(Diptera: Culicidae). Richard C Wilkerson/+, Thomas V Caffigan, Jo& Bento Lima* ... Species C and D were found by RAPD-PCR to be sympatric at. Costa Marques ...... Wilkerson RC, Parsons TJ, Klein TA, Gaffigan TV 1995. Discovery by ...
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Identification of Species Related to Anophe/es (Nyssor/iynchus) albitarsis by Random Amplified Polymorphic DNA-Polymerase Chain Reaction (Diptera: Culicidae) Richard C Wilkerson/+,
Thomas V Caffigan,
Jo& Bento Lima*
Department of Entomology, Walter Reed Army Institute of Research,Washington, DC 20307 U.S.A. *U.S. Army Medical Research Unit - Brazil, Instituto de Biologia do ExCrcito, Rua Francisco Manuel 102, 2091 l-270 Rio de Janeiro, RJ, Brasil
Species-specific Random Amplified Polymorphic DNA-Polymerase Chain Reaction @?A PD-PCR) markers were used to identify four species related to Anopheles(Nyssorhynchus) albitarsisLynchArribtrlzaga from 12 sites in Brazil and 4 in Venezuela. In a previous study (Wlkerson et al. 1995), which included sites in Paraguay and Argentina, thesefour species were designated “‘A “, B ‘ “, “C” and “D “. It was h.ypothesizedthat speciesA is An. (Nys.) albitarsis,species B is undescribed, species C is An. (Nys.) marajoaraGalvco and Darnasceno and species D is An. (Nys.) deaneorumRosaFreitas. SpeciesD, previously characterized by RAPD-PCR f rom a small samplefrom northern Argentina and southern Brazil, is reported here from the type locality of An. (Nys.) deaneorum,GuajarciA&rim, State of Rondhnia, Brazil. Species C and D were found by RAPD-PCR to be sympatric at Costa Marques, State of RondGnia, Brazil. Species A and C have yet to be encountered at the same locality. The RA PD markers for species C were found to be conserved over 4,620 km; from Iguape, State of ,%o Paulo, Brazil to Rio Socuavo, State of Zulia, Venezuela. RAPD-PCR was determined to be an effective meansfor the identijcation of unknown specimenswithin this species complex. Key words: Anopheles (Nyssorhynchus)albitarsis -Anopheles(Nyssorhynchus)marajoara - Anopheles (nyssorhynchus)deaneaorum- RAPD PCR - identification
The AlbitarsisComplexof Anopheles subgenus Nyssorhynchus (Albitarsis Subgroup of Linthicum 1988)is knownto includea numberof crypticspecies(Kreutzeret al. 1976,Steineret al. 1982,Rosa-Freitas et al. 1990,Naranget al. 1993). Most recently,usingRandomAmplifiedPolymorphic DNA - PolymeraseChain Reaction(RAPDPCR), Wilkersonet al. (1995) demonstrated the existenceof four speciesin the AlbitarsisComplex in Brazil, Argentina and Paraguay. Members of this complexhave been incriminatedas importantvectorsof malariaparasites(reviewed by Rosa-Freitas et al. 1990)but,becausemembers of the complexare difficult to identify by morphology,it is not knownwhich speciesmight be responsible for transmission in a givenarea. Our objectivewasto identifl membersof theAlbitarsis Complexby RAPD-PCR from additionallocalities and obtainrearedassociated siblingvoucher The views of the authors do not purport to reflect the views of the supporting agencies. +Correspondingauthor. Present address:Museum Support Center, Smithsonian Institution, Washington DC 20560. Received 2 January 1995 Accepted 8 August 1995
specimensfor furture morphological studies. Becuaseof its simplicity,the useof morphological charactersis desirableas an aid to researchin vectorbiology,biogeography, malariaepidemiology,malariadrugandvaccinetrialsandultimately developmethodologies andstrategiesfor malaria control.In addition,RAPD-PCRwasinvestigated as a possiblemeansfor routineidentificationsin lieu of morphologicalidentificationkeys. At present,thereare threevalid speciesin the complex:An. (nys.) albitarsis Lynch-Arribtizaga, 1878(Paraguay,sotithernBrazil and Argentina); An. (nys.) deaneorum Rosa-Freitas,1989 (State of Rondbnia,Brazil); and An. flys.) marajoara GalvCioandDamasceno,1942 (CostaRica to Bolivia). Wilkersonet al. ( 1995)designated thetaxa, characterizedby RAPD-PCR, as speciesA, B, C and D. They hypothesizedthat speciesA is An. (Nys.) albitarsis sensu stricto, species B is undescribed, speciesC isAn. (Nys.) marajoara and speciesD is An. (ivys.) deaneorum. RAPD genetic markersfound by Wilkersonet al. (1995) wereusedto ident@ specimens collectedat additional sitesin Brazil and Venezuela.A summary of the distributionof the speciesand photodocumentationof thesemarkersis presented(Figs l16; TablesI-III).
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RAPD (Williams et al. 1990, Welsh & McClelland1990)isa PCR-based techniquewhich has proven to be a quick and effectivemeansof identifyinggeneticmarkers. This techniquepermits numerousmarkers to be assayedon DNA extractedfrom a singlemosquito. Insteadof using primer pairsas in traditionalPCR, RAPD reactionsusea singleshortprimer (usually10bases in length) of randomlychosensequence.For a RAPD band to be produced,the primer needsto matcha bindingsitethat is within approximately 2-3 kilobasepairs of anotheroppositelyoriented bindingsitesothat the singleoligonucleotide can prime replicationin both the forwardand reverse directions. A typical RAPD reactionproduces multiple amplificationproducts,each representing a discretegeneticlocus,whichcanbe resolved easilyby agarosegel electrophoresis. RAPD-PCR has shownpromisefor use in a wide variety of organismsincludingbacteria,higherplants,vertebrates,and invertebratesincludingmosquitoes and other insects. It hasbeenusedas a tool for geneticmapping;strain, speciesand population identificationand systematics (Hadryset al. 1992, Chapcoetal. 1992,Blacket al. 1992,Karnbhampati et al. 1992, Williams et al. 1993, Bowditchet al. 1993,Perringet al. 1993, Gawel&Bartlett 1993, Wilkersonet al 1993,Wilkersonet al. 1995). MATERIALS
AND METHODS
Identijkation - Peytonet al. (1992) providea definition of the subgenusNyssorhynchus.Speciesin theAlbitarsisComplexhavehindtarsomere 5 without a basaldark bandand vein 1A mostly pale-scaled(ArgyritarsisSection)and sternumI with a distinctrow of white scaleson each side with posterolateralscaletufts beginningon segmentsIII or IV. The sisterspeciesAn.braziliensis (Chagas)has tufts beginningon segmentII [see Linthicum (1988) for additionalimmaturecharacters]. Specimen preservation - Adultsandlarvaeused in DNA analysiswereinitially frozenin liquidnitrogenandtransported to our laboratoryon dry ice wherethey were maintainedat -70°C. Larval and pupal exuviae were collectedfrom individually rearedadultsand preservedin 80% ethanoland held for slidemounting.Each individuallyreared specimenwasgivena uniquecodenumberthatassociatedit with other progenyof a singlefemale andalsoassociated it with a pin-mountedadult. Sourceof specimens- Collectionlocalitiesof Fl progenybroodsusedin this studyare givenin TableI alongwith thosepresentedin Wilkersonet al. (1995). Associatedspecimensretained for morphogicalstudyare depositedin the National Museumof Natural History,SmithsonianInstitu-
RC Wilkerson
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tion, Washington DC; US Army MedicalResearch Unit, Rio de Janeiro;InstitutoOswaIdoCruz, Rio deJaneiro;andtheNucleodePesquisa Taxonomica e Sistematica emEntomologiaMedica(NUPTEM) at the Universidade de so Paulo. Experimentalprocedures- DNA Isolation. Individual larvae or adultswere ground with a plasticpestlein microcentrifugetubesin 200 pl extractionbuffer (1OOmMTris pH 8.0, 1OOmM EDTA, 1OOmMNaCl); proteinascK was then addedto 200 pg/ml andSDSto 0.5%. After incubationat 55OCfor 3 to 12 hr, RNasewasaddedto a final concentrationof 100 pg/ml and incubated at roomtemperaturefor 30 min. The solutionwas extractedonce with an equalvolume of phenol/ chloroforrn/isoamyl alcohol(25:24:1, equilibrated with 10 mMTris pH 8.0,lmM EDTA) by heating to 55OCfor 10min with periodicmi?iingof phases. Afterbrief centrifugationin a microcentrifuge, the supernatant was extracted with chloroform/ isoamylalcohol(24: 1) asabove. The supernatant was collected, 2 volumes(4OOpl)95% ethanol wereaddedto it, and the solutionstoredat - 20°C for 15min to precipitatethe DNA. The DNA was pelleted(15,900 x g in a microcentrifugefor 4 min), washedwith 700 @70% ethanol,dried under vacuum,and dissolvedin 100 pl 10 mM Tris pH 7.5, 1mM EDTA. Typical yieldswere 0.5 to 6.5 pg DNA per individual. RAPD PCR ampi@ cation. Detailed proceduresare discussedin Bowditchet al. (1993). Total reactionvolumesof 25 u.lwere usedwith the followingfinal concentrations:11 mM Tris-HCl (pH 8.3); 50 mM KCl; 1.9 mM MgC12;0.1 mg/ml BSA; 0.1 mM eachof dATP, dCTP, dGTP and TTP; 0.24 pmol/pl (6 pmol) primer; 0.2-4.0 ng/p.ltemplateDNA; and 0.02-0.06 U/p1 (0.5-1.5 units) Taq DNA polymerase.PCR conditions.A Perkin-ElmerCetus model480 thermocyclerwasusedfor all reactions with the following parameters:one min denaturation at 94OCfollowedby 45 cyclesof onemin at 94OC,annealingone min at 35OCand elongation two min at 72OC,all with minimum ramp times. Agamsegel electrophoresis. Usingstandardmethods (Sambrooket al. 1989), amplificationproductswereanalyzedusinga USA Scientific“Extra Wide”mini horizontalsystem(cat.no. 3488-0000) which uses100 ml 1.5% agaroseminigelswith 0.8 pg/ml ethidium bromide run at 50 volts/25 milliampsfor about3 hr in TBE (89mM Tris base, 89mM boric acid and 2mM EDTA, pH 8.3). Amplificationproductswere observedandphotographedusing short wave (3 12 nm) ultraviolet light. Molecularweightstandards wereprovided by lambda DNA digested with Hind III, and @X174DNA digestedwith Hae III (New England
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Biolabs). The approximatemolecularweight of amplificationproductswascalculatedusinga program written for Lotus l-2-3 by AF Cockbum, United States Department of Agriculture, Gainesville,Florida,USA. Oligonucleotide primers. All primerswere 10basesin length. Primers were purchased from Operon Technologies, AlamedaCA. Primersdiscussed in the text have the following sequences (5’-3’): B16, TTTGCCCGGA; 805, TGCGCCCTTC; DO1, ACCGCGAAGG; C07, GTCCCGACGA; B02, TGATCCCTGG; AOl, CAGGCCCTTC; C 19, GTTGCCAGCC; C 16, CACACTCCAG; Cl 5, GACGGATCAG; Bll, GTAGACCCGT; A12, TCGGCGATAG; A08, GTGACGTAGG. Basedon the resultsof Wilkersonet al. (1995) (Figs2- 13) thefollowingprimerswereusedto amplify species-specific markersfor identificationof unknownspecimens: Sp A, primersAOl, B16, (2 markers);Sp B, primersC07, B02, Cl5 (4 markers); Sp C, primersAOl, C16, C19, DO1(7 markers);and SpD, primersA12, DO1(3 markers).In addition,the presenceor absenceof sharedbands producedby primersB05 (speciesA,C), Cl9 (speciesA,B,D) andDO1 (speciesCD) werealsotaken into consideration (TableII). In orderto makedirectcomparison withpreviousresults,knownspecimenswere run with all unknowns.To minimize the numberof reactionsneededfor an identilication, a “bestguess”of a speciesidentitywasfirst madebasedon publishedand unpublished analysesof morphological characters[e.g.larval seta3C branchedinAn. (1vys.)deaneorum (Rosa-Freitas 1989)or lengthofbasaldarkbandonhindtarsomere 2 and the relativelengthof the prehumeraldark spot(unpublished data)], or basedon what is alreadyknownaboutthe distributions of the species (e.g.speciesA is only recordedfrom southern Brazil, Bolivia,Paraguay andnorthernArgentinawhile speciesB hasnotbeenrecordedfrom westernBrazil). A species-specific primer wasthen usedfor the suspected species.Thosematchingwereverified with one or two additionalspecies-specific primers. Thosethatdidn’t matchweretestedwith a next “best guess”. Figure 14 showsspeciesC primer Al2 run with a seriesof specimens from Venezuela(lanes4-18) anda singlespecimen from theStateof Bahia,Brazil(lane3). Theknownspecimenof speciesC is in lane2 from the typelocality of An. (1vy.s.) marajoara, Ilha de Marajo, Stateof Par-a,Brazil. The specimenin lane 3 that did not matchwas then run with speciesB primerswith whichit didmatch(notshown).Figure15showsa seriesof specimens from the type localityof An. (nys.) deaneorum, Guajarh-Mirim, Stateof Rondonia,Brazil(lanes3-lland13)which,whenrunwith
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species D primerA12, comparedpositivelywith a knownspecies D from Guaira,Stateof SaoPaulo, Brazil (lane2). The specimens that did not match (lanes12and 14-19)fromPeixotodeAzevedo,State of Mato Grosso,Brazil are amongthoserun with species C primerCl9 shownin Fig. 16 (lanes3-9). RESULTS
Based on genetic markers determined by Wilkersonet al. (1995) (Figs 2-l 3; Table II) we were able to identify the four known speciesbelongingto the AlbitarsisComplexfrom an additional 12 sitesin Brazil and4 in Venezuela(Table I; Fig 1). An additionaleight individualsof speciesA, 41 of speciesB, 108 of speciesC and26 of speciesD were identified. All (exceptnine speciesC) representseparateprogenybroodswith associatedlarvalandpupalskinsandpinnedadults. To carry out identificationswe usedten of the 12 primersusedby Wilkersonet al. (1995) (TableII; Figs 2-13). These primers producedcorrelated markersas follow: A(3), D(3), B(4) and C(7). Thesedatavalidatepreviousresults(Wilkersonet al. 1995)with somevariation(Table III). Although it is preferable to use absolutely “fixed’ markersfor identifications,in reality this is probablynot possiblewhen one uses larger samplesand/orwidely separatedpopulations. It
Fig. 1: summ~ of geographicaldistributionof the four known speciesin the Albitarsis Complex de&mined by RAPD-PCR. Presumed identities (see text) of species are as follow: A = albitarsis;B = undescribed;C = marajoara atad,D = deanwrum.
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TABLE I Summary of collection localites for Albitarsis Complex species identified by Random Amplified Polymorphic DNA - PolymeraseChain Reaction State/ Province
Locality
Coordinates
Date
Species(n)
Costa Marques
12”28’S 64’16W
28-III-92
C(l6)> D(3)
13
Guajara-Mirim Belem
lO”5O’S 65”2OW 1’27’s 48”29W
26-VII-92 8-X-92
~(23) B(2)
12
0’56’s 47”06W lO”23’S 54”54W
16-X-92 20-N-93
B(5) C(29), B( 12)
Bahiaa
Primavera Matupa, Peixoto de Azevedo Itaquara
13’26’s 39”56W
30-I-93
B(9)
15
C earaa
Fortaleza
3”43’S 38”3OW
8-III-93
B(l1)
11
C earha
Parapaiba
Santa Catarinaa
Massaranduba
3’25’s 39”13W 26”35’S 48’58113
9-III-93 8-I-93
B(13) A(8)
10 26
Rio de Janeiroa
Morro da Panela Manaus Ilha Comprida
22”58’S 43’2 1W 2”53’S 60”15W
19-193 16-XII-93
B(1) C(34)
18 7
24’42.8’s 47”3 1.6W 24’36.8’s 47O53.1W
g-II-89 26-I-92
C(l3) A(30), B(19)
24 25
22”39.05’S 53”Ol w l”24’S 47’11 W
1o-II-92 VIII-93
A(1) C(8), B(1)
17 6
C(43)
Map Ref.
BRAZIL RondBniaa Rond6niaa Paraa Paraa Mato Grossoa
Amazonasa Sso Paul0 Sso Paul0 Sgo Paul0
6 km SW Registro
Par6
Ponte Melo Peixoto Capanema
ParB
Ilha de Marajo
1”OO’S49”3OW
VIII-91
Parana
Santa Helena
24”56’S 54”23W
30-I-92
Parana
nr. Guaira
24”04’S 54” 15W
Espirito Santo
Aguia Branca
18”59’S 40”44W
1-II-92 20-I-92
Zuliaa Zuliaa
Rio Socuavo Tres Bocas
8”54’N 72”38W ?8”54’N 72”38W
22-IX-92 24-IX-92
Cojedesa
Finca Rosa Blanca
9.6”N 68.9”W
Barinasa
Castilla
8”25N 70”38W
Alto Parana
Rio Acaray
Alto Parana Alto Parana
Hernandarias nr. National Airport
Misiones Corrientes Corrientes
A(5), D(1) A(l2), B(l), D(4) B(1)
8 9 14
5 20 19 16
VENEZUELA C(16)
1
26-VI-9 1
C(1) C(l0)
2 4
14-IX-92
C(2)
3
25’29’s 54”42W
4-II-92
A(9)
22
25”22’S 54”45W (not known)
6-R-92 8-II-92
A(8)> B(6) B(9)
21 23
Posadas 9Okm W Posadas
27”23’S 55”53W
30-I-92
A(12)
27
(not known)
3 l-I-92
A(1 )> D(2)
28
Corrientes
27’28’s 59’50117
3 l-I-92
A(9), D(2)
29
Corrientes
1lkm W Mercedes Santa Tome
29”12’S 58”05W 28’33’s 56’03717
1-II-92 2-II-92
A(5)
Corrientes
A(8)
31 30
Buenos Aires
Baradero
33”48’S 59”3OW
6-E-92
A(22)
32
PARAGUAY
ARGENTINA
a: collection localities presented here for the first time, others appear in Wilkerson (1995).
is not criticalfor the effectiveuseof RAPD markers,to have100%fixed markers,butto havenearly fixed markersthat are correlated. As long as all theexceptionalmissingmarkersdonotcorrespond in a singleindividual,there shouldbe no conht-
sion aboutassigningindividualsto their respective geneticallydefined taxa. A comparisonof the frequency of diagnostic markers between Wilkersonet al. (1995) andthepresentstudyshow somedifferences.The 0.38 kbpbandproducedby
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Fig. 2: RAPD amplifications of Albitarsis Complex speciesA with primer B16. Arrow on right indicates fragment of approximately 1.75 kbp. Lane 1, DNA size standard; 2-3, Baradero, Argentina; 4, Registro, Brazil; 5, Santa Tome, Arg.; 6, Posadas, Arg.; 7, Corrientes, Arg.; 8, Mercedes. Arg.; 9, near Santa Helena, Brazil; 10, near Guaira, Brazil; 11, Ponte Melo Peixoto, Brazil.
Fig. 3: RAPD amplifications of Albitarsis Complex speciesA with primer B05. Arrow on left indicates fragment of approximately 1.75 kbp. Lane 1, DNA size standard; 2-3, Baradero, Argentina; 4, Registro, Brazil; 5, Santa Tome, Arg.; 6, Posadas, Arg.; 7, Corrientes, Arg.; 8, Mercedes, Arg.; 9, near Santa Helena, Brazil; 10, near Guaira, Brazil; 11, Ponte Melo Peixoto, Brazil.
Fig. 4: RAPD amplifications of Albitarsis Complex specieswith primer W 1. Lanes2-5, speciesA; 6-9, speciesB; lo- 14, speciesC; 15-l 8, speciesD. Arrows on letI indicatetiagmentsofapproximately 2.32 kbp (all species)and 1.42 kbp (speciesA and B); arrows on right indicate fragments of approximately 0.96 kbp (speciesD), 0.80 kbp (speciesC and D) and 0.55 kbp (speciesC). Lane 1, DNA size standard (see Fig. 2); 2-3, Baradero, Argentina; 4, 6-7, Registro, Brazil; 5, 17, 18, near Guaira, Brazil; 8-9, 15, Hemandarias, Paraguay; 10-12, Ilha de Marajb, Brazil; 13-14, Iguape, Brazil; 16,90 km W Posadas,Arg.
primer CO7 and the 1.88 kbp band producedby primerB02 were missingin a smallpercentage of specimensreportedhere for the first time. Also, the 1.41, 1.22 and 0.85 kbp bandsproducedby primer A01 for speciesC were found in slightly lower frequenciesthan before. WiIkersonet al. (1995) reportedthat only oneindividualoutof 6 1 hadtwo of thethreebandsmissing.In thepresent study,twoof 108alsolackedthesesametwobands
( 1.22and0.85 kbp). However,theotherfour species C markerstestedfor by primers C16, DO1 and C 19 werepresentin both individuals. The 1.OOkbp markerproducedby primer C 15 in speciesB wasthe only one foundto be absent in a significantproportionof individualscompared to the previousstudy(lOO%, 15/15vs 68.3%, 28/ 4 1). In addition,a 1.41 kbp band producedby thisprimerwhich wasnot recognizedasa poten-
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Fig. 5: RAPD amplifications of Albitarsis Complex speciesB with primer C07. Arrow indicates tiagment of approximately 0.41 kbp. Lane 1, DNA&e standard(see Fig. 2); 2-5, Registro, Brazil; 6- 10, Hemandarias,Paraguay; 11, &uia Branca, Espirito Santo, Brazil.
12
3
4
5
6
7
8910
2
3
4
5
6
7
8
9
1011
Fig. 6: RAPD amplifications of Albitarsis Complex speciesB with primer B02. Arrow indicates fragment of approximately 1.88 kbp. Lane 1, DNA size standard(see Fig. 2); 2-5, Registro, Brazil; 6-l 0, Hemandarias,Paraguay; 11, &uia Branca, Esphito Santo, Brazil. 11
12
13 14 15
16 17
18 19 20
Fig. 7: RAPD amplificationsof Albitarsis Complex specieswith primer A0 1. Lanes 2-11, speciesC; 12-19, speciesA Arrows on lefi indicatefragmentsof approximately 1.41, 1.22 and 0.85 kbp (speciesC); arrow on right indicatesfragmentof approximately 2.03 kbp (speciesA). Lanes 1,20, DNA size standard(see Fig. 2); 2-11, Iguape, Brazil; 12-15, Baradero, Argentina; 16-17, Registro, Brazil; 18- 19, Hemandarias, Paraguay.
tially usefklspecies-specific markerbecauseof its inconsistent brightness,wasactuallyconsistently presentand recognizablein all individualstested. A possiblehomologous1.41 kbp band was seen in four of fifteen speciesC, butfurthertestingwas not doneto verify its size. Twenty-three progenybroodsfrom the type locality of An. (1vys.)deaneorummatchedfor all three markerspreviouslydeterminedfor species D. Even thoughthe initial speciesD samplefrom northernArgentinaandsouthernBrazilwassmall (n = 9), these markerswere also presentin the
larger sample(n = 26) from two localitiesin the Stateof Rondonia,Brazil. A distanceof approximately2,400 km was previouslyreported(Wilkersonet al. 1995) betweenpopulationsof speciesC. With the addition of specimensof speciesC from Venezuela, the maximum distancewe have found between populations exhibitingthe samediagnosticmarkersis nowapproximately4,620 km (Iguape,State of SaoPaulo,Brazil toRio Socuavo,Stateof Zulia, Venezuela). SpeciesC and D were sympatric at Costa
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Fig. 8: RAPD amplifications of Albitarsis Complex specieswith primer C19. Lanes2-7, speciesC; 8-11, speciesA; 12-15, speciesB; 16-19, speciesD. Arrows on left indicate fragments of approximately 1.172 and 0.88 kbp (speciesC); arrow on right indicates fragment of approximately 1.30 kbp (speciesA, B and D). Lanes 1 and 20, DNA sizestandard(seeFig. 2); 2-4, Ilha & Marajb, Brazil; 5-7, Iguape, Brazil; 8, Posadas,Argentina; 9, Baradero, Arg.; 1O-13, Registro,Brazil; 14-l 5, Hemandarias, Paraguay; 16-17,90 km W Posadas,Arg.; 18-19, near Gush, Brazil.
Fig. 9: RAPD amplifications of Albitarsis Complex speciesC with primer C16. hw indicatesfi-agmentof approximately 0.993 kbp. Lanes 1 and 14, DNA size standard(see Fig. 2); 2-5, Ilha de Marajb, Brazil; 6-9, Iguape, Brazil; 10-13, Capanema, Brazil.
Marques, Stateof Rondonia,Brazil. These two specieshad beencollectedpreviouslyin the same humanandbovinebait collections(Klein & Lima 1990). With thisfinding,all possiblepairsof species,exceptspeciesA and C, havebeenfoundto besympatricat somelocality.The sitenearGuaira, State of Parana, Brazil remains the only site sampledwith three of the speciesoccurringtogether(A, B, D). DISCUSSION
LJsing RAPD-PCRwe havebegunto solveparts
ever,the of the biologirelatior ultscancal ant not be _ _~~~~_~___ ______----_-_, ,-_-- iscussed in Wilkersonet al. 1995). Many have attempted to correlatemalaria transmissionwith behavior and/or relative length of the dark portion of hindtarsomere2 (reviewed by Rosa-Freitas& Deane 1989and Rosa-Freitaset al. 1990). Some of the studiesdemonstrateda correlation,while mostwere ambiguous.These attemptshad their basisin earlyobservations regardingmalariatrans-
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Fig. 15: screenof unknown Albitarsis Complex specimenswith speciesD primer Al 2. Lanes 1 and 20, DNA size standard(see Fig. 2); Lanes 3-11, and 13, Guajarb-Mirim, Rondhia, Brazil, type locality of An. (jyys.) deaneorum; lanes 12 and 14-19, Peixoto de Azevedo, Mato Grosso,Brazil. 1
2
3
4
5
6
7
8
9
10 11 12
13
14
15 16
17 18 1920
Fig. 16: screenof unknown Albitarsis Complex specimenswith speciesC primer C 19. Lanes 1 and 20, DNA size standard(see Fig. 2); lane 2, Ilha de Maraj6, Para, Brazil, type locality ofh. flys.) marqoaru; lanes3-19, Peixotode Azevedo, Mato Grosso, Brazil. Includesspecimensnot matching markers for speciesD primer Al 2 given in Fig. 15.
haveofferedsomeinsightinto understