Immunocytochemical localization of some aminergic and peptidergic neurosubstances in the cephalic neurosecretory system of the dragonfly, Tramea virginia (Rambur) (Anisoptera: Libellulidae)

2008·2.1 MB·English

by  Patankar, N V

Odonatologica37(2): 119-130 June I,2008
Immunocytochemical localizationof some
aminergicandpeptidergicneurosubstances
inthe cephalicneurosecretory system of the
dragonfly, Tramea virginia (Rambur)
(Anisoptera: Libellulidae)
N.V.Patankar and D.B.Tembhare*
DepartmentofZoology,NagpurUniversityCampus,Amravati Road,Nagpur-440033,India
ReceivedNovember6,2006 / RevisedandAcceptedOctober15, 2007
Animmunocytochemicalstudyshowedthepresenceof7neurosubstance-like ma-
terials: FMRFamide,neuropeptide-Y(NPY),substance-P,serotonin,gastrin,chole-
cystokinin(CCK)andvasoactiveintestinalpeptide(VIP)inthemedian,lateral,ventral
andopticneurosecretory cellsgroups(MNC,LNC, VNCand ONC,respectively) in
thebrain andin thecorporacardiaca (CC)oftheadult, T.virginia.Inthe MNCcell
typeAshowed NPY- andserotonin- while Band C cell types showedNPY-, serot-
onin-,substanceP-and CCK-likepositiveimmunoreaction. TheB cell typeinLNC
showed FMRFamide-,NPY-and serotonin- and theC celltype showedonly NPY
and serotonin-like positiveimmunoreaction. InVNC group,theBcell typeshowed
substanceP-andgastrin-,whilethe Ccell typeshowedsubstanceP-andgastrin-and
VIP-like positiveimmunoreaction. Band C cell typesofONCgroupshowed sub-
stanceP-and serotonin-like positiveimmunoreaction. TheCCshowedonlyNPY-like
positiveimmunoreactive intrinsicneurosecretorycells.Thefunctional significanceof
these myotropic and vertebrate gastrointestinalhormone-like substancesin the ce-
phalicneurosecretorysystemofT.Virginiaisdiscussed.
INTRODUCTION
Recently, anumberofworkers(KRAMER, 1984;RAABE, 1989;GADE, 1992;
NIJHOUT, 1994;TEMBHARE,1995;GADEetal., 1997;NASSEL,1999,2000,
2002; PREDEL, 2001; SCHOOFS etal.,2001, NICHOLS, 2003; TAGHERT &
VEENSTRA,2003)havereviewedtheaccumulatedinformationonlocalization.
*Address forcorrespondence:Prof.D.B.Tembhare,44 VijayaNagar,South Ambazari Road, Nag-
pur 440022, India; — [email protected]
120 N.V.Patankar&D.B.Tembhare
isolation, characterizationandfunctionalsignificance ofseveralneurosubstances
in theinsects.
Apartfromseveral histomorphological, histochemical, ultrastructuralandex-
perimental studiesexploring the structureand functionsofthe neuroendocrine
system in variousodonates(ARVY & GABE, 1962;SCHALLER & MEUNI-
ER, 1968;GILLOTT, 1969;THAKARE&TEMBHARE,1975a, 1975b;TEM-
BHARE & THAKARE, 1976;TEMBHARE, 1980,2002) anda study on the
immunocytochemical localizationof some vertebratepeptide hormones in the
nervoussystem ofAeshnacyanea(ANDRIES etal., 1991),nothorough immu-
nocytochemical studieshaveyetbeenmadeonthelocalizationofthe aminergic
and peptidergic neuroactivesubstances in theneurosecretory cells(NSC) inthe
brainofthe dragonflies. Thepresentstudywas, therefore, undertakentolocalize
the presenceof someaminergic andpeptidergic neurohormone-likesubstances
in theNSC inthebrain andinintrinsicneurosecretory cells(INC)andextrinsic
neurosecretoryaxonsinthecorporacardiaca(CC)ofthedragonfly, Trameavir-
ginia.
MATERIAL AND METHODS
HISTOLOGICALTECHNIQUES —Adultdragonflieswerecollected fromthe universitycam-
pus, dissected immediatelyin Bouin’sfluid andfixed for 18-24hours. The materialwasdehydrated
inethanol,cleared in xyleneandembedded in paraffinwaxat58-62°C. Thesectionswerecutat 4-5
pmthickness, fixed toMayer’salbumenized slidesandstained withBargman’sChrome alum,Hema-
toxylinPhloxin (CHP)orEwen’sAldehydeFuchsin (AF).
