Pedigree Analysis Of Some Hereditary Diseases in The Successive ... - SCIENTIFIC...

THE SCIENTIFIC TEMPER ISSN 0976 8653

VOL -VII, No. 1&2, January-2016

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Pedigree Analysis Of Some Hereditary Diseases in The SuccessiveFive Generations Of A Family Of Punjab With Special Reference To

Syndactyly

Dr. (Mrs.) Sushil GuptaAssociate ProfessorDev Samaj College for WomenFirozepur City-152002 (Punjab) India.(Received December 2014, revised and accepted August 2015)HemantGarg,SGRD Dental College and Research Institute, Amritsar,India

ABSTRACT

Syndactyly [from Grek roots ‘Syn’-together+ ‘Dactylus’- [finger ]=fingerstogether is a condition where tow or more than two digits are fused together.It occurs normally in some mammals, such as siamang but is unusualcondition In humans.Syndactyly can be simple, complex orcomplicated.Syndactyly can be possible out come of a large number of rareinherited and developmental disorders.It can be present over 100 differentdisorders where they are minor features compared to other characteristics ofthese disorders. Pedigree Analysis of some hereditary diseases in thesuccessive five generations of a family of Punjab [INDIA] with specialreference to Syndactyly was carried out. Some other inheritable charactersobserved were Griegcephalopolysyndactyly, presence of Flat Feet, Early teethfall, Eary greying of hair, Presence of bow legs, Heart problems, Osteoprosis,Baldness , Asthma, Gout,Ulcerative colitis,Eye defects,upper palate missingand Stammering defect. They may be due to some changes in the samechromosomes which causes Syndactyly or not.This may form basis for furtherstudies.

Keywords: Syndactyly,Griegcephalopolysyndactyly, Osteoporosisand Ulcerative colitis.



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INTRODUCTION

Syndactyly ( from Greek roots “syn”-(together)+ “dactylos”- (finger) = fingerstogethers, is a condition where two or more thantwo digits are fused together. It occurs normallyin some mammals, such as siamang but is anunusual condition in humans.

Syndactyly can be simple or complex. Insimple syndactyly, adjacent fingers and toes arejoined by soft tissue. In complete syndactyly,the skin is joined all the way to the tip of thefingers. In incomplete syndactyly the skin is onlyjoined part of the distance to the finger tip.

In complex syndactyly, the bones orcartilage of the adjacent digits are fused. Thekangaroo exhibits complex syndactyly.

Complicated syndactyly occurs as a partof syndrome ( such as apert’s syndrome) andtypically involves more digits and withcomplex syndactyly there may be abnormalitiesof nerves, vessels and tendons. Syndactylyresults from the failure of the programmed cell

living member of the family who is consideredreliable.

Individuals were examined in their housesfor the digital and other defects. Photographsof the most affected members with syndactylywere taken with a camera. X-rays were alsodone of few available members. Informationregarding other defects was collected by askingoral questions from them.

death that normally occurs between digits, mostoften this is due to genetic defects. There areseveral forms of syndactyly, each of thesewhere the genetics is understood, is caused byan autosomal dominant gene. Syndactyly is alsopossible out come of a large number of rareInherited and developmental disorders. It canbe present over100 different disorders wherethey are minor features compared to othercharacteristics of these diseases.

An incidence of occurrence of syndactylyin decreasing order of frequency is as follows:-

Between the middle and ring fingers(57%)

Little and ring fingers (27%) Middle and index fingers (14%) Thumb and index fingers (3%)Males are affected twice as often females,

(http.Syndactyly) complete simple syndactylyof all digits with polysyndactyly involving bothhands and feet is rare.[Hosalkar et al,2000]

SUBJECTS AND METHOD

No single individual served as the probandin this kindred since various affected memberswere brought to our attention during the courseof other genetic studies being conducted in thetowns of Punjab. The pedigree (fig.1 ) wasstructured through interviews with nerely 75%of the living members in the last fourgenerations. In the information on the first twogenerations was supplied by the one of the

RESULTS

SLIDES A,B,C,D and E showing The pedigree chart of the family with



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A. Slide showing inheritance of Syndactyly, Asthama, Griegcephalo polysyndactyly anddistalphalanyxinturned.

B. Slide showing inheritance of heart problems, ulcerative colitis, colour Blindness, upperpalatemissingstammering andgout.



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C. Slide showing inheritance of osteoporosis, Early Teeth Fall, Eye Defects and Baldness.

D. Slide showing inheritance of Flat Feet and GreyHairs.



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E. Slide showing inheritance of BowLegs.



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inheritance of different charactersIn the male member of Gen 1 terminal

phalangeal bones of all fingers of both handswere inturned (bent fingers). This characterappeared only in one member of Gen. 1 and allothers were normal.(SLIDE A)

In generation II female no. 3 of the familyinherited this character of bent fingers from herfather. Some other defective characters e.g. flatfeet, early teeth fall, early graying of hair, legturned in ward, osteoporosis etc. appeared inher. She died due to heart attack. Theinheritance pattern of these characters wasobserved in successive three generations.Syndactyly appeared in Gen.III and inheritedin Gen IV & V.(SLIDE A,B,C,D and E)

Syndactyly was observed in the membersof Gen. III-1,2,5,7 no. of males(SLIDE A)

Gen. IV- 7,11,13,19,21(SLIDE A)Gen. V- 21,22,34,36(SLIDE A)The 1,2 individuals of generation III and

22 male of Gen. V died immediately after theirbirth due to greigcephalopolysyndactyly.(SLIDE A)

5 no male of Gen III showed completesimple syndactyly in between 3 and 4th fingersof left hand.

7 no male of Gen III showed completeComplex syndactyly in between 3 and 4 th

fingers of left hand(PHOTO FILE 011.jpg) butcomplete complex syndactyly in 3rd and 4th

fingers of right hand (PHOTO FILES010.jpg,012.jpg,013.jpg AND 014.jpg) .Syndactyly of 4th and 5th toes of right foot wasalso observed. Small toe always remainedunder 4th toe (photo)

7 number male of Gen IV showed completesimple syndactyly in between fingers of lefthand (PHOTO FILES 007.jpg and009.jpg) butcomplete complex syndactyly in between 3rd

and 4th fingers of right hand.(PHOTO FILES008.jpg and 009.jpg, X-RAY 4X6d.jpg) Smalltoe was present under 4th toe.

11 number male of Gen IV showedcomplete simple syndactyly in between 3 and4th fingers of left hand.(PHOTO FILES015.jpg,016.jpg and 017.jpg)

13 number male of Gen IV showedcomplete simple syndactyly in between fingersof right hand syndactyly was also observed in4th and 5th toes of right foot. To separate thefingers his hand was successfully operated.

19 number male of Gen IV showedcomplete Simple syndactyly in between 3 and4th fingers of Right hand(PHOTO FILE004.jpg) and Complete Complex Syndactly inbetween 3rd and 4th fingers of left hand(POTOFILES 001.jpg,002.jpg and 003.jpg). Right footalso showed syndactyly between 4th and 5th toes.

21 number female of Gen IV showedcomplete complex syndactyly in betweenfingers of both hands. She was operated toseparate the fingers but operation was notsuccessful.

21 number male of Gen V showedcomplete simple syndactyly in between 3 and4 th fingers of left hand(PHOTO FILES018.jpg,019.jpg and 020.jpg).

34 number female of Gen V showedcomplete complete syndactyly between 3rd 4th

fingers of right hand.Some other inheritable characters appeared

in the female of gen. II are also heritable andobserved in the subsequent generations. Theymay be due to some changes in the samechromosomes which is causing syndactyly ornot. This needs further investigations.Observations are given below –

(1) Flat Feet –Character appeared in themembers of



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Gen II-3 (SLIDE D)Gen III- 4,5,6,7,8,9&10 (SLIDE D)Gen IV- 5,7,10,11,12,14,18,19,& 20

(SLIDE D)Gen-V- 33 (SLIDE D)

(2) Early teeth fall character wasobserved inGen II-3 (SLIDE C)Gen III- 3,4,5,6,8,9 & 10 (SLIDE C)

(3) Early Graying of hair was observedin the members ofGen II- 3(SLIDE D)Gen III- 3,4,5,6,7,8,9 and 10 (SLIDE D)Gen IV- 1,2,4,5,6,7,12,13,15,18 ,19 and 20 (SLIDE D)Gen V-31,33 (SLIDE D)

(4) Legs turned in wards(Bow Legs)appeared in the members ofGen II-3 (SLIDE E)Gen III-3.4.5.6.7.8.9 &10 (SLIDE E0Gen IV-1,2,3,4,5,6,7,9,10,11,12,13, 14,

16,18,19 &20. (SLIDE E)Gen V-11,12,32,33,34.(SLIDE E)

(5) Heart problem was observed in themembers ofGen II- 3 no female died to heart attack.

(SLIDE B)

Gen III-4,5,6 No males died due toheart attack .8,9, no male and 10th nofemale are suffering from heart problem.(SLIDE B)

(6) Osteoporosis appeared in the members ofGen II- 3 number female (SLIDE C)Gen III 3,4,5,6,8,9,& 10 (SLIDE C)

(7) Baldness character appeared inmember ofGen IV-11.(SLIDE C)

(8) Asthma was observed inGen II 3 number female.(SLIDE A)GenIII-4,6,10 (SLIDE A)Gen IV-5,9(SLIDE A)

(9) Ulcerative colitis appeared inGen IV-4, 14. (SLIDE B)

(10) Gout was observed inGenIV-10 (SLIDE B)

(11) Eye- defects were observed inGen IV- 2, 3,7,14,15,16,17 (SLIDE C)Gen V- 15,17,33,34(SLIDE C)

(12) Upper plate missing in one member ofGen- V-17 (SLIDE B)

(13) Stammering defect in one member ofGen IV-18 (SLIDE B)

DISCUSSION

On the basis of study of 63 pedigree Bellproposed a classification of syndactyly basedprimarily on the localization of the webbing.[Bell,1953]

Type A1- Webbing occurs between thesecond and third toes with no involvement ofthe hands.

Type A2 - Webbing between the third andfourth fingers

Type B1 - Webbing between the fourth &fifth toes.

Type C - Webbing occurs between threeor more digits in one or more extremities.

In some pedigrees, however multipledigital anomalies were present and these wereconsidered combinations of the above typessuch as A1 with A2 or A2 with B1.

After reviewing the literature and studyingadditional families five types of syndactylywasconcluded.[Temtamy,1966]

Type 1- Syndactyly of the third and fourthfingers and the second and third toes.



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Type II - Syndactyly between third &fourth fingers and fourth and fifth toes.

Type III - Syndactyly involves the fourthand fifth fingers only.

Type IV - Complete syndactyly of allfingers.

Type V- Syndactyly is associated withmetacarpal and metatarsal synostosis.

Each of these types is inherited as anautosomal dominant malformation withvariable expressivity and incompletepenetrance .[Cross et al,1967]

Syndactyly with metacarpal 4-5 fusionwith X linked recessive inheritance was alsoreported.[Orel,1928;Holmes et al,1972]Inheritable syndactylism is associated withgenetic defects involving particular candidateregions on the second chromosome.[Kozin,2001]

Five type of syndactyly have beenidentified in humans.[Flatt,2005] Thecorresponding loci associated with these typesand their common phenotypical expression areas follow-

Type 1 - 2q34-2q36, Webbing occursbetween middle and ring fingers and/or secondand third toes.[Bosse et al2000]

Type II – 2q31, Involves long and ringfingers but has a sixth finger merged inbetween.[Sarfarazi et al,1995]

Type III – 6q21-23, Small finger is mergedinto the ring finger.[Sarfarazi et al1995]

Type IV – 7q36, involves all fingers andtoes.[Sato et al,2007]

Type V – 2q31-q32, similar to type I butthe metacarpals and metatarsals may also befused.[Sato et al,2007]

Synpolydactyly and HOXD13 polyalaninerepeat were reported and it was found thataddition of 2 alanine residues is without clinical

consequences.[Malik et al,2007] Type IIsyndactyly is characterized by webbingbetween 3/4 fingers and 4/5 toes with partialor complete digit duplication with in thedactylus web Three loci have been identifiedat chromosomes 2q31,22q 13.31 and 14q 11.2q12 and have been designated as SPD1, SPD2,SPD3 respectively.[Apert,1956 and]Akarsu etal,1956] Unique expansion mutations in apolyalanine repeat(PolAR) of HOXD 13 havebeen implicated in SPDI families.

Syndactyly between the third and fourthfingers and between the fourth and fifth toes,with partial or complete digit duplication in thesynpolydactyly webIn most cases thiscondition is caused by mutatious in the HOXD13 gene which is located on chromosome2q31.[Akarsu et al,1996;Muragaki et al,1996and Goodman et al,1997] Classical SPD hasbeen shown to be due to different sizedexpansions of an imperfect trinucleotide repeatsequence encoding a 15 residue-N terminalpolyalanine tract in HOXD-13. An atypicalform of SPD, associated with a novel footphenotype is related with two differing intragenic deletions in HOXD 13.[Goodmanetal,1998] A distinct form of SPD which is notaccompanied by an expansion of the HOXD13 polyalanine tract was also reported and itseems to co-segregate in the family with anapparently balanced translocation t (12:22)(p11.2, q13.3). A Chinese pedigree withcongenital synpolydactyly showing partial orcomplete webbing between 3&4 fingers wasanalysed and syndactyly type I,II and III weremapped to 2q34-36, 2q31-q32 and 6q21-23.2respectively. This condition is inherited as anautosomal dominant trait with reducedpenetrance.[Deebeer et al,1996;1998 and2000]Syndactyly type II is named synpolydactyly



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(SPD). Expansion of polyalanine tract inHOXD 13 gene is known to causesynpolydactyly. HOXD 13 gene locates in theHOXD complex. Nine homologous genes(HOXD1-D3-D4-D8-D9-D10-D11-D12-D13)of HOXD complex locate on chromosome 2 inorder of HOXD 1 to HOXD 13, among whichHOXD13 is closest to the centromere.Deletions and duplications in HOXD complexor its upstream regulator factors have beenidentified to cause hand heteroplasia andconsequently lead to the abnormalities of fingernumber or abnormalities of configuration.Synpolydactyly (SPD) locus in the Chinese hanpopulation is in the region of chromosome2q31-q32 but a different casual gene can beinvolved.[Qin et al,2003] A pedigree of Belgianfamily, a father and his two daughters all havea complex type of SPD associated withmetacarpal and metatarsal synostoses. Theseindividuals also have an abnormal karyotypet(12.22),(p11.2,q13.3). In these members ofBelgian family which cosegregate witht(12.22),(p11.2, q13.3) chromosomaltranslocation.[Desmet,1996] 12 break point ofthis translocation maps to 12p11.2 betweenmarkersD12S1034 and D12S1596 wereprepared for the chromosome which showed

that mutation in the HOXD 13 gene is notresponsible for the phenotype and presented aphysical map of the region around the 12p11.2break point. A search for expressed sequenceswith the contig have so far revealed one CpGisland, Seven anonymous ESTs and threepreviously characterized genes, DAD,R KRAGand HTZ, all of which were found not to bedirectly disrupted by the translocation. Thegene represented by EST R72964 was foundto be disrupted by translocation20.

Greigcephalopolysyndactyly syndrome[Biesecker,2008] is caused by the loss offunction mutation in the GL 13 transcriptionfactor genes and is inherited in an autosomaldominant pattern. The disorder is allelic to thePallister Hall Syndrome and one form ofAcrocollasal Syndrome.[Williams et al,1997]More than 75% of patients with clinicallyrecognizable GCPS who have been evaluatedin NIH study have been found to havemutations in GL13.[Johnston et al,2003]Mutations shown to cause GCPs includenonsense, missense and spilicing mutations andtranslocations, deletions andinsertions.[BIESECKER,2008; and Johnston etal,2003]

ACKNOWLEDGEMENTS:

It is my great privilege to acknowledge Miss.Sonia (Prof. in Computer Sciences) of Dev

Samaj College who rendered her services forthe preparation of manuscript and Pedigreechart.

BIBLIOGRAPHY

1. Hosalkar, S. H., Kulkarni,A.D., Yagnik,M.G.,Shah,H., Gujar,P. (2000) complete simpleSyndactyly of all digits with polydactyly. A rare casereport . http:// www.bhj.org/journal/4201.

2. Bell, j. (1953) On hereditary digital anomalies (Thetreasury of human inheritance . vol . v )Combridgeuniv press London .



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3. Temtamy , S.A. (1966) Genetic factors in handmalformations. Unpublished ph, D Thesis.JohnHopkins Univ.

4. Cross, E. Hollard, D. Lerberg,B. Victor, A.,McKisck(1967). Type II syndactyly. Am. J. Hum.Genet. 368-380.

5. Orel, H., K.[1928] Beitrage zurverebungsWissenschafr. Z. Gez.Anat. 14:244-252.

6. Holmes, L. B., Wolf E., Miettineer, O.V.I.S.(1972).A metacarpal 4-5 fusion with X- linked recessiveinheritance. Am.J.Gene.24:562-568.

7. Kozin, S.H.(2001) Syndactyly. Syndactyly volume1, Issue 1, pages1-13.

8. Flatt, A.(2005). “Webbed fingers” PMID 16200145.

9. Bosse, K., Betz, R.C., Lee, Y.A. et. al.(2000).Localization of a gene for type I to chromosome2q34-q36. Am. J. Hum. Genet. 67(2):492-7. doi :10.1086/303028PMID10877983.

10. Sarfarazi, A. et.al.(1995). Localisation of thesyndactyly type II (synpolydactyly) locus 2q31region identification of tight linkage to HOXD8intragenic markers. Hum. Mol. Genet. 4 : 1453. doi: 10.1093/hmg/4.8.1453.PMID7581388.

11. Sato, D., Liang, D., Wu, L. et.al.(2007). Asyndactylous type IVlocus maps to 7q36. Am. J.Hum. Genet. 52(6) : 561-4. doi : 10.1007/10038-007-150-5PMIG17476456.

12. Malik, S.,Girisha,K.M., Muhammad,W., Akhilesh,K. R., Shuba R. P., Sayednl H., Wasim A, Manuela.C and Karl-Heinz.,[2007). Synpolydactyly andHOXD13 polyalanine repeat: addition of 2 alanineresidues is without clinical consequences. MedicalGenetics. 8: 78 dvi :10, 1186/1471-2350-8-78.

13. Apert , E (1956) Delacrocephalo syndactyly lie. Bull. Soc .Med . Hop . Paris 23 :1310.

14. Akarsu, N.A. , stoilov, I., yilmaze, E., sayli, B.S.,Sarfaverzi, M. (1996) Genomic structure of

HOXD13 gene : a nine polyalanine duplicationcauses synpolydactyly in two unrelated families.Hum Mol Genetic 5: 945-952.

15. Muragaki, Y., Mundlos, S., Upton, J., Olsen,B.R.,(1996). Altered growth and branching patternin synpolydactyly caused by mutations in HOXD13. Science, 272 : 548-551.

16. Goodman, F.R., Mundlos,S., Murgaki, Y.,et.al.(1997) Synpolydactyly phenotypes correlatewith the size of expansions in HOXD 13 polyalaninetract Proc. Natl. Acad. Sci. USA 94 : 7458-7463.

17. Goodman ,F.R., Giovannucci Uzielli, M.L., Hall,C.,Winter, R.M. , scrambler, P. J. (1998) Deletions inHOXD13 segregate with an identical novel footmalformation in two unrelated families. Am J HumGenet 63: 992-1000.

18. Debeer,P., schoenmakers, E.F.P.M., Thoelen, R.,Fryns, J.P.,Van de Ven, W.J.M. (1996) physicalmapping of the +(12:22) translocation breakpointin a family with a complex type of 3/3/4synpolydactyly. Cytogenet cell Genet 81 : 229-234.

19. Debeer, P., schoenmakers ,E.F.P.M.,De Smet LanDe Ven, W.J.M., Fryns, J.P.[1998]: co-segregationof an apparently balanced reciprocal t(12:22)(11.2:q13.3) with a complex type of 3/3/4synpolydactyly associated with metacarpalmetatarsal and tarsal synostoses in three familymembers .Clin. D’ysmorph. 7 : 225-228.

20. Debeer, P., schoenmakers, E.F.P.M., Thoelen,R.,Holvoet,M., Kuittinen ,K.,. Fabry,G., Fryns,J.P.,Goodman,F.R., and van de ven,W.J.M. (2000)physical map of a 1.5 Mb region on 12p 11.2harbouring a synpolydactyly associatedchromosomal break point. European journal ofhuman genetics. 8, 561-570.

21. Qin, W., Shu, A.L., Xing, Q.H., Yang, M. S., Feng,G.Y., He, L.(2003). Genetic analysis of a Chinesepedigree with congenital synpolydactyly. 30(10):973-7.



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INTRODUCTION

The Eastern Ghats are sporadic mountainranges along the east coast of Peninsular India,running parallel to the Bay of Bengal about1750 km from Odisha through Andhra Pradeshto Tamil Nadu and also transitory in a few partsof Karnataka state. Some perennial riversoriginated from the Western Ghats and cutthrough the Eastern Ghats ranges, particularly

Bryodiversity of Eastern Ghats (India)

Afroz Alam*1, Krishna Kumar Rawat2, Praveen Kumar Verma3 and Sonu Yadav4

1 Department of Bioscience and Biotechnology, Banasthali University- 304 022 (Rajasthan), India2CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow (Uttar Pradesh), India3Forest Research Institute, New Forest, Dehradun 248006 (Uttarakhand), India4Department of Botany, University of Lucknow, Lucknow 226 007 (U.P.) IndiaCorresponding author’s email:[email protected]

ABSTRACTEastern Ghats contribute considerably to both species richness and endemicityof the India. However, the forests of Eastern Ghats are comparatively under-studied, under-explored and have received less consideration for conservationcompared to the fairly better-known Western Ghats, the hot spot ofbiodiversity. Eventually, Eastern Ghats is left with inadequate data for severalgroups of organisms. The data insufficiency is prominent, particularly in thecase of cryptogams, especially Bryophytes. In this chapter an attempt hasbeen made to fill this lacuna to some extent by providing an updated checklistof bryophytes of southern most part of Eastern Ghats, comes under TamilNadu, based on several published old and recent sporadic reports along withavailable literature on web sources.

Keywords: Bryophyta, Distribution, Diversity, Eastern Ghats, TamilNadu

Mahanadi, Godavari, Krishna and Kaveri, toend their flow into the Bay of Bengal. The rocksof Eastern Ghats are supposed Gondwanaorigin and made up of granite gneiss,charnokites, metamorphic gneiss khondalites,and quartzite. Numerous hill ranges andhillocks of Eastern Ghats locally divided intothree parts viz. northern Eastern Ghats (mostly



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in Odisha and northern Andhra Pradesh),middle Eastern Ghats (Andhra Pradesh) andsouthern Eastern Ghats (Tamil Nadu). Thevegetation of Eastern Ghats typicallyencompassed with deciduous and scrub forestsscattered with grassy open canopies. In thisarticle bryodiversity of Eastern Ghats has beenprovided.Characteristics of Eastern Ghats:

Eastern Ghats are older than Western Ghats.The elevation of Eastern Ghats is lower thanthe Western Ghats. They are located between11o 30' and 22o N latitude and 76o 50' and 86o

30 E longitude in a North-East to South-Weststrike. It covers a total area of around 75,000sq. km. Sirumalai and Karanthamalai hills ofTamil Nadu lies in the southern most part ofthe Eastern Ghats. North of Kaveri River ishigher Kollimalai, Pachaimalai, Shevaroy,Kalrayan Hills, Palamalai and mettur hills innorth Tamil Nadu. The average elevation of themountain range is about 600 m and the utmost

peak is Shevaroy Hills that reaches up to aheight of 1700 m. Eastern Ghats hold a richrange of tropical forests, including pockets ofsoggy deciduous, evergreen and semi-evergreen forests (Champion and Seth, 1968).

One of the biggest characteristics of EasternGhats lies in its being extremely fertile. In fact,the Ghat is said to be the watershed of manyrivers as the Ghat gets higher average waterfall.Due to higher rainfall, the fertile land resultsin better crops. Often referred as “Estuaries ofIndia”, Eastern Ghats gift its inhabitant thepopular profession of fisheries as its coastalarea is full of fishing opportunity. Like WesternGhats, thus, Eastern Ghats also carry a heap ofecological importance. All these distinctivenessof Eastern Ghats guarantees an exciting bio-physical environments and wealthybiodiversity.

