Table Of ContentOdonatologica35(3):225-231 SeptemberI,2006
Effect ofpaper mill effluent
onthe cephalicneurosecretory and
midgutproteaseactivities in thelast instar
larva of Bradinopygageminata (Rambur)
(Anisoptera: Libellulidae)
R.J.Andrew¹,E. Balmik²and L.Kodhe²
1DepartmentofZoology,HislopCollege,Civil Lines,Nagpur-440001 (MS),India
2 DepartmentofZoology,SSESA’sScienceCollege,Congressnagar,
Nagpur-440012(MS),India
ReceivedAugust21, 2004/Revised andAcceptedJanuary20, 2006
Thelast instarlarvaeweretreatedwith sub-lethal concentrations ofpapermill ef-
fluent (PME)for 5days.PMEinhibits thesynthesis ofneurohormones intheA-type
cellsofthemedial group ofthebrain and intheintrinsic neurosecretorycells ofthe
corpora cardiaca. PME also causeshistomorphologicalchangesin thecorpora al-
lataandsuppress proteaseactivityofthemidgut.Thesefindingssuggestthatvarious
metamorphicand intermediarymetabolic alterationscaused bythePMEtreatment
ismodulated bythechangesin thesynthesisandsecretion oftheneurohorraones of
thecephalicneurosecretorycomplex.
INTRODUCTION
Various industrialeffluentscontaminatedwithuntreatedwaste are gradually
proving tobeagreathazardforterrestrialandaquaticecosystem.Thepaperand
pulp millisawetprocessing industry, dischargingeffluentsinthesurroundingwa-
terbodies.Thepapermilleffluent(PME) isrich inavarietyoforganicandinor-
ganicmaterial toxic totheenvironment(KSIBIetah,2003). ReleaseofPMEhas
resultedinseriousthreatstovariousaquaticorganisms,includinginsects(GHOSH
&KONAR, 1980;CHANDRASEKARAN etah, 1989).In©donatelarvae, the
PMEarrestsmoulting (SUBRAMANIAN & VARADARAJ, 1993),alters the
biochemicalcomponents ofthehaemolymph (VARADARAJ etah, 1993),disin-
tegratestheeggchorionandterminatesembryogenesis(ANDREW etah, 2006).
KHAN(1983) foundthatodonatelarvaearecharacteristicofclean,non-polluted
water.BUSS etah, (2002) classified themas a groupwhichcan toleratemoderate
226 R.J.Andrew, E,Balmik &L.Kodhe
levelofpollution. Variousexperimental studies on theselarvaehaveelucidated
that theneurohormonesof thecephalic neurosecretorycomplex not only con-
trolmetamorphosis butmost ofthemetabolicpathways mediatethrough these
neurohormones(TEMBHARE& ANDREW, 1991a, 1991b, 1991c).
Thepresentinvestigation hasbeenundertakentostudytheeffectofPMEonthe
secretoryactivity ofvariouscomponentsofthecephalic neurosecretorycomplex
viz.theneurosecretorycellsofthebrain,thecorporaallata,thecorporacardiaca
andtheventral(ecdysial) glands andalso its effecton midgut proteaseactivity.
MATERIAL AND METHODS
PenultimateinstarlarvaeofBradinopygageminata(Ratnbur)werecollected fromthegardenpond
ofSSESA’s ScienceCollege,Nagpur,andacclimatized andreared inthelaboratoryataphotoperiod
of 10hlightat25+5°C. Theywerefedadlibitumonmosquitolarvae. Aftermoulting,thelastinstar
larvaewereseparatedand reared individuallyin smallplastic containers. The 96h LC 50value for
PMEwasfoundto be40%. Twosub-lethal concentrations of20% (group-1)and 30%(group-2)of
PMEwerepreparedasperthemethod ofSUBRAMANIAN &VARADARAJ(1993).
5-dayoldlastinstarlarvaewereusedascontrolandexperimentofeachgroup. Sixlarvaeweresacri-
fiedonthesecond,thirdandfifthdayoftreatment.Thecephalicneurosecretorycomplexwasdissected,
fixedandprocessed forwax-blockpreparations.Thesectionsof6pm thickwerecutandstainedwith
AldehydeFuschsin(AF)andChromalum HaematoxylinPhloxin (CAHP)(PANOV, 1980).Whilethe
midgutprotease activity wasdetermined by themethodofISHAAYAetah,(1971).
RESULTS
Paper milleffluenthasaprofound effectonmostofthecomponentsofthece-
phalic neurosecretorycomplex(Tab. I).
