no job is assigned to any machine so the assignment(σ ) at the root node(level 0)of the following branching tree is a null set and the corresponding lower bound vσ is also 0, as shown is Figure 1:

Figure1. Branching tree at the root node Further branching: The three different sub-problems under the root node are shown in the Figure 2, the lower bound for each of the sub-problems is shown by the right side of it.

0 Figure 2. Branching tree after from pϕ Calculation for lower bound:

1 Lower bound for p11

= + ( min ). ………. (7) vσ ∑ cij ∑ ∑ cij ij, ∈ A iX∈ jy∈ Where σ = {(11)}, A = {(11)}, X= (2, 3), Y= (2, 3).

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Then

= + ( min ) ………. (8) v11 c11 ∑ ∑ cij i∈(2,3) j∈(2,3)

= c11 + c23 + c33 = 19+16+13=48.

1 Lower bound for p12 σ = {(12)}, A = {(12)}, X= (2, 3), Y= (1, 3). Then

= + ( min ) ………. (9) v12 c12 ∑ ∑ cij i∈(2,3) j∈(1,3)

= c12 + c21 + c31 = 28+11+12 = 51.

1 Lower bound for p13 σ = {(13)}, A = {(13)}, X= (2, 3), Y= (1, 2). Then

= + ( min ) ………. (10) v13 c13 ∑ ∑ cij i∈(2,3) j∈(1,2)

= c13 + c21 + c31 = 31+11+12 = 54. Further branching: Further branching is done from the terminal node which has the least lower bound. At

11 1 1 this stage, the nodes p11,,p 12 and p13 are the terminal nodes. Among these nodes, the node p11 has the least lower bound. Hence, further branching from this node is shown in Figure 3. The lower bound of each of the newly created node is shown by the right side of it.

1 Figure 3. Branching tree after from p11

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Calculation for lower bound:

2 Lower bound for p22 σ = {(22)}, A = {(11), (22)}, X= (3), Y= (3). Then

= + + ( min ) ………. (11) v22 c11 c22 ∑ ∑ cij i∈3 j∈3

= c11 + c22 + c33 = 19+17+13 = 49.

2 Lower bound for p23 σ = {(23)}, A = {(11), (23)}, X= (3), Y= (2). Then

= + + ( min ) ………. (12) v23 c11 c23 ∑ ∑ cij i∈3 j∈2

v23 = c11 + c23 + c32 =19+16+15=50.

1 1 2 2 Further branching: At this stage, the nodes p12 , p13 , p23 and p22 are the terminal nodes. Among these

2 nodes, there is one node with the least lower bound of 49. So the node p22 which is at the bottom-most level

is considered for further branching. Since this node lies at (n− 1)th (level k=2) of the branching tree, where n is the size of the assignment problem, optimality is reached. The corresponding solution is traced from the

2 root node to the node p22 along with the missing pair of job and operator combination (3, 3), so optimal solution is presented through the following table. Job Machine Cost 1 1 19 2 2 17 3 3 13 Hence total cost = 19+17+13 = 49 Rs.

Example-2. A methods engineer wants to assign four new methods to three works centers. The assignment of the new methods will increase production and they are given below. If only one method can be assigned to a work center, determine the optimum assignment: Increase in (production) unit

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A B C 1 10 7 8 Methods 2 8 9 7 3 7 12 6 4 10 10 8

Solution: First we will convert maximum matrix into minimum matrix, for this subtracting all the elements of given matrix from maximum element (12), then we have minimum matrix as following:

A B C 1 2 5 4

2 4 3 5 3 5 0 6

4 2 2 4

Now we will introduce a dummy column for making a square matrix, which is balanced assignment problem is given below:

A B C D 1 2 5 4 0 2 4 3 5 0 3 5 0 6 0 4 2 2 4 0

Initially no method is assigned to any production so the assignment (σ ) at the root node of the following branching tree is a null set and the corresponding lower bound vσ is also 0, as shown in Figure 4.

Figure 4. Branching tree at the root node Further branching: The four different sub-problems under the root node are shown in the Figure 5, the lower bound for each of the sub-problems is by the right side of it.

0 Figure 5. Branching tree at the root node pφ

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Calculation for lower bound:

1 Lower bound for p11

= + ( min ). ………. (13) vσ ∑ cij ∑ ∑ cij ij, ∈ A iX∈ jy∈ Where σ = {(11)}, A = {(11)}, X= (2, 3, 4), Y= (2, 3, 4). ⎛⎞ vc11=+ 11 ∑∑⎜⎟ Minc ij ………. (14) ij∈∈(2,3,4)⎝⎠ (2,3,4)

= c11 + c24 + c34 + c44 =2+0+0+0=2.

1 Lower bound for p12 σ = {(12)}, A = {(12)}, X= (2, 3, 4), Y= (1, 3, 4). Then

v12 = c12 + c24 + c34 + c44 ==5+0+0+0=5. ……….. (15)

1 Lower bound for p13 σ = {(13)}, A = {(13)}, X= (2, 3, 4), Y= (1, 2, 4). Then

v13 = c13 + c24 + c34 + c44 =4+0+0+0=4. ………. (16)

1 Lower bound for p14 σ = {(14)}, A = {(14)}, X= (2, 3, 4), Y= (1, 2, 3). Then

v14 = c14 + c22 + c32 + c42 =0+3+0+2=5. ……….. (17) Further branching:-Further branching is done from the terminal node which has the least lower bound. At

11 1 1 1 this stage, the nodes p11,,p 12 p13 and p14 are the terminal nodes. Among these nodes, the node p11 has the least lower bound of 2. Hence, further branching from this node is shown as Figure 6. The lower bound of each of the newly created node is shown by the right side of it.

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1 Figure 6. Branching tree after branching from p11 Calculation for lower bound:

2 Lower bound for p22 σ = {(22)}, A = {(11), (22)}, X= (3, 4), Y= (3, 4). Then

v22 = c11 + c22 + c34 + c44 =2+3+0+0=5. ……….. (18)

2 Lower bound for p23 σ = {(23)}, A = {(11), (23)}, X= (3, 4), Y= (2, 4). Then

v23 = c11 + c23 + c34 + c44 =2+5+0+0=7. ………… (19)

2 Lower bound for p24 σ = {(24)}, A = {(11), (24)}, X= (3, 4), Y= (2, 3). Then

v24 = c11 + c24 + c32 + c42 =2+0+0+2=4. ……….. (20)

Further branching: Further branching is done from the terminal node which has the least lower bound. At

1 1 1 2 2 2 this stage, the nodes p12 , p13 , p14 , p22 , p23 and p24 are the terminal nodes. Among these nodes, two nodes

2 have the least lower bound of 4. So the node p24 which is at the bottom most level is considered for further branching from this node is shown as Figure 7. The lower bound of newly created node by the right side of it.

Asian J. Exp. Sci., Vol.27, No. 2, 2013; 57-71

2 Figure 7. Branching tree after branching from p24 Calculation for lower bound:

3 Lower bound for p32 σ = {(32)}, A = {(11), (24), (32)}, X= (4), Y= (3). Then

v32 = c11 + c24 + c32 + c43 =2+0+0+4=6. ……….. (21)

3 Lower bound for p33 σ = {(33)}, A = {(11), (24), (33)}, X= (4), Y= (2). Then,

v33 = c11 + c24 + c33 + c42 =2+0+6+2=10 ...... (22)

1 1 1 2 2 3 3 Further branching: At this stage, the nodes p12 , p13 , p14 , p23 , p22 , p32 and p33 are the terminal nodes.

1 3 Among these nodes, there is one node p13 with the least lower bound but not bottom-most node and p32 is

3 th bottom-most node. Hence, further branching from p32 node. Since this node lies at (n− 1) level of the branching tree, where n is the size of the assignment problem, optimality is reached. Hence, optimal solution is

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Method Production Unit 1 A 10 2 B 0 3 C 12 4 D 8 Hence total units =10+0+12+8=30.

Example-3. Secretary of a school is talking bids on the city’s four school bus routes. Four companies have made the bids as detailed in the following table. Company Bids A B C D C1 7000 8000 ------C2 --- 7000 --- 7000 C3 6000 --- 5000 --- C4 ------7000 8000

Suppose each bidder can be assigned only to one route. Use the assignment model to minimize the school’s cost of running the four bus routes. Solution: First we are taking highest cost ( ) for unassigned routes. The problem can be represented as A B C D C1 7000 8000 7 8 C2 7000 7000 7 7

C3 6000 5000 6 5 C4 7000 8000 ⇒× 1000 7 8

Initially no company is assigned to any bidder, so the assignment (σ ) at the root node (level 0) of the following branching tree is a null set and the corresponding lower bound vσ is also 0, as shown in Figure 8.

Figure 8. Branching tree at the root node Further Branching: The four different sub-problems under the root node are shown in Figure 9. The lower bound for each of the sub-problems is shown by the right side of it.

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0 Figure 9. Branching tree after branching from pφ Calculation for lower bound:

1 Lower bound for p11 σ ==={()11} ;Ax{() 11} ; (2,3,4), y = (2,3,4). ………. (23) vcccc11=+++=+++= 11 22 33 43 775726.

1 Lower bound for p12 σ ===={()12} ;Ax{() 12} ; (2,3,4), y (1,3,4). ………. (24) vcccc12=+++=+++= 12 24 33 43 8757 27.

111 1 Further Branching: At this stage, the nodes p11,pp 12 , 13 and p 14 are terminal node. Among these nodes,

1 the node p11 has the least lower bound. Hence, further branching from this node is shown in Figure 10. The lower bound of newly created nodes by the right side of it.

1 Figure 10. Branching tree after branching from p11

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Calculation for lower bound:

2 Lower bound for p22 σ =={()22} ;Axy{() 11 ,(22)} ; == (3,4), (3,4). ………. (25) vcccc22=+++=+++= 11 22 33 43 775726.

2 Lower bound for p24 σ =={()24} ;Axy{()() 11 , 24} ; == (3,4), (2,3). ………. (26) vcccc24=+++=+++= 11 24 33 43 7 7 5 7 26.

1 1122 2 Further Branching: At this stage, the nodes p12,,pppp 13 14, 22, 23 and p24 are terminal node. Among these

2 nodes, two nodes have least lower bound of 26. The node p22 is the first least lower bound so that further branching considering from this node shown in Figure 11. The lower bound of newly created nodes by the right side of it.

2 Figure 11. Branching tree after branching from p22

Calculation for lower bound:

3 Lower bound for p33

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σ =={()33} ;Axy{() 11 ,(22),(33)} ; == (4), (4). ………. (27) vcccc33=+++=+++= 11 22 33 44 775827.

1112 2 3 3 Further Branching: At this stage, the nodes p12,,ppp 13 14, 23 , p24 , p33 and p34 are terminal node. Among 2 these nodes, the node p24 has least lower bound but this is not bottom most and after branching from this 3 3 node we have nodes p32 and p33 . These nodes have highest lower bound so that this not for further 3 branching. Hence p33 is second least lower bound and bottom most node at level three. Since this node lies at (n− 1)th level of the branching tree, where n is the size of the assignment problem, optimality is reached. Hence optimal solution is Company route Cost C1 A 7000 C2 B 7000 C3 C 5000 C4 D 8000

Hence total cost= (7+7+5+8) ×1000=27000 Rs. Conclusion In this paper, Branch and Bound technique is used to solve assignment problem. This method is applicable for all kind of assignment problems, whether maximize or minimize objective function. This technique is easy to apply and consume less time comparative to another techniques. Acknowledgement: The authors are thankful to Dr. Sandeep gupta for his valuable help in preparation of the paper. References Goel B.S., Mittal, S. K. (1982): Operations Research. (Pragati Prakashan). 2405-2416. Dantzig G. B., Ford, L. R., Jr. and Fulkerson D. R., (1956): A Primal-Dual Algorithm for Linear Programs, in Linear Inequalities and Related Systems. Annals of Mathematics Study No. 38, (Eds) Kuhn and Tucker, (Princeton University Press, Princeton). Ford L. R., Jr. and Fulkerson D. R. (1957): A Simple Algorithm for Finding Maximal Network Flows and an Application to the Hitchcock Problem. Canadian Journal of Mathematics. 9, 210-218. Hamdy A. Taha (2007): Operations Research, an introduction. (Pesarson Publication). Kuhn H.W. (1955): The Hungarian Method for the Assignment Problem, Naval Research Loqistics Quarterly, 2,83-97. Bazarra M.S., Jarvis J. J. and Sherali H.D. (2005): Linear programming and network flows, Wiley. Munkres, J. (1957): Algorithms for the Assignment and Transportation Problems. Journal of the Society for Industrial and Applied Mathematics. 5,32-38.