Approximately 2600 angiosperms,gymnosperms, pter idophytes and 160cultivated plants are known from the Eastern

Figure 1: Map of Peninsular India showing expand of Eastern Ghats (graceindia.info)



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Ghats (Nayaka, 2013), which also includes over530 tree species, 1800 medicinal and 450endemic plants (Jonathan, 2006). Thebiodiversity wealth in the region can beillustrated with an example from six hillcomplexes of the southern Eastern Ghats inwhich 143 lianas and 272 tree species arereported (Muthumperumal, 2010). The forestsof Eastern Ghats are relatively under-studiedand have received less attention forconservation compared to the relatively better-known Western Ghats (Srinivasalu, 2008).Ultimately Eastern Ghats is left withinsufficient data for several groups oforganisms. The data deficiency is prominentin the case of cryptogams, especiallybryophytes and lichens (Singh and Sinha,1997).

In case of lichen, progress is notable andrecently 26 species of lichens recorded as newfor Andhra Pradesh, and 40 species were newlyadded to the lichen flora of Shevaroy Hills(Nayaka 2013). While, as far as bryophytes areconcerned, only sporadic attempts have beenmade in past (Dash et al., 2007; Dash et al.2009; Alam et al., 2012) but mainly fromOdisha part of Eastern Ghats. However,regarding bryophytes considerable andconsolidated assessment of bryophytes of allthe regions of Eastern Ghats is still desirable.

Bryophytes are one of the importantcontributors to the global plant diversity byvirtue of being pioneers of terrestrialecosystem. They represent a somewhatheterogeneous assemblage of plants, includingliverworts, hornworts, and mosses that impartlush greenery, a verdant cover, spongy bed orcarpet in every possible habitat. Irrespectiveto their small size, they encompass major

components of the biomass and photosyntheticproduction in forest ecosystems (Frego, 2007).Bryophytes are also widely used as metal-accumulator and bio-indicators of environmentfor their unique morphology and anatomicalresponses as some species are extremelysensitive to pollutants like lichens and revealvisible symptoms even in the presence of veryminute quantities of pollutants (Alam andSrivastava, 2009; Sahu et al., 2007). They haveseveral biological features, making themparticularly suited to serve as study organismin macro-evolutionary, population genetics andecological research. Bryophytes are used inmedicines, household purposes, horticulture,agriculture, fuel in industries and as ecologicalindicators throughout the world (Glime 2006;Nath and Asthana 2005). Bryophytes are thesecond largest group of plants, with about25,000 species worldwide (Buck and Goffinet2000). The plants are distributed in Eastern andWestern Himalayas, Southern India (Westernand Eastern Ghats) and Central India (Nath andAsthana 2005).

Although in India the work of bryophyte’sdiversity is on a call and most of the well knownregions have been explored such as EasternHimalayas, Western Himalayas, Nilgiri andPalni hills (South India), and Central India(Singh, V. B. 1966; Singh, D. K. 1997;Srivastava, 1998; Parihar et al., 1994; Alam,2011; Gangulee, 1969-1980). Now it is criticalto explore those areas of the country whereexploration regarding bryodiversity is lessknown such as Eastern Ghats. Work is goingon this direction, and several regions of TamilNadu come under Eastern Ghats have beenexplored recently (Alam and Srivastava 2011;Alam et al. 2011).



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2. MATERIALS AND METHOD

On the basis of few past and recent studies, asan initiative effort, the following checklist ofbryophytes of Eastern Ghats has been provided.The checklist is based on almost all availabledocumentation (Gangulee, 1969-1980; Pariharet al., 1994; Lal, 2005; Alam and Srivastava,2007; Alam, 2011; Alam and Srivastava, 2012;Verma et al., 2013).

CHECKLIST OF BRYOPHYTESBRYOPHYTA (MOSSES)A. ORDER HYPNALES (M. Fleisch.) W. R.Buck & Vitt.I. Meteoriaceae Kindb.a. Diaphanodon Renauld & Cardot.1. Diaphanodon procumbens (Müll. Hal.) Ren.& Cord.b. Meteorium (Brid.) Dozy & Molk.2. Meteorium brevirameum (Müll. Hal.) Broth.c. Aerobryidium M. Fleisch3. Aerobryidium punctlatum (Müll. Hal.) Dixon4. Aerobryidium filamentosum (Hook.) M.Fleisch.II. Entodontaceae Kindb.d. Entodon Müll. Hal.5. Entodon plicatus Müll. Hal.e. Erythrodontium Hampe6. Erythrodontium julaceum (Schwaegr.) Par.III. Pterobryaceae Kindb.f. Floribundaria M. Fleisch.7. Floribundaria floribunda (Doz. et Molk) M.Fleisch.g. Meteoriopsis Broth.8. Meteoriopsis squarrosa (Hook.) M. Fleisch.h. Symphysodon Dozy & Molk.9. Symphysodon perrottetii Mont.IV. Miyabeaceae Enroth et al.i. Homaliadelphus Dixon & P. de la Varde10. Homaliadelphus targionianus (Mitt.) Dix. &Verd.

V. Neckeraceae Schimp.j. Neckera Hedw.11. Neckera goughiana Mitt.12. Neckeropsis exserta (Scwaegr.)Broth.NeckeraceaeVI. Sematophyllaceae Broth.k. Sematophyllum Mitt.13. Sematophyllum caespitosum (Hedw.)Broth.14. Sematophyllum subhumile (Müll. Hal.) M.Fleisch.VII. Hylocomiaceae M. Fleisch.l. Thamnium Müll. Hal.15. Thamnium schmidii (Müll. Hal.) Jaeg.VIII. Thuidiaceae Schimpm. Thuidium Bruch & Schimp.16. Thuidium cymbifolium Doz. et Molk.17. Thuidium glaucinum (Mitt.) Bosh. et Lac.IX. Hypnaceae Schimp.n. Isopterygium Mitt.18. Isopterygium albescens (Hook.) Jaeg.19. Isopterygium serrulatum M. Fleisch.X. Stereophyllaceae W. R. Buck & Irelando. Entodontopsis Broth.20. Entodontopsis wightii (Mitt.) W.R. Buck& Ireland.B. ORDER BRYALES Limpr.XI. Bartramiaceae Schwägr.p. Bartramia Hedw.21. Bartramia dicranacea Müll. Hal.22. Bartramia leptodonta Wils.q. Breutelia (Bruch & Schimp.) Schimp23. Breutelia dicranacea (Müll. Hal.) Mitt.r. Philonotis Brid.24.Philonotis pseudofontana (Müll. Hal.) Jaeg.XII. Bryaceae Schwägr.s. Anomobryum Schimp.25. Anomobryum schmidii (Müll. Hal.) Jaeg.t. Brachymenium Schwägr.



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26. Brachymenium bryoides Hook. exSchwaegr.27. Brachymenium buchananii var. cuspidatumu. Bryum Hedw.28. Bryum alpinum With.XIII. Mniaceae Schwägr.v. Mielichhoferia Nees & Hornsch.29. Mielichhoferia schmidii Müll. Hal.x. Mnium Hedw.30. Mnium shynehophorum Hook.y. Pohlia Hedw.31. Pohlia flexuoasa W.J. Hook.C. ORDER HOOKERIALES M. Fleisch.XIV. Hypopterygiaceae Mitt.z. Hypopterygium Brid.32. Hypopterygium tenellum Müll. Hal.D. ORDER ORTHOTRICHALES DixonXV. Orthotrichaceae Arn.aa. Ulota D. Mohr33. Ulota schmidii (Müll. Hal.) Mitt.ab. Zygodon Hook. & Taylor34. Zygodon cylindrocarpus Müll. Hal.ac.Macromitrium Brid.35. Macromitrium nilgherrense Müll. Hal.E. ORDER GRIMMIALES M. Fleisch.XVI. Ptychomitriaceae Schimp.ad. Ptychomitrium Fürnr.36. Ptychomitrium tortula (Harrey) Jaeg.XVII. Ptychomitriaceae Schimp.ae. Ptychomitrium Fürnr.37. Ptychomitrium tortula (Harrey) Jaeg.F. ORDER DICRANALES H. Philib. ex M.Fleisch.XVIII. Ditrichaceae Limpr.af. Ditrichum Hampe38. Ditrichum heteromallum (Hedw.) Hamp.ag. Pleuridium Rahb.39. Pleuridium denticulatum (Müll. Hal.) Mitt.XIX. Fissidentaceae Schimp.ah. Fissidens Hedw.40. Fissidens ceylonensis var. ceylonensis

41.Fissidens ceylonicus Dozy et al.42. Fissidens wilsoni Mont.43. Fissidens anomalus Mont.XX. Dicranaceae Schimp.ai. Campylopodium (Müll. Hal.) Besch.44.Campylopodium nodiflorum Müll. Hal.aj.Campylopus Brid.45. Campylopus introflexus (Hedw.) Brid.XXI. Leucobryaceae Schimp.ak. Leucobryum Hampe46. Leucobryum nilgherense Müll. Hal.G. ORDER POLYTRICHALES M. Fleisch.XXII. Polytrichaceae Schwägr.al. Pogonatum P. Beauv.47. Pogonatum aloides (Hedwig.) PalisotBeauv.H. ORDER POTTIALES M. Fleisch.XXIII. Pottiaceae Schimp.am. Bryoerythrophyllum P.C. Chen48. Bryoerythrophyllum recurvistrum (Hedw.)Chenan. Hyophila Brid.49. Hyophila involuta (Hook.) Jaeg.50. Hyophila kurziana Ganguleeao. Trichostomum Bruch.51. Trichostomum orthodontum (Mitt.) Broth.I. ORDER FUNARIALES M. Fleisch.XXIV. Funariaceae Schwägr.ap. Funaria Hedw.52. Funaria hygrometrica Hedw.aq. Entosthodon Schwägr.53. Entosthodon perrottatii Müll. Hal.ar. Physcomitrium (Brid.) Brid.,54. Physcomitrium curgens Broth.

PHYLUM MARCHANTIOPHYTA(LIVERWORTS)

CLASS: JUNGERMANNIOPSIDAA. ORDER: FOSSOMBRONIALESSchljakovI. Fossombroniaceae Hazsl.



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a. Fossombronia Raddi1. F. cristula Aust.2. F. foreaui Udar et S. C. Srivast.3. F. himalayaensis Kashyap4. F. wondraczekii (Corda) Dum.II. Pallaviciniaceae Mig.b. Pallavicinia Gray5. P. crispatus (Mont.) Steph.B. ORDER: METZGERIALES ChalaudIII. Metzgeriaceae H. Klinggr.c. Metzgeria Raddi6. M. consanguinea Schiffn.7. M. himalayensis Udar et S. C. Srivast.8. M. indica Udar et S. C. Srivast.9. M. nilgiriensis Udar et S. C. Srivast.IV. Aneuraceae H. Klinggr.e. Aneura Dumort.10. A. maxima (Schiffn.) Steph.11. A. pinguis (L.) Dumort.f. Riccardia Gray12. R. levieri Schiffn.13. R. multifida (L.) Gray14. R. perssonii S. C. Srivast et Udar15. R. tenuicostata Schiffn.C. ORDER: PORELLALES SchljakovV. Porellaceae Caversg. Porella L.16. P. campylophylla (Lehm. & Lindenb.)Trevis17. P. perottetiana (Mont.) TrevisVI. Radulaceae Müll. Frib.h. Radula Dumort.18. R. kurzii Steph.19. R. meyeri Steph.20. R. tabularis Steph.VII. Frullaniaceae Lorch in G. Lindaui. Frullania Raddi21. F. acutiloba Mitt.22. F. arecae (Spreng.) Gottsche23. F. campanulata Taylor24. F. ericoides (Nees) Mont.

25. F. muscicola Steph.26. F. neurota Talyor27. F. tamarisci var. obscura (L.) Dumort.VIII. Lejeuneaceae Caversj. Cololejeunea (Spruce) Schiffn.28. C. cordiocarpa (Mont.) Steph.k. CheilolejeuneaC. imbricata (Nees) S. Hatt.l. Lejeunea Lib.29. L. aloba Sande Lac.30. L. discreta Lindenb.31. L. flava (Sw.) Nees32. L. neelgherriana Gottsche33. L. perrottetii Steph.34. L. tenirrima Lindenb.35. L. wightii Lindenb.m. Leucolejeunea A. Evans36. L. xanthocarpa (Lehm. & Lindenb.) A.Evansn. Lopholejeunea (Spruce) Schiffn.37. L. sikkimensis Steph.38. L. subfusca (Nees) Steph.n. Microlejeunea Steph.39. M. ulicina (Taylor) A. Evanso. Schiffneriolejeunea Verd.39. S. polycarpa (Nees) Gradst.IX. Lophocoleaceae Vanden Berghen inRobynsp. Lophocolea (Dumort.) Dumort.40. L. bidentata (L.) Dumort.41. L. heterophylla (Lehm.) Nees42. L. muricata (L.) Dumort.q. Heteroscyphus Schiffn.43. H. argutus (Reniw., Blume & Nees)Schiffn.44. H. orbiculatus Abha Srivast. et S.Cc.Srivast.45. H. palniensis Abha Srivast. et S. C. Srivast.X. Scapaniaceae Mig.r. Gottschelia Grolle46. G. schizopleura (Spruce) Grolle



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XI. Plagiochilaceae Müll. Frib. & Herzog inMüllers. Plagiochila (Dumort.) Dumort.47. P. elegans Mitt.48. P. indica Mitt. & Steph.49. P. nepalensis Lindenb.50. P. peradenyensis Schiffn.XII. Cephaloziaceae Mig.t. Cephalozia (Dumort.) Dumort.51. C. hamatiloba Steph.XIII. Cephaloziellaceae Douinu. Cephaloziella (Spruce) Schiffn.52. C. kiaerii (Austin) ArnellXIV. Calypogeiaceae Arnellv. Calypogeia Raddi53. C. azurea Stotler & Crotz.XV. Family Jungermanniaceae Rchb.,w. Jungermannia L.54. J. lanceolata L. emend Schrad.55. J. truncata Nees56. J. pyriflora Steph.XVI. Geocalycaceae H. Klinggr.x. Notoscyphus Mitt.57. N. darjeelingensis Udar and A. Kumar58. N. pandei Udar and A. Kumar59. N. paraoicous Schiffn.XVII. Lepidoziaceae Limpr. in Cohny. Bazzania Gray60. B. tridens (Reinw., Blume et Nees) Trevis.D. ORDER MARCHANTIALES Limpr. inCohnXVII. Cleveaceae Caversz. Athalamia Falconer61. A. pusilla (Steph.) KashyapXVIII. Aytoniaceae Caversa1. Asterella P. Beauv62. A. khasyana (Griff.) Pande et al.

a2. Mannia Opiz63. M. foreaui Udar & V. Chnadraa3. Plagiochasma Lehm. & Lindenb.64. P. cordatum Lehm. & Lindenb.65. P. rupestre (J.R. Frost. & G. Frost.) Steph.a4. Reboulia Raddi66. R. hemisphaerica (L.) RaddiXVIV. Lunulariaceae H. Klinggr.a5. Lunularia Adans.67. L. cruciata (L.) Dumort.XVV. Marchantiaceae Lindl.a6. Marchantia L.68. M. paleacea Bertol.69. M. palmata NeesXVVI. Dumortieraceae D. G. Longa7. Dumortiera Nees70. D. hirsuta (SW.) NeesXVVII. Targioniaceae Dumort.a8. Targionia L.71. T. hypophylla L.XVVIII. Ricciaceae Rchb.a9. Riccia L.72. R. fluitans L.73. R. grollei Udar

HORNWORTPHYLUM (DIVISION)ANTHOCEROTOPHYTA CLASS ANTHOCEROTOPSIDA Jancz. exStotl. & Crand.-Stotl.E. ORDER ANTHOCEROTALESLimpricht in Cohn.XVIX. Anthocerotaceae Dumort.,a10. Anthoceros L.74. A. angustus Steph.75. A. subtilis Steph.

3. RESULTS AND DISCUSSION

On the basis of above checklist, the occurrenceof 9 orders, 25 families, 43 genera and 54

species of mosses in eastern ghats can beelucidated (Fig. 2). The order Hypnales is most



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diversified with 10 families having 15 generaand 20 species followed by order Dicranaleswith 6 families (7 genera and 10 species) andorder Bryales is next to them consisting of 3families (9 genera and 11 species). The mostprominent family is Pottiaceae Schimp. (3genera and 4 species) followed byOrthotrichaceae Arn. (3 genera and 3 species).Genus Fissidens Hedw. having the maximum4 species Interestingly, the moss flora of theregion has significant similarities with theWestern Ghats (Verma et al, 2011) exhibitingmore or less similar environmental conditionsand microclimate as well as niche. The studyrevealed that like Western Ghats, Eastern Ghatsis a remarkable place for the diversity of mossesand need further exploration in future also thatwould provide some more interesting results.In case of Liverwort diversity, it is representedby 3 orders, 24 families, 36 genera and 75

species (Fig. 2). The order Porellales ismaximally diversified with 13 families. Thelargest and most diversified family isLejeuneaceae with 6 genera and 12 species.Likewise, the most diversified genus isFrullania (7 species) followed by Lejeunea (6species). The order Marchantiales isrepresented by 7 families with 10 genera and13 species. Aytoniaceae is the most diversifiedfamily and represented by 4 genera. Whileamong genera, Marchantia, Riccia andPlagiochasma are most diversified with 2species each.Hornworts are of least occurrence in this regionof Eastern Ghats and order Anthocerotales (Fig.2) is just represented by a single family with issole genus Anthoceros (2 species).



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4. CONCLUSION

Eastern Ghats, a long range of variable habitats,have received very lesser attention than the andWestern Ghats in terms of diversity study,conservation and protection planning. In thisstudy bryodiversity of the region has beencompiled. It has been reveled that habitatdestruction without any prior knowledge ofbryodiversity eliminates bryophytes earlierthan many other phanerogams in Eastern Ghatsbecause they are more sensitive toenvironmental change. Some species straightaway require protection and conservation tosave them from extermination. Eastern Ghatsis filled with lush green vegetation and highlydiversified habitats. The peculiar conditions of

these areas occur at high frequency and density,providing an excellent environment for growthof bryophytes; they may serve in in situconservation of these plants. After aconsolidated assessment of bryodiversity ofEastern Ghats, one can know the changes inthe habit, habitat, growth rate and pattern of aparticular species and their classification underthreatened plant’s categories of IUCN, and canbe used to predict the growth potential ofbryophytes during climatic change in the area.Selected rare, Endangered and threatened(RET) bryophyte species may be grown in anatural conservatory for their in situconservation.

6. REFERENCES

Alam, A. and S. C. Srivastava (2007). Liverwort diversityin Palni hills (Tamil Nadu), India, A checklist.NELUMBO (Botanical survey of India). 51:99-122.

Alam, A. and S. C. Srivastava (2009). Marchantiapaleacea Bert. As an indicator of heavy metalpollution. Indian Journal of Forestry 32(3):465-470.

Alam, A. and S. C. Srivastava (2012). Hepaticae ofNilgiri hills, Western Ghats India (India), Vol.I- Terrestrial diversity. LAP-Lambert AcademicPublishers, Germany

Alam, A., S. Vats and K. K. Behera (2012). Exormothecaceylonensis Meijer - a threatened liverwort inIndia, rediscovered in Palni Hills, Tamil Nadu,Journal of Threatened Taxa 4(5): 2593–2595

Alam, A. (2011). Diversity and altitudinal distributionof terrestrial liverworts (Hepaticae) in Nilgirihills, Tamil Nadu, India. Proceedings NationalAcademy of Sciences, India, Sect.B, 81: 206-217.

Buck, W.R. and B. Goffinet (2000). Morphology andclassification of mosses. In: Shaw, A.J., B.Goffinet (eds.). Bryophyte Biology. CambridgeUniversity Press, University of Cambridge,UK.

Champion, H.G. & S. K. Seth (1968). A Revised Surveyof the Forest Types of India. Govt. of IndiaPress, Delhi.

Dash, P. K., Mahapatra, P. K., Kar, M., Dhal, N. K. andN. C. Raut (2007). Bryoflora of Similipalbiosphere reserve with special reference to

5. ACKNOWLEDGEMENTS

The authors are grateful to Prof. Aditya Shastri,Vice Chancellor, Banasthali University,Rajasthan (India) for providing the necessaryfacilities. One of the authors (AA) is also

thankful to Ministry of Environment andForest, New Delhi for providing financialsupport during the tenure of AICOPTAX andDr. Geeta Asthana, Bryology Unit, Universityof Lucknow for her support.



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liverworts and hornworts. Similipal BiosphereReserve 2 (1&2): 32-37.

Dash, P.K., Sahu, D.K. and D.K. Saxena (2009).Bryoflora of Baphlamali hill in Eastern Ghatsof Orissa, India. EPTRI - ENVIS Newsletter15 (1): 2-6.

Frego, K.A. (2007). Bryophytes as potential indicatorsof forest integrity. Forest Ecology andManagement 242: 65–75.

Gangulee, H. C. (1969-1980). Mosses of Eastern India& Adjacent regions, Fascicles 1-8, Calcutta.

Glime, J. M. (2006). Bryophyte Ecology (PhysiologicalEcology). Vol. I. Published online by <http://www.bryoecol.mtu.edu>

Jonathan, K. H (2006). Estuaries, Mangroves, CoralReefs and Lagoons, ENVIS–SDNP Newsl.Spec. Issue, pp. 8–9.

Lal, J. (2005). A Checklist of Indian mosses. Bishen SinghMahendra Pal Singh, Dehra Dun (India), pp.1-164.

Muthumperumal, C. and Parthasarathy, N (2010). Alarge-scale inventory of lianas diversity intropical forests of south eastern ghats. Syst.Biodivers 8: 289–300.

Nath,V. andA. K. Asthana (2005). Studies on IndianBryophytes. In: Pushpangadan, P., S. Kumar& V.K. Kochhar (eds.). 50 years of NationalBotanical Research Institute. 277-288pp.

Nayaka, S., A. M. Reddy, P. Ponmurugan, A Devi andD. K. Upreti (2013). Eastern Ghats’biodiversity reserves with unexplored lichenwealth. Current Science 104 (7): 821-825.



23

INTRODUCTION

Some yeast strains, such as Rhodosporidiumsp., Rhodotorula sp. and Lipomyces sp. canaccumulate intracellular lipids as high as 70%of their biomass dry weight. The majority ofthose lipids are triacylglycerol containing long-chain fatty acids that are comparable toconventional vegetable oils.(Ratledge & Wynn,2002). More importantly, some of thoseoleaginous species show the ability to

Study of Rhodotorula glutinis growth and lipid production using lowcost substrates

Maj Neerja Masih and E.S. CharlesIncharge, Department of Biotechnology,Isabella Thoburn College, Lucknow. [email protected]

ABSTRACT

The present day challenge for biodiesel production is that as feedstock variedvegetable oils such as soybean oils, rap seed oils, palm oils and waste cookingoils are being used making it uneconomical. Microbial oil could representan alternative to produce bio-oils for biodiesel production. In the presentstudy in our lab red oleaginous yeast Rhodotorula glutinis was used forlipid production using various low cost substrates as sole carbon source.Rhodotorula glutinis culture was obtained from NCIM Pune and wassubcultured and maintained on YPD medium. The growth conditions wereoptimised and dry cell weight and lipid content were estimated using fruitpeel and sugar cane residue hydrolysate. The study can be further extendedusing different other substrates and result in cost effective high density cellculture.

Key words: biodiesel, microbial lipid, low cost substrate, Rhodotorulaglutinis.

metabolize pentoses, demonstrating thepotential to produce TAG from lignocellulosicbiomass and other cheap materials (Meesterset al., 1996, Papanikolaou et al., 2002). Recentdemand for biodiesel worldwide has turnedTAG into an ever-growing and substantialconsumption resource( Easterling et al., 2009,Angerbauer et al., 2012), The basic physiologyof lipid accumulation in microorganisms has



24

been well studied. It is known that lipidproduction requires medium with an excess ofsugars or similar components (e.g., glycerol,polysaccharides, etc.) and limited othernutrients, usually nitrogen. Thus, oleaginouspotential is critically affected by the carbon-to-nitrogen (C/N) ratio of the culture and otherfactors like aeration, inorganic salt presence,etc.The costs of microbial oil production arecurrently higher than those of vegetable oil butthere are many methods to drastically improvethe techno-economics of microbial oil

production processes. In particular, theexploration of lignocellulose-basedcarbohydrates as feedstock may greatly lowerthe costs (Pirrozi et al., 2012)In this study, wehave studies the growth of Rhodotorula glutinisand the lipid production with low costsubstrates such as whey and fruit peel juice.The studies are important due to the economicimportance of this yeast as potential strain forlipid production to be used in biodeiselproduction.

2. MATERIALS AND METHODS

2.1. Organism, media and chemicalsRhodotorula glutinis culture was obtained fromNCIM Pune and was subcultured formaintenance on YPD slants using slant streakmethod.Yeast extract (0.5%) was prepared in basalmedia of composition 0.3% (NH4 )2 SO4 ,0.1% KH2 PO4, 0.05% MgSO4 ·7H2 O and5% inoculum was added and incubated atdifferent temperatures and pH.The experiments were carried out to check thegrowth at varying pH and temperature.All otherchemicals and reagents were bought locally andwere of analytical reagent grade.2.2. Biomass yieldFive gram yeast were inoculated in 100 ml ofYPD medium and shook with 120rpm for morethan 12 hrs. Ten millilitres of cultured yeastwas transferred in to a test tube and centrifugedwith 4500 rpm for 20 minutes. Supernatant wasremoved and the pellet was collected andinoculated into 5 % (w/v), 10 % (w/v) and 15% (w/v) in the above mentioned basal media.Yeast growth was measured every two hrs for14 hrs at 540 nm wave length. Based on the

data the microbial growth curves were plottedfor each strain.2.3. Analytical method used for measuringand analyzing yeast growthYeast growth kineticsFive ml of cultured medium sample was takenin every 12 hours time interval until the36solution optical density was measured at 540nm wave length. A blank solution was used forreference reading. The results were recordedand used in plotting growth curve of the yeastcells.Biomass estimationAt the end of the yeast growth, the biomasswas harvested centrifuging the culture mediumat 4500 rpm for 20 minutes. Duringcentrifuging the supernatant and pellet wasseparated. The remaining pellet was dilutedwith 5 ml distilled water and its optical densitywas measured at 540 nm.Dry weight determinationThe wet cells were collected by centrifugationand washed with the same volume of distilledwater. Cell dry weight was obtained from wet



25

cells from a 20 ml culture broth after beingdried at 105 æ% C overnight.Lipid extractionThe lipid was extracted by the method of Soxletmethod, 1995. The lipid content was expressedby the percentage of lipid in the DCW.

These data suggested that a wide range ofsubstrate concentrations were suitable forgrowth of this strain with no significantsubstrate inhibition. However, drastic growthrate loss was found for the culture when glucoseconcentrations were higher than 200 g/ l.3. 2. Effect of different pHTo determine the effect of pH on growth profileof R. glutinis batch experiments wereperformed in conical flasks with pH rangingfrom 5 to 8. Fig 2 shows the plots of cell cultureoptical density against fermentation time.These results indicated that the optimum pHfor this strain was 6 with no growth at pH 8.Correspondingly the lipid content was alsofound to increase.

Table 1.Time Optical Density

10g/l 50g/l 100g/l 200g/l 400g/l0 0 0 0 0 02 0.1 0.1 0.1 0.05 0.054 0.15 0.2 0.15 0.1 0.066 0.2 0.3 0.2 0.12 0.068 0.25 0.4 0.25 0.15 0.0610 0.3 0.5 0.3 0.16 0.0612 0.35 0.6 0.35 0.18 0.06Effect of initial glucose concentration on growth of Rhodotorula glutinis.

Table 2

Carbon Source Glucose Pomegranate peel Sugar cane dustDry cell weight 13.4 g/l 7.2 g/l 10.2 g/lLipid content 1.2 g/l 0.6 g/l 9.8 g/lDry cell weight and Lipid Content (12 hrs) using different substrate as sole carbon source.

Treatment of fruit peel

The fruit peels were dried, ground andhydrolysed as per Wang el al.

3. RESULTS3.1. Effect of different SubstrateconcentrationTo determine the effect of substrateconcentration on growth profile of R. glutinisbatch experiments were performed in conicalflasks with glucose concentration ranging from10 to 400 g/ l. Table 1 shows the plots of cellculture optical density against fermentationtime. These results indicated that the yeast grewwell on glucose as a sole source of carbon andenergy at a concentration up to 150 g /l. Whenthe glucose concentration reached 200 g/ l, cellgrowth was greatly repressed and more severeinhibitory effects were observed at even higherglucose concentrations.



26

3.3. Effect of different SubstratesThe growth pattern was also observed usingfruit peel of pomegranate and sugar caneresidue after juice extraction as substrate. Itwas observed that the yeast was able to show

hydrolysate for the biosynthesis of oils and thecell growth. The present study can economisethe production of microbial lipid which is beingconsidered as an important raw material for bio-diesel production. The soxlet method has itsdisadvantage that only extractable lipids couldbe estimated but nevertheless it gave a generalidea of the relationship between the biomassand lipid content(Li et al.(2007), Zhao etal.(2005)).This study can be extended to furtherstudy the use of other lingo-cellulosic waste inachieving a high density cell culture of thiseconomically important yeast.

ACKNOWLEDGEMENTS:

Author’s are grateful to the UGC for the Minor Research Project sanctioned to Dr. E S Charles.

Pirozzi, D., Yousuf, A., Zuccaro, G., Aruta, R., andSannino, F. (2012). “Synthesis of biodieselfrom hydrolyzates of Arundo Donax,” Environ.Eng. Manag. J. 11(10), 1797-1801.

Ratledge C, Wynn JP. The biochemistry and molecularbiology of lipid accumulation in oleaginousmicroorganisms. Adv Appl Microbiol2002;51:1–51

Soxhlet method (AOAC, 1995)Ruling Wang,Jiancai Wang,

Ronghua Xu,

Zhen Fang,

and Aizhong Liu. Oil Production by theOleaginous Yeast Lipomyces starkeyi usingDiverse Carbon Source, (2014) BioResources9(4), 7027-7040.

Y. Li , Zongbao Zhao , Fengwu Bai, Enzyme andMicrobial Technology 41 (2007) 312–317

Zhao ZB. Toward cheaper microbial oil for biodiesel.China Biotechnol 2005;25(2):8–11.

growth in the absence of glucose when thesesubstrates were used. The growth wasmaximum for sugar cane residue and was lessfor pomegranate under all other similarconditions.

4. DISCUSSION

Since Rhodotorula glutinis is one of the yeaststrains that can accumulate intracellular lipidsas high as 70% of their biomass dry weight, inan effort to achieve a high density cell culture,this study was carried out. The fruit peel ofpomegranate and sugar cane which is a wasteproduct of fruit juice industry were found tohave potential to act as low cost substrates forbiomass production of this yeast. Wang et al.,has found similar results with the oleaginousyeast Lipomyces starkeyi utilizing diversecarbon sources including glucose, xylose,glycerol, and willow wood sawdust (WWS)

REFERENCES:

Angerbauer, C., Siebenhofer, M., Mittelbach, M., andGuebitz, G. M. (2008). “Conversion of sewagesludge into lipids by Lipomyces starkeyi forbiodiesel production,” Bioresour. Technol.99(8), 3051-3056.

Easterling, E.R., French, W.T., Hernandez, R., and Licha,M. (2009). “The effect of glycerol as a soleand secondary substrate on the growth and fattyacid composition of Rhodotorula glutinis,”Bioresour. Technol. 100(1), 356-361.

Meesters PAEP, Huijberts GNM, Eggink G. High-cell-density cultivation of the lipid accumulatingyeast Cryptococcus curvatus using glycerol asa carbon source. Appl Microbiol Biotechnol1996;45:575–9.

Papanikolaou S, Chevalot I, Komaitis M, Marc I. Singlecell oil production by Yarrowia lipolyticagrowing on an industrial derivative of animalfat in batch cultures. Appl MicrobiolBiotechnol 2002;58:308–12.



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INTRODUCTION

District Faizabad, the erstwhile capital ofAwadh, lies at 26 47’N and 82 08’E, its heightabove the mean sea level is 113 meter. Faizabadis a part of the eastern Uttar Pradesh, border ofthe district is bounded by Gonda, Basti,Ambedkar Nagar, Sultanpur and Barabanki.River Ghaghra is the main wet source of thedistrict and contributes as important tributariesof the river Ganga which enters the Faizabad

Conservation Needs of Freshwater Fin-Fish Genetic Resources

P N Tripathi, Ved Prakash Tripathi and Swapnil Raj DubeyZoology Department, K S Saket P G College, Ayodhya, Faizabad-224123E-mail: [email protected]

ABSTRACT

The present communication deals with the general picture of fishand fisheries status of river Ghaghra and other wetlands of districtFaizabad and adjoining regions and the status of fish production fromriver stock, and also with the problems of fish farmers that they faceduring fish culture on their fish farm. During observation it is noticedthat fish production from river is continuously decreasing due tooverfishing and lack of well planned programme and a continuousdecrease in fish production causing loss of biodiversity. During presentinvestigation some other reasons are also discussed which areresponsible for decline of production.

Key words: Rohu, Bhakur, Nain, Ghaghra, , Quantitative estimation

district near Badagaon. The river Ghaghraflows from west to east and covers the entirelength of district. It is one of the most importantrivers for the capture fishery of the district. Thetwo other rivers namely Madha and Bisuee arealso flowing through Faizabad join at the borderof the district and make one river known asTons (Tamsa) in Ambedkar Nagar. TheGhaghra river flows on the northern border and



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its main tributaries are Tirwa, Pikai and Torari.In addition to Ghaghra, Madha, Bisue river andtheir tributaries lakes, ponds and wetlandsspreads through the district. There are somereports on the biodiversity of different part ofUttar Pradesh by Menon (1949), Sinha andShiromany (1953), Chaudhary and Khandelwal(1960) Singh (1964) Srivastava (1967), Pandey(1977). To fill up this lacuna and to present a

general picture of the existing fish and fisheriesof the river and cultured water bodies of thedistrict, an intensive survey was carried outfrom local market as well as some collectingcenter of the district. The present work is theresult of two year field observation wherepresent status and conservation needs arediscussed.

MATERIAL AND METHODS

The climatic condition of this region is markedby mild cold during winters and intensive heatduring summers. The monsoon season is Julyto September. The rainfall fluctuated year toyear. Average rainfall is about 1200 mm. Thecollection of fish was made regularly fromdifferent localities at Ghaghra, Madha, Bisueerivers and from culture ponds for speciescomposition study. Collection of fishes weremade with the assistance of local fishermenoperating cast net, drag net, mahajal, gill net,hooks and line and fry collection nets. Few

fishes were purchased from the local fishmarket for collection. For fisheries study theinformation were collected in the field duringsurvey from district fisheries department andAgricultural University. To study the fish production of variousfood fishes from river Ghaghra, regular surveywas done on local fish market Niyanwa duringyear 2008 and other information about lastyear’s from1980 to 2005 is based on theinformation of local fishermen and RajkumarManjhi was chief resource to collect these data.

OBSERVATION

The fishing activities last duringMarch to June. The fresh water fisheriesresources of Faizabad district consist of cultureand capture fishery. In culture system mainlycarp fishes are found in culture ponds.

The listed species are commerciallyimportant. All the major indigenous carp andcommon carp (Cyprinus carpio) are foundthroughout the water and few cat fishes andEels are found in the river water. The highlyspecialized gear and crafts are not used forfishing; angling, trapping and netting are usedin ponds and reservoir. The fishermen orcontractor wants to catch maximum fish havingthe least or no consideration for catching the

undersized fish, and uses dragnets and gill netsof various size and code number. Flat bottomedwooden boats of different dimensions are usedfor fishing. Sometimes iron sheet flat bottomedboats and dugout wooden cannons are also usedbut all these crafts are non-mechanized.

During market survey of someimportant food fishes in the year (2008) it isnoticed that the highest production of thecommon carp fishes yield was during May i.e.300, 200, 240 kg per day for Rohu, Bhakur,Nain respectively and production of Mangur,Padhin, Tengara is also highest during May,which is 150, 50, 50 kg per day respectivelyand for common, silver and grass carp it is 50,



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TABLE-1Average production of common carp during 2008 (Kg/day)

Rohu Bhakur NainJanuary 150 75 50February 150 75 50March 250 100 100April 250 80 150May 300 240 200June 200 200 200July 100 70 60August 100 70 60September 100 70 65October 150 72 63November 150 80 70December 150 80 70



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TABLE-2Average production of exotic carp during 2008 (Kg/day)

Common Carp Grass carp Silver carpJanuary 40 100 80February 40 100 80March 50 250 150April 60 250 150May 50 300 200June 50 200 200July 40 100 60August 40 100 60September 45 100 60October 50 125 70November 50 125 70December 50 140 75



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TABLE-3Average production of some fishes during 2008 (Kg/day)

Mangur Padhin TengaraJanuary 120 40 40February 130 42 40March 130 50 48April 150 60 50May 150 50 50June 100 50 50July 120 40 45August 100 40 40September 125 45 40October 130 50 42November 135 50 40December 132 45 40



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TABLE-4Average production of some fishes during different year (Quintal/month)

Year Rohu Bhakur Nain Mangur Padhin Tengara1980 400 280 30 10 30 301985 340 230 30 15 30 301900 300 200 25 20 30 241995 150 100 25 24 25 202000 90 60 25 28 24 202005 70 40 25 30 21 212008 52 35 20 30 20 20



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200, 300 kg per day respectively. The averageproduction of these fishes during variousmonths of year 2008 is shown in Table-2, 3, 4.

According to Rajkumar Manjhi (a 55year old, traditional fisherman) the productionof the carp fishes from river Ghaghra is 52, 35,25 quintal per month at this time instead of 400,

280, 30 quintal per month for Rohu, Bhakur,Nain respectively. These observations indicatea marked decline in the availability of the fishesover the year. Some fishes are endangered ornear to threatened, the conservation strategiesneed to be developed to protect these fishesbefore it is too late.

DISCUSSION

India is very rich in fishery resources. The aimof fish culture is mainly the production of fishfor human food. Culture of Rohu, Bhakur andNain with Grass carp, Common carp and Silvercarp is popular in this region. Though Pandey(1977) had suggested the culture of smaller livefishes like Channa striatus, A. testudineus andH. fossilis but not much success has beenachieved in this direction. But facing theincreased demand of fish there is an urgent needto explore the possibilities to adapt othercultivable species in the aquaculture system.Now a days when there is excessive pressureon scientist to introduce new species in list ofcultivable fish species. We also favor andrecommend mono or mixed culture of fewnutritive and economic important fishes.Fish biologist always try to introduce exoticspecies into different environments. Now adays it has become easier to move a speciesfrom its natural habitats to anotherenvironment. But a study by an internationalteam (University Paul Sabatier of Toulse, IRDin Paris, CNRS) showed that the humaninfluence involved establishing a non nativespecies into new environment had not beenfully recognized (Leprier et. al 2008), theyinvestigated 1,000 rivers with more than 10.000fresh water fish species and reported thepopulation was largely down to humaninterference. It shows that a particular

environments are not always adapted forhosting new arrivals, they tested threehypothesis- “ the biotic resistance”, “the bioticacceptance” and human activity and suggestthat high diversity of fresh water fish in thehost ecosystem acts as barrier to establishmentof non-native species population and for givenecosystem non-native diversity follows that anative species because favorable ecologicalconditions for the latter are also suitable forthe newly arrived species. Thus the studyshould be useful in introducing the exoticspecies in other environments. Some fishes viz.Mystus vittatus is very important fish with highnutritive and ornamental value, by introducingit in aquaculture we provide an opportunity tofish former because it fetches higher price inurban and rural market. Quality fish seed is abasic input for the success of the fish cultureprogramme Sultan (2004). There are only oneGovernment fish seed hatchery (Saryuhatchery) situated at Faizabad- Sultanpur roadanother is situated at Faizabad-Lucknow roadnamely Anas fish seed center. Some vendorscome from other state to provide Indian andexotic carp’s seed, some seeds are collected byfishermen from the rivers. These entire centersproduce the seeds of carps because of greatdemand of such seed by formers, but thesecenters unable to provide sufficient quality andquantity of fish seed in comparison to need of



34

the area and due to lack of good quality of seedthe fish farmer faces different kinds of problemin their farm. They receive 600-700 seeds onpacking of on thousand seed from Governmentseed centre. So they spend more money for seed.

During survey various reasons arenoticed for low production. Local fishermentold that Ghaghra become a great source offisheries in 1960-70. Their parents captured 7-8 quintals fishes per day, but 7-8 quintal fish isthe capacity of market at present time. Somefishes came into the boat without netting onlyby striking the bamboo in river water; this thingexplains how many fishes were found in theriver. There are two main reasons to decreasethe quantity of fishes in Ghaghra, first theconstruction of Bandha on the river, the fishesfalls down through the flow of water and do notcome back to this region and the second reasonis outside fishermen, they continuously do thefishing of all size fishes so the fishes die beforethe development. Due to above reason thepopulation of fishes continuously decreases inthis region. The fishermen tell that the fisheshave decreased 1/50 times during last 25 years.

The water bodies are shrinking veryfast, the average atmospheric temperaturereaching up to 45 0c in this region, thus lakesand ponds dry-up by March-April every year,with a result the aquatic fauna get killed or fallprey to carnivores animals and human beingsas it can easily be removed using small sizednets. The natural habitat of the fishes are underthreat, so there is a need to protect the waterbodies and to conserve the fishery resources.The Government ponds are in critical stage,its rent increases every year and area decreasesevery day. During observation it seems that thenumber of ponds is decreasing and ponds areconverting into meaningless pits. Poisoning andfishing by the villagers is regular problem for

fish farmers. Some fishermen take away all thefishes from ponds in few hours during winternight. Moderate and heavy fishing havedetectable effect on the stock. In case of overfishing, the fishing pressure is too great and itmay result harmful effects. Heavy fishingreduces greatly the relative abundance of theadult individuals in the stock which destroysthe reproductive capacity of the stock whichleads to considerable decline of thecommercially more desirable species andcauses increase in turn of commercially lessdesirable species.

Limning, manuring and fertilization ofponds are not done by the scientific methods.Stocking of fingerling of carp is done @ 6000-10000/ha/Year or more in comparison to@5000-6000/ha/year. The fish formers have noidea about modern technique and they try oldmethods, which directly affect the fishproduction, so there is a need to establish somefish culture help centre in these areas whichprovide necessary information to fish farmerto increase the fish production. During present investigation it seemsthat few years ago Ghaghra was a great sourceof fishes in this region, but continuous fishingand lack of well planed programme to conservethe fish resources a continuous decrease in thefish production is noticed. At present time theculture fishery is great source of fishes butunable to fulfill the requirement and fish farmerfaces different kinds of problem duringcultivation. Loss of fish biodiversity directlyaffected the food resources of human beings andsocio economic condition of fishermen and fishfarmer. The climatic changes and condition ofwet sources also play major role, so to save thefish biodiversity there is urgent need to developsome Government programme.



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ACKNOWLEDGEMENTS

The author is thankful to Sri Raj Kumar Majhifor providing market data of fishes, and also toDr Rajesh Dayal and Dr PP Srivastava (bothfrom NBFGR, Lucknow) for going through the

Manuscript. The financial assistance receivedfrom U G C, New Delhi F. No. - 38-99/2009(SR) is thankfully acknowledged.

REFERENCES

Chaudhary, H.S. and O.P. Khandelwal 1960, fish surveyof Nanital district, Vigyan ParishadAnusandhan Patrika, 3:139-145.

Jayaram, K.C. 1981, the fresh water fishes of India,Pakistan, Bangladesh, Burma and Sri-lanka-aHandbook, published by Director, Zoologicalsurvey of India.

Leprieur. F. , Olivier Beauchard, Simon Blanchet, ThierryObrdorff and Sebatien Brosse 2008, FishInvasions in the world’s River system: whennatural process are blurred by human activity.Plos Biology Vol.6 No.2e. 28.

Menon, A. G. K. 1949. Fishes of Kumaon Himalaya. J.Bombay Nat. Hist. Soc, 43:44-45.

Sinha, B. M. and P. P. Shiromany 1964. The fishes ofDoon Valley, Icthyologica, 3:85-92.

Srivatava, G. J. 1967. Fishes of Eastern Uttar PradehVishwavidalaya Prakashan, varansi, pp.163.

Sultan, S., 2004. Impact of the development of fishculture through FFDA in Banda district of UttarPradesh, Fishing Chimes, 24(7): 22-25.



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ABSTRACT

The feasibility and efficacy of relaxation techniques with non psychoticindividuals, there is still paucity of researches on the effects ofrelaxation training applied on schizophrenic patients. Currenttheoretical speculation and research evidences suggest that elevatedanxiety level, stress & tension and poor anxiety coping skills may beimportant aspects of the schizophrenic process. This study evaluatedthe effectiveness of progressive muscular relaxation in the treatmentof persons with schizophrenia who experienced significant anxietyand stress with residual symptoms despite their adherence tomedication. 05 medicated schizophrenic female patients were takenfor treatment. After twenty two weeks of training, subjects reportedlower level of anxiety and stress.

Progressive Muscular Relaxation in Schizophrenic Patients :A Pilot Study

Poonam Singh*,Seema Rani Sarraf** Pranay Kumar Tripathi*** and Chandini Gupta* Guest Lecturer, Department of Applied and Clinical Psychology, M.J.P. Rohilkhand Uni., Bareilly, U.P., India.**Guest faculty, Department of Psychology, M.G. kashi Vidyapith, Varanasi (U.P.)***Sr. Lecturer, Department of Psychology,K.S. Saket P.G. College, Ayodhya, Faizabad (U.P.)**** Student, Department of Applied and Clinical Psychology, M.J.P. Rohilkhand Uni., Bareilly, U.P., India.

INTRODUCTION

Progressive relaxation therapy is a form ofrelaxation therapy which focuses for its effectsupon successive tensing and relaxing of eachof the 15 major muscle groups. It wasdeveloped by American physician Edmund

Jacobson in the early 1920s (Jacobson, 1938).Jacobson argued that since muscle tensionaccompanies anxiety, one can reduce anxietyby learning how to relax the muscular tension.PMR entails a physical and mental component.



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The physical component involves thetensing and relaxing of muscle groups over thelegs, abdomen, chest, arms and face. With theeyes closed and in a sequential pattern, atension in a given muscle group is purposefullydone for approximately 10 seconds and thenreleased for 20 seconds before continuing withthe next muscle group.

The mental component focuses on thedifference between the feelings of the tensionand relaxation. Because the eyes are closed,one is forced to concentrate on the sensationof tension and relaxation. The patient is told tosimply focus on the feelings of the tensedmuscle. Because of the feelings of warmth andheaviness are felt in the relaxed muscle after itis tensed, a mental relaxation is felt as a result.With practice, the patient learns how toeffectively relax and deter anxiety when itbecomes at an unhealthy level where an anxietyattack would otherwise occur (Craske &Barlow, 2006). Jacobson trained his patientsto voluntarily relax certain muscles in theirbody in order to reduce anxiety symptoms. Healso found that the relaxation procedure iseffective against ulcers, insomnia, andhypertension. There are many parallels withautogenic training, which was developedindependently. The technique has also proveneffective in reducing acute anxiety in peoplewith Schizophrenia (Chen et al. 2009).

Relaxation method: - Progressive relaxationinvolves alternately tensing and relaxing themuscles (Wolpe &Lazarus, A.A.1966). Aperson using PMR may start by sitting or lyingdown in a comfortable position. With the eyesclosed, the muscles are tensed (10 seconds) andrelaxed (20 seconds) sequentially throughvarious parts of the body. The whole PMRsession takes approximately 30 minutes. As thisis a technique, practice with PMR does make

perfect and will usually not work effectivelyas it should the first couple of times.

Schizophrenia: - Schizophrenia is a form ofpsychosis that shifts sufferers from reality toan often terrifying world of delusions,confusion, and hallucinations. Often thesymptoms of schizophrenia are described as“positive” or “negative.” The prevalence ofschizophrenia is approximately 0.85 percent ofthe population worldwide and is fa irlyconsistent across race and geographicalregions. Men and women are equally affected.Average age of onset in men is 15 to 25 yearsof age, while in women it is 25 to 35 years ofage. The symptoms are organized into threemajor categories: positive symptoms, negativesymptoms, and cognitive impairment.

Posit ive symptoms includehallucinations, delusions, thought disorders,and bizarre behaviors. Hallucinations are mostcommonly auditory, usually experienced asvoices talking to or about the person. Delusionsare false beliefs and tend to be paranoid,grandiose, or bizarre in nature. Disorganizedspeech is presumed to be a manifestation of anunderlying thought disorder. The flow of ideasis illogical and may range from being mildlyconfusing to incomprehensible. Words may bestrung together based on sound rather thanmeaning, or entirely new words may be created.Bizarre behavior may be observed as repetitivemovements, unusual mannerisms, odd ways ofdressing, and disregard for social norms.

Negative symptoms include flat affect(facial expression), avolition, and apathy. A flataffect is one revealing little emotion orexpression. Generally, persons withschizophrenia seem emotionally disconnectedand tend to be socially withdrawn. Avolitionand apathy are characterized by a lack ofmotivation and poor grooming and hygiene.