TableI
The effect of PMEon the synthesis of neurosecretory material ofthe cephalic neurosecretory
complexandmidgutproteaseactivityin thelarva ofBradinopygageminata
%PEM days A-cell cc INC Proteaseactivity- (OD)
C E c E C E C E
Group-1 2 +++ +++ +++ +++ +++ ++ 0.85±0.02 0.42±0.02
(20%) 3 ++ ++ +++ ++ ++ + 0.78±0.06 0.32±0.04
5 +++ - ++ + +++ - 0.80±0.05 0.25±0.03
Group-2 2 +++ + +++ +++ +++ ++ 0.84±0.02 0.43±0.06
(30%) 3 ++ - +++ + ++ - 0.91±0.05 0,28±0.02
5 +++ - ++ - +++ - 0.85±0.06 0.18±0.08
Amount ofneurosecretory material: +++ completelyfilled; — ++moderatelyfilled;— +poorly
filled;—absent; —CC: corpora cardiaca; — INC: intrinsicneurosecretorycells; — C:control;—
E:experimental;— (OD)opticaldensity
Proteaseactivityin Bradinopygageminatalarva 227
NEUROSECRETORYCELLS OFTHEBRAIN
Therearethreetypesof neurosecretorycellsinthebraindependingupontheir
staining affinityandcytomorphological characteristics.TheA-cellstain darkly
andis pyriforminshape; theB-cellstainwiththecounterstainandare spherical
whereastheC-cells are amphibious instainingaffinityandellipticalinshape. Of
these, PMEpredominantly interferedwiththe secretory activity ofthe A-cells
of themedial group(Figs 1-6).
Incontrolofboththegroups, theperikarya oftheA-cellis filledwithneuro-
secretory materialwhichis alsoevident along the axons ofthe cells indicating
Figs 1-6.Section ofbrain throughthemedial group ofneurosecretorycells (AF,x1000):(1)con-
trol onfirst day(5-daysold);notethe abundance ofneurosecretory material(NSM)in theA-cells
(arrow);— (2)20%PMEtreated larvaafter2days.Note reduction inthesynthesis ofNSM; — (3)
20%PMEtreated larva after5 daysexhibitingnegligibleamountofNSM; —(4)control onthird
day(8-daysold larva)showingsynthesisofNSM; —(5)30% PMEtreated larvaafter2daysshow-
ingactiverelease ofthe axonscontainingNSMin theaxonsand pathway;— (6)30%PMEtreated
larvaafter5days:note emptyA-cellswithoutNSMintheperikarya andaxons.
228 R.J.Andrew, E.Balmik &L.Kodhe
active synthesis and release of neurosecretorymaterial (Figs 1, 4). But within
twodaysofPMEtreatment ingroup-1 thereisamarkeddeclineinthesynthesis
ofneurosecretorymaterial by thesecells (Fig. 2),whereasingroup-2, thereis a
suddenrelease ofneurosecretorymaterialandtheaxons are filledwiththe neu-
rosecretory material(Fig. 5).
WithincreaseintheconcentrationofPMEandperiod ofexposure,thereis a
corresponding decrease in the synthesis ofneurosecretory material(Figs 2, 5).
By the fifth day, the A-cells andtheiraxons are completely devoidof neurose-
cretory materialinbothgroupsofPMEtreatedlarvaeindicatingblockage ofits
synthesis (Figs 3,6)(Tab. I).
THECORPORACARDIACA
Thecorporacardiacaisaneuroendocrineaswellas aneurohaemalorgansince
it containsintrinsic neurosecretorycells, andalso stores theneurosecretoryma-
terial produced by the neurosecretory cellsof the brain.The corporacardiaca
of the control is filledwith a large amount of neurosecretorymaterialand the
perikarya of the intrinsicneurosecretory cell isalso loadedwith stainableneu-
Figs7-9. Sectionsofcorporacardiaca (AF, x460):(7)control showingabundance ofNSM; — (8)
20%PMEtreated(group-1)larva after3daysshowingdecreasedamountofNSM;- (9)30%PME
treated (group-2)larva after5daysshowingnegligibleamountofNSM.
Figs10-11.Sectionsofcorporaallata(CAHP,x460):(10)controlexhibitinglargenucleiandthesyn-
cytialcondition ofthegland;—(II) 30%PMEtreatedlarva after5daysshowingcellular condition
oftheglandwithsmall nucleus and scantycytoplasm.
Protease activityinBradinopygageminatalarva 229
rosecretorymaterialindicatingactive synthesis ofneurohormones(Fig. 7). But
in PMEtreatedlarvaeofbothgroupsthereis a gradual decreaseinthe amount
ofneurosecretorymaterialinthebody of corporacardiacaandintrinsic neuro-
secretory cells(Fig. 8). By the fifthday, thecorporacardiaca andintrinsiccells
containnegligible amount ofneurosecretorymaterial(Fig. 9).
THECORPORA ALLATA
Thecorporaallataiscomposed ofalargenumberofsmall, sphericalepitheli-
al cellswithaprominent, centrally placed nucleus(4.7+0.4pm diameter).Inthe
control larva, the corporaallataexhibits syncytial conditionsince thecellwalls
areindistinct(Fig. 10).InlarvaetreatedwithPME, thereisamarkedincreasein
thecellwallformationalong withreductioninthesize of thenucleus(3.85±0.34
pm diameter)andbythefifthday inboththegroupsoftreatment,thehistomor-
phology ofcorporaallataexhibits acomplete cellularstructure(Fig. 11).
THEVENTRALGLANDS
Theventralglands are composed ofpolygonal epithelial cellswitha largenu-
cleusandnongranularcytoplasm. Histomorphological changes arenotobserved
intheglands ofcontrolandtreatedlarvaeof boththe groups.
MIDGUTPROTEASEACTIVITY
Themidgut proteaseactivity incontrol larvaemeasures 0.85+0.2OD at 280
nm. By the second day thePMEcauses asignificant declineinthemidgut pro-
tease activity andby thefifthdayof treatment,it drops downto0.25+0.09and
0.18+0.08 OD at280nmin thefirstand secondgroups,respectively (Tab. I).
DISCUSSION
InOdonata, the neurosecretorycells of thebrain exhibita cyclical secretory
activityduring the development ofthe lastinstarlarvaandtheneurohormones
secretedbythemedialneurosecretorycellsarepredominantly involvedinthemeta-
morphosisofthelarvaintoanadultdragonfly(SCHALLER, 1959;SCHALLER
&CHARLET,1970;TEMBHARE&ANDREW, 1994).Inlarvaofthedragon-
fly Trameavirginia, theA-cells secretealtatotrophic neurohormoneto stimulate
thecorporaallataand prothoracicotropic neurohormoneto stimulatethe ven-
tralglands (TEMBHARE & ANDREW, 1994). In B. geminata, A-cells exhibit
activesynthesis andsecretionof neurohormonesbutinPMEtreatedlarvae, the
cellsgraduallystop theirsecretoryactivity. Inodonates, theactivecorporaallata
exhibitsyncytial condition, whereasinactivecorporaallataare cellularwithdis-
230 RJ.Andrew, E.Balmik& L.Kodhe
tinctcellwalls(SCHALLER &CHARLET, 1970;TEMBHARE& ANDREW.
1994).AfterPMEtreatment,thecorporaallatabecomeinactiveandchange from
syncytial to cellular.The present observationsontheA-cellsandcorporaallata
indicatethat PMEprobably causes inhibitionofsynthesis and secretion ofal-
latotrophic neurohormonebytheA-cells, whichinturn results intheregression
ofthecorporaallata.
The corporacardiaca is devoidofstainableneurosecretorymaterialin PME
treatedlarva because the corpora cardiaca acts as a neurohaemalorgan and
stores the neurosecretory materialproduced by the neurosecretory cells ofthe
brain (TEMBHARE& THAKARE,1976;ANDREW, 1989;TEMBHARE&
ANDREW, 1994).Thecessationofsecretoryactivity bytheA-cellsofthebrain’s
neurosecretorycellsresultsinthe depletionofthestoredneurosecretorymaterial
in thecorporacardiaca. ThePMEalso causes depletion in thesecretary activ-
ityofthe intrinsicneurosecretory cellsofthe corporacardiaca.VARADARAJ
etah, (1993) reported thatPMEcauses asignificant declineinthe haemolymph
sugar level. This is probably dueto the non-production ofneurohormonesby
the intrinsiccells, which controlhaemolymph sugar levelinthe dragonfly larva
(TEMBHARE & ANDREW, 1991c).
Theventralglands areactive atthe beginningand end oftheinter-moultpe-
riod in the lastinstar larvaofOdonata(SCHALLER & HOFFMANN, 1973;
TEMBHARE& ANDREW. 1994). Thepresentstudy didnot observeany his-
tological changes inthecontrolandexperimental larvaeprobably becausethese
experiments were conductedin themid-intermoultperiod (5-10 days) whenthe
ventralgland is inaninactive state.
Theproteaseactivity ofthemidgutisalsoaffectedby thePMEtreatment since
proteaseactivityiscontrolledby thesecretionoftheA-cellsofthemedialneuro-
secretorycells ofthebrain(TEMBHARE& ANDREW. 1991a)and PMEhasa
direct inhibitory effect on thesecellsas observedinthepresent study.
In larvae, PME treatment causes major changes in the metamorphic (SU-
BRAMANIAN& VARADARAJ, 1993)and intermediary metabolicprocesses
(VARADARAJ etah, 1993). Thepresentreportindicates thatthesechanges are
manifesteddueto theprofound negative effectof thePME onthesecretory ac-
tivityofthecephalic neurosecretorycomplex.
ACKNOWLEDGEMENT
RJA isthankful tothe University GrantsCommission forpartialfinancial assistance under the
minor researchproject.
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