Immunocytochemical demonstration of some vertebrate peptide hormone-like substances in the midgut endocrine cells in Tramea virginia (Rambur) (Anisoptera: Libellulidae)

2006·2.1 MB·English

by  Patankar, N V

Odonatologica35(2): 151-158 June 1,2006
Immunocytochemicaldemonstration
ofsomevertebratepeptidehormone-like
substances in themidgutendocrinecells
in Tramea virginia (Rambur)
(Anisoptera: Libellulidae)
N.V.Patanka*r and D.B.Tembhare
DepartmentofZoology,NagpurUniversityCampus,Amravati Road,Nagpur-440033,India
Received September2,2005 / RevisedandAcceptedNovember 16,2005
Thepresentimmunocytochemicalstudyrevealsthepresence ofwell-defined endo-
crinecells,intermingledwiththecolumnarcellsoftheepitheliuminthemidgutregion
ofthealimentarycanal ofT.virginia.Themidgutendocrine cellsareof2types,the
open-typemidgutendocrine cells(OMEC)withalongtubuleopeningintothelumen
ofthemidgutandclose-typemidgutendocrine cells(CMEC)whicharesphericalin
shapeand devoidofextendingtubules.Various gastrointestinal hormone-like sub-
stancesarelocalized inrespectivetypesofmidgutendocrine cellsindifferentregions
ofthemidguti.e.anterior, middle and posterior.TheNPY, FMRFamideand 3-en-
dorphinwerelocalized in theopen-typewhile substanceP, gastrin,CCKand VIPin
theclose-typemidgutendocrine cells. Themidgutendocrine cellsinT. Virginiadiffer
fromeach otherintheirlocation,cytomorphologicalandimmunocytochemicalchar-
acteristics representingdifferent typesofendocrine cells. Functional significanceof
these myotropicandvertebrate gastrointestinalhormone-like substancesinthemid-
gutendocrine cellsofT. Virginiaisdiscussed.
INTRODUCTON
Withthehelpofelectronmicroscopic andimmunohistochemicaltechniques, the
endocrinecellsinthemidgut ofseveralinsectspecies havebeenlocalizedshowing
immunoreactivity withantibodiesof variousvertebratepeptidergic andaminer-
gichormonalsubstances (FUJITAetah, 1981;BROWN, 1986;SCHOLS etah,
1987;CRIMetah, 1992;LUNDQUISTetah, 1994;NIJHOUT. 1994;LEHANE,
*Addressforcorrespondence:D.B,Tembhare,44VijayaNagar,South AmbazariRoad,Nagpur-440
022,India; — e-mail;[email protected]; — [email protected]
152 N.V.Patankar&D.B.Tembhare
1998;VEENSTRA etal., 1995;LANGE, 2001; WANGet al., 2001; NEVES et
al.,2002). Although thefunctionalsignificance ofthemidgutendocrinecellsand
their secretory materialis still obscure, some evidenceshavebeenaccumulated
suggesting theirinvolvementin thecontrolof transportand digestionof food
materialby inducing peristaltic contractionintheforegut andstimulatingrelease
of digestive enzymes fromcolumnarcellsintothelumenof midgut,respectively
(FUJITA etal., 1981;NIJHOUT, 1994;MURALEEDHARAN,1995;NACH-
MANetal., 1997;FUSEetal.. 1999;PRABHU& SREEKUMAR, 1999;WEI
et al., 2000;HILL&ORCHARD.2005).
BesidessomeinformationonAeshnacyanea(ANDRIES&TRAMU, 1985b),
no comprehensive efforts have beenmade to demonstratecytomorphological
characteristics andimmunocytochemical localizationof various vertebratehor-
mones in Odonata.The present study was, therefore, undertaken to elucidate
cytomorphology andimmunoreactivityof themidgut endocrinecellsin Tramea
virginia.
MATERIALANDMETHODS
HISTOLOGICALTECHNIQUES. - Adultdragonflieswerecollected fromtheuniversitycam-
pus premisesand theiralimentarycanals weredissectedand fixed immediatelyfor 18-20h inaque-
ousBouin’sfluid,dehydratedinethanol,clearedinxyleneandembedded inparaffinwaxat58-62°C.
The 4pmthick sectionswerestained with Ehrlich’s Hematoxylin-Eosinand Heidenhain’s Iron-He-
matoxylin-OrangeG.
IMMUNOCYTOCHEMICALTECHNIQUES. - Forimmunocytochemicalstudies,the mid-
gutwasfixed incold Bouin’sfluid,Zambonin or4%paraformaldehydefixativesfor24h.Thereafter,
overnighttreatment ofcoldsucrosesolution wasgivenforcryoprotection oftissue and frozen sec-
tionsof 15pm thickness werecut onthe cryostat(Lieca).The sectionswereaffixed topoly-L-lysin
coated orgelatinsubbedslides.
Theslideswerepreserved indeepfreezerand proceededforimmuno-staining.
TheStreptavidin-biotin-peroxidase(Sigma)method wasemployedduringthepresentstudy. The
sectionswerewashedin PBS(pH-7.4)for 15min andtreated with 1%bovineserumalbumin (BSA)
inPBScontaining0.3%Triton X-100.ThepolyclonalantibodiesagainstFMRFamide(Incstar;Cat,
No.20091,dilution-1:1000),neuropeptideY(NPY)(Sigma;Cat No.N-9528,dilution-1:800),p-en-
dorphin(Sigma;Cat.No.El520,dilution 1:1000),substance-P(ICN;Cat,No. 11820,dilution-1:1000),
gastrin(Sigma;G-0785,dilution-1:800),cholecystokinin(CCK)(Sigma;Cat.No.C-2581,dilution-1;
800)and vasoactiveintestinal peptide(VIP)(ICN; Cat.No. P-2026,dilution-1:800)werediluted in
PBS inrespectiveconcentrations containing0.3%TritonX-100 and 1%BSA and thesectionswere
thenincubated for2h at25°Cwith eachantibodyseparately.ExcessantibodywasrinsedinPBSand
incubated withbiotinylatedsecondaryantibodyfor40min,followed byStreptavidin-peroxidasecon-
jugate(Sigma).3-Amino-9-ethylCarbazole(AEC101)wasusedaschromogentovisualizethereddish
brownreactionproduct.Sectionswereagainwashedindistilled watertostopreactionandmounted
in glycerolgelatin.The specificities and crossreactivities oftheaboveantiseraweretestedbyliquid-
phasepreabsorptionofthediluted antiserawith respectiveconcentrations separatelyandwasfound
that it abolished all immunostainingsinthemidgutsectionsofT.virginia.
Midgutendocrine cellsin TrameaVirginia 153
RESULTS
Themidgut wallis composed ofan outerlayer oflongitudinal muscles, mid-
dlelayer of circularmusclesand innerlayer ofepithelium consisting two types
ofcells; thetall columnarcells andtinyregenerative cells. Besidestheabovecell
types, the immunocytochemical studies reveal the presence of endocrinecells
intermingled withthecolumnarepithelial cells. They can beclassified intotwo
principal types; the openandclose-types of midgut endocrine cells.Theopen-
-type midgut endocrinecellsare closely associated withtheinnersurfaceof the
circularmusclelayer and provided withthe distalcytoplasmic tubule opening
intothe lumen, while, the close-type midgut endocrinecells are devoidof the
distalcytoplasmic tubularprocess (Fig. 1). Themidgut endocrinecells vary in
location, shape, sizeandimmunoreactivity fromeachotherandcanbeclassified
invarioussub-types(Tab. I).