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In addition to the positive and negativesymptoms of schizophrenia , cognitiveimpairment with deficits in attention span,memory, and information processing is oftenpresent. Persons with schizophrenia experiencevarying constellations and severities ofsymptoms resulting in a range of impairedfunctioning. Sometimes anxiety plays asignificant role in symptoms of schizophrenia.Cutler and Siris (1991) observed in a sampleof 45 patients diagnosed with schizophrenia,in which 11 met criteria for panic disorder.Cassano etal. (1998) reported that co-morbidityof anxiety symptoms were related to insight inpatients with schizophrenia. Huppert and Smith(2005) found anxiety disorders are common inschizophrenic patients and the presence of these

symptoms may influence the core psychoticsymptoms and quality of life.

Progressive Muscular Relaxation hasbeen widely used and its effectivenessestablished with numerous patient populations.However there has been limited application ofrelaxation techniques in the treatment ofschizophrenic patients and little researchregarding its efficacy. This neglect may be dueto the dominance of pharmacological treatmentand less use of psychotherapy, the severity ofthe disorder, or the inadequacies of previousattempts to understand and manageschizophrenia. The objective of this study wasto examine the efficacy of progressiverelaxation training on anxiety in patients withschizophrenia.

METHOD

ParticipantsThe study was comprised of the patientssuffering from Schizophrenia. The sampleconsisted of 05 female schizophrenic inpatientsof age range 35- 50 years. Patients wererecruited from the female inpatient ward ofMental hospital, Bareilly. Diagnoses weremade according to DSM-IV-TR criteria withcase study format. Inclusion criteria were oneof the previously cited diagnoses and thepresence of anxiety and psychotic symptoms,duration of illness was greater than 10 yearsand patients were hospitalized and onpharmacological treatment for last 5 years.There is absence of positive symptoms andpresence of restlessness, tension and free-floating anxiety along with negative symptomsof schizophrenia. Exclusion criteria wereorganic illness involving the central nervous

system, current substance abuse and /or pastand current alcohol dependence, and clinicalevidence of mental retardation.MeasuresThe test instruments that were selected for thepresent study were Brief Psychiatric RatingScale (BPRS) and Global Assessment ofFunctioning (GAF) Scale. The BPRSdeveloped by Overall and Gorham (1962), iswidely used, relatively brief scale that measurespsychotic and non psychotic symptoms inindividual with a major psychiatric disorder.GAF has been developed by the AmericanPsychiatric Association DSM-IV-TR (2000).GAF has 10 items and BPRS has 18 items ofassessment. It has taken approximately onehour to conduct clinical interview and theassessment on the scales. The effectiveness ofthe treatment techniques will be assessed on



40

the basis of the difference between Pre and Posttreatment assessment scores on GAF andBPRS.ProcedurePatients were approached to participate in thestudy and only those who volunteered and gaveinformed consent were included in the study.Baseline observations were made and any typeof psychotherapy was not provided during thisduration. Subjects were observed, interviewed,diagnosed and rated on the measures used inthe study. A warm empathetic and genuinetherapeutic relationship was established with

the patients. Patients of the present study havereceived training in PMR. The treatmentcontinued for 22 weeks, and sessions werethrice a week on alternate days initially andreduced to twice a week from 15 week. Eachsession was of around 45 minutes. At thetermination of therapy subjects were assessedon BPRS and GAF scales. They were guidedto continue the relaxation practice and indulgein social interactions. The patients werefollowed up for 2 weeks to monitor theeffectiveness of the treatment.

RESULT AND DISCUSSION

Patients were observed twice a week for aninitial 2 week period in their ward. The baselineperiod was followed by Progressive MuscularRelaxation training. The BPRS scores ofanxiety and tension during baseline,intervention and follow-up are presented in thetable 1 & 2.A perusal of table 1 and 2 reveals that all caseswere showing extremely severe to moderatelysevere anxiety and tension in baselineobservation. There was no difference in theiranxiety and tension during initially phase ofintervention up to 6 weeks. Gradually patientsshow improvement from week 15 and there ismuch decrease of anxiety and tension by 21st

week.The BPRS and GAF scores of the patients afterthe treatment and follow-up of two weeks havebeen presented in table 3Table 3 reflected that patients have shownobservable improvement on BPRS and GAF

Scores at the termination of the treatment andduring follow-up. In the present study areduction of anxiety and tension scores onBPRS and an increase in GAF rating was seen.The heightened motor activation andrestlessness of the patients were reduced. Therewas observable decrease in anxiety scoresindicate decreasing over concern, fear andworry of the patients. GAF Scores of thepatients were better after treatment.

The result of present study is consistent withChu et al (2009) finding that the degree ofanxiety improvement was significantly higherin the progressive muscular training group thanin the control group of acute schizophrenicpatients.It can be concluded that progressive musclerelaxation training can effectively alleviateanxiety in patients with schizophrenia.



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Table 1 BPRS scores of Baseline, Intervention and Follow-up of Schizophrenic Patients on Anxiety

CASES CASE 1 CASE2 CASE3 CASE4 CASE5PROCEDUREDURATIONBaseline Week 1 6 5 6 7 5

Week 2 6 5 7 6 6Intervention Week 3 6.0 5.3 6.0 7.0 5.7

Week 6 5.7 5.0 5.3 6.3 5.7Week 9 5.7 4.7 5.7 6.0 5.3Week 12 4.7 3.7 5.0 5.7 4.7Week 15 4.3 3.3 4.3 4.7 4.0Week 18 3.7 3.0 2.7 4.7 3.3Week 21 2.3 2.0 2.3 3.3 2.0

Follow-up After 1 week 3 2 2 3 2After 2 week 3 2 2 3 3

Table 2 BPRS Scores of Baseline, Intervention and Follow-up of Schizophrenic Patients on Tension

CASES CASE 1 CASE2 CASE3 CASE4 CASE5PROCEDUREDURATIONBaseline Week 1 7 5 5 7 6

Week 2 6 6 6 7 6Intervention Week 3 6.0 6.0 6.0 7.0 6.0

Week 6 5.7 5.7 5.3 6.7 5.7Week 9 5.3 5.7 5.3 6.0 5.3Week 12 5.3 5.0 5.0 5.7 5.3Week 15 5.0 4.7 4.3 5.0 4.7Week 18 4.7 4.0 3.3 4.3 4.0Week 21 3.0 3.7 3.3 3.7 3.3

Follow-up After 1 week 3 4 3 4 3After 2 week 3 3 2 4 4

Table 3 BPRS and GAF scores of Schizophrenic Patients

Sl. CASES BPRS SCORES GAF SCORESNo ANXIETY TENSION

Pre- Post Follow- Pre Post Follow- Pre PostTherapy Therapy up Therapy Therapy up Therapy Therapy

W1 W2 W1 W21 Case 1 6 2.3 3 3 6 3.0 3 3 42 522 Case 2 5 2.0 2 2 6 3.7 4 3 38 483 Case 3 7 2.3 2 2 6 3.3 3 2 28 384 Case 4 6 3.3 3 3 7 3.7 4 4 32 455 Case 5 6 2.0 2 3 6 3.3 3 4 38 52



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REFERENCESCassano, G.B., Pini, S., Saettoni, M., Rucci, P., and

Dell’Osso, L. (1998). Occurrence and clinicalcorrelates of psychiatric comorbidity in patientswith psychotic disorders. J. Clin. Psychiatry.59, 60-68.

Chen WC; Chu H; Lu RB; Chou YH; Chen CH; ChangYC; O’Brien AP; Chou KR. (2009), “Efficacyof progressive muscle relaxation training inreducing anxiety in patients with acuteschizophrenia.”, Journal of Clinical Nursing18 (15): 2187–96, doi:10.1111/j.1365-2702.2008.02773.x, PMID 19583651.

Craske & Barlow (2006), Worry, Oxford UniversityPress, Inc., p. 53, ISBN 0-19-530001-7

Cutler, J.L. and Siris, S.G. (1991). Panic likesymptomatology in Schizophrenic and

Schizoaffective patients with post psychoticdepression: observation and implications.Compr. Psychiatry. 32, 465-473.

Jacobson, E. (1938). Progressive relaxation. Chicago:University of Chicago Press

Huppert, J. D. and Smith, T. E. (2005). Anxiety andSchizophrenia: The interaction of subtypes ofAnxiety and Psychotic symptoms. CNS Spectr.10 (09), 721-731.

Wolpe, J. & Lazarus, A.A. (1966). Behavior therapytechnique. New York: Pergamon Press.



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INTRODUCTION

Growth reflects the adaptive property of thespecies. It is the change in size over time. Thisis one of the most intensively studied aspectsof fishery biology. Study of age composition,rate of growth per year is essential inunderstanding the dynamics of fish populationand for prediction of fishery stocks. The annualvariation in the fishery of aquatic bodies

Age and Growth Related Investigations on Major Carps in the RiverineEnvironment of River Ghaghra at and Around Faizabad

Ramendra Kumar Dwivedi**, Ved Prakash Tripathi, Nagendra Pratap Singh And P.N. Tripathi*Zoology Department, K S Saket PG College, Ayodhya,(Dr Ram Manohar Lohia Awadh University), Faizabad-224001E-mail: **[email protected], * [email protected]

ABSTRACT

A considerable population of India is suffering from malnutrition. Thisproblem can be solved up to some extent when we add fishes in our food.Fishes are good source of protein and vitamins. To ensure the availability offishes to masses we have to improve the production in our available waterresources. Rivers in India are a good source of freshwater fish production.Ghaghra is an important river of North India. It has sufficient water aroundthe year. The present investigation was carried out to observe the comparativegrowth of Cirrhinus mrigala,Labeo rohita,Labeo calbasu and Catla catlain riverine environment of Ghaghra.The back-calculation method wasadopted to assess the age and growth of these fishes. It was observed thatthe growth rate of Labeo calbasu is maximum among above four fishes.

Key words: Cirrhinus mrigala, Catla catla,Labeo rohita,Labeo calbasu,Majorcarps, Ghaghra, Ayodhya, age and growth

depends upon the growth pattern of fishes.Fishes bear on their scales permanent marks inthe form of annulus rings. These rings areformed every year. These marks are faithfulevidence not only of the age but of the growthas well at the end of each year of the life. Thisfact forms the basis of estimating year to yearchanges in growth and also of back-calculating



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body length. A direct relationship thus existsbetween the annuli and the growth pattern ofthe individual. Body lengths may bedetermined, assuming certain relationship toexist between the scale size and the body. Itmust be mentioned that growth pattern andgrowth rate are highly species-specific. Thusscale is best way for calculating the growth offishes.

Ghaghra is an important river of NorthIndia. It has sufficient water around the year.

Major carps are most edible fishes of NorthIndia and mostly cultured in small water bodies.Study of major carps in riverine environmentis necessary for exploitation of fresh waterresources. As per available literature though thestudy on age and growth drew attention fisherybiologist in other parts of the country but thisregion has remained ignored. Therefore, to fillgap in our knowledge the present investigationwas carried out.

MATERIALS AND METHODS

We studied fishes in river Ghaghra andcollected the key scales of selected fishes fromselected places and washed in water with tipof finger and brush. After that it was dried withthe help of Blotting paper and made 50 slidesof fishes in different age group by using abovemethod. Photo of these slides were taken fromNBFGR which is given. With the help ofmagnifying glass, Scale and Calculator, wederived the Total scale radius and distance offirst, second, third and so on annulus from focusthis is showed in observation as L1, L2 L3 etc.The L1 shows distance between focus and firstannual ring, L2 shows distance between focusand second annual ring and so on.By using formula, which is given below, wecalculated the back-calculated length ofselected fishes in different age group.

SS

LL

1 1

SS

LL

1 1

Where S1 = length of scale radius toannulus X.

S = length of total scale radius

L SS

L11

L SS

L11

OrL1= length of fish when annulus X was formed.L= length of fish when scale sample wasobtained.By using back- calculated length of differentage group, we derived the growth rate of fishand plotted graph.

OBSERVATION

Labeo rohitaDuring the period of observation we

examined seven fishes of Labeo rohita in zeroage group in river Ghaghra. The lengths ofthese fishes were 12.5, 14.1, 15.5, 16.4, 17.2,18.3, 20.1 cm respectively.

Three fishes were examined in firstyear age group and found their lengths were22.4, 24.3 and 25.2 cm respectively. The l1 ofthese three fishes were 0.25, 0.4 and 0.41cmrespectively.



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We examined 17 fishes in second yearage group. The lengths of these fishes were26.1, 27.5, 28.31, 29.21, 31.1, 31.21, 32.5,33.48, 34.25, 35, 36.2, 37.3, 38.1, 39.5, 41.2,45.72, 46.2 cm respectively. The l1 of thesefishes were 0.41, 0.42, 0.43, 0.44, 0.45, 0.46,0.48, 0.5, 0.35, 0.36, 0.37, 0.38, 0.38, 0.4. 0.45,0.45, 0.45, 0.46 cm respectively. The l2 of thesefishes were 0.61, 0.62, 0.63, 0.64, 0.65, 0.66,0.68, 0.7, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.65,0.65, 0.66 cm respectively.

Five fishes were observed in threeyear’s age group and found that the length were47.5, 48.3, 49.45, 50.1, 51.2 cm respectively.The l1 of these fishes were 0.45, 0.45, 0.5, 0.5,0.55 cm respectively, l2 were 0.65, 0.65, 0.7,and 0.75 cm respectively and l3 were 0.8, 0.81,0.83, 0.85, 0.85 cm respectively.

Fourth year’s investigation revealedthat the length of nine fishes were 53.5, 54.1,55.3, 57.2, 59.1, 61.5, 62.1, 63.3, 64.6 cm. Thel1 of these fishes were 0.5, 0.55, 0.55, 0.55, 0.6,0.6, 0.65, 0.65, 0.65 cm respectively. The l2 ofthese fishes were 0.7, 0.75, 0.75, 0.75, 0.8, 0.8,0.85, 0.85, 0.85 cm respectively. The l3 of thesefishes were 0.8, 0.85, 0.9, 0.9, 1.0, 1.0, 1.05,1.1 cm respectively. The l4 of these fishes were0.9, 0.95, 1.0, 1.0, 1.0, 1.1, 1.1, 1.2, 1.2 cmrespectively.

We examined nine fishes in five yearage group, their length were 67.7, 69.9, 70.1,71.3, 73.2, 74.2, 76.3, 77.1, 79.8 cmrespectively. The l1 of these fishes were 0.65,0.65, 0.65, 0.65, 0.7, 0.7, 0.7, 0.7 cmrespectively. The l2 of these fishes were 0.85,0.85, 0.85, 0.85, 0.9, 0.9, 0.9, 0.9, 0.9 cmrespectively. The l3 of these fishes were 1.0,1.0, 1.0, 1.0, 1.1, 1.1, 1.15, 1.15, 1.2 cmrespectively. The l4 of these fishes were 1.1,1.2, 1.2, 1.2, 1.25, 1.25, 1.3 cm respectively.The l5 of these fishes were 1.2, 1.25, 1.25, 1.3,1.3, 1.35, 1.4, 1.45, 1.5 cm respectively.

We examined four fishes in sixth yearage group, their length were 81.2, 82.5, 84.1,85.2 cm respectively. The l1 of these fishes were0.75, 0.75, 0.8, 0.8 cm respectively. The l2 ofthese fishes were 0.95, 0.95, 1.0, 1.0 cmrespectively. The l3 were 1.25, 1.25, 1.3, 1.3,cm respectively .The l4 were 1.35, 1.35, 1.4,1.4, cm respectively. The l5 were 1.5, 1.5, 1.55,1.55 cm respectively. The l6 were 1.55, 1.55,1.6, 1.65 cm respectively (Fig-4).

Cirrhinus mrigalaDuring the period of observation we

examined seven fishes of Cirrhinus mrigala inriver Ghaghra in zero age group and found theirlength were 21.1, 23.1, 24.5, 26, 27.1, 28.2,30.1 cm respectively.

We examined four fishes in first yearage group, their length were 31.2, 32.5, 33, 35.6cm respectively. The l1 of these fishes were 0.8,0.85, 0.85, 0.9 cm respectively.

We examined five fishes in second yearage group, their length were 36.4, 37.1, 39.7,40.64, 41.63 cm respectively. The l1 of thesefishes were 0.45, 0.45, 0.45, 0.5, 0.55 cmrespectively. The l2 of these fishes were 0.62,0.63, 0.65, 0.65, 0.65 cm respectively.

We examined five fishes in third yearsage group, their length were 42.2, 43.1, 45.35,48.2, 49.1 cm respectively. The l1 of these fisheswere 0.45, 0.45, 0.45, 0.5, 0.55 cm respectively.The l2 of these fishes were 0.65, 0.65, 0.65,0.7, 0.75 cm respectively, the l3 of these fisheswere 0.75, 0.8, 0.85, 0.9, 0.95 cm respectively.

We examined nine fishes of fourthyears age group, length were 50.7, 52.1, 53.3,55.4, 57.1, 58.25, 60.0, 62.01, 63.1 cmrespectively. The l1 of these fishes were 0.55,0.55, 0.55, 0.6, 0.6, 0.6, 0.65, 0.65, 0.65 cmrespectively. The l2 of these fishes were 0.75,0.75, 0.75, 0.8, 0.8, 0.8, 0.85, 0.85 cm



46

respectively. The l3 of these fishes were 0.95,1.0, 1.0, 1.0, 1.0, 1.05, 1.10, 1.15, 1.2 cmrespectively. The l4 of these fishes were 1.0,1.1, 1.1, 1.15, 1.2, 1.25, 1.3, 1.35, 1.4 cmrespectively.

We examined four fishes of fifth yearage group, length were 65, 66.6, 69.2, 71.05cm respectively. The l1 of these fishes were 0.7,0.7, 0.75, and 0.75 respectively. The l2 of thesefishes were 0.9, 0.9, 0.95, 0.95 cm respectively.The l3 of these fishes were 1.15, 1.15, 1.2, 1.2cm respectively. The l4 of these fishes were1.35, 1.35, 1.4, 1.4 cm respectively. The l5 ofthese fishes were 1.4, 1.45, 1.45, 1.5 cmrespectively.

We examined three fishes of sixthyears age group, length were 73.5, 75.1, 78.5cm respectively. The l1 of these fishes were 0.8,0.8, 0.81 cm respectively. The l2 were 1.0, 1.0,1.1cm respectively. The l3 were 1.2, 1.2, 1.25cm respectively. The l4 were 1.3, 1.3, 1.35 cmrespectively. The l5 were 1.4, 1.4, 1.45 cm andthe l6 were 1.5, 1.55, 1.55 cm respectively.

We examined five fishes of seventhyears age group, length were 80.5, 82.2, 83.2,85.5, 86.2 cm respectively. The l1 of these fisheswere 0.85, 0.85, 0.85, 0.9, 0.5cm respectively.The l2 were 1.2, 1.2, 1.2, 1.25, 1.25 cmrespectively. The l3 were 1.3, 1.3, 1.35, 1.4, 1.4cm respectively. The l4 were 1.4, 1.4, 1.45, 1.5,1.5 cm respectively. The l5 were 1.5, 1.5, 1.55,1.6, 1.6 cm respectively. The l6 were 1.6, 1.6,1.65, 1.7, 1.7 cm and l7 were 1.65, 1.7, 1.7,1.75, 1.8 cm respectively.

We examined two fishes of eight yearsage group. The length of these fishes were 88.5and 90.2 cm respectively. The l1 of these fisheswere 0.9, 0.9cm respectively. The l2 were 1.25,1.25 cm respectively. The l3 were 1.4, 1.42 cmrespectively. The l4 were 1.5, 1.52cmrespectively. The l5 were 1.6, 1.62 cm.The l6

were 1.7, 1.73 cm.The l7 were 1.8, 1.83 cm andthe l8 were 1.85, 1.9 cm respectively(Fig-2).Catla-catla

During the period of observation ofCatla-catla in river Ghaghra, we examinedthree fishes of the zero year’s age group. Thelength of these fishes were 23.1, 24.5 and 25.1cm respectively. We examined two fishes in oneyear age group, the length of these fishes were28.4 and 30.1 cm. The l1 of these fishes were0.2 and 0.21 cm.We examined 03 fishes in twoyear’s age group the length of these fishes were32.2, 34.1, 36.2 cm . The l1 of these fishes were0.22, 0.24, and 0.3 cm. The l2 of these fisheswere 0.40, 0.42, 0.5 cm respectively.

We examined 12 fishes in three year’sage group, the length of these fishes were 38.7,40.1, 41.4, 43.18, 43.5, 44.2, 45.3, 46.4, 48.2,49.3, 52.4, 53.3 cm respectively. The l1 of thesefishes were 0.4, 0.5, 0.5, 0.55, 0.56, 0.56, 0.58,0.6, 0.65, 0.7, 0.75, 0.8 cm. The l2 of thesefishes were 0.6, 0.7, 0.7, 0.7, 0.72, 0.72, 0.75,0.8, 0.85, 0.9, 0.95, 1.0 cm. The l3 of thesefishes were 0.8, 0.9, 0.9, 1.0, 1.5, 1.05, 1.1,1.0, 1.05, 1.1, 1.15, 1.2 cm respectively. Weexamined 06 fishes in four years age group,the length of these fishes were 55.3, 56.2, 58.2,60.3, 61.1, 63.3 cm respectively. The l1 of thesefishes were 0.85, 0.9, 0.95, 1.0, 1.05, 1.1 cm.The l2 of these fishes were 1.05, 1.1, 1.15, 1.2,1.25, 1.3 cm. The l3 of these fishes were 1.25,1.3, 1.35, 1.4, 1.45, 1.5 cm. The l4 of thesefishes were 1.45, 1.5, 1.55, 1.6, 1.65, 1.7 cmrespectively.

We examined 03 fishes in five year’sage group. The length of these fishes were 64.4,65.5, 67.2 cm. The l1 of these fishes were 1.15,1.15, 1.2 cm. The l2 of these fishes were 1.35,1.35, 1.4 cm. The l3 of these fishes were 1.55,1.55, 1.6 cm. The l4 of these fishes were 1.75,1.75, 1.8 cm. The l5 of these fishes were 1.8,



47

1.85, 1.9 cm respectively. We examined fourfishes in six year’s age group, the length ofthese fishes were 70.1, 72.2, 75.5, 76.2 cm. Thel1 of these fishes were 1.2, 1.21, 1.21, 1.25 cm.The l2 of these fishes were 1.4, 1.41, 1.45 cm.The l3 of these fishes were 1.6, 1.61, 1.61, 1.65cm. The l4 of these fishes were 1.8, 1.81, 1.82,1.85 cm. The l5 of these fishes were 1.9, 1.91,1.92, 1.95 cm. The l6 of these fishes were 1.95,1.96, 1.97, 2.0 cm respectively.

We examined 02 fishes in seven year’sage group, the length of these fishes were 80.1,82.2 cm. The l1 of these fishes were 1.25, 1.3cm. The l2 of these fishes were 1.45, 1.5 cm.The l3 of these fishes were 1.65, 1.7 cm. The l4of these fishes were 1.85, 1.9 cm. The l5 of thesefishes were 1.95, 2.0 cm. The l6 of these fisheswere 2.0, 2.1 cm. The l7 of these fishes were2.1, 2.2 cm respectively (Fig-1).Labeo calbasu

During the period of observation ofLabeo calbasu in river Ghaghra, we examined05 fishes in zero age group, their length were20.3, 23.1, 24.3, 25.5, 28.3 cm respectively. Weexamined 08 fishes in one year age group, theirlength were 30.3, 31.2, 32.3, 33.6, 35.5, 36.6,38.2, 40.1 cm. The l1 of these fishes were 0.5,0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.58 cmrespectively.

We examined four fishes in two year’sage group, their length were 45, 47.7, 48.3, 50.1cm. The l1 of these fishes were 0.6, 0.62, 0.64,0.65 cm. The l2 of these fishes were 0.7, 0.72,0.74, 0.75 cm respectively. We examined 06fishes in three year’s age group, and theirlengths were 51.2, 52.3, 53.1, 54.4, 55.0, 56.0cm. The l1 of these fishes were 0.7, 0.71, 0.72,0.73, 0.75, 0.75 cm. The l2 of these fishes were0.8, 0.81, 0.82, 0.83, 0.85, 0.85 cm. The l3 ofthese fishes were 0.9, 0.91, 0.92, 0.93, 0.95,0.95 cm respectively.