IMMUNOCYTOCHEMICAL TECHNIQUES —Forimmunocytochemicalstudies,the brain
wasfixedincoldBouin’sfluid,Zambonin or4%paraformaldehydefor24hours.Thereafter,overnight
treatmentincoldsucrosesolution wasgivenforcryoprotectionofthetissue.Frozensectionsof 15pm
thickness werecut onacryostat(Leica). The sectionswerefixed topoly-L-lysin coatedor gelatin
subbed slides. Theslideswerepreserved inadeepfreezerand proceededforimmuno-staining.
TheStreptavidin-biotin-peroxidase(Sigma)methodwasused. Thesections werewashed in PBS
(pH-7.4)for 15min andtreated with 1%bovineserumalbumin (BSA)in PBScontaining0.3%Tri-
tonX-100.ThepolyclonalantibodiesagainstFMRFamide(Incstar;Cat.No.20091,dilution-!;1000),
neuropeptideY(NPY)(Sigma;CatNo.N-952B,dilution-1:800),serotonin (5-HT)(Zymed;Cat.No.
080077,dilution-L1000),substance-P(lCN;Cat.No.11820,dilution-1 TOGO),gastrin(Sigma;G-0785,
dilution-l:800),cholecystokinin(CCK)(Sigma;Cat.No.C-2581,dilution-1:800)and vasoactivein-
testinal peptide(VIP)(ICN; Cat.No. P-2026,dilution-1:800)werediluted inPBS in therespective
concentrations containing0.3% TritonX-100and 1%BSA.Thesections werethen incubated for2
hrs at 25°Cwith eachantibodyseparately. Excessantibody wasrinsed in PBS and incubated with
biotinylatedsecondaryantibodyfor40min,followedbyStreptavidin-peroxidaseconjugate(Sigma).
3-Amino-9-ethylCarbazole (AEC101)wasusedasachromogentovisualize thereddish brownre-
actionproduct.Sectionswerewashedin distilledwatertostopthereaction and mountedin glycerol
gelatin.Thespecificitiesandcrossreactivities oftheaboveantiseraweretestedbyliquid-phaseprea-
bsorptionofthediluted antiserawiththerespectiveconcentrations separately,which inhibited posi-
tive immunoreactions inthebrain sectionsofT. virginia.
Immunocytochemistryin TrameaVirginia 121
RESULTS
HISTOMORPHOLOGYOFTHE
CEPHALICNEUROENDOCRINE SYSTEM
Thecephalic neurosecretorysystemoftheadultT. virginia, consistsoftheneu-
rosecretary cells(NSCs) inthe brain,anda pair ofcorpora cardiaca(CC).
NEUROSECRETORYCELLSINTHEBRAIN - Therearefivepaired groupsofneu-
rosecretory cellsinthebrain:themedial(MNC),mid-dorsal(MDNC)andlateral
groupsintheprotocerebrum, a ventralgroup(VNC)inthetritocerebrumandan
optic group(ONC)inthebasalregion oftheoptic lobes(Fig. 1)(TEMBHARE
& ANDREW, 1995;PATANKAR,2004). Theaxons ofeach groupof MNC,
MDNC, LNC and VNC form themedial(MNSP), mid-dorsal(MDNSP), lat-
eral(LNSP)andventral(VNSP) neurosecretorypathways respectively. Theyrun
dorso-ventrally and emergeas apair offine nerves, thenervi corporis cardiaci
(NCC,) fromtheventero-posteriorregion ofthebrain.Thetwo NCCrunparal-
leltoeachotheroneithersideof the aorta, dorsalto theoesophagus andenter
theanteriorend oftheCC. Theaxons oftheONCs formthe optic neurosecre-
tory pathways (ONSP), whichrun in an antero-posterior directiontowardsthe
medullainternaandterminateinthe closevicinityofthe innerchiasma within
the optic lobeswherethey appearto storeandreleasetheneurosecretory mate-
rial.
CORPORA CARDIACA — The CC is a paired, elongated, fusiformglistering
white body lying just
beneath the brain on
themid-dorsalregion
of the oesophagus,
intimately associated
with the dorsal aor-
ta. Anteriorly thetwo
lobesarefreefromone
anotherbut fused to
formasinglelobepos-
teriorly. Apair oflat-
eral nerves, the nervi Fig. I. Diagrammaticrepresentationofthecephalicneurosecretory
cardiostomatogastrici
system depictingthe distribution ofneurosecretory cell groups and
connects the CC ven- their neurosecretory pathways in the brain of Tramea Virginia.
trally to the hypocer- LNC: lateral neurosecretory cells; — LNSP: lateral neurosecretory
ebral ganglion. Inter- pathways; - MDNC;mid-dorsal neurosecretory cells;- MNC:me-
dialneurosecretory cells;— MNSP:medialneurosecretorypathways;
nally, theCC consists
- ONC:optic neurosecretory cells; - ONSP:optic neurosecretory
of alarge number of pathways; - VNC: ventral neurosecretory cells; - VNSP: ventral
extrinsic axonal end- neurosecretory pathways.
122 N.V.Patankar&D.B.Tembhare
ingsoftheNSCs ofthebrain, someintrinsicneurosecretorycells(INC)dispersed
randomly (Fig. 2)andafewordinaryneurons.
IMMUNOCYTOCHEMISTRY
TheNSCswereclassifiedintoA, BandCcellsbasedontheirstainingaffinities
withthechrome-alumhematoxylin phloxine (CHP)and aldehyde fuschin (AF)
stains(Tab. I).
FMRF-AMIDE POSITIVENEUROSECRETORYCELLS - Threetypes of B cell in
the LNC, onetypeof Band one typeofC cellin the VNCandtwo typesofB
andthreetypesofCcellintheONC,alongwiththeiraxonsinthebrain,showed
strongimmunoreactivity withthe FMRFamideantisera(Figs 3-5).
THE NEUROPEPTIDEYPOSITIVENEUROSECRETORYCELLS - InthebraintheCell
bodiesofoneA,eight
BandfourCcelltypes
intheMNCandsix B
and four C cell types
in the LNC showed
strong reaction with
the neuropeptide-Y
antibody (Figs 3, 6,
7). TheINCintheCC
also exhibitedpromi-
nent neuropeptide-Y
positive immunoreac-
tion(Fig. 8).
THE SUBSTANCE-P
POSITIVE NEUROSE-
CRETORY CELLS -
Ten Aand two C cell
typesintheMNCand
all B andC cell types
intheVNCandONC,
along with theirneu-
rosecretory pathways,
showedintenseimmu-
noreaction with the
substance-P antisera
Fig.2.Sectionthroughthecorporacardiaca ofTrameavirginiashow- (Figs 9-11).
ingtheintrinsicneurosecretory cells. — AO;aorta; — CCC: corpora
cardiaci connectives;—CB-INC:chromophobicintrinsicneurosecre- THESEROTONINPOS-
torycells; —CL-INC:chromophillicintrinsicneurosectretory cells;— ITIVE NEUROSECRE-
NSM-neurosecretorymaterial. — [Scalebar: 1000pm] TORY CELLS - The
Immunocytochemistryin Tramea Virginia 123
TableI
Cytomorphologicalcharacteristics ofthecerebralneurosecretorycellsin TrameaVirginia
s# CytomophologicalCharacteristics NeurosecretoryCell Type
A-cell B-cell C-cell
1.0 STAININGAFFINITIES
1.1 Chrome-alum haematoxylinphloxine Blue black Red Blueblack inclusion.
(CHP) red cytoplasm
1.2 Aldehydefuchsin(AF) Dark purple Greenish Purpleinclusion,brown
cytoplasm
2.0 SHAPE
Pyriform Spherical Oval
3.0 SIZE
3.1 Cell diameter(pra) 1063±027 960± 080 18621056
3.2 Nuclear diameter(pm) 572±032 540± 027 920±036
4.0 DISTRIBUTION
4.1 Medial group 40-45 25-30 8-10
4.2 Mid-dorsal group - 10- 15 10- 12
4.3 Lateral group - 15-18 4- 6
4.4 Ventral group - 15-18 10-12
4.5 Opticgroup - 8- 12 10-12
serotonin-positive immunoreactivity was observeddistinctlyinoneA,two Band
two CcelltypesintheMNC,fourB celltypesinthe LNCandsix B celltypesin
theONC andtheiraxons inthebrain (Figs 12-14).