THE OPEN-TYPEMIDGUTENDOCRINE CELLS(OMEC)
TheOMECare oval, conicalorellipticalin shapeandeach cellbears abunch
of dendrite-likeprocesses apicallyand along tubularprocess distallyleading to
thelumen. They showed immunoreactionswiththe NPY,FMRFamideand(3-
endorphin antisera.TheNPY-positive OMECare observedin large numberin
the anteriorand middleregions of the midgut (Fig. 2) and the FMRFamide-
positive OMEC are densely distributedintheanteriorandposterior regions of
Fig. 1.Diagrammaticrepresentationofthemidguthistologicalstructure showing-co-
lumnarepithelialcells(CEC),regenerativecells(RC)andopen-typemidgutendocrine
cells (OMEC) and close-type midgutendocrine cells(CMEC) and other histological
structures.[LM=longitudinalmuscles, — CM=circularmuscles,— BM=basement
membrane,—PM=peritrophicmembrane, —D=dendrites,—TP=tubularprocesses]
154 N.V.Patankar& D.B.Tembhare
Table I
Cytomorphologicaldetails ofmidgutendocrine cells with respect to different vertebrate peptide
hormone-like substancesin Tramea virgina
Midgut Antibodies Size(pm)
Endocrine applied Location
Cells Cell Nucleus Dendrite Tubule
diameter diameter length
OMEC NPY 13.5±2,78 6.80± 1.92 18.23±2.36 70.52 ±7.73 AMG,
MMG,
FMRFamide 14.82± 1.78 7.25± 1.40 52.36±4.95 AMG,
PMG
(3-Endorphin 18.15± 1.72 7.08± 2.44 11.26±2.36 44.26± 5.04 AMG
CMEC VIP 6.38± 2.32 3.12± 1.94 MMG
Gastrin 7.26±2.12 3.56±2.36 AMG
Substance P 8.52+ 2.10 4.46± 1.92 MMG
CCK 10.74+ 2.42 5,18± 2.64 MMG
(AMG-Anteriormidgut;MMG-Middlemidgut;PMG• Posteriormidgut).
the midgut (Fig. 3)whilethe (3-endorphin immunopositive OMECare confined
to theanteriorpartof themidgutonly (Fig. 4).
THECLOSE-TYPE MIDGUTENDOCRINECELLS (CMEC)
TheCMECare spherical inshape butdevoidofany kind ofprocesses. They
showedimmunoreactionwiththeVIP, gastrin, substance Pand CCKantisera.
TheVIP,substancePandCCK- positive CMECare observedin themiddlere-
gion(Figs 5, 7& 8) while thegastrin-positive CMECin the anteriorregion of
themidgut(Fig. 6).
DISCUSSION
Themidgut endocrinecells are well distinctinthe dragonfly, Trameavirginia
andonthebasisofshape,sizeandimmunoreactivity to theantibodiesof various
vertebratehormones, theycanbe classifiedintovariouscell-types similarto that
inotherspecies ofinsects(MONTUEGA etal., 1989; SIVASUBRAMAN1AN,
1992;ZITNAN etal., 1993;VEENSTRA etal., 1995; NACHMANetal., 1997;
PREDEL etal., 1999,2001;NICHOLSetal.,2003;HILL&ORCHARD,2005).
MONTUEGA etal., (1989)noticed ten typesof midgut endocrine cellsimmu-
noreactiveto ten differenttypesofneuropeptides in Aedesaegypti whileduring
thepresentstudy seventypesofmidgut endocrinecellsimmunoreactiveto seven
differenttypes of neuropeptides canbe recognized in Tramea virginia.
TheNPY, FMRFamideand 3-endorphin immunoreactiveopen-type endo-
Midgutendocrine cellsin Tramea Virginia 155
crine cells are well distinct in T. virginia.
The neuropeptide Y (NPY) immunoreac-
tivepeptidewasdemonstratedinDrosophila
melanogaster(BROWNetal., 1999).FMR-
Famide-like immunoreactionwas reported
in the midgut inthe larvaeof Helicoverpa
zea(CRIMetal., 1992),beeMelipona quad-
rifasciata anthidiodes(NEVES et al.,2002)
and locust Locusta migratoria (LANGE,
2001). The immunocytochemical locali-
zationof (3-endorphin-like substance has
beennoticedintheguttissuesofPeriplaneta
americana(SCHOLSet al., 1987).
Usingvariousradioimmunoassay, immu-
nohistochemicaland immunofluoresence
techniques, it hasbeen foundthat FMR-
Famide related peptides not only modu-
latemidgut contraction(NICHOLS, etal.,
1999) but are also directly involvedin the
controlofdigestive process (NEVES etal.,
2002) and gut motility (RANKIN,2001).
HILL&ORCHARD(2003) foundthatthe
gut tissue and associated nervous system
ofLocustamigratoria, containedFMRFa-
mide-likeimmunoreactive(FL1) material
throughout thefivelarvalinstarsandadult
stageinbothmaleandfemaleandrevealed
Figs 2-4. Immunocytochemistry of the
the significant differencein thecontent of
midgutendocrine cells:(2)OMECstained
ELI in anteriorand posterior midgut by with NPYantibody(arrows) *450; — (3)
radioimmunoassay withrespect to the tis- OMECstainedwithFMRFamideantibody
suesize. (arrows) x450; - (4)OMECstainedwith
Similarly,gastrin, CCK,substancePand (3-Endorphinantibody(arrows) x400.
VIP immunnoreactiveclose-type midgut endocrinecells areobserved in T Vir-
ginia.Thevertebrate-likegut-neuropeptides viz. gastrin, CCK,substancePand
VIP -likematerialshavebeendescribedinthemidgutof someinsects(ANDRIES
&TRAMU, 1985a, 1985b;LUNDQUIST etal„ 1994).
Severalmyotropic factorswerereported inthemidgut endocrinecellsofthree
lepidopteran species: Manducasexta, Agrotis segetumand Spodoptera exempta
(YI et al., 1992). These peptides have FMRFamideC-terminusand thushave
homology withFMRFamide.RecombinantDNA techniques revealedagenein
Drosophila melanogasterthatencodestwo additionalmembersofthegastrin/CCK
family.Theseare termedasdrosulfakininIandII (NICHOLS etal., 1988).
156 N.V.Patankar &D.B.Tembhare
Figs5-8.Immunocytochemistryofthemidgutendocrine cells:(5)CMECstainedwithVIPantibody
(arrow)x400; —(6)CMECstainedwith Gastrinantibody(arrow)*400; —(7)CMECstainedwith
Substance Pantibody(arrows) x400; — (8)CMECstained withCCK antibody(arrows) x400.
Accordingtothecurrentresearchfindings,Gastrin/CCK-like immunoreactivity
isattributed, atleastinpart,tothesulfakinins(NICHOLSetal., 1988;FONAGY
etal., 1992;PREDEL etal., 1999). Sulfakininshavebeen shownto stimulatedi-
gestive enzyme secretionin theweevil, Rynchophorus ferragineus (NACHMAN
etal., 1997). Intheopinion of someworkers, neuropeptides, such as sulfakinins,
myosuppressins andallatostatinsalterdigestive enzymeactivityinvitro(NACH-
MAN etal., 1997;FUSEetal., 1999;WEI etal.,2000; HARSHIN1 etal., 2002;
HILL&ORCHARD,2005).
ACKNOWLEDGEMENTS
ThanksareduetoProf.N.K.SUBHEDAR,DepartmentofPharmaceutical Sciences.NagpurUni-
versity,forprovidingtraininginimmunocytochemicaltechniques.The“UniversityResearchFellow-
ship”awarded toN.V.Patankarby theNagpurUniversity, isacknowledgedgratefully.
REFERENCES
ANDRIES, J.C.&G.TRAMU, 1985a. Ultrastructure and immunohistochemical studyofendo-
crine cellsin themidgutofcockroachBlaberus craniifer(Insecta:Dictyoptera). CellTissue
Res. 240: 323-332.
ANDRIES,J.C.&G.TRAMU,1985b.Distribution patternofmammalian-likepeptideiramunore-
activecellsinthemidgutofAeshnacyanea(Insecta:Odonata). Experientia42: 500-503.
Midgutendocrine cellsin Tramea Virginia 157
BROWN, M.R.,J.W.CRIM&A.O.LEA, 1986.FMRFamide-,andpancreaticpolypeptide-likeim-
munoreactivityofendocrine cellsin midgutofamosquito.Tissue Cell 18:419-428.
BROWN, M.R.,J.W.CRIM.,R.C.ARATA.,H.N.CAI.,C.CHUN&P.SHEN, 1999. Identifica-
tionoftheDrosophillabrain-gutpeptiderelatedtothe neuropeptideYfamily. Peptides20:
1035-1042.
CRIM,J.W.,A.C.JENKINS&M.R.BROWN, 1992. Amosquitoneuropeptideinmoth larva(Heli-
coverpazea)relatedtoFMRFamideimmunoreactivity. TissueCell24:537-545.
FONAGY, AL.,L. SCHOOFS,P.PROOST,J.VANDAMME&A.DELOOP, 1992. Isolationand
primarystructureoftwosulfakinin-likepeptidesfromthefleshfly,Neobellieria bullata.Comp.
Biochem. Physiol.(C) 103: 135-142.
FUJITA,T.,R.YUI.,T.IWANAGA.,J.NISHI1TSUTSUJI-UWO.,Y.ENDO&N.YANAIHARA,
1981. Evolutionaryaspects of“Brain-gutpeptides”:An immunohistochemical study. Pep-
tides 1\ 123-131,
FUSE, M.J.,R.ZHANG, E. PARTRIDGE, R.J.NACHMAN, I.ORCHARD,W.G.BENDENA
&S.S.TOBE, 1999. Effectsofanallatostatin andamyosuppressinonmidgutcarbohydrate
enzymeactivity inthecockroach,Diplopterapunctata.Peptides.20: 1285-1293.
HARSHINI,S.,R.J.NACHMAN&S.SREEKUMAR, 2002.Inhibition ofdigestionenzymerelease
byneuropeptideinthe larvaeofOpisinaarenosella (Lepidoptera:Cryptophasidae).Comp.
Biochem. Physiol. (B) 132: 353-358.
HILL,S.R.&I.ORCHARD, 2003.FMRFamide-related peptidesin thegutofLocustamigratoria
L.:acomparativemapand developmentalprofile.Peptides24: 1511-1524.
HILL,S.R. &I.ORCHARD, 2005. Invitroanalysis ofthe digestiveenzymes amylaseand alpha-
glucosidasein the midgutsof Locusta migratoria L. in response to the myosuppressin,
SchistoFLRFamide. J. InsectPhysiol.51: 1-9.
LANGE, A.B., 2001.Feedingstateinfluences thecontentof FMRFamide- and tachykinin-related
peptidesin endocrine-like cellsofthemidgutofLocustamigratoria.Peptides22:229-234.
LEHANE,M.J., 1998. TheMidgut.In: F.W.Harrison & M.Locke,[Eds],Microscopicanatomyof
invertebrates,1IB,pp.725-746,Wiley-Liss,New York.
LUNDQUIST,C.T.,F.L.CLOTTENS., G.M.HOLMAN.,J.P.RIEHM.,W.BONKALE&D.R.
NASSEL, 1994. Locustatacykinin immunoreactivity in theblowflycentral nervoussystem
andintestine. J. comp.Neurol. 341:225-240.
MONTUENGA,L.M.,M.A.BARRENECHEA.,P.SESMA.,J.LOPEZ&J.J.vASQUEZ, 1989.
Ultrastructureand immunocytochemistryofendocrine cellsinthe midgutofthe desertlo-
cust,Schistocerca gregaria(Forskal).CellTissue Res.258: 577-583.
MURALEEDHARAN,D., 1995.Endocrinologyofmidgut.In:D.Muraleedharan&M. Jacob,[Eds],
Recentadvances ininsectendocrineresearch, pp.99-115,Ass.Adv.Ent.,Trivandrum,India.
NACHMAN,R.J.,W.GIARD.,P.FAVREL.,T.SURESH.,S.SREEKUMAR& G.M.HOLMAN,
1997. Insect myosupressinsandsulfakinins stimulate releaseofthe digestiveenzymea-amy-
laseintwoinvertebrates: thescallopPectenmaximus andinsectRynchophorusferruginous.
Neuropeptidesin developmentand aging, [B.E. Beckwith,A.Saria.,B.M.Cronwall.,C.A.
Sandman&F.L. Strand,Eds], Ann. N.Y.Acad. Sci. 814: 335-338.
NEVES,C.A.,L.L BHERING.,J.E.SERRAO& L.B.GITIRANA,2002.FMRFamide-like mid-
gutendocrine cellsduringthemetamorphosisinMeliponaquadrifasciataanthidioides (Hy-
raenoptera:Apidae).Micron. 33:453-460.
NICHOLS,R., 2003.Signalingpathwaysand physiological functions ofDrosophilamelanogaster
FMRFamide-related peptides.Annu. Rev.Ent. 48:485-503.
NICHOLS,R.,S.A.SCHNEUWLY&J.E.DIXON, 1988.Identification andcharacterization ofa
Drosophilahomologuetothevertebrate neuropeptidecholecystokinin.J. biol. Chem. 263:
12167-12170.
NICHOLS,R.,J.McCORMICK&I.LIM, 1999.RegulationofDrosophilaFMRFamideneuropep-
158 N.V.Patankar& D.B.Tembhare
tide geneexpression. J.Neurobiol. 39:347-358.
NIJHOUT, H.F., 1994. Insecthormones. Princeton Univ. Press.
PRABHU,V.K.K.&S.SREEKUMAR, 1999. Endocrine regulationoffeedinganddigestioninin-
sects.In: R.K.Bhola,[Ed.],Advances ininsectendocrinologyandphysiology, pp. 1-15,Kaly-
ani.NewDelhi.
PREDEL,R., R.J.NACHMAN &G.GADE,2001. Myostimulatoryneuropeptidesincockroach-
es:structures,distribution,pharmacologicalactivitiesandmimeticanalogs.J.InsectPhysiol.
47: 311-324.
PREDEL,R.,W.BRANDT,R.KELLNER,J.RAPUS,RJ.NACHMAN&G,GADE, 1999.Post-
translational modificationsoftheinsectsulfakinins:sulfation,pyroglutamate-formationand
O-methylationofglutamicacid. Ear. J. Biochem.263: 552-560.
RANKIN, S.M.&S.M.SEYMOUR,2001.FMRFamide-like materialin theearwig,Euborellia an-
nulipes,and itsfunctional significance.Arch. Insect Biochem. Physiol.47: 189-197.
SCHOLS, D„P.VERHAERT, R. HUYBRECHTS., H. VAUDRY, S. JEGOU& A.DE LOOP,
1987. Immunocytochemicaldemonstration of proopiomelanocortin-and otheropioid-re-
lated substances and aCRF-like peptidein thegut oftheamerican cockroach,Periplaneta
americana L. Histochemistry 86: 345-351.
SIVASUBRAMANIAN,P., 1992. Localization ofFMRFamide-like immunoreactivityin thelarval
midgutofthefly,Sarcophagabullata. Comp. Biochem. Physiol (C) 102:555-560.
VEENSTRA,J.A.,G.W.LAU,H.- J.AGRICOLA&D.H.PETZEL 1995. Immunohistologicallo-
calization ofregulatorypeptidesinthe midgutofthe female mosquitoAedesaegypti.His-
tochem. CellBiol. 104: 337-347.
WANG, P,J.T.CONRAD &M.SHAHABUDDIN, 2001.Localization ofmidgut-specificprotein
antigensfromAedes aegypti(Diptera:Culicidae)usingmonoclonal antibodies. J.med Ent.
38: 223-230.
WEI,Z.,G.BAGGERMAN., R.NACHMAN., G.GOLDSWORTHY., P.VERHAERT., A. DE
LOOP&L.SCHOOFS,2000, Sulfakininsreducefoodintakeinthedesertlocust,Schistocer-
cagregaria.J. InsectPhysiol. 46: 1259-1265.
YI,S.-X.,L. TIRRY&D.DEGHEELE, 1992.Presence ofmyotropinsinlarval midgutextracts of
lepidopteraninsects: Manducasexta, Agrotissegetumand Spodopteraexempta. J. Insect
Physiol. 38: 1023-1032.
ZITNAN,D.,I.SAUMAN&F.SEHNAL, 1993.Peptidergic innervation andendocrine cellsofin-
sectmidgut.Arch. InsectBiochem. Physiol. 22: 113-132.