We examined four fishes in four year’sage group, their length were 60.3, 61.0, 62.2,64.4 cm. The l1 of these fishes were 0.8, 0.85,0.9, 0.9 cm. The l2 of these fishes were 0.9,0.95, 1.0, 1.0 cm. The l3 of these fishes were1.0, 1.05, 1.1, 1.1 cm. The l4 of these fisheswere 1.1, 1.15, 1.2, 1.25 cm respectively. Weexamined 04 fishes in five year’s age group,their length were 66.6, 67.7, 68.7, 70.0cm. Thel1 of these fishes were 0.9, 0.95, 0.95, 1.0 cm.The l2 of these fishes were 1.0, 1.05, 1.05, 1.1cm. The l3 of these fishes were 1.14, 1.15, 1.15,1.2 cm. The l4 of these fishes were 1.2, 1.25,1.25, 1.36 cm. The l5 of these fishes were 1.3,1.35, 1.4, 1.45 cm respectively (Fig-3).Back calculationDuring the period of investigation scale sampleof 56 specimens of Labeo rohita in riverGhaghra, ranging in length from 12.5 to 88.7cm and belonging to 0 to 7 age classes wereexamined. The maximum number of specimensbelonged to age class two. The mean backcalculated lengths (in cm) obtained by theanalysis of pooled key scale samples were 29.18,41.53, 55.2, 62.96, 72.83, 79.82, 83.17 for firstto seventh age classes respectively (Fig. 1).

During the period of investigationscale sample of 35 specimens of Catla catla inriver Ghaghra, ranging in length from 23.1 to82.2 cm and belonging to 0 to 7 age classeswere examined. The maximum number ofspecimens belonged to age class three. Themean back calculated lengths (in cm) obtainedby the analysis of pooled key scale sampleswere 29.89, 40.53, 51.93, 62.25, 67.86, 72.04,78.41 for first to seventh age classesrespectively (Fig. 2).

During the period of investigationscale sample of 44 specimens of Cirrhinusmrigala in river Ghaghra, ranging in lengthfrom 21.1 to 90.2 cm and belonging to 0 to 8



48

age classes were examined. The maximumnumber of specimens belonged to age classfour. The mean back calculated lengths (in cm)obtained by the analysis of pooled key scale

samples were 29.13, 42.60, 53.60, 62.66, 69.65,76.36, 82.22, 86.97 for first to eight age classesrespectively (Fig.3).

Fig1-Scale of Catla catla Fig2- Scale of Cirrhinus mrigala

Fig-3Scale of Labeo calbasu Fig-4 Scale of Labeo rohita



49



50

Fig. Growth rate of cirrhinus mrigala in ghaghra



51

During the period of investigationscale sample of 31 specimens of Labeo calbasuin river Ghaghra ranging in length from 20.3to70 cm and belonging to 0 to5 age classes wereexamined. The maximum number of specimens

was 40.53 cm, l3 was 51.93 cm, l4 was 62.25cm, l5 was 67.86 cm, l6 was 72.04 cm and l7was 78.41 cm respectively (Fig.)

Johal (1981) studied the growth ofLabeo rohita from Gobindsagar andRangmahal. He recorded the growth rate (incm) in Gobindsagar was 23.81, 16.64, 13.24,10.48, 6.30, 4.54 and 3.06 cm for one to sevenage classes respectively. He also recorded thegrowth rate (in cm) in Rangmahal was 28.65,14.78, 11.36, 10.25, 10.58, 4.52, 2.31 and 3.56cm for one to eight age classes respectively.

According to my investigated thegrowth rate of Labeo rohita in river Ghaghrawas 29.18 cm,41.53 cm,55.2 cm,62.96cm,72.83 cm,79.82 cm and 83.17 cm from oneto seven year’s age group respectively(Fig.)

Rao and Rao (1972) examined thegrowth of Labeo calbasu in river Godavari. Hefound that the l1 of this fish was 20.22 cm, thel2 was 30.18 cm, l3 was 38.21 cm, l4 was 45.15cm, l5 was 50.94 cm, l6 was 54.73 cm, l7 was61.62 cm respectively.

Johal and Kingra (1988) also workedon growth of Labeo calbasu in Jaismand Lake.They found that the l1 of this fish was 14.81cm, l2 was 21.27 cm, l3 was 27.82 cm, l4 was33.36 cm, l5 39.14 cm, l6 was 41.34 cmrespectively.

We worked on growth of Labeocalbasu in river Ghaghra.The l1 of this fish was33.55 cm, l2 was 41.02 cm, l3 was 48.32 cm, l4was 56.38 cm and l5 was 64.71 cmrespectively(Fig.)

belonged to age class one. The mean back-calculated lengths (in cm) obtained by theanalysis of pooled key scale sample were 33.55,41.02, 48.32, 56.38, 64.71 for first to fifth ageclasses respectively (Fig.4).

DISCUSSION

Jhingran (1957, 1959) investigated the agegrowth of Cirrhinus mrigala in detail from theriver Ganga at Buxar (Bihar). He described thatdecline in the feeding intensity during summerand spring months are responsbile for theformation of annual rings. Tandon and Johal(1983a) described the age and growth of mirrorcarp Puntius sarana from the river Ghaggar,Rajasthan and Sukhana Lake, Chandigarh. Thespawning mark formed during March-May hasbeen considered valid for age determination.

We studied the growth of Cirrhinusmirgala from river Ghaghra. We recorded thegrowth rate (in cm) which was 29.13, 13.47,11.3, 8.76, 6.99, 6.71, 5.86 and 4.75 cm forone to eight age classes respectively (Fig.)

Johal (1982) worked on growth ofCatla catla in Gobindsagar, Rangmahal andHarike. They found that the l1 in Gobindsagarwas 25.89 cm. The l2 in Gobindsagar was 45.33cm, in Rangmahal 52.11 cm, in Harike 48.77cm. The l3 in Gobindsagar was 60.75 cm, inRangmahal 65.56 cm, in Harike it was 64.73cm. The l4 in Gobindsagar was 77.99 cm, inRangmahal 77.15 cm, in Harike it was 74.34cm. The l5 in Gobindsagar was 90.49 cm, inRangmahal 85.77 cm, in Harike 82.44 cm. Thel6 in Gobindsagar was 96.15 cm, in Rangmahal91.97 cm, in Harike 88.85 cm. The l7 inGobindsagar was 99.31 cm, in Rangmahal85.58 cm, in Harike 94.67 cm respectively.We worked on growth of Catla catla in riverGhaghra. We found that the l1 was 29.89 cm, l2



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BIBLIOGRAPHY

Alikunhi, K. H. 1957 Fish culture in India. Farming Bull.ICAR, New Delhi, (20) : 144 pp.

Beckman, D. W., Stanley, L. A., Render, J. H. and Wilson,C. A. 1990 Age and growth of black drum inLouisiana waters of the Gulf of Mexico. Trans.Am. Fish. Soc., 119 : 537-544.

Gupta,S.D.and Jhingran,A.G. 1973 Ageing of Labeocalbasu through its scale,

J.Inland Fish Soc.India, 5:126-128.Jhingran, V. G. 1957 Age determination of the Indian

major carp, Cirrhina mrigala (Hamilton) by meansof scales. Nature (Lond), 79(4557) : 468-469.

Jhingran, V. G. 1959 Studies on the age and growth ofCirrhinus mrigala (Ham.) from the river GangaProc.Natl.Inst. Sci.India,25B(3):107-137.

Jhingran. V. G. 1968 Synopsis of biological data on catla,Catla catla (Hamiltion, 1822). FAO Fish Synop.(32) : Rev : pag,var.

Jhingran. V. G. and Khan. H.A. 1979 Synopsis ofbiological data on the mrigal, Cirrhinus mrigala(Hamilton, 1822). FAO Fish. Synop., (120), pp.78.

Johal, M. S. and Tandon, K. K. 1983 Age, growth andlength-weight relationship of Catlacatla andCirrhinus mrigala (Pisces) from Sukhna Lake,Chandigarh (India). Vest. cs. Spolec. zool., 47 :87-98.

Johal, M. S. and Tandon, K. K. 1985 Use of growthparameters in Labeo rohita (Pisces, Cyprinidae).Ibid., 49 : 101-107.

Johal, M. S. and Tandon, K. K. 1987b Age and growthof Cirrhinus mrigala (Pisces : Cypriniformes)from northern India. Ibid., 51 : 252-280.

Johal, M. S. and Tandon, K. K. 1987c Annulus formationin Cirrhinus mrigala (Hamilton) and Labeo rohita(Hamiltion). Pb. Fish. Bull., 11 (1): 83-89.

Johal, M. S. Kingra, J. S. and Chahal, J. S. 1987Harvestable size of Labeo calbasu based on growthdata using scales. Abst. Natonal Symposium ofRccent Trends in Fish Biology, Department ofZoology, Magadh University, Bodh-Gaya,December 1-5, 1987.

Kamal, M. Y. 1969 Studies on the age and growth ofCirrhinus mrigala (Ham.) from commercialcatches at Allahabad. Proc. Natl. Acad. Sci. India,35B (1) : 72-92.

Kamal, S.,Sarkar,U.K.,Ponniah,A.G. and Lakra,W.S.2002.A comperative evaluation of age and growthof cyprinid fish Labeo calbasu (Ham)fromdifferent wild populations. In:Life history traits offreshwater fish population for its utilization inconservation NBFGR-NATP publicationNo.4,AA-4.

Khan, H. A. and Jhingran, V.G. 1975 Synopsis of thebiological data on rohu, Labeo rohita (Hamilton,1822). FAO Fish. Synop., (111) : 100pp.

Kingra, J. S. and Johal, M.S. 1992. Age and growth ofCirrhinus mrigala (Hamilton) from JaisamandLake. Bioved, 3(2) : 131-136.

Natarajan, A. V. and Jhingran. A. G. 1963 On the biologyof Catla Catla (Hamilton) from the river yamuna.Proc. Natl.Inst. Sci. India 29B ()3) : 326-355.

Rao, G. M. and Rao, H.L. 1972 On the biology of Labeocalbasu (Hamilton) from the river Godavari. J.Inland Fish Soc., India, 4: 74-86.

Singh, S., Sharma, L.L.and Saini, V.P.1998 Age andgrowth and harvestable size of Labeo rohita (Ham.)from the lakeJaisamand.Rajasthan,India.Indian.J.Fish.,45(2):165-175.

Tandon, K. K., Johal, M.S. and Kaur, J. 1989b. Remarkson the age and growth of Labeo calbasu (Pices :Cyprinidae) from Rajasthan, India. Lbid., 53: 153-160.



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During extensive Survey of localmarket in 2014 at Hathras, Aligarh and Agra(U.P.). Several fruits of Ananas (AnanasComocus Roxb) was found infected. The fruitsexhibited Whitish-Blue and brownish patches,which later on, embraced the whole fruit. Inthe advance stage, the diseased fruits wereirregularly depressed due to intensive tissuemaceration. Such fruits were collected from themarket. The extent of rotting ranged from 15-18% a time, the whole consignment is rendered

Fruit Rot of Ananas

Sheetal Sarswat* and R.B. SharmaDepartment of BotanySarswati P.G.College, Hathras (U.P.)* Research Scholar - Mewar University Chittorgarh (Raj.)

unfit for human consuption. The effect tissueswere soft, Blueish and watery. Under humidconditions brown blue mass of spores appearedon the surface of rotten tissues and emitted badsmell. (Fig.1)

To isolate the pathogen diseased fruitswere surface sterilized with 0.1% Hgcl2. andcut in to small bits, which plated on P.D.A. andCzapek’s agar media and incubated at280C+20C. The fungi was isolated asPanicillium Italicum Thom and thepathogenicity was tested with replicates byartificial inoculation method of Tandon andMishra (1969). Inoculated fruits were incubatedat 280C+20C. Corresponding controls weremaintained.

The fungi produced soft rot on fruitswithin 4-5 days and reisolation from the yieldedthe same organism.

The authors are thankful to Dr.A.N.Roy, Department of Botany, Agra College,Agra and U.G.C. for financial assistance.

REFERENCES :Tandon R.N. and Mishra A.N.(1969). Indian

Phytopathology. test by knife injury method2nd phytopathology 20:1-12.



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55

Population Dynamics of Sarus Crane (Grus antigone antigone, Linn.)in and around Alwara Lake of district - Kaushambi (U.P.), India

Shri Prakash1 and Sunil Kumar21Department of Zoology, KAPG College, Allahabad - 211001 (U.P.)Email: [email protected] of Zoology, KAPG College, Allahabad - 211001 (U.P.)Email [email protected]

ABSTRACT

Survey of Sarus Crane (Grus antigone antigone, Linn.) was carriedout within a period of one year (from Jan. 2013 to Dec. 2013) in and aroundAlwara Lake of district Kaushambi (U.P.), India. Crane population wasrecorded as single, pairs, pairs with juvenile, juvenile and flocks. Populationsize was 60.3±6.2 (16-80). Population was significantly influenced by localclimatic temperature than humidity. Only single and flocks more significantlycorrelated positive to climatic temperature. Maximum number of 723 Craneswas recorded with a density of 1.8/ hectare including all age groups. Duringthe survey adult pairs were recorded with a maximum percentage of 40.1preceded by flock (23.9%) among all the observed age groups.

Inspite of extreme temperature and humidity cranes were observed enjoyingthis land form. Besides this openness of land, numerous marsh wet landincluding the lake, alterations of crop fields i.e. Kharif, Rabi and Jayad ,availability of food resources, variation in aquatic plants make the crane lifestyle compatible to this habitat. Hence the objective of this study was toexplore conservational and distributional aspects of crane and its habitatwhich can be shared in public awareness programmes.

Key words : Conservation, population dynamics, Kaushambi, Grusantigone, Sarus Crane, Alwara lake.

INTRODUCTION

Sarus Crane is the only residentbreeding crane in India and its population iscomparatively more in Uttar Pradesh than other

states. Hence, it is declared as State Bird bythe Government of Uttar Pradesh for itsconservation. It is the world’s biggest flying



56

water bird. It is widely distributed withmaximum number in Uttar Pradesh than itsadjacent states of Bihar and Madhay Pradesh(Gole, 1989) followed by Gujarat andRajasthan.

In Uttar Pradesh, they are locatedmainly in district Mainpuri, Etawah, Etah,Kaushambi and Aligarh (Sunder, 2000). Amongthese only one species of crane has beenrecorded in and around the Alwara Lake,District Kaushambi. Biodiversity indices ofaquatics as well as inland are sparinglyproductive and seasonal. The variations ofrainfall, temperature and the Yamuna riverflood are the abiotic factors which greatlyattributes to its aquatic biodiversity. It is an oldmarshy riparian wetland surrounded byagricultural and nonagricultural land. The smallseasonal marshy lands were considerablynoticed around the lake among crops land. Thelarge openness around the lake was noticed dueto seasonal flooding by Yamuna river. Theseare the factors that provide optimum habitatfor the Sarus Cranes. Prakash et al, 2014reported 487 cranes from its three differenttransect and agrued that from ecological pointof view site is very favourable for Sarus Crane,Grus antigone distribution. They furtherreported that the highest population of SarusCrane was reported in Transect II (207) duringthe survey probably due to availability ofsufficient food, mainly small molluscs,crustaceans, worms and grains present in

cropland vegetation, e.g., paddy fields andother kharif crops.

In India, there is an alarming crash inthe population of this species especially in UttarPradesh. The loss of natural habitat seems tobe the most important cause for this drasticchange or reduction in number. Therefore thepresent study is an attempt to explore the censusof crane population and its correlations toclimatic parameters, natural habitat and Alwaralake.

Study areaThe lake under exploration is a natural

lake and now a part of important wetland. Thestudy area was divided into three majortransects of landscape on the basis of itsvastness, diversity of flora and fauna and typeof water body (wetland or taal, Image 1).

The lake is surrounded by agriculturalfields and connected with river Yamunatowards transect III and terminal part ofKishanpur lift canal towards transect I. Thusthe habitat in the form of wetland is perennial,although during summer the water level fallsdown. During rainy season water depth goesupto about 20 meters and during summer thedepth comes down to about 15 meters. Lakehas derived its name from village Alwara.Locally it is called Alwara Taal. Alwara lakeis a naturally formed lake and covers about 400hectare. The surrounding villages are PaurKashi Rampur in East, Tikara in North andShahpur in South and river Yamuna in west.


The authors used binocular, camera,motorbike, chappu boat, field stick etc. forvarious purposes. The findings are based uponthe work conducted between January toDecember, 2013. Investigations were

conducted in morning hours between 6.0am to9.0am and evening hours between 2.0pm to5.0pm during our routine field trips. All theobservations were made while moving throughthe chappu boat and walking along the



57

croplands, mudlands, natural areas using 7.35and 8.40 binoculars. Sights and calls were thedevices to enlist a bird at a particular site.Identification of different species was aided byusing standard guides. Besides actual sightings,inquiries from local people were also made toensure the estimate of existing population andtheir perceptions about the existence of theCrane. Census was avoided during rainy days.Population comparison was determined as asingle, pair, pair with one juvenile, pair withtwo juveniles and flocks comprising adults andjuveniles. The encounter rate of Sarus cranepopulation was calculated by the relationship:

Systat 12 (Wilkinson, 1982) softwarewas used for the statistical analysis of data.The local climatic temperature and humiditywere recorded by mercury degree centigradethermometer and alcoholic hygrometerrespectively. Their monthly mean±standarderror variations were tabulated.Geography of Alwara Lake

By road the lake is 85 km away fromAllahabad district, 45 km from Manjhanpurhead quarter of Distric-Kaushambi and 290 kmfrom Lucknow, capital of Uttar Pradesh. Itsnearest railway station is Bharwari at a distance

of 50 km and nearest airport is Bamrauli,Allahabad at a distance of 75 km. It is situatedbetween the latitude 25O24’05.84’’N –

25025’10.63’’N and longitude 81O11’39.49’’E-81O12’57.95’’E.

Climate around Alwara LakeIts weather is tropical to subtropical

with some variations over the year. Winteroccurs between the months of late October toearly March. Mid December to mid January isseason of severe cold and irregular appearanceof fogs are the characteristic feature of winter.Usually Spring occurs from mid February toend of April. Summer approaches in the monthof April and ends in late June. It is marked byhigh velocity of winds known as loo in themonth of May and June. Rainy- Approximately350 mm rainfall observed annually from lateJune to early October. Irregularity of rainfallwas also noticed year wise which influencedthe landscape ecology of the lake. Autumn- Itcommences in mid October & ends in lateNovember. Temperature shows with highfluctuation over the year and noticeddeterminant parameters of this landscape.Maximum temperature is 480C to 500C duringsummer and minimum 010C to 50C duringwinter.


The population of sarus crane was recorded assingle, pairs, pairs with juvenile, juvenile,flocks in their respective numbers and agegroups (Image 2, 3, 4). Their monthly variationsare tabulated in Table 1 and statistical graphsare shown in Figure 1-4. The climatic variation(Atmospheric temperature and humidity)recorded monthly are shown in Table 2. A totalnumber of 723 (16-80) cranes were recordedduring the survey period of 2013 which was

more than previously reported 487 by Prakashet al., 2014 during Sep, 2011-Dec, 2012. Theenhanced crane population was due to morenesting sites (marshes) in the study area andshort distance immigration of flocks acrossYamuna river. Similar short distance migrationin Sarus Crane has been also reported by Nandi,2006. Since the adult pairs were recorded inmaximum number 292 (4-40) with 40.1%preceded by flocks 169 (5-20) with 23.9%



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Table 1. The overall monthly variations of Sarus Crane in and around Alwara lake during 2013.S. Months Single No. of No. of Juvenile Flock Total Density = Pairs Pairs+J Flocks

No. Adult in Adult in Adult Cranes TotalCrane No. No. No.

Pair Pair+J observed /Hectare

1. Jan.,2013 0 4 3 4 5 16 0.0 2 1 1

2. Feb. 3 18 9 17 8 55 0.1 9 3 1

3. Mar. 9 22 12 14 12 69 0.2 11 4 2

4. Apr. 12 26 9 8 17 72 0.2 13 3 3

5. May 16 30 9 6 19 80 0.2 15 3 3

6. Jun. 19 34 3 1 20 77 0.2 17 1 4

7. Jul. 10 38 0 0 18 66 0.2 19 0 2

8. Aug. 7 40 0 0 17 64 0.2 20 0 2

9. Sep. 4 38 3 0 16 61 0.2 19 1 2

10. Oct. 5 20 30 1 15 71 0.2 10 10 2

11. Nov. 4 16 36 3 15 74 0.2 8 12 2

12. Dec. 0 6 3 2 7 18 0.0 3 1 1

13. Total 89 292 117 56 169 723 1.8 146 39 25

14. Range 0.0-19.0 4-40 0-36 0-17 5-20 16-80 0.0-0.2 2.0-20.0 0-12 1-4

15. Average 7.4± 24.3± 9.8± 4.6± 14.5± 60.3± 0.16± 12.1± 3.5± 2.1±

+SE 1.734 3.5 3.3 1.6 1.4 6.2 0.02 1.8 1.1 0.9

Table 2. The overall monthly climatic variation in and around Alwara Lake during 2013.

S. No. Temperature (oC) Humidity (%)

1. Jan., 2013 5.3±2.1 89.3±2.52. Feb. 12.4±2.5 84.9±3.23. Mar. 26.2±2.5 79.1±5.24. Apr. 35.4±2.0 58.9±4.05. May 40.0±2.2 57.8±4.36. Jun. 45.8±3.6 59.6±5.87. Jul. 30.5±4.8 84.0±9.08. Aug. 30.0±1.0 92.8±2.69. Sep. 29.3±1.3 94.6±1.610. Oct. 25.9±1.7 87.7±2.511. Nov. 12.3± 2.5 82.0±9712. Dec. 10.0±2.3 91.3±1.8



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Table 3. Analysed values (r) of Pearson Correlation between the different age group of Sarus crane andclimatic factors during 2013.

Single No. Adult No. Adult+ Juvenile Flock Total Density Tem. Hum.in Pair J in Pair /hectare (oC) (%)

Single 1.000 No. Adult in 0.622* 1.000Pair No. Adult +J -0.154 -0.319 1.000in Pair Juvenile -0.028 -0.303 0.083 1.000 Flock 0.807** 0.845*** 0.041 -0.389 1.000 Total 0.733** 0.688* 0.350 0.040 0.861*** 1.000 Density/ 0.650* 0.788** 0.274 -0.114 0.887*** 0.958*** 1.000hectare Tem. (oC). 0.919*** 0.806** -0.222 -0.207 0.894*** 0.751** 0.752** 1.000 Hum. (%) -0.853*** -0.207 -0.016 -0.220 -0.529 -0.549 -0.380 -0.662* 1.000Significant at *P<0.05, **0.01, ***0.001

Figure 1. Bar Graph shows the observed monthly variations among the different age group ofSarus Crane during 2013.



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Figure 2. Line Chart shows the Monthlyvariations of total count crane with standard

error in Alwara lake during 2013

Figure 3. Pie chart represents the percentilevariation of Sarus Crane population in Alwara

lake during 2013.

Fgure 4. Pearson Correlation Matrix with linear regression line shows the significant correlation amongall age groups of Sarus Crane and climatic factors of Alwara lake during 2013.



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Image 1. The location of the study area

Image 2. Shows the single adult Sarus Crane in Alwara Lake.



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Image 3. Shows the adult pair of Sarus Crane in Alwara Lake.

Image 4. Shows the adult pair with juvenile of Sarus Crane in Alwara Lake.



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(Figure 3, Table 1); their numbers have beensupportly increased in the month of August andMay respectively. The increment in pairs isencouraged by the breeding site (marshes)around the lake (Lahiri, 1955 and Borad et al.2001). Similarly flocks increased due toopenness around the lake in the summer seasonfrom May-July (Livesey, 1937). Pearsoncorrelation values (r) also strongly supportedthe relations between maximum temperatureand flocks at P<0.001 and flocks weresignificantly related with pairs atP<0.001(Figure 4,Table 3). Therefore, it couldbe hypothesized that flocks took short distanceimmigration to this favourable site acrossYamuna river during summer season andimparted in pairing habit during the late rainy

season for preparation of breeding beforewinter season (August to October).

The atmospheric temperature (5.3 -45.8o C; Table 2) of Alwara lake supportedmore significantly to the population of singlecrane at P<0.00 (Figure, Table 3) and flocks atP<0.001than pair at P<0.01 but discouraged toadults with juvenile and juvenile. Thiscorrelation occurred due to general biologicalresponse of birds against climatic temperature.Perhaps the density/hectare was encouraged byatmospheric temperature at P>0.01. Thiscorrelation was seen due to the openness andflora of the lake. The atmospheric humiditysignificantly showed negative effect only to thesingle crane population at r=-0.853 (P.0.001)than insignificant to other individuals of cranepopulations (Table 3 and Figure 3, 4).

CONCLUSION

The observed variations among all age groupof Sarus Crane were seasonally correlated inand around Alwara lake. Their populationpattern was significantly influenced bytemperature than humidity. While other factors

Gole, P. (1989). The Status and Ecological Requirementsof Sarus Crane. Phase I. Ecological Society, Pune,India. 45 pp.

Lahiri, RK (19955). Breeding of Sarus Crane (Antigoneantigone, Linn.) in captivity. Journal of theBombay Natural History Society. 53:1:130-131.

Nandi, P.K. (2006). Protection of habitat of Sarus craneGrus antogone in Bhoj wetland, India, pp. 309–310. In: Boere, G.C., C.A. Gal Braith & D.A.Stroud (eds.). Water Birds Around The World. Thestationery office, Edinburgh, UK.

Prakash, S., Narain, S. & Kumar, S. 2014 .Conservation of the threatened Sarus Crane Grus

like openness, agricultural land and seasonalmarshes dominated over the climatic factorswhich resulted in increased population ofCranes during 2013 in and around Alwara lake.

REFERENCES

Ali, S. (1941). The Book of Indian Birds. The BombayNatural History Society, Bombay, 457pp.

Aryal, A., T.K. Shrestha, D.S. Sen, B. Upreti & N.Gautam (2009). Conservation regime and localpopulation ecology of Sarus Crane (Grus antigoneantigone) in west-central region of Nepal. Journalof Wetlands Ecology 3: 1–11; http://dx.doi.org/10.3126/jowe.v3i0.2224

Borad, CK, Mukherjee and Parasharya, BM (2001). Nestsite selection by the Indian Sarus Crane in thepaddy crop agroecosystem. BiologicalConservation 98:1:89-96.

Choudhary, H. (1999). Black-tailed Crake Porzanabicolor: a new species for Nepal. Danphe 8(2/4): 4.



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antigone (Linnaeus, 1758) around Alwara Lake inKaushambi District, Uttar Pradesh, India. Journalof Threatened Taxa . 6: 5726-5730. http://dx.doi.org/10.11609 (accessed on 5 July 2014)

Sundar, K.S.G. (2000). Distribution, demography andconservation status of the Indian Sarus Crane (Grusantigone) in India. Journal of the Bombay NaturalHistory Society 97(3): 319–339.

Wildlife Institute of India (1999). Training workshopon wetland research methodology measuring andmonitoring biological diversity. WII, Dehra Dun,5–10pp.

Wilkinson, L. (1982). SYSTAT: Statistical analysis andgraphical presentation of scientific and engineeringdata, version 12, University of Illinois, Chicago.Systat Software, Inc. Washington, 225pp.



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INTRODUCTION

Proteases are group of enzymes, catalyze hydrolysisof polypeptide chain into smaller peptide chain orfree amino acids (Abirami et al., 2011).

Partial purification and characterization of protease enzyme from soilborne bacteria

1Arun Kumar Sharma, 1Vinay Sharma*, 2Jyoti Saxena, 1Bindu Yadav, 1Afroz Alam and 1Anand Prakash1Department of Bioscience and Biotechnology, Banasthali University, Rajasthan, India2Department of Biochemical Engineering, Bipin Tripathi Kumaon Institute of Technology,Dwarahat, Uttrakhand*Corresponding Author: [email protected]

ABSTRACT

Protease enzyme from bacterial isolates AKS-4 and AKS-6 was successfullypurified by ammonium sulfate precipitation. Purified enzyme fractionateobtained from 80% salt saturation showed highest protease activity. Proteaseactivity of crude lysate was increased after purification with 80% saltconcentration. Purified protease fractions (80% salt saturation) obtained frombacterial isolate-AKS-4 and AKS-6 were named as P-1 and P-2, respectivelywith protease activity of 35.07 U/mL and 34.46 U/mL. Extracellular proteasefrom isolate AKS-4 was purified to 9.29 fold by (NH4)2SO4. Enzymefractionates P-1 and P-2 showed increased activity at neutral and alkalinepH range while enzyme activity was reduced under acidic pH. P-1 and P-2demonstrated increased protease activity at higher temperature range (50ºC, 60 ºC). The impact of different divalent metal ions and organic solventson protease activity of both enzyme fractions was investigated. Highestprotease activity by both enzyme fractions was obtained with calcium chlorideand n-butanol.

Key words: Bacterial isolates, purified enzyme, ammonium sulfateprecipitation, crude lysate, temperature.

Proteases are of the most imperative industrialenzymes synthesize and produced by a widerange of microorganisms, including fungi,



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bacteria, yeasts, molds, and are also found indifferent animal tissues and in plants. Bacterialproteases are generally extracellular, active atbroad pH range, and easily produced in largequantity (Walsh et al., 1970). Bacillus producesa broad range of extra-cellular enzymes,including proteases. Several Bacillus specieswere known as protease producers i.e., Bacillusmojavensis, Bacillus cereus, Bacillusmegaterium, Bacillus subtilis and Bacillussterothermophilus (Asker et al., 2013).A submerged fermentation in which nutrientsare present in solubilized form in large quantityof water with good aeration rate and agitationspeed in a fermenter. In this methodenvironmental factors such as temperature andpH can be controlled very well. Impeller offermenter control mixing and allows equaldistribution of microbial cells, products, toxicwaste products, nutrients and oxygen throughtfermentation broth. This method isrecommended for the production ofextracellular enzymes from microbes (Gervais

et al., 1996). Protease has application in laundryindustry, where they are used to remove proteinbased stains from clothing (Asker et al., 2013).About 50% of the total industrial enzymes iscontributed by proteases so they are veryimportant enzyme (Rao et al., 1998). Theyoccupy an important position due to their broadapplications in leather, pharmaceutical, food,detergent, laundry, photography and agricultureindustries. They are also used in brewing,peptide synthesis, meat tenderization, cheesemaking, bioremediation and medical diagnosisand as treatment for wound and inflammation(Anwar and Saleemuddin, 1998; Gupta et al.,2002). The exploitation of an enzyme forvarious industrial purposes depends on itsdistinctive characteristics such as optimum pH,temperature, enzyme stability in presence ofvarious organic solvents, metal ions, mode ofaction of enzyme, etc. (Zhang et al., 2011).Therefore, the aim of present study was topurify and characterize the protease enzymefrom soil borne bacteria.


2.1. Isolation, screening and identificationof proteolytic bacteria from soil

Soil samples 1 and 2 were collected frombotanical garden and medicinal plant gardenof Banasthli University, respectively. Bacteriawere isolated by serial dilution agar platetechnique earlier described by Kader et al.(1999). In order to identify protease producers,bacteria were inoculated in gelatin agarmedium plates followed by incubation of platesat 37ÚC for 2 days. Protease producers wereidentified based on formation of clear zonearound their colonies after flooding the plateswith HgCl2 solution (HgCl2 5.0 gm,

concentrated HCl 20 mL and distilled water 100mL). HgCl2 reacted with unhydrolyzed gelatinto produce opacity making the clear zoneseasier to see (Abdel Galil, 1992).

2.2. Protease productionOne mL of 24 h old Luria broth culture wasinoculated in 100 mL of protease productionmedium containing (% w/v): CaCl2 0.01;K2HPO4, 0.05; yeast extract 0.02; peptone 1.0;MgSO4 0.01; glucose 0.1; pH 7.0 (Quadar etal., 2009). Crude enzyme extract was preparedby centrifugation of fermentation broth at 8000rpm, 4 ºC for 15 minutes.



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2.3. Measurement of enzyme activity

Protease activity in the crude enzyme extractwas determined according to the method ofCarrie Cupp-Enyard (2008) by using casein assubstrate.

2.4. Partial purification of proteaseCrude enzyme extracts were used forpurification. Different concentration ofammonium sulfate (30%, 70% and 80%) wastaken for partial purification of protease. For30% salt saturation, solid ammonium sulfatewas added to enzyme extract with stirring andit was left for 3 hours at 4 ÚC. The precipitatewas removed by centrifugation at 10,000 rpmat 4ºC for 15 minutes. The pellet wasreconstituted in minimum amount of 50 mMpotassium phosphate buffer (pH 7.5) andprotease activity in pellet was determined.Additional ammonium sulfate was added to thesupernatant in order to bring the saturation to70% and it was left for 3 hrs at 4 ÚC. Again itwas centrifuged to obtain pellet. Enzymeactivity was measured in pellet and additionalammonium sulfate was added to thesupernatant in order to bring the saturation to80% and enzyme activity at 80 % saturationwas determined. Fold purification andpercentage yield was determined usingfollowing formula.Fold purification =

Specific protease activity of each fractionSpecific protease activity of crude protein extract

Percent yield =Protease activity of each fraction

Protease activity of crude protein extract ×100

2.5. Measurement of activity of partiallypurified protease

The reaction mixture was containing 1 mL of0.65% case in solution and 0.2 mL of crude

enzyme extract were placed at 37 ºC for 30minutes thereafter 1mL of Trichloroacetic acidsolution was added to stop the enzymaticreaction. After standing for 15 minutes forprecipitation reaction to occur, it was filteredusing Whatmann’s No 1 filter paper. 0.4 mL offiltrate was added in 1 mL of sodium carbonatesolution followed by addition of 0.2 mL of 2fold diluted Follin Ciocalteus phenol reagent.Above mixture was incubated in dark at roomtemperature for 30 minutes for the developmentof blue color. The concentration of tyrosinereleased from casein by protease action wasmeasured at 660 nm against a reagent blankusing tyrosine standard. Tyrosine was taken infollowing concentration range for thepreparation of standard curve: 27.5 µM, 55 µM,110 µM, 220 µM and 275 µM. One proteaseunit was defined as the amount of enzymerequired to releases 1 µM of tyrosine per minuteper mL at 37ºC, pH 7.5 (Mohapatra et al.,2003). All the experiments were done intriplicates and mean values are presented.The enzyme activity (U/mL) was calculatedby following formulaEnzyme activity (Units/mL) =

µmole tyrosine equivalent releases x 2.2(Total volume of assay)

Volume of enzyme taken (0.2 mL) xIncubation time (30) x 1.6

2.2= Total volume of assay (in milliliters).0.2= Volume of enzyme used (in milliliters).1.6= Volume of sample taken in cuvette forabsorbance.Specific activity is the enzyme activity in onemg of total protein (expressed in μmol/min/mg).

The specific enzyme activity (U/mg) wascalculated by following formula

Specific enzyme activity (U/mg) =Enzyme activity (U/mL)

Total protein content (mg/mL)



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2.6. Characterization of partially purifiedenzyme

Partially purified enzyme from both bacterialisolates (AKS-4 and AKS-6) obtained after 80% salt saturation was used for characterizationstudy. The effect of various parameters onenzyme activity and stability was measured.

2.6.1. Effect of pH on protease activity

The impact of pH on the activity of purifiedenzyme was studied by incubating reactionmixture at different pH range (4-9) ofpotassium phosphate buffer (50 mM) at 37 ºC.pH of buffer was adjusted using 1N HCl and1N NaOH. Upon completion of incubationperiod enzyme activity was determined.

2.6.2. Effect of temperature on proteaseactivity

The impact of temperature on the activity ofpurified enzyme was studied by incubating thereaction mixture at different temperature range(40 ºC, 50 ºC, 60 ºC and 70 ºC). Uponcompletion of incubation period enzymeactivity was measured.

2.6.3. Effect of metal ions on enzyme stability

The impact of various metal ions atconcentration of 5mM on protease stability wasstudied by pre-incubation of enzymepreparation for 2 hrs at 37 ºC in the presenceof divalent metal ion solution (Ca2+, Mg2+,Mn2+, Ba2+, Cu2+, Li2+ and Zn2+). Thereafter,protease assay was performed at 50 ºC and pH8.0. The enzyme activity was determinedwithout adding metallic ions, considered as thecontrol for determining relative enzymeactivity. Relative enzyme activity wasdetermined using following formula (Sharmaet al., 2014).Relative enzyme activity (U/mL) =

Enzyme activity of control (without metal ion)Enzyme activity with metal ion

2.6.4. Effect of organic solvents on enzymestability

The impact of various organic solvents (10%,v/v) on protease stability was studied by pre-incubation of enzyme preparation for 2 hrs at37 ºC in the presence of organic solventsolution. Thereafter, protease assay wasperformed at at 50 ºC and pH 8.0. Relativeenzyme activity was determined by consideringthe activity of the enzyme without any organicsolvents as 100%.


3.1. Isolation, screening and production ofproteaseFrom a total of 2 collected soil samples, 12bacterial colonies were isolated. Among 12isolates, 4 isolates from soil sample 1 and 4isolates from soil sample 2 were shown clearzone around streaked lines. Then these 8bacterial isolates were selected for protease

production in fermentation broth. Four bacterialisolates from soil sample 1 were named asAKS-1, AKS-2, AKS-3 and AKS-4respectively. Four bacterial isolates from soilsample 2 were named as AKS-5, AKS-6, AKS-7 and AKS-8 respectively. Isolates AKS-4 andAKS-6 was showing highest protease activityso protease from these isolates were selectedfor purification and characterization study.



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3.2. Partial purification of enzymeResults presented in Table 1 showed that(NH4)2SO4 concentration of 80% totallyprecipitated protease enzyme. In the crudeenzyme preparation, the enzyme extracted frombacterial isolates AKS-4 and AKS-6 wasdemonstrated 24.46 U/mL and 33.61 U/mLenzyme activity, respectively. Protease activitywas increased by enzyme fractions of bacterialisolates AKS-4 and AKS-6 obtained afterpurification with 80% (NH4)2SO4concentration. It was 35.07 U/mL and 34.46U/mL, respectively. Partially purified enzymefractions obtained from 80% (NH4)2SO4concentration from bacterial isolate-AKS-4 andAKS-6 was named as P-1 and P-2.

Similar purification process was done byAbirami et al. (2011). Umayaparvathi et al.(2013) reported purification of protease fromBacillus cereus SU12 with ammonium sulfateprecipitation. Highest specific activity wasobtained after 80% salt concentration. Oke andOnilude, 2014 reported purification of proteaseby (NH4)2SO4 precipitation, gel filtrationchromatography and ion exchangechromatography. Sharma et al. (2014) purifiedprotease by ammonium sulfate and reportedthat 6.65-fold purification was achieved frominit ial crude lysate during (NH4)2SO4precipitation (50-70%).

3.3. Effect of pH on enzyme activity

Results presented in Table 2 showed that theoptimum pH for protease activity of enzymefractions P-1 and P-2 was 8.0. The enzymeactivity for P-1 and P-2 was 21.76 U/mL and12.50 U/mL, respectively. Enzyme activity wasincreased at neutral and alkaline pH whileprotease activity was reduced under acidic pH.Alkaline nature of protease indicates it’ssuitability in alkaline environment of variousindustries.Balachandran et al. (2012) reported proteaseactivity over a wide pH range and it was foundthat enzyme was most active at pH 8-9. Asker

et al. (2013) reported bacterial protease was78% active in the pH range of 7.0-8.0. Joo etal. (2003) reported that Bacillus clausiiprotease was stable in the broad pH range of4.0-12.0 while maximum activity was obtainedat pH 12.0. Abirami et al. (2011) reportedoptimum pH for highest protease activity fromPenicillium janthinellum. It was 6.5.3.4. Effect of temperature on enzyme activityTable 3 demonstrated results of impact oftemperature on protease activity of purifiedenzyme fractions (P-1 and P-2). Both fractionsP-1 and P-2 showed highest protease activitywhen incubation temperature of protease assaywas 50 ºC. The protease activity was increasedat higher temperature range. The enzyme

Table 1: Summary of Partial purification of protease extracted from bacterial isolatesAKS-4 and AKS-6.

Percentage Absorbance Protease Protein Specific Fold Percentagesaturation of at 660nm activity content activity purification yield(NH4)2SO4 (U/mL) (mg/mL) (U/mg)

AKS-4 AKS-6 AKS-4 AKS-6 AKS-4 AKS-6 AKS-4 AKS-6 AKS-4 AKS-6 AKS-4 AKS-6Crude enzyme 0.80 1.10 24.46 33.61 4.99 4.33 4.90 7.66 1.0 1.0 100 100extract30% 0.53 0.206 16.31 6.28 0.22 0.54 74.13 11.62 15.12 1.51 66.68 18.6870% 0.632 0.215 19.27 6.55 0.78 0.25 24.70 26.2 5.04 3.42 78.78 19.4880% 1.15 1.13 35.07 34.46 0.77 0.08 45.54 430.75 9.29 56.23 143.37 102.52



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activity was found in following descendingorder: 50 ºC > 60 ºC > 70 ºC > 40 ºC.Balachandran et al. (2012) reported proteaseactivity over a broad range of temperature from4 to 50 ºC. Highest activity was obtained at 4-8 ºC. Enzyme activity was completely lost at37 ºC. Bajaj et al. (2011) reported that proteaseactivity was tested over a wide range of

temperature 60-90 ºC. Asker et al. (2013)investigated effect of different temperature onBacillus megaterium protease activity and Itwas found that protease activity was highestwhen incubation temperature was 50 ºC.Abirami et al. (2011) reported highest proteaseactivity at temperature between 30-40 ºC.3.5. Effect of metal ions on enzyme stability

Table 2: Effect of pH on the activity of partially purified enzyme fractions (P-1 and P-2).pH Absorbance at 660nm Concentration of Protease

liberated tyrosine (µM) activity (U/mL)P-1 P-2 P-1 P-2 P-1 P-2

4 0.16 0.07 26.66 11.66 4.88 2.135 0.21 0.13 36.16 21.66 6.61 3.967 0.68 0.18 114.66 31.00 20.98 5.678 0.71 0.41 118.66 68.33 21.76 12.509 0.55 0.21 92.33 36.33 16.89 6.64

Table 3: Effect of temperature on the activity of partially purified enzyme fractions (P-1 and P-2).

Temperature Absorbance at 660nm Concentration of Protease activity (ºC) liberated tyrosine (µM) (U/mL)

P-1 P-2 P-1 P-2 P-1 P-240 0.2 0.11 34.00 19.00 6.22 3.4750 1.96 1.04 326.66 174.83 59.78 31.9960 1.85 0.42 309.33 70.00 56.60 12.8170 1.62 0.33 271.33 56.00 49.65 10.24

Table 4 revealed results of impact of variousmetal ions on protease stability of purifiedenzyme fractions (P-1 and P-2). Proteaseactivity from P-1 was increased in presence offollowing divalent metal ions Ca 2+

, Mg2+, Mn2+

and Li2+. A decrease in the protease activity wasmeasured when enzyme was preincubated withBa2+, Cu2+ and Zn2+. Highest enzyme activityby both fractions (P-1 and P-2) was observedin presence of Ca 2+. It was 59.78 U/mL for P-1 and 47.33 U/mL for P-2, respectively.Sharma et al. (2014) reported that Ca2+

, Mn2+

and Mg2+ increased enzyme activity by 36%,

22% and 8%, respectively while other metalions Cu2+, Zn2+, Na+ and K + had a negativeeffect on protease activity. Uddin et al. (2015)reported that MgSO4 increased the proteaseactivity while β-mercaptoethanol decreasedenzyme activity. NaCl did not change theenzyme activity. Asker et al. (2013) reportedthat Mg2+, Ca2+ and Cu2+ increased proteaseactivity from purified bacter ial enzymefractionate by 110, 112 and 109%, respectively.3.6. Effect of organic solvents on enzymestability



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Results presented in Table 5 showed thathighest enzyme activity by both fractions (P-1and P-2) was obtained in presence of n-butanol.Table 5 revealed that protease was found stablein various organic solvents. Methanol, ethanoland n-butanol increased protease activity byboth protein fractions (P-1 and P-2) whiledecrease in the enzyme activity was measuredin presence of acetone.

Karbalaei-Heidari et al. (2013) reported thatpurified protease was completely stable in ethylacetate, toluene, n-hexane and chloroform at10% and 50% (v/v) and moderate stable inethanol and DMSO at 50% (v/v). Rahman etal. (2006) reported stability of protease indifferent organic solvents. Solvents such as n-dodecane, n-decane, isooctane and n-

Table 4: Stability of partially purified enzyme fractions (P-1 and P-2) under various divalent metal ions.

Metal ions Absorbance at Concentration of liberated Protease Percentage relative660nm tyrosine (µM) activity (U/mL) enzyme activityP-1 P-2 P-1 P-2 P-1 P-2 P-1 P-2

Control 0.9 0.88 150 146.66 27.45 26.84 100 100CaCI2 1.96 1.55 326.66 258.66 59.78 47.33 217.77 176.36MgSO4 0.98 0.75 163.33 125 29.89 22.87 108.88 85.22MnCI2 1.18 1.45 196.66 241.66 35.99 44.22 131.11 164.77BaCI2 0.85 0.88 142.66 147.5 26.10 26.99 95.11 100.56CuCI2 0.85 0.87 141.66 145 25.92 26.53 94.44 98.86LiSO4 0.99 0.99 166 165 30.37 30.19 110.66 112.5ZnSO4 0.77 0.70 128 116.66 23.48 21.35 85.55 79.54

Table 5: Stability of partially purified enzyme fractions (P-1 and P-2) under various organic solvents.

Organic Absorbance at Concentration of liberated Protease Percentage relativesolvents 660nm tyrosine (µM) activity (U/mL) enzyme activity

P-1 P-2 P-1 P-2 P-1 P-2 P-1 P-2Control 0.35 0.33 58.33 55.66 10.67 10.18 100 100Methanol 0.31 0.46 51.66 76.66 9.45 14.03 88.61 137.81Ethanol 0.43 0.55 71.66 92.33 13.11 16.89 122.91 165.98n-butanol 0.99 0.86 166.33 143.33 30.43 26.23 285.27 257.66Acetone 0.25 0.28 43 46.66 7.86 8.54 73.74 83.88

hexadecane (25%, v/v) increased proteaseactivity by 85%, 83%, 78% and 59%,

CONCLUSION

From the present study, it is concluded thatprotease extracted from soil borne bacterialisolates AKS-4 and AKS-6 was purified byammonium sulfate precipitation. Thecharacterization of partially purified proteasewas done. Protease activity was maximum atpH 8.0 of reaction mixture, indicates alkaline

nature of protease. Purified protease is highlyactive at temperature near 50 ºC. Enzymeactivity from protein fractionate P-1 and P-2was increased in presence of calcium chloride.Methanol, ethanol and n-butanol have positiveimpact on protease activity. Furthercharacterization of protease can be carried out

respectively after 30 minutes preincubation.



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to determine its suitability in various industrialprocesses. The purified protease can be used

ACKNOWLEDGEMENTSMany thanks to Professor Aditya Shastri, Vice-Chancellor, Banasthali University, Rajasthan forproviding research facilities.

Mohapatra BR, Bapuji M, Sree A (2003). Productionof industrial enzymes (amylase,carboxymethylcellulase and protease) bybacteria isolated from marine sedentaryorganisms. Acta. Biotechnol. 23: 75-84.

Oke MA, Onilude AA (2014). Partial Purification andCharacterization of Extracellular Protease fromPedicoccus acidilactici. Niger. J. Basic Appl. Sci.22 (1&2): 19-25.

Quadar SA, Shireen E, Iqbal S, Anwar A (2009).Optimization of protease production from newlyisolated strain of Bacillus sp. PCSIR EA-3. IndianJ. Biotechnol. 8: 286-290.

Rahman RNZRA, Geok LP, Basri M, Salleh AB (2006).An organic solvent-stable alkaline protease fromPseudomonas aeruginosa strain K: Enzymepurification and characterization. Enzyme Microb.Tech. 39: 1484-1491.

Rao MB, Tankasale AM, Ghatge MS, Desphande VV(1998). Molecular and biotechnological aspects ofmicrobial proteases. Microbiol. Mol. Biol. Rev.62: 597-634.

Sharma KM, Kumar R, Vats S, Gupta A (2014).Production, partial purification andcharacterization of alkaline protease from Bacillusaryabhattai K3. Int. J. Adv. Pharm. Biol. Chem.3(2): 290-298.

Uddin ME, Rahman M, Faruquee HM, Khan MRI,Ahammed T, Alam MF, Alam MS, Sarkar MKI (2015).Identification, Partial Purification and Characterizationof Protease Enzyme from Locally Isolated SoilBacterium. Am. Res. J. Biosci. 1 (3): 1-9.

Umayaparvathi S, Meenakshi S, Arumugam M,Balasubramanian T (2013). Purification andcharacterization of protease from Bacillus cereusSU12 isolated from oyster Saccostrea cucullata.Afr. J. Biotechnol. 12 (40): 5897-5908.

Walsh KA, Wilcox PE, Perlmann GE, Lorand L, (Eds.),(1970). Methods in Enzymology, vol. 19 AcademicPress, New York, pp. 31–226.

Zhang Y, Zhao J, Zeng R (2011). Expression andcharacterization of a novel mesophilic proteasefrom metagenomic library derived from Antarcticcoastal sediment. Extremophiles 15 (1): 23-29.

for various purposes in food, pharmaceuticaland detergent industry.

REFERENCESAbdel Galil OA (1992). Fermentation of protease by

Aspergillus fumigates and Penicillium sp. J. King.Saud. Univ. 4(2): 127-136.

Abirami V, Meenakshi SA, Kanthymathy K,Bharathidasan R, Mahalingam R, PanneerselvamA (2011). Partial Purification and Characterizationof an Extracellular Protease from Penicilliumjanthinellum and Neurospora crassa. Eur. J. Exp.Biol. 1(3): 114-123.

Anwar A, Saleemuddin A (1998). Alkaline proteases: Areview. Bioresour. Technol. 64: 175-183.

Asker MMS, Mahmound MG, Shebwy KE, Aziz MSA(1998). Purification and characterization of twothermostable protease fractions from Bacillusmegaterium. J. Genet. Eng. Biotechnol. 11: 103–109.

Balachandran C, Duraipandiyan V, Ignacimuthu S(2012). Purification and characterization ofprotease enzyme from actinomycetes and itscytotoxic effect on cancer cell line (A549). AsianPac. J. Trop. Biomed. S392-S400.

Bajaj BK, Sharma P (2011). Thermotolerant extracellularprotease from a newly isolated Streptomyces sp.DP2. New Biotech. 28 (6): 725-732.

Cupp C, Enyard (2008). Sigma’s Non-specific ProteaseActivity Assay-Casein as a Substrate. J Vis. Exp.19: 899.

Gervais PA, Marechal, Molin P (1996). Water relationto solid state fermentation. J. Sci. Ind. Res. 55 (5-6): 343-357.

Gupta R, Beg QK, Lorenz P (2002). Bacterial alkalineproteases: molecular approaches and industrialapplications. Appl. Microbiol. Biotechnol. 59: 15-20.

Joo HS, Kumar CG, Park GC, Paik SR, Chang CS(2003). Oxidant and SDS-stable alkalineprotease from Bacillus clausii I-52: productionand some properties. J. Appl. Microbiol. 95 (2):267-272.

Kader AJ, Omar O, Feng LS (1999). Isolation ofcellulolytic fungi from the Bariohighlands,Sarawak. ASEAN Review of Biodiversity andEnvironmental Conservation (ARBEC).

Karbalaei-Heidari HR, Shahbazi M, Absalan G (2013).Characterization of a novel organic solventtolerant protease from a moderately halophilicbacterium and its behavior in ionic liquids.Appl. Biochem. Biotechnol. 170 (3): 573-86.



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Undoubtedly, modern scientific andtechnological innovations in almost all thebranches of science elicited a revolutionaryimpact on the society, throughout the world.

Throwing some light on the Darkness of Millions of the Suns:Historiography of Biotechnology Communication

and Scientific Temper in Indian Perspective

Uma Prasad RaiCentre for People’s Biotechnology, Latghat, Azamgarh – 276 136Uttar Pradesh, India

ABSTRACT

Biotechnology in recent years emerged as a cascade of tools of immensepossibilities and exhibited substantial role in the service of humans inparticular in agriculture, biomedical and drug development sector.Simultaneously the biotechnological approaches have been severely criticizedby the scientists, Politicians, Administrators, sociologists and even thecommon public. General conception against the newest branch of Bio scienceis gradually being formed and a traditional society as we have in Indiaconstitutes the rationale. Many times the grounds of the criticisms are preinformative and the critics are imaginary and non scientific. Therefore, inbetter service to the society biotechnologists need to acquire the faith of thePeople of the country which can be done only by serving the society enablingthe real benefits. The intermittent interrelation ships of science, scientistsand the society in different centuries along with the significant historiographyof scientific communication among masses and scientific temper in Indianperspective have been discussed. Besides, the historiography and therelevance of the Centre for People’s Biotechnology in National andInternational context have been discussed

Keywords: Historiography / Science / Scientific Temper / BiotechnologyCommunication / India

On one hand, the science has provided severaltools to lead a luxurious and prosperous life,manage several incurable diseases, distances,communication strategies, energy solutions,



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employment opportunities and multi sectarianeconomy. According to a report from ChuckEasley, a Stanford Faculty, seven millions(7,209,700) of researchers were there in theworld who have contributed to science throughpublications or their doctorate thesis in the year2007. On the other hand, the backlashes andthe detrimental manifestations of science in thesociety are enumerated. Green house gasmediated global warming, deterioratingenvironmental health, water and sanitationcrisis, vector borne diseases, depleting forestcovers, endangered species diversity, soilinfertility, agricultural sustainability, insectresistance against insecticides, microbial drugresistance, poverty and hunger, nutritionaldeficiency, social security, natural calamitiesand episodic events of awashes, droughts, earthquakes, waste and sewage management,depleting natural resources, etc. have beenrecognized as the serious challenging threat tothe mankind. The recent scientific andtechnological developments in various sectorsenabled us to elucidate management strategieswhich a few decades earlier were largelyconsidered to be an arduous task to concernedmanagers, policy makers and scientists. For themost of its parts the mitigation strategies, inview of scientists as well as sociologists,require public participation on the gross rootlevel and an integrated effort of administrators,politicians, policy makers, corporate leadersetc. However, during episodic events generalpublic and even the Government authoritieslook forward towards scientists.

The foregoing raises a very pertinent question– who should throw light on the existingdarkness, spread over the society? Obviously,the answer is ‘Scientists’. On ethical grounds,the scientific community cannot escape from

the responsibility. It would not be out of contextto describe two incidents to elaborate theconceptual dilemma and status of scientifictemper in India. The first one, it was the middleof the last quarter of the twentieth Centurywhen the then Prime minister, Late RajivGandhi invited the attention of the countrymento prepare for progression of the country totwenty first century. Meanwhile, in the lastdecade of the last century on a miraculous dayGanesha and almost all the idols of the LordShiva, Kali, Krishna etc. felt hungry and urgedmilk from his followers and began to engulfmilk throughout the world. People rushed totemples with pots filled with milk. The matterof fact came to the author’s notice. Afterreaching the nearby temple it was noticed thatthe crowd was behaving his own way withoutany order or sequence. The milk offered to thegods was going to the gutter through thetemple’s outlet. It was the matter of belief andthe tendency of crowd. Scientifically, theincident was an excellent exhibition ofvisionary illusion and principle of surfacetension. The matter of great concern was that aradio (AIR) Journalist interviewed a Scientistfrom highly reputed research institute situatedat Lucknow, saying that the God has acceptedhis offered milk and emptied his pot filled withmilk. The second incident was the occasion ofBhoomi Poojan ceremony of the NationalAcademy of Sciences, India’s New Buildingin the year 2001. The Eminent Professor, M.G. K. Menon, the then President of theAcademy was offering his worship on behalfof the Academy before laying the foundationstone and Professor S. K. Joshi the then D. G.CSIR was standing adjacent to him chantingthe Durga Saptasati Mantras, A senior Scientist,Perhaps retired Professor of Botany (AU) cameto one of the organizers (Author) and asked



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“what is the sense of Boomi Poojan ceremonyin the Science Academy?” and moved forward.The two stories are sufficient enough todescribe the realistic scenario of scientifictemper in Indian Scientists and society whichconstitutes the basis and rationale for theestablishment of Centre for People’sBiotechnology.

The term “Science” is simply defined as“Systematic knowledge”. The systematicknowledge constitutes empirical, theoreticaland practical knowledge about the naturalworld. The profounder of the knowledge iscalled scientist. The term ‘scientist’ is a newword and was first of all coined by WilliamWhewell in the Nineteenth century.

Prior to the first use of the term scientist, theywere called natural observers and philosophers,for example Paracelsus, Thales, Aristotle etc.The history of modern science dates back tothe scientific revolution of 16th and 17th centuryEurope. During 18th to 20th century evolutionaryhistory of science exhibited progressiveconceptualization of realistic and convincingtheories replacing the misconceptions andobsolete theories. The European Scientificrevolution followed by the industrialization andurbanization manifested lasting effects on thesocio-economy of the globe. Consequently,Europe emerged as the excellent educationalhub of scientific knowledge as well as socialSciences and humanities. The pre independenceIndian leaders were educated and influencedby the western scientific revolution andrenaissance. The inductive conceptual impactsof the leadership on the educated society can’tbe completely ruled out. The discussion so farinferred that the Indian society accepted thescientific innovations mediated positive and

negative manifestations but failed to accept andadopt scientific temper as a life style. It isattributable to deep rooted improper traditionalknowledge.

The early Natural Observers, Philosophers andscientific community faced great difficulties incommunication with the groups of workers orindividuals. They almost worked in isolation.The communication was very slow andaccounted from several months to years.Probably, the urge of the need ofcommunication led to establish the firstScientific Society, “Academia SecretorumNaturae” meaning Academy of the mysteriesof Nature in Naples in the year 1560 byGiambottista della Porta. The Academy waseventually banned by Pope Paul V. Later, incommencing years a number of Academiescame to its existence in various countriesnamely, Academia dei Linceiv in Rome (1603);Academia del Cemento Florence (1657); RoyalSociety of London (1660); Academies dessciences, France; Academie derWissenschaffen, Berlin(1700) etc. During Late18th and early 19th century universities evolvedas excellent convergence centres of democraticvalues and views of pioneers of knowledge ofscience and humanities. In twentieth centurythe Paradigm of science changed and showedinclination towards society to bridge the gapof communication between common peopleand scientific community. During the Britishrule in India, The National Academy ofSciences, India, Allahabad was established inthe year 1930 by the eminent Physicist Prof.Meghnad Shaha to propagate Science andscientific temper in the country. In1935,National Institute of Science India (NISI) wasestablished with almost similar objectives ofNASI. Later in the year 1970 NISI was renamed



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Fig. Showing Biotechnology Exhibition / Communication program Organized by CPB during KumbhMela 2001at Allahabad

Fig. Showing Biotechnology Exhibition / Communication program Organized by CPB during KumbhMela, 2001at Allahabad



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Fig. Showing Field photograph of Jatropha Curcus



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Figure - Sowing Biodiesel from seed to sale

as Indian National Science Academy (INSA).In independent India, our first Prime MinisterPandit Jawahar Lal Nehru, was the firstPolitician to invite the attention of the scientificcommunity to propagate science and scientifictemper among masses. Consequently, abooming trend in the establishment of scienceeducation and research in both, public andprivate sectors of the country was recorded.

In post independent India substantial fundswere invested by the government for science,agriculture, Engineering and medical educationand research which was relatively career drivenand job oriented. The component of socialservice was largely neglected. Medium of theeducation mainly being English developed asense of superiority among the educated youth,ultimately leading to a communication gapbetween the societies they represented. During

the Sixth five year plan (1980-85), PlanningCommission of India identified Biotechnologyas whole, and molecular biology and geneticengineering in particular as a potent tool to copeup with the agricultural and health requirementsof the Indian population and provisions forNational Biotechnology Board were made.In the Year1986, Department of Biotechnologywas instituted upgrading the NationalBiotechnology Board (Bhargawa 2009).

Indian Biotechnological research system isalmost within the framework of specificscientific and Technological system which isspecialized in biotechnological approaches(Seuba and Correa 2010). Almost all thedisciplines adopted Biotechnologicalapproaches for example, AgriculturalBiotechnology, Environmental Biotechnology,Medical Biotechnology, Pharmaceutical



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Fig. Showing Field Photograph during “Intensive Aquaculture Training and DemonstrationProgram 2007 - 08 organized by Centre for People’s Biotechnology, Azamgarh.



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Biotechnology etc. and so on. Interdisciplinaryand fundamental Biotechnological researchinstitutions have not been established so far.Likewise, promotional and communicationactivities to legitimize biotechnology amongthe people are largely neglected and conductedby the concerned specialties.

Meanwhile, in the year 1987, Dr. M. P.Parmeshwaran on the basis of his twenty yearsexperience in Kerala People’s Sciencemovement ignited the flame of the NationalPeople’s Science Movement. During thesedays, the author was working as a researchscholar in Zoology Dept. of AllahabadUniversity and joined the National Movementled by Dr. M. P. Parmeshwaran, Dr. ViondRaina, Prof. Anil Sadgopal, Dr. Dinesh Abroll,Dr. P. K. Mandal, S.K. Bhattacharya etc. Themovement attracted considerable attention ofscientists, Doctors, Engineers Students andeven the common public. However, for certainunavoidable circumstances the movement inUttar Pradesh and Bihar could not get thedesired success in spite of attracting substantialgathering of the crowd. The All India People’sScience Network, two years later in 1989organized a People’s Science Congress at SaltLake Stadium Calcutta (Kolkata) and theNational Working Committee reviewed thereasons of failure in the two states andattributed it to specialized social, political andeconomic factors as well as high level ofilliteracy in the both states. The committeefurther resolved to initiate National LiteracyMission. Consequently, All India Gyan VigyanSamiti came to its existence. The main reasonbehind the failure of the program adaptabilityand wide acceptability of PSM in Uttar Pradeshand Bihar, in author’s opinion, were almostsimilar because of the socioeconomic similarity

and was due to the fact that though beinginformative programs were relatively moreconceptual and theoretical rather thanexemplifying its applicability among the peoplein economic or day to day life terms.

A few years back, in the year 1984 theScientific Advisor to the Prime Minister,Member Planning Commission and renownedEmeritus Scientist Prof. M. G. K. Menon whilediscussing in a workshop on Ganga pollutionorganized by the National Academy ofSciences, India, Allahabad emphasized the needof public participation in pollution control ofthe holy river. In later years, at many scientificgatherings, many opportunities to interact withthe views of Prof. Menon, Prof. S. K. Joshi (D. G, CSIR ), Dr. T. N. Khushu, Dr. V. P. SharmaDirector Malaria Research Centre (NMIR), Dr.P. N. Tondon (AIIMS), Dr. Manju Sharma(Secretary, DBT), etc emphasized the need ofpublic participation in scientific mitigationprograms. The central theme of thedeliberations underlined the need of masscommunication, public awareness and theirparticipation in scientific, technological andsociological developments. The contribution,commitment and views of the above mentionedscientific legends inspired me to a conceptualcentral dogma “the ultimate objective ofscience is society” which eventually evolved afirm determination to work for the great causeof science and society.

In the beginning of last decades of the 20th

century (1992) in a National Symposium on“Science and Technology beyond 2000 A.D.”organized by the National Academy ofSciences, India, Allahabad, Prof. M. G. K.Menon in his historical concluding lecturereviewed the great scientific discoveries of the



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century and drew a frame work of the futuristicscenario of revolutionary prospect of sciencein the 21st century. Further, he pointed outtowards immense possibilities of science andtechnology and named the 21st century as the“Century of Biotechnology”. However, Prof.Menon identified that ethical, social andeconomical factors, lack of wide acceptabilityand adaptability shall act as a major limitingfactor in the traditional Society of the country.Therefore, in order to combat with the situationhe emphasized the need of wide propagationmeasures to popularize the biotechnology forthe cause of human welfare and nationalinterest. On the occasion of concluding session,social scientific responsibility realized by theauthor was the main driving force to decide tocontribute in the area of biotechnologycommunication and research throughinstitution building to bring out the realisticscenario of the societal considerations of thegeneral public. The name “Centre for People’sBiotechnology” was coined in Sir PadampatSinghania library Hall of The NationalAcademy of Sciences, India, Allahabad.

By this time, the general discussions inacademia, political, scientific andimplementing authorities began and werearound arduous task of scientificcommunication among masses and thegovernment authorities were looking towardsengaging voluntary social workers andorganizations. Due to the workload of researchand National Literacy Mission, I temporarilypostponed the idea of CPB. Mean while, theUttar Pradesh People Science Movementwitnessed the untimely and premature demiseof the state president BGVS Professor P. K.

Mandal on April 19th 2000. A few days later,on 30th June, Centre for People’s Biotechnologywas established at Azamgarh. In the same year,perhaps in October, Emeritus Professor and ExPresident of The National Academy of SciencesDr U. S. Srivastava asked me to organize anexhibition of biotechnology during the forty(30+10) days of occasions of Kumbh festivalat Allahabad in the year 2001. I further feltprivileged and inspired when Dr. ManjuSharma, Secretary, DBT; Professor M. G. K.Menon; Dr S. K. Joshi, Director General,Council of Scientific and Industrial Research;Professor P. N Tandan, All India Institute ofMedical Sciences New Delhi; Dr. V. P SharmaEx Director Malaria Research Centre(Deceased), New Delhi; Dr. Anand Kumar IAS,Director DBT; Dr. K. K. Tripathi, PrincipalScientist, DBT etc. accompanied the inauguralfunction.

It would be pertinent to mention the continuousencouragements and critical suggestionsreceived from Dr. Anand Kumar IAS, the thenDirector, DBT and Dr. Piyush Goel Seniorscientific Officer, DBT. Lastly, on 16th March2005, The Centre for People’s Biotechnologywas registered under Societies Registration Act,1860 at Azamgarh, Uttar Pradesh, India and theCPB came to its final existence to servehumanity which was awaited since long.

The sociological problems, sites ofinterventions and achievements of the CPB, inChallenge – intervention – and result (CIRapproach), are under progression and shall bediscussed elsewhere. In Conclusion, improperawareness of biotechnology, prejudice and



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superstitious beliefs, lack of people’sacceptability and adaptability andmisconceptions about biotechnologicalapproaches in various specialties among thesociety are potent limiting factors and thereforeestablishment of the Centre for People’s

Biotechnology with an objective of conductinglocal, regional, global societal research andcommunication strategies, is the demandingeffort of present and futuristic Science and shallthrow adequate light on the darkness of morethan seven millions of suns.

REFERENCES:

Bhrgawa, M.P., 2009 “Biotechnology in India thebeginnings. Biotechnology

Journal Vol. 4 pp 313- 318Department of Biotechnology, 2001. Biotechnology – A

Vision (Ten Year Perspective)Seuba, Xavier and Correa, Carlos 2010. Biotechnology

in India: Its policy and

Normative framework edited by ACC10, PublicationService – www.acc10 cat/

publications, Collaboration: European Business andTechnological Centre –

www.ebtc.euSixth five Year Plan, Government of India, New Delhi,

1981Dpartment of Biotechnology 2005. Annual Report 2004-

2005



83

INTRODUCTION

Blood parameter of fishes is verysensitive indicator of stress, water pollutantsand toxicants. The biochemical disturbancescaused by environmental toxicants has beenaccepted as a major research frameworkconcerning fish toxicology. Effect of industrialeffluent on blood of catfish have been well

Glucose level influences breeding cycle in catfishes

V.K. PandeyDepartment of Zoology, K.S.S .P.G. College,Ayodhya, Faizabad, 224001 (UP)

ABSTRACT

The effect of sub-lethal concentration of paper mill effluents on glucoselevel in blood of Heteropneusts fossilis has been recorded. The glucosecontent of blood was measured. The changes were observed to be statisticallysignificant (P<0.05, P<0.01 in all the three phases of breeding cycle)

Key words – Effluents, Catfish, Blood, water pollution.

described by many workers (Grant and Mehrle1973, Sastry and Sharma 1980, Bhaleroa et al1986, Dhanapakiam and Ramasamy 2001). Thepresent work has been undertaken to observethe change in the blood-glucose level due toexposure in differnet concentration of milleffluents for 96 hours in Heteropneustes fossilis.


The common catfish Heteropneustesfossilis (125 ± 0.25 gram / 6.8 ± 0.15 inch) werecollected locally and were acclimatized tolaboratory conditions for 10 days in a largeaquaria containing 125 Litres of tap water undernatural photoperiod and temperature. Fisheswere fed daily with wheat flour pillets and driedshrimps. Mill effluents of Sanjay paper mill,

maghar, santkabir Nagar,UP , India, wasselected as a test toxicants for biochemicalstudies.Fishes were put in the group of 50animals.GROUP A – Control were placed in tap waterGROUP B – Fishes were exposed to 0.5 and0.8 sublethal concentration of effluents



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The glucose level in the fish bloodplasma was estimated by standard method ofTOLLIN WU as suggested by Oser (1976).

In the present study SD, SE,Confidence Limit and Regression CorrelationCoefficient were calculated and tested properlyby statistical method of Snedecor (1961)


Control Group (A) of test fish showedthe maximum value in the spawning phase ascompared to the pre and post spawning phaseof the breeding cycle (fig 1) but in ExperimentalGroup (B) of test fish the estimation of glucosecontent in the blood were observed to beincreased very significantly in all the phasesof breeding cycle and changes were observedto be statistically significant (P<0.05, P<0.001).

The spawning phase is the most activephase of the life cycle because in this phaserapid growth and proliferation of gonadialtissue occurs resulting in the formation ofgametes therefore higher supply of nutrientsmaterial is required. Under stress condition thefishes requires extra energy for this purpose,gluconeogenesis and glycogenolysis inducedto meet out the energy demand of stressed

BloodControl FishSPME, 0.5 of 96 hrs LC50SPME, 0.8 of 96 hrs LC50 ........

60-

50-

40-

30-

10-

A B C

Fig.1- Glucose content of blood of control and experimental H. fossilis during Pre-spawning (A), spawning(B) and post spawing (C) phases values represent mean SE of monthly observation through the concernedphase. All experimental values significantly different from corresponding control value [p<0.05].



85

fishes (Wedmeyer et al, 1967, Love 1980,Pandey et al, 2003, Mishra et al, 2003,Upadhyay et al, 2005, Achyutha et al, 2006).

In the present study the hyperglycemiaappears to be the main reason for the increaseglucose level under the stress of toxic effluents.This increasing is due to glycolysis and this

took place as a response to stress andtransformations to glucose for enrgyrequirement by fish. Various workers havebeen reported the increase in blood sugarcontent in variety of fishes under environmentalcontaminants (Lockhart et al 1972, Dalela etal 1981, Pandey et al 2010.

REFERNCES

Achyutha Devi, J, Piska, Ravishankar (2006) Effect offluoride on tissue protein of fresh water catfishClarias batrachus (linn). J. Aquatic Bio 21(1):184-187

Bhaleroa, V; Pandya, S.S. and Rao, K.S. (1986) Leadnitrat induced stress on Channa punctatus at sublethal level. Hematological parameters J .Hydrobiol. 2 (3) : 1-9

Dalela, R.C., S. Rani, V. Kumar and S.R. Verma (1981)in vitro hematological alteration in a fresh waterteleost Mystus vitatus following sub acute exposureto pesticide and their combination. J. Environ.Biol. 2(2):79

Dhanapakiam.P. and V.K. Rama samy (2001).Toxic effectof copper and zinc mixtures on some hematologicaland biochemical parameters in common carp.Cyprinus carpio (Linn). J. environ. Biol ; 22 (2) :105-111

Grant B.f, and P.m. mehrle (1973) Endrintoxicosisin rainbow trout (Salmo gardneri). J. fish. Res.Board. Canada. 30 : 31-40

Lockhart, W.L., J.F. Uthe, A.R., Kenney and P.M.,Meherley (1972) Methyle mercury in northern pike(Esox Lucius): Distribution elimination and somebiochemical characteristic of contaminated fish.J. Fish Res. Bd. Canada 29 : 1519-1523

Love, R.M. 1980a.The chemical Biology of fishes,1.Academic press, London/New York.

Mishra, R.N., Pandey, V.K. and Swaroop, A. (2003)Effect of subletha value of LC50 of mill effluenton liver carbohydrate of Heteropneustes fossilis(Bl) in relation to reproductive cycle. J. Liv. World.10(3):21-25

Oser, B.L. (1976) Hawk’s physiological chemistry.TataMcgraw Hills publishing Co. Ltd. New Delhi.

Pandey, V.K., Mishra, R.N. and Swaroop, A. (2003)Effect of paper mill effluent on the mortality andbehavior of Indian catfish Heteropneustes fossilis(Bl). Acta Ecol. 25(1):51-55

Pandey, V.K., Mishra R.N., Upadhyay, Shipra,Swaroop, Anand (2010) Toxicity of paper milleffluents effects liver, protein and amino acidsduring annual breeding cycle of Heteropneustesfossilis (Bl) J. The Scientific Temper(1):pp 131-133

Pandey, V.K., Upadhyay, Shipra, Singh, Indu (2010)Effect of sub lethal concentration of sugar milleffluent on protein and amino acid level in theblood of Clarias batrachus(linn) in relation toreproductive cycle. Abstract SN. 43 AquaticPollution :Endocrine disruption in fish and otheraquatic organism.

Sastry, K.V. and Sharma, K (1980) mercury inducedHematological and biochemical anomalies inophiocephalus (channapunctatus). Toxicol. Letters5:245-249

Snedecor, G.W. (1961) Statistical method Allied PacificPvt. Ltd. New Delhi.

Upadhyay, Shipra, Singh, Indu and Pandey, V.K. (2005)Effect of sub lethal concentration of sugar milleffluent on protein and amino acid level in ovaryof catfish Clarias batrachus in relation toreproductive cycle. Flora Fauna11(2):155-157

Wedmeyer, G.A., Mayer, F.P. and Smith, L. (1976)Environmental stress and fish diseases. Diseasesof Fish Book 5, (Eds : Srieszko, S.E. and H.R.Axelrod) T.H.F. Publication, Neptune. 192pp



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INTRODUCTION

Many workers notably Goel (1975),Chakravorty and Sinha (1982), Imtiyaz andAshok (2010), Kozaric et al (2011), Kuzir et al(2012) and Tlak et al (2013) have studied theQualitative distribution of ACP activity in theintestine of fishes and birds. Sastry and Gupta(1978), Sastry and Malik (1979), Gupta andSastry (1981) and Dalela et al (1982) discussedvarious effects of heavy metals, toxins anddrugs on the distribution of phosphatase in theintestine of fishes. Pollution of aquaticenvironment is a serious problem that shouldbe taken for higher concern. Aquatic pollutionundoubtedly affects fish health and survival.

Histoenzymological Observations on Acid Phosphatase Activity in thePosterior Intestine of HGCL2–Treated Fish, Channa striatus

DhirenderDepartment of Zoology, JNVU, Jodhpur -342001, Rajasthan, India.E-mail: [email protected]

ABSTRACT

Present studies incorporate enterotoxic effects of HgCl2 on the relativedistribution of acid phosphatase (ACP) activity in the posterior intestine offish, Channa striatus. It was observed that there appears to be slightlyincreased ACP activity accompanied by histolytic changes in the columnarepithelial cells lining of villi and intestinal glands of the posterior intestineof treated carnivorous fish. However, in control, these changes were invisible.

Key words: ACP, Channa striatus, HgCl2

Heavy metals are common pollutants of theaquatic environment. These metals are slowpoisoning and slow degradable substanceswhich could have toxic effect for prolongedperiod. Mercury is one of the heavy metalsfound in nature. Some amount of mercurypresent in water enters into the digestive tractof fish through ingestion and food chain andcan produce structural and functionaldisturbances. Interestingly, Mercury and itscompounds accumulate in different tissues ofaquatic organisms including fish (Dhanekar etal 1987). Mercurial compounds are well knownfor causing toxic effects in fish.



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MATERIAL AND METHOD

The living fresh water carnivorous fish, Channastriatus (weighing 160 gram approx.) werecollected from Umaid Sagar, Jodhpur. Theywere kept in aquarium for about 24 hoursbefore subjecting them to experimentaltreatment. For determining the effect of HgCl2on the distribution of acid phosphatase in theposterior intestine of Channa striatus, 1 ml of0.0001% approx solution of mercuric chloridewas injected into the abdominal cavity of the

carnivorous fish. After 12 hours the fish wasdissected out and the small pieces of posteriorintestine were fixed in 10% neutral chilledformalin for 12-14 hours at 40C. Frozensections of the posterior intestine were cut 5-10 µ with the help of freezing microtome andwere processed for the demonstration of acidphosphatase activity using Gomori’s method(1952).


It was observed that ACP activity in theposterior intestine of HgCl2 –treated C. striatusdisplayed relatively increased as compared tocontrol. In the posterior intestine, HgCl2 -induced toxicity resulted in the surfaceepithelial layer of terminal and lateral regionsof intestinal villi seemed to erode followed bycytoplasmic extrusion of the contents. Theconnective tissue of submucosa andmuscularies became loose and disorganized.Lumen of the intestinal villi showed strongACP activity as indicated by distribution oflarger numbers of lysosomal granules (Plate-1, Figs. 1 and 2). On the other hand, in controlexperiment, ACP rich lysosomal granules wereless concentrated in the brush border ephithelialcells of intestinal villi where mild depositionwas found. Intestinal glands showed mild ACPactivity as denoted by low concentration oflysosomal granules along their inner borders.Whereas, lumen of the villi, submucosa and

muscle layers exhibited scattered lysosomalactivity (Plate-1, Figs. 1a and 2a). Thefunctional signification of acid phosphatase indifferent parts of the intestine of fishes havebeen stated by many workers notablyChakravorty and Sinha (1982), Imtiyaz andAshok (2010), Kozaric et al (2011), Kuzir et al(2012) and Tlak et al (2013). The effects ofvarious entero-toxins, heavy metals and drugsin the intestine of fishes have been stated bymany workers notably Sastry and Gupta (1978),Sastry and Malik (1979) and Gupta and Sastry(1981). Recently, Debora et al (2014) reportedslight elevation of acid phosphatase activity inthe intestinal parts of juvenile dourado,Salminus brasiliensis when fed differentconcentration of bovine colostrum. My presentobservations confirm the findings of theseworkers with regard to elevation of ACPenzymic activities and structuraldisorganization of intestine tissue.

ACKNOWLEDGEMENT

The author is grateful to the Head, Department of Zoology, J.N.V.University for providingnecessary facilities.



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Figs. 1 and 2. C S of a part of posterior intestine of HgCl2 -treated Channa striatus magnified 150x and600x respectively. Note erosion of the borders of intestinal villi (IV).Figs. 1a and 2a. C S of a part of posterior intestine of Channa striatus magnified 150x and 900xrespectively. Note ACP reaction on the borders of villi (IV), submucosa (SM), and serosa (SER) showpositive activity.



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REFERENCES

Chakravorty, P and Sinha, G.M. (1982) : Detection andlocalization of alkaline and acid phosphatases inthe digestive system of the adult Catla catla(Hamilton), an indian freshwater major carp byhistochemical methods J . Gegenbaursmorphologisches Jahrbuch, 128(2), 799-808.

Dalela, R.C., Rani, S. And Verma, S.R. (1982): Acidphosphatase activity in tissues of Notopterusnotopterus chronically exposed to phenoliccompounds. Proc. Indian Acad. Sci. 91(1), 7-12.

Debora, B.M., Wiolene, M.N., Thaline M.P.C.,Jose,E.P.C and Raul,M (2014) Histochemistry ofintestine enzymes of juvenile dourado Salminusbrasiliensis fed bovine colostrum. Inernational J.Biological, Biomolecular, Agricultural, Food andBiotechnological Engineering. 8(9), 1031-1035.

Dhanekar, S., Shrivastava, S., and Rao K.S. (1987):Experimental studies on accumulation of mercuryin different fish tissues. J. Perspectives inHydrobiology. 3(18), 87-89.

Goel K.A. (1975): Histochemical study of the activity ofacid and alkaline phosphatases and lipase in thegastro-intestinal tract of Cirrhinus mrigala. J. ActaHistochemica, 54(1): 48-55.

Gupta, P.K. and Sastry, K.V. (1981): Effect of mercuricchloride on enzyme activities in the digestivesystem and chemical composition of liver andmuscles of the catfish, Heteropneustes fossilis.Ecotoxicology and Environmental saftey, 5(4),389-400.

Imtiyaz, H.M., and Ashok, C (2010) : HistochemicalDistribution of Lipase and Acid Phosphatase inthe intestinal tract of the snow trout, Schizothoraxcurvifrons HecKel.

Kozaric, Z., Petrinec, Z. , Kuzir, S., Gjurcevic, E andBazdaric, B (2011): Histochemical Analysis ofDigestive Enzymes in the intestine of adult large -scaled Gurnard (Lepidotrigla cavillone, Lacepede1801) J. Anatomia, Histologia, Embryologia.40(4),314-320.

Kuzir, S., Gjurcevic, E., Nejedli, S., Bazdaric, B. andKozaric, Z. (2012): Morphological andHistochemical study of intestine in wild and rearedEuropean eel (Anguilla anguilla L.). Fishphysiology Biochemistry. 38, 625-633.

Sastry, K.V. And Malik, P.V. (1979): Studies on the effectof dimecron on the digestive system of a freshwater fish, Channa punctatus . Archives ofEnvironmental contamination and Toxicology.8(4), 397-407.

Sastry, K.V. and Gupta, P.K. (1978): Histopathologicaland enzymological studies on the effects of chroniclead nitrate intoxication in the digestive system ofa freshwater teleost , Channa punctatus .Environmental Research. 17(3), 472-479.

Tlak, G., Nejedh, S. And Kozaric, Z. (2013):Histochemical distribution of digestive enzymesin the intestine of the common two-banded SeaBream, Diplodus vulgaris. J. Anatomia, Histologia,Embryologia. 42(3): 161-167.



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The diseases play an important role inthis great balancing act of nature. Accordingto smith et al . (1964) there are more than 250known diseases of fruits and vegetables of themmore than 150 diseases are caused by fungi.The data collect from some studies carried outin India on post –harvest diseases of fruits andvegetables, put the average loss at 20-30percent (Mehta 1975). The injured Averohacarom bola. L. gets infected by fungal formsduring Transit and storage the vegetable iscommercial product utilized for edible. Asurvey in market and storage was made Hathrasand Aligarh.the extent of the rotting rangedfrom 18-25% a time, the whole consignmentis rendered unfit for human consumption. Thediseased fruits were collected separately insterilized in polythen bags and brought tolaboratory for carrying out the presentinvestigation. The effected tissues were soft, brown,black and watery. Under atmosphericconditions Reddish mass of spores appearedon the surface rotten tissues. (fig1) To isolate the pathogen diseased fruitswere surface with 0.1% Hgcl2. And cut in to

A Severe Fruit Rot In Market

Prempal *and R.B. SharmaDepartment of botany, Sarswati P.G. College, Hathras (U.P)*Research scholar Mewar University (Raj.)

small bits which plated on P.D.A. and czspek.sagar media and incubated at 280c+ 20c. Thefungi were isolated and the pathogenicity wastested with. Replicates by artificial inoculation methodof tendon and mishra (1969). Inoculated fruitswere incubated at 280C+ 20C. correspondingcontrols were maintain. The fungi productedsoft rot on fruits within 4-5 days and re-isolation from the yielded the same organism.

FUSARIUM OXYSPORUM ROT:- The Pathogen caused soft rot ofkamrak only through injury, which develop inthe form small circular green whithish greenpatches of 3rd day of incubation, increased inparimeter with the increase in incubationperiod, ultimately forming irregular with darkreddish cavity below the infected light brown–red rind. Internally the tissues were found tobe macerated. The looked peach colour andemitted pungent smell. The pathogen decayedabout 28-32% fruits tissues withen 8 days ofincubation.



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FUSARIUM SOLOMI ROT:- The fungus also developed soft rot.The incident tissues turned dark brown, blackand water soaked with pungent small exudedfrom the rotten tissues about 28-30% rot wasrecorded on 8th day of incubation.

TRICHODERMA VIRIDE ROT :- The pathogen caused soft rot ofkamrak only through injury, which develop inthe form of small circular green whitish greenpatches of 3rd day of incubation, increased inparameter with the increase in incubationperiod, ultimately forming irregular with darkgreen cavity below the infected light brown-red ring. Internally the tissues were found tobe macerated. The looked peach color and

emitted bad smell. The pathogen decayed about20-22% fruit tissue within 8days of incubation.

ASPERGILLUS FLARUS ROT:- Induced soft rot black rot that spreadrapidly to spoil nearly half of the incubatedfruits within 8 days and developed irregularshallow depression accompanied with secretionof black-yellow symptoms associated with thedisease. The pathogen spoiled about 25% fruitswithin 8 days. Latest work on fruit and vegetable rotgiven here. Pre-harvest rot of vegetable andfruits (Singh and Surbali 2001), occurrence ofvegetable rots in market (Sharma and SomKumar 2007) Sharma and Robin (2008), Verma(2009) soft rot of khira (Sharma and Khan2011), New market disease of Ramphal(Sharma et al 2012).

ACKNOWLEDGEMENT The authors are grateful to Dr. A.N.Roy , head Department of Botany , Agra College, Agraand to U.G.C. for financial assistance.

Sharma N.D. and Robin Gogoi (2008) first reportsclerotium rolfsii On Tulsi (ocimum sanctem ) fromindia Indian Phytopathology 61(3):370.

Singh Y.P. and Surbali G (2001). Pre harvest Rot ofvegetable and fruits. Ind. Phytopathology 53:492.

Tandon R.N. and Mishra A.N. (1969) Pathogen city testby knife injury Ind Phytopathology 20:1-12.

Verma V.S.(2009) Incident of post harvest fungal rot ofkin now Maudarin and their management IndianPhytopathology 62(3):335-336.

REFERENCES

Prasad S.S and Bilgrami R.S.(1973) at differenthumidities & temperature the percentage IndianPhytopathology, 26:517-552

Sharma, R.B., Sandeep Kumar and I. khan (2012) newmarket Disease of Ramphal Bio Science Guardian2(1):177

Sharma R.B. Som kumar (2007) occurrence vegetablerots in market. J. of curr. Bio Science 1(1):61-65

Sharma R.B. and I. Khan (2011) soft rot of Khira BioScience Guardian 1(2):512.



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INTRODUCTIONAvian seasonal breeders remain reproductivelyactive mostly for a shorter duration. During theperiod high calcium kinetics occurs in layingbirds to supply the material to eggshell (Dacke,2000; Sugiyama & Kushuhara, 2001). Gonadalsteroids are known to affect largely the serumcalcium (Ca) level in birds (El-Ghalid, 2009).In seasonal breeders, increasing photoperiod isan important cue for onset of breeding (Williamset. al., 1987; Beebe et. al., 2005). This adaptationenables the animals to ensure their offspring areborn when climate is moderate and food isabundant (Atsumi et. al., 2016).

Changes in Total Serum Calcium Level and Shell Calcium Content inBank Myna, Acridotheres ginginianus (Latham) During Breeding Season

Somnath Bose1 and Preeti Singh2

1. Department of Zoology, Ganpat sahai P. G. College, Sultanpur-228001 [[email protected]]2. Department of Zoology, Rana Pratap P. G. College, Sultanpur-228001

ABSTRACTBank Myna is a wild Passerine seasonal breeder of North India. Highervalues of serum Ca had been documented in early breeding season whichgradually reduced in late breeding season. Eggshell Ca content closelycorrespond the rise and fall in serum Ca level. Highest serum Ca level of15.13+0.146mg/dl and eggshell Ca content of 357.25+0.310mg/g wererecorded in first week of June.

Keywords: Serum Calcium, Eggshell Calcium, Bank Myna, Seasonalbreeder, Photoperiod

The Bank Myna (Acridotheres ginginianus) isa commonest and abundantly present wildPasserine seasonal breeder of Northern andCentral Indian plain. The bird is seen nearhuman habitation, found to feed upon kitchenscrap (Ali & Ripley, 2007; Bose & Das, 2012)and behave as if tamed. Breeding female layegg in clutch of 3 eggs mostly, however duringbreeding peak, the clutch size increases, 3-5(Bose & Das, 2012).Except few reports (Bose& Das, 2012; Bose & Das, 2015), seasonalchanges in total serum calcium in accordancewith reproductive activity of wild birds of IndianSub-continent have been grossly neglected. The



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objective of present study was to record changesin total serum calcium level of wild Bank Myna

while serum separated above it. Apical part ofsyringe was then cut with a sterilized sharpblade and serum was taken up for estimationby micropipette. Total serum calcium (Ca) wasestimated by the method described byMoorehead and Biggs (1974) on Erba Chem-5plus V2 semi Automatic Photometer using ErbaCa kits. The birds were freed before dusk neartheir habitat.Eggs are collected from weep-hole nests onweekly basis from May last week to Augustfirst week. In all 5-eggs were taken in accountevery week. Care has been taken to collect only

Month Week Total Serum Ca mg/dl Shell Ca content mg/gMay 1st 13.76+0.251 NA

2nd 14.50+0.369 NA3rd 14.85+0.137 322.53+0.1534th 14.83+0.283 338.37+0.235

June 1st 15.13+0.146 357.25+0.3102nd 14.86+0.193 324.43+0.1363rd 14.78+0.259 310.38+0.2754th 14.52+0.315 307.29+0.252

July 1st 13.86+0.297 295.39+0.1762nd 13.23+0.284 285.73+0.2683rd 12.76+0.258 276.47+0.1844th 11.52 +0.196 268.54+0.264

August 1st 10.95+0.261 260.29+0.1932nd 10.27+0.293 NA3rd 9.68+0.243 NA4th 8.95+0.273 NA

Values are mean + SD of 5 observationsNA= Eggs were not laid during the period

and its relationship with eggshell content duringentire breeding period.

MATERIAL AND METHOD

Observations were initiated in early breedingseason from May first week and continued tillthe end of August. Every week 5 adult BankMyna (Weight 55-65 gms) were locallycaptured in urban Sultanpur (Awadh), wellbefore dawn, from weep holes (commonest siteof their nesting) with the help of a bird catcher.0.5ml of blood sample was collected from awing vein of each bird, under ether anesthesia,using sterilized disposable PVC syringes 30G5/16 (0.30X 8 mm) needles. The syringes withblood samples were kept vertically up for 4hthat allowed the coagulated part to settle down,

Table-1

OBERVATION



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one egg from one nest. Eggshells were removedand membranes separated carefully. Shellswere rinsed with distilled water several timesfollowed by drying in hot air oven at 100oC for24h and crushed. 1gm of crushed shell is ashedby adding 2ml of Nitric acid solution (60-61%v/v) and then kept under 100oC for 24h.Dry samples were oxidized by 2ml of hydrogen

peroxide (30-31% v/v) followed by drying ay100oC for 6h. The dry sample was dissolved in2.0ml of 0.05 M HCl solution (Ogawa et. al.,2004). Amount of Calcium was determined byusing atomic absorption spectrometer (280 FS-AA, Swati Safesecure Equipments Pvt. Ltd.)according to the method of Gimblet et. al. (1967).

DISCUSSION

During the entire breeding period, Bank Mynaserum total Ca level remained high. A gradualrise from 13.76+0.251mg/dl (in first week ofMay) to 15.13+0.146mg/dl (in first week ofJune) had been documented. Increasingphotoperiod promote onset of breeding in avianseasonal breeders (William et. al., 1987; Beebeet. al. , 2005). Dawson et. al. (2001) suggestedthat seasonal breeding in birds involvephotoperiodic control in accordance withendogenous circannual rhythmicity. Increasedsexual activity of female birds accompaniesincreased circulating estrogen level (Whiteheadand Fleming, 2000). El-Ghalid (2009)mentioned that gonadal steroids largely affectthe serum calcium level in birds. However,good degree of reduction in serum total calciumhad been recorded during late breeding season.Bose and Das (2012) reported no furthergonadal recrudescence in Bank Myna duringJune with constant long photoperiod causedphotorefractoriness. Bank Myna serum totalcalcium value of 8.95+0.273mg/dl in last weekof August is in consonance with 8.16+0.12mg/dl of adult Sudanese Geese (Bakhiet et. al.,2006).

The calcium content of Bank Mynaeggshell range between 260.29+0.193 to

357.25+0.310mg/g of shell weight. However,higher values (368-415mg/g) of calcium ascomponent of eggshell have been reported bySchaafsma et. al. (2000) in commerciallyavailable Slovakian chicken eggshell. Highereggshell calcium content was evident in earlybreeding season with respect to late season. Thevalue gradually reduced towards the end ofbreeding season and it closely correspond thereduction in serum total calcium. Moreover,less number of eggs per clutch in late breedingseason had also been reported (Bose and Das,2012). During the laying period, high calciumkinetics occur in birds to supply the materialto eggshell (Dacke, 2000; Sugiyama &Kushuhara, 2001). Dhande et. al. (2006)recorded 17.66+0.38mg/dl of serum calciumin breeding Grey Quail. However, a maximumof 15.13+0.146mg/dl had been recorded inbreeding Bank Myna. Dhodt and Hochachka(2001) documented that Passerines acquirecalcium shortly before and during laying, thuseat extraneous calcium from the surrounding.Ahmed et. al. (2013) mentioned that in layerhens (Bovan) the source of dietary calcium hadno significant effect on eggshell thickness,weight and ash.



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ACKNOWLEDGEMENTAuthors are thankful to Principal, Ganpat Sahai P. G. College, Sultanpur and Rana Pratap P. G.College, Sultanpur for providing laboratory facilities.

REFERENCES

Ahmed, N. M., Atti, K. A. A., Elamin, K. M., Dafalla,K. Y., Malik, H. E. E. and Dousa, B. M. (2013).Effect of Dietary Calcium Source on LayingHens Performance and Egg Quality. J. Anim.Prod. Adv., 3(7):226-231.

Ali, S. and Ripley, S. D. (2007). Handbook of the Birdsof India and Pakistan. Oxford University Press,New York. Vol. 5.

Atsumi, Y., Yorinaga, E., Ota, W. and Yoshimura, T.(2016). Different Photoperiodic Responses in FourLines of Japanese Quail. J. Poult. Sci., 53: 63-66.

Bakhiet, A. O., SultanAli, M., Sharif E. Al and Badwi,S. M. A. El. (2006). Some Biochemical values inthe Young and Adult Sudanese Geese Anser anser.J. Anim. Vet. Adv. 5: 24-26.

Beebe, K., Bentley, G. E. and Hau, M. (2005). Aseasonally breeding tropical bird lacks absolutephotorefractoriness in wild, high photoperiodicsensitivity. Funct. Ecol., 19(3): 505-512.

Bose, S. and Das, V. K. (2012). Distribution, Habit andReproductive Activity of Bank Myna,Acridotheres ginginianus (Latham) in relation toNatural Photoperiod. J. Appl. Biosci., 38(1): 52-56.

Bose, S. and Das, V. K. (2015). Seasonal Changes inTotal Serum Calcium, Inorganic Phosphate Leveland Gonosomatic Index of Bank MynaAcridotheres ginginianus (Latham) with Referenceto Natural Photoperiod. Int. J. Zoo. Invest., 1(1):33-39.

Dacke, C. K. (2000). The Parathyroids, Calcitonin andVitamin D. In “Sturkie’s Avian Physiology’ 5thEdition, ed. Wittow, G. C., Academic press, pp.473-488.

Dawson, A., King, V. M., Bentley, G. E. and Ball, G. F.(2001). Photoperiodic Control of seasonality inBirds. J. Bio. Rhythms, 16(4): 365-380.

Dhande, R. R., Suryawanshi, S. A. and Pandey, A. K.(2006). Seasonal changes in plasma calcium andinorganic phosphate level in relation to parathyroid

structure in grey quail Coturnix coturnix coturnixLinnaeus. J. Environ. Biol. 27: 123-128.

Dhondt, A. A. and Hochachka, W. M. (2001). Variationsin calcium used by birds during the breedingseason. The Condor, 103: 529-598.

El-Ghalid, O. A. H. (2009). Exogenous Estradiol: Bloodprofile, productivitve and reproductiveperformance of female Japanese Quails at differentstages of production. Asian J. Poult. Sc. 3(1):1-8.

Gimblet, E. G., Marney, A. F. and Bonsnes R. W. (1967).Determination of calcium and magnesium inserum, urine, diet, stool by atomic absorptionspectrophotometry. Clin. Chem. 13: 204-214.

Moorehead,W.R. and Biggs H.G. (1974). 2-amino-2methyl-1 propanol as the alkalizing agent in animproved continuous flow cresolphthaleinComplexone procedure for calcium in serum.Clinical chemistry 20,pp.1458-60.

Ogawa, H., Uehara, M., Kuwayama, T., Kawashima, M.and Tanaka, K. (2004). Changes in Calcium ,Magnesium and Phosphorous Contents of Eggshellduring stay in Oviduct Uterus in the Guineafowland the chicken. J. Polt. Sc. 41: 236-240.

Schaafsma, A., Pakan, I., Hofstede, G. J. H., Muskiet, F.A. J., Van Der Veer, E. and De Vries P. J. F. (2000).Mineral, Amino Acid , and Hormonal Compositionof Chicken Eggshell Powder and the Evalution ofits use in Human Nutrition. Poult. Sc. 79: 1833-1838.

Sugiyama, T. and Kusuhara, S. (2001). Avian calciummetabolism and bone function. Asian-AustralianJ. Anim. Sci. 14: 82-90.

Whitehead, C. C. and Fleming, R. H. (2000).Osteoporosis in cage layers. Poult. Sc. 79: 1033-1041.

Williams, T. D., Dawson, A. and Nicholls, TJ. (1987).Sexual maturation and moult in juvenile StarlingsSturnus vulgaris in response to different daylights.The int. J. Avian Sci. 131: 135-140.

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