488 A. aquasalis Eggs Compared in Venezuela by SEM V Maldonado et al. MATERIALS ... width of eggs (ventral view), width of the deck at .... MO, CRUnlev t.
Mem Inst OswaldoCruz, RiodeJaneiro,Vol. 92(4): 487-491, Jul./Aug.1997
487
AnophelesaquasalisEggsfromTwoVenezuelanLocalities Comparedby Scanning ElectronMicroscopy V Maldonado,HJ Finol*,JCNavarro+ Institutode ZoologiaTropical,Laboratoriode Biologiade Vectores,Facultadde Ciencias,UniversidadCentral de Venezuela,Apartado47058, Caracas1041-A, Venezuela*Centrode MicroscopiaElectr6nica,Facultadde Ciencias,UniversidadCentralde Venezuela,Apartado47114, Caracas104l-A, Venezuela
Anopheles(Nyssorhynchus) aquasalis, is themaincoastalvectorof malariafrom northeasternVenezuelato southeastern Brazil. Severalauthorshavearguedthat An. aquasalisis a highlypolymorphic specieswhileothersindicatedthat it is a complexof closelyrelatedspecies.Thisinvestigation compared the morpholog7 of An. aquasalis eggsfrom Sinamaica(Zulia State)and Yaguaraparo(Sucre State),thewestandeastof Venezuela, respectively. Wewereabletoseparateeggsfrom thetwolocalities usingdiscriminant analyses basedon ratiosandpercentages of anteriorandposteriortuberclesmeasuredb), scanningelectronmicroscopy. Theresultsof thisworksuggestthat An. aquasalishashigh intraspecificvariation. Key words:Anopheles aquasalis- scanningelectronmicroscopy- eggs- Venezuela
Anopheles
aquasalisCurryis
the main coastal vector of malaria from eastem Ven-
ezuelato southernBrazil (Berti et al. 1993). Its coastal distributionrangesfrom Nicaraguato southernBrazil, Ecuador(althoughthereare no collection records from this country in the Smithsonian's collection), TrinidadandTobago and the LesserAntilles (Knight & Stone1977, Faran & Linthicum! 981, Zimmerman1992).Taxonomically, this speciesbelongsto the OswaldoiSeries
Rangel et al. 1995), polytene chromosomes (Kitzmiller & Chow 1971, Moncada &Conn 1992),mitochondrialand ribosomalDNA (Conn etal. 1993)andexternalmorphology (Cova-Garcia 1964, Faran 1980, Gabald6n & Escalante 1986,
Linley et al. 1993b). Someauthorssuggested that An. aquasalisis a singleentity,althoughSteineret al. (1982) and Connet al. (1993) suggestedthat An. aquasaliscould representa complex of species.
intheAlbimanus Section ofAnopheles, subgenus Nyssorhynchus (Faran 1980, Faran& Linthicum 1981, HarbachI994). Given the latitudinalrangeof An. aquasalis, one might expectthat differentpopulationsexist with morphologicalandbehavioralvariationsac-
cordingto localconditions. Thefollowingarevariantsdescribed asspecies relatedtoAn. aquasalis: An. emilJanus Komp,An.guarujaensis Ramos,An. guaraunoAnduze,An. delta Anduze(Knight& Stone 1977), and An. deltaorinoquensisCovaGarcia, which are now regardedas synonyms (Farani980). An. aquasalishasbeenexaminedforpolymorphismsin proteins(Steineret al. 1981,Vele 1993,
This studywas supportedby CDCH 03.023.95 and Conicit RP-VII-240060
to J.C. Navarro
+Correspondingauthor. Fax:+58-2-605.2204. E-maihjnavarro
strix.ciens.ucv.ve//
Received12 April 1996 Accepted4 April 1997
Cova-Garcia (1964) and Gabald6n and Escalante(1986) reporteddifferencesin eggmorphologywith light microscopy,and Moncadaand Conn(1992) andConnet al. (1993) recommended thatthemorphologyof the eggsshouldbe investigatedby meansof scanningelectronmicroscopy (SEM) to determinethe intraspecificvariationin An. aquasalis.Accordingly,Linley et al. (I993b) described theeggsofAn. aquasalisin populations from Brazil (Rio de Janeiro), Suriname (Paramaribo) and Venezuela (Moron and Carlo Rico), representingthe first SEM descriptionof eggmorphologyof An. aquasalis.The objectives of the currentinvestigationwere twofold: (1) to provide comparative SEM descriptionsof An. aquasaliseggsfrom two additionallocalitiesin Venezuela, Sinamaica, Zulia State (nonvector population)andYaguaraparo,SucreState(vector population),in thenorthwestandnortheast,respectively; (2) to conductstatisticalanalysesof chorionicmeasurements in orderto determineeggdifferencesbetweenthe two populations,thuscontributingmoreinformationabouteggmorphology of An. aquasalisin Venezuela.
488
A. aquasalisEggsComparedin Venezuelaby SEM V Maldonadoet al. MATERIALS
AND
METHODS
of eggsmay partially dependon the size of the Sourcesof specimens - Bloodfed femalesof female,which at the sametime is influencedby An. aquasaliswere collectedfrom animalbait in the conditionsduringlarval development(food To avoidsuchintwo localitiesof Venezueladuringthedry season: supply,crowding,temperature). fluences,we usedderivedcharacters(percentages Sinamaica (SI), Zulia State, 71 andYaguaraparo (YA), SucreState, andratios)in the discriminantanalysis(Linley et al. 1993a). Females were identified to specieswith the keysof Cova-Garciaand Sutil (1977) and Faran(1980). Femalesweretransferredindividuallyto glass vialscontainingdampfilter paperat thebottomto maintaina highrelativehumidity.Vialswerecovered with tulle, fixed with rubberbandsand trans-
ported to the laboratoryin a humidified cooler. Femaleswerekeptat 80% rh, until they laid theireggs.
Nine different attributes of the deck tubercles
werestudied(eggs-13for eachlocalityandfemale, at random)and five charactersof the micropylar apparatus (eggs=9for eachlocalityand female,at random).Micrographstaken and attributesstudiedweresimilartothosepreviouslymadeby Linley etal. (1993b)for eggsofAn. aquasalis.SEM measurements weretakenfrom digital images(stored on the harddisc)usingthe PhilipsAnalysisPro-
Preparation for scanningelectronmicroscopy gram 2.02.007. Discriminantanalyseswere carriedout to in- Eggsof 44 femalesfromeachlocalityweregiven differences betweentheSI andYA popu24 hr to embryonateafteroviposition.Thosefrom vestigate lations with Statgraphics PlusVersion6.1 Software Zulia Statewereair-driedandindividuallyplaced (Statistical Graphics Corporation, Rockville,MD). in the requiredpositionon double-facedadhesive tape adheredto stubs,usingan extra fine brush The descriptivemorphologicalterminology is (000). Silvertincturewasplacedon the edgesof basedon Harbachand Knight (1980). the adhesivetape in orderto ensureelectricand thermalconductivity;and the sampleswere coveredwithplatinum-palladium in anEicoEngineering Model 1B.2 sputtercoater.The sampleswere observed usinga PhilipsModel XL-20 scanning electronmicroscope operatedbetweenI 0 and 15 kV. Two permanentrecordswerekept:an image on the harddiscsystemandphotographic records. Eggs from SucreStatewere transportedin small vialsandfixed24 hr afterovipositionin alcoholic Bouin's(ethyl alcohol80%, 150ml; concentrated formalin,60 ml; glacialaceticacid,15 ml andpicric acid crystals,1g). Completedehydration was
initiatedwith 80%ethanolusingtwochanges (I 0 raineach)to removepicricacidandincreasing the ethanolconcentration by 5% to 100% (10 min each).Subsequently, thesamples wereplacedon stubs and coated as above.
Data collectionand analyses- Length and width of eggs(ventralview), width of the deckat the anteriorandposteriorends,and lengthof the floats and ribs on each float were measured from
eggs(n-22 for eachlocalityand female,at random) whichwereplacedon slidesin Canadabalsamandphenolto maintaintheirshape.Afterhardeningof themounting medium,samples werecovered with Hoyer's mediumand observedundera
Wild ModelM 11 lightmicroscope equipped with an ocular micrometer.
Unstandardized
values of all variables were
takenintoconsideration sincetheseattributes possessdifferentdimensions (areas,ratios,and percentages). Evidencefrompreviousstudiesonmosquitoes(Steinwascher 1984)indicatedthatthesize
RESULTS
The generaldescriptionof the eggs of An. aquasaliscoincideswith that indicatedby Linley et al. (1993b);thus,emphasis is placedonthecomparisonbetweenthesetwo populations. Comparisonsof the measuresbetweenSI and populations- The mean size of eggs (length andwidth) waslargerin YA than SI. Floats in YA occupiedonaverage82% of the lengthof theegg, while in S! theycoveredonly 70.8%. Anterior tubercleswere largerthan middle and posteriortubercles(TableI), andwere surroundedby smaller,
moreabundant tubercles (lessthan1pm2)(Fig.1). Anteriortubercles fromSI (Fig. 1a, b) weresmaller thanthosefromYA (Fig. 1c, d) (Table I). Middle tubercles wererounderasindicatedby largerform factors(Table I). Posteriortubercleswere irregular with few, surroundingsmallertubercles,some of whichwere connected to tuberclesby meansof bridges(Fig. 2a, b). Middle and posteriortubercles fromYA werelargerthanthosefrom SI, and tubercledensityhad higherin eggsfrom SI thanthosefrom YA (Table I). Discriminantanalysis- The discriminantanalysis of data on tubercles indicated that the anterior
and posteriortubercleswere the more important variablesandprovidedexcellentdifferentiationof theeggsfrombothlocalities.Summaryof thediscriminantanalysesis indicated in Table II. The variantsmeasured in themicropylarapparatusand the anterior/posterior deck width ratio and float attributesdid notprovideany significantdifferen-
tiationof eggsfromthe two localitiesusingdiscriminantanalysis.
MernInst OswaldoCruz,Riode Janeiro,Vol. 92(4), Jul./^ug.1997 TABLE
489
I
Attributes theeggs twopopulations
aquasalisin Venezuela
Mean(+SD)forpopulation Yaguaraparo
Attribute
Linear dimensions(n=22)a Egg length Egg width Lenght/widthratio Anterior/posteriordeckwidthratio
Sinamaica
434.91 + 31.86 218.61 + 26.03 2.01 + 0.20 1.26+0.5
403.64 + 30.48 182.46 + 28.70 2.28 + 0.48 1.35+0.21
356.07 + 28.80 82.04 + 6.01
284.24 70.81
Floats
Meanfloat length(of thetwofloats) Meanfloat lengthas % egglength .Meannumberof ribs (of thetwofloats) Meanfloat length/mean numberof ribs Tubercles(n= 13)
Anterior decktubercle density b Mean anterior deck tubercle area c
Meananterior decktubercle formfactord Middle decktubercledensity Mean middle deck tubercle area
Meanmiddledecktubercle formfactor Posterior deck tubercledensity
Meanposteriordecktuberclearea Meanposteriordecktubercle formfactor Micropyle (n--9) Totalarea of micropylarapparatus Collar area of micropylarapparatus Diskarea of micropylarapparatus Disk area as % total apparatusarea Numberof sectorsin micropylardisk Diameter micropyle
29.41 +4.60 12.36 2.00 65.16+19.10 1.48 + 0.53 0.53 +0.10 81.96 + 13.44 1.04 0.33 0.59 + 0.22 82.77 + 25.54 1.I 1 + 0.39 0.50+0.13
404.01 + 78.40 263.08 + 51.43 140.93 + 30.90 34.83 2.93 6.56 + 0.73 2.79 + 0.63
28.51 8.50
21.41 13.39
2.44 1.59
92.12 + 17.62 1.12 0.22 0.56 + 0.09 109.40 18.53 0.65 + 0.08 0.58 + 0.07 93.12 21.08 0.78 + 0.20 0.55+ 0.11
409.66 265.42 144.46 35.23 6.44 3.26
75.69 + 50.55 + 32.00 + 4.25 + 0.73 + 0.47
a: alllinear measurements ini.tm;b:number inanarea of400i.tm2;c:all areameasurements in i.tm2;d: form factor= 4 xnxarea/perimeter (indexofroundness); SD:standard deviation
Tubercles, anterior deck,different females. Fig.la, b:Sinamaica. Fig.lc, d:Yaguaraparo. Bar= 10gm.
490
A. aquasalisEggsComparedin Venezuelaby SEM V Maldonadoet al.
Tubercles, posterior deck.Fig. 2a:Sinamaica. Fig. 2b:Yaguaraparo. Bar= 10Ism. TABLE
II
Summaryof discriminantanalysis
Discriminantanalysis
Micropylarapparatus Ratioandpercentages measured
Eigenvalues
Chi-square
df
p
0.3848038 0.8521945
4.2322601 24.654846
6 4
0.64528 0.67831
6.6260463
34.536688
by opticalmicroscopy Tubercles
14
0.00172
df: degreeof freedom;p: probability
DISCUSSION
The discriminantanalysisdifferentiatedthetwo localitiesstudied,supportingthe idea of intraspecific variation,asobservedby Steineret al. (1981), MoncadaandConn(1992) andRangelet al. (1995). Linley et al. (1993b) describedthe eggs of An. aquasalisfrom Venezuela(CR andMO) as having shorterfloats,largerdecksnot taperedposteriorly, and a greaterwidth in relationto egg length. Also, the tuberclesof the anteriordeck seenunder
highmagnificationdifferentiatedpopulations. Our results agree with these observations.Attributes that contributed more to differentiation
included
the anteriorandposteriortubercles,with the anteriot tuberclescontributingmoreto the differentiation of the two analyzedpopulations. Thepopulations of An. aquasalisanalyzedhere (SI andYA) andby Linley et al. (1993b) (MO and CR) (Fig. 3) spanthe Venezuelancoastfrom east to west. Intraspecificdifferencesamongthe four populationsare basicallyin the lengthandwidth of the egg,the floats,andanteriortubercles.This
MO, CR Unlev nt ISl, YA =This Fig. 3: map of Venezuelashowinglocationof Anopheles aquasalis populations analyzedby scanning electronmicroscopy.
portant geographicalbarriers since the MioPliocene), provides biogeographical supportto the characteristics couldbe suggested as diagnostic hypothesisthat in VenezuelaAn. aquasalisis a markersin eggstaxonomyof An. aquasalisby species withhighintraspecific variation.Thesame SEM. However, thesecharactersdid not show evicouldbe saidaboutAn. aquasalisfrom Trinidad,
denceof morphologicalsharpdiscontinuities betweenVenezuelansamples of malariavectors(YA) andnonvectors (MO, CR andSI) populations. This
whichis a continentalislandsharingthe samegeo-
continuityof the Venezuelancoast,(with no ira-
differences in the coastal Venezuelan and Trinidad
logicaloriginastheVenezuelanCordillerade la Costa(coastalrange).Fromitscloseproximityand results and the more or less continuous coastal habirecentisolation(changeof sealevel in the latest tat of An. aquasalisandtheapparentgeographical glaciation) onewouldnotexpectto find anymajor
Mem Inst OswaldoCruz, Riode Janeiro,Vol. 92(4), Jul./Aug.1997
populations ofAn.aquasalis, suchasthosereported by Connetal.(1993)amongVenezuelan (MO, SF)/ TrinidadJan (PS) and Brazilian(M J, RJ) populations.
Also,ourresultsandthereportsof Connet al. (1993) and Moncadaand Conn (1992) on Sucre State populations(YA, Santa Fe and Guayana)
compared with westandcentralpopulations (SI,
491
XXXIV. A revision of the Albimanus sectionof the
subgenus Nyssorhynchus ofAnopheles. Contrib Am Entomol Inst 15:1-215.
FaranME, LinthicumKJ 1981. A handbookof the Amazonian species of Anopheles (Nysxorhynchus) (Diptera:Culicidae).MosqSyst13:1-81. Gabald6nA, Escalante C 1986.Estudiode loshuevos deAnopheles emilianus entresregiones de Venezuela. Dir. Malariol. Saneam.Ambient. Maracay.Mim-
MO, CR and PuertoCabello) might suggestthat eographed Document.8 pp. Cova-Garcia'sinterpretation of the eggmorphol- Harbach RE 1994. Review of the internal classification of thegenusAnopheles (Diptera:Culicidae):The ogy (An. emilianus)wasmistaken. foundation for comparative systematics andphyloThis investigationsupportsthe hypothesisof geneticresearch. Bull EntomolRes84:331-342. intraspecific variationin An. aquasalisanddemonstratesthe usefulnessof studyingthe morphol- HarbachRE, KnightKL 1980. Taxonomists'glossar)' of mosquitosanatom.v.PlexusPublishing,Inc., ogyofanophelineeggswith SEM. Similarstudies Madton, New Jersey,413 pp. shouldbe done throughoutthe range of the spe- KitzmillerJB, ChowGW 1971.The salivaryglandchrociesto determinethe continuityof structuralvarimosomesof An. aquasalis.Rev Bras Malariol antsand confirm the existenceof morphological DoencasTrop 23: 65. differences
that would allow the characterization
of differentpopulations, includingvectorcompetencestudies.It is recommended thatfutureanalyses take into consideration the effects of seasonal
KnightK, StoneA 1977.A catalogof themosquitoes of the world.ThomasSay Foundation,Vol. VI. 2nd. ed. Entomol.Soc.Amer, 611 pp.
LinleyJR,KaiserPE, CockbumAF 1993a.A descriptionandmorphometric studyof theeggsof species of theAnopheles quadrimaculatus complex(Diptera:
variation(wet anddry seasons) anddifferenthabitats(eggsfrom freshandbrackishwaterpopulaCulicidae).MosqSyst25.' 124-147. tions)on eggmorphology.Finally,polymorphism LinleyJR, LounibosLP, ConnJ 1993b.A description in proteinsandmitocondrial andribosomalDNA andmorphometric analysis of theeggsof fourSouth studiesmustbe doneincludingSI, YA populations American populations ofAnopheles (Nyssorh.vnchus) and comparewith the anothersVenezuelanand aquasalis (Diptera:Culicidae).MosqSyst25: 198Sudamerican
localities.
214.
MoncadaA, ConnJ 1992.A polytenechromosome study
ACKNOWLEDGMENTS
offourpopulations ofAnopheles aquasalis fromVen-
ezuela. Genome 35:327-331 To inspectors Humberto Montafiez and Jesus Amaristafor fieldassistance. To Centrode Microscopia RangelY, VeleG, ChadeeDD 1995.Population genetic Electr6nica,FacuttaddeAgronomia,UCV, (CeMEFA), analysis of Anopheles aquasalisin Venezuelaand and Centro de MicroscopiaElectr6nica,Facultadde Trinidad.JAm MosqControlAssoc11: 347. Ciencias, UCV. To Hermes Pifiango (Laboratorio de SteinerWWM, Kitzmiller JB, OsterburDL 1981. On BiologiadeVectores)andCarlosChedas(CeMEFA) for thegeneticidentityandevolution of themalariavecvaluablecontribution.To Dr Alan Wheeler (University torsAnophelesaquasalisCurry and Anopheles of Keele, U.K.) for final translaterevision. emilianus Komp,p. 75-90.In R Pai,JB Kitzmiller,
T Kanda(eds)Cytogenetics andgeneticsof vectors.
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