THEGASTRINPOSITIVE NEUROSECRETORY CELLS - One B and one C cell
typeinthe MNC,threeBandC celltypesintheLNC andfiveBandoneC cell
typesin the VNC showedthe immunoreactionwiththe gastrin antibody (Figs
15-17).
THECCK POSITIVENEUROSECRETORY CELLS - Two B and C cell types of
MNCandthreeCandall B celltypeintheVNC displayed astrongimmunore-
actionwith theCCK antibody(Figs 18-19).
THEVIPPOSITIVENEUROSECRETORY CELLS - Six Band fourCcell typesin
theVNC andallB celltypesintheONC showedtheimmunoreactionwithVIP
antibody (Figs 20-21).
DISCUSSION
Thehistomorphological organization ofthe cephalic neuroendocrinesystem
in the adultdragonfly, Tramea virginia was described by TEMBHARE & AN-
DREW(1994) and PATANKAR(2004)and isbasically similartothatinAeshna
cyanea (CHARLET, 1972a, 1972b;TEMBHARE,1980)and Orthetrumchrysis
124 N.V.Patankar& D.B.Tembhare
Immunocytochemistryin Tramea Virginia 125
(TEMBHARE &THAKARE, 1976).Thepresent study haslocalizedthe pres-
enceofFMRFamide-, NPY-,serotonin-, substanceP-,gastrin-, CCK-andVIP-
likesubstances intheNSCsof differentgroupsinthe brainandCC oftheadult
T. virginia.
TheFMRFamide-likepositive immunoreactionhas been noticedintheNSC
inthebrainofseveralinsectspecies. Inthelocust, SchistocercagregariaMYERS
& EVANS(1985)tested BPPantiseranot specific fortheCterminalhexapeptide,
along with liquid preabsorption experiments with BPPand FMRFamide, and
suggested thattheendogenous peptide antigen containedinthestainedneurones
maybelong to the pancreatic polypeptide family or to the FMRFamidefamily.
WHITEetal.(1986) characterizedneuropeptide-FMRFamide-like immunoreac-
tivityinthefruit fly;Drosophila melanogasterandsuggested itssignificance during
development. EICHMULLERetal. (1991)analysed thebrainandsuboesopha-
geal ganglion ofthehoneybee ApismelliferaL. immunocytochemically andob-
servedFMRFamide-likeimmunoreactivity inthecellsprojectingto theCCfrom
themedian, lateralandsuboesophageal ganglion, suggestngtheirneurosecretory
nature. Definitiveevidencefor thepresenceofFMRFamideininsects was pro-
videdbyKINGAN etal.(1990)whoextractedandpurified thispeptide fromthe
centralnervous system ofthehawkmoth, Manducasexta. HEWES etal. (1998)
analyzed the effectof eightFMRFamide-relatedpeptides expressed by neuro-
secretory cells onnerve-stimulatedcontraction(twitch tensions) of Drosophila
larval body wall muscles encoded by Drosophila FMRFamidegene. They sug-
gested thatFMRFamidefunctionsas aneurohormonetomodulatethestrength
ofcontraction atthelarvalneuromuscularjunction inwhichthe functionalrole
of the seven peptides appearto be functionally redundant. ROBB & EVANS
(1990) studied the quantitativedistributionof FMRFamide-likepeptides inthe
nervous system and in theirputative target sites in the locustSchistocerca gre-
gariaand suggested thatFMRFamide-likepeptides in thelocustfunctionboth
as circulatingneurohormonesandas locallyreleasedneuromodulatorsorneuro-
transmitters. RANKIN&SEYMOUR(2001), immunocytochemically observed
amaterialsimilartothatofFMRFamideintheneurosecretorysystem oftheear-
wig,Eubeorelliaannulipes andsuggested itsfunctionalsignificance. Recently, NI-
CHOLS(2003),reviewedstructures,precursors, organizations, distributionsand
Figs3-21. Immunocytochemistryoftheneurosecretorycellsinthebrain andcorporacardiaca (CC)
ofTramea virginia:(3-5)brainNSCstainedwithFMRFamideantibody:(3)B-LNC andLNSP (ar-
row),(4)C-VNC,(5)B andC-ONCandONSP(arrow); - (6-8)NSCstained withNPYantibody:
(6)BandC-MNC, inthebrain,(7)C-LNC,inthebrain,(8)ExtrinsicaxonalfibersinCC(arrow); —
(9-11)NSCinthebrainstainedwith SubstancePantibody:(9)BandC-MNC,(10)BandC-VNC,
(11)Band C-ONC; — (12-14)NSCinthe brainstainedwith Serotoninantibody:(12)A,BandC-
MNC, (13)B-LNC,(14)B-ONC; — (15-17)NSCin thebrainstainedwith Gastrinantibody:(15)
BandC- MNC, (16)C-LNC, (17)B and C-VNC; - (18-19)NSCinthe brainstainedwith CCK
antibody:(18)A,BandC-MNC,(19)B andC-VNC; —(20-21)NSCinthebrainstainedwithVIP
antibody:(20)B andC- VNC, (21)B andC-ONC. —[Scalebars: 1800;im]
126 N.V.Patankar&D.B.Tembhare
activitiesofFMRFamiderelatedpeptides(FaRPs) encodedby themelanogaster
FMRFamide(dFMRFamide), myosuppressin (Dms) andsulfakinin(Dsk)genes
andpredictedtheirsignalingpathways andbiologicalfunctions. DUTTLINGER
etal. (2003) observedthestructureof the FMRFamidereceptorandthe activ-
ity ofthecardioexcitatory neuropeptide ina mosquito. Theyconcludedthat its
structureandactivity are conservedinthemosquito,whichmayhelp toincrease
the frequency of spontaneouscontractionsofthe larval heart, thus increasing
heartrate.
REMY etal. (1988)demonstratedaneuropeptide relatedto mammalianneu-
ropeptide Y(NPY) inLocusta migratoriainvarious neurosecretorycells which
also display anFMRFamide-likeimmunoreactivity inthecephalic andthoracic
nervous systems. They inferredthat inlocustNSC thesetwoantiserarecognize
twodistinctantigenic sitesbelonging eitherto alargepolypeptide, orto twodis-
tinctneuropeptides. SETTEMBRINIetal. (2003)observed NPY andNPY-Y1
receptor-like immuno-reactivity initsreceptorsandcolocalizationwithcCK-LI,
inthecentralnervoussystem ofTriatomainfestans andanalysed its distribution
patterns,suggesting thatpeptideandreceptoraremainly involvedintheprocess-
ing ofinformationcoming fromsensory receptors
SCHURMANN & KLEMM (1984), observed serotonin immunoreactive
neurons in the brain of the honeybee and concluded that the serotonin- and
catecholamine-containing neuronsoftenoccur togetherinthe samebrainareas.
KLEMM etal.(1986) observedneuronsreactive toantibodiesagainst serotonin
inthestomato-gastric nervous system andin thealimentary canaloflocustand
crickets and suggested thatserotonin actsas a neurotransmitterand/or neuro-
modulatoronintestinaland somesomaticmusclesandglandularcells, andalso
actsas a neurohormonereleased fromthe neurohaemalsites intothe body flu-
id. GRANGER etal.(1989) observed serotonin-immunoreactiveneuronsinthe
brain ofManducasexta during larval development and larval-pupal metamor-
phosis andsuggested thepossible relationship ofserotonintocerebralneuroen-
docrinefunctionsduringthepostembryonic development.AccordingtoNASSEL
(1995), in Drosophilamelanogasterthese peptides, liketheirmammaliancounter
parts, actasneuromodulators(neurohormones) through asecond-messenger and
facilitaterelease of hormones, such as prothoracicotropic (PTTH), allatotropic
(ATH), allatostatic(ASH)andeclosion hormone(EH), PREDELetal. (2001a,
2001b) discussedthe structure, distribution, pharmacological activitiesand mi-
metic analogs of the myoinhibitory and myostimulatory neuropeptidesin the
American cockroach, Periplaneta americana.Thedistributionof cholinacetyl-
transferase(CHAT); GABA, histamineandoctapamine andserotoninhavebeen
reported inthelarvalchemosensory system ofDrosophilamelanogaster(FRAN-
CIOSE etal., 2002) suggesting theirroleas neurotransmitters.NASSEL(2002)
suggested thattheneuropeptides in thenervous system of Drosophila andother
insects actas a neuromodulatorandneurohormones.LANGE (2004) observed
ImmunocytochemistryinTramea Virginia 127
thatserotoninincreasesthe frequency and amplitude ofspontaneouscontrac-
tionsandleadsto anincreaseinthebasaltonus oflocustoviducts suggesting the
neurohormonalrole forserotoninin thecontroloflocustoviducts. MARTINI
etal.(2004), observed thepossibleregulation by serotonin(5-HT) to detect the
presenceofanaquoporin-like waterchannelinthemalphigian tubules(MT)of
thehematophagous insectRhodniusprolixus, andsuggested thattheup-regulated
expression ofMTMIPmRNA aftera bloodmealis probably due to theaction
of5-HT via. acyclic AMPdependent pathway.
SEVERINI etal. (2002) stated thattachykinins havebeen recognized to have
a varietyofeffects inphysiological andpathological conditions, andthereisevi-
dencesuggesting intrinsicneuroprotective and neurodegenerative properties of
theseneuropeptides. Thisreviewprovides an update onthecurrent bodyofknow-
ledgeregarding theoccurrence anddistributionoftachykinin intheanimalking-
domandthephysiological andpharmacological actionsoftachykinins, outlining
theimportance ofthislarge peptide family. LUNDQUIST & NASSEL, (1990)
demonstratedneurons displaying substanceP-(SPLI), FMRFamide-(FLI), and
cholecystokinin-like (CCKLI) immunoreactivity inthethoraco-abdominalgan-
gliaofthe dipterans Drosophila melanogasterand Calliphora vomitoria.
Gastrin/CCK-likeimmunoreactivity havebeenobservedintheNSCandCCof
variousinsects. DUVE& THORPE (1984) studied itsdistribution in theretro-
cerebralcomplex ofCalliphora vomitoriaandobservedthatCC containsCOOH-
-terminalspecific gastrin/CCK-like materialwithintheintrinsic cells andinthe
neuropile, suggesting its co-existencein thecellsofCC. HANSEN et al.(1987)
observed COOH-terminalspecific gastrin/CCK-likematerialinCC-CAcomplex,
suggesting theappearanceoftheseantibodiesearlyinevolutionwithinneuralel-
ements andbeingconserved during phylogeny. ANDRIES etal. (1991) studied
antiseragastrin/CCK, VIPimmunohistochemically inthebrain,suboesophageal
ganglion and corpora cardiaca of Aeshna cyanea and found multiple peptide
immunoreactivities.TAMARELLE& VANHEMS,(1997) characterizedanew
neurosecretory cell-type inthe locust Schistocerca gregaria pars intercerebralis,
immunolabelledwith an antiserumagainst a vertebratepeptide related to gas-
trin-cholecystokinin (CCK-8 (s)), both in situand in primary cell cultures. On
thebasisoftheir number, sizeandlocalizationthey suggested thepossibility of
CCK-likeneurosecretorycellscorresponding to aneurosecretorycelltypewhich
has not to datebeen identified at the fine structural level. HEWES& TAGH-
ERT(2001) scanned therecently completed Drosophila genomesequence forG
protein-coupled receptorssensitive to bioactivepeptides (peptide GPCRs) and
also scannedfor genesencoding potential ligands. They described 22bioactive
peptideprecursorsandfoundthatatleast32Drosophila peptidereceptorsappear
to haveevolved fromcommonancestors of 15monophyletic vertebrateGPCR
subgroups (e.g.,theancestralgastrin/cholecystokinin receptor),shedding lighton
theevolutionary history ofpeptideGPCRs andproviding a template forphysi-
128 N.V.Patankar&D.B.Tembhare
ological andgenetic analyses ofpeptide signalinginDrosophila.
Thepresentpaperprovides informationbasedonimmunocytochemical stud-
iesregarding thepresenceof some vertebratehormone-, neurotransmitter-and
neuromodulator-likesubstances inthecerebralneurosecretorysystem whichmay
representco-localizationwiththe intrinsicinsect neuropeptides andmay exerta
vitalrolein thelifeofan insect.
ACKNOWLEDGEMENTS
We aregratefultoProf.N.K.SUBHEDAR forprovidingtrainingin immunocytochemicaltech-
niquesand permittingtowork inhis laboratory.The“UniversityResearch Fellowship”awardedto
NVPbytheNagpurUniversityisalsoacknowledged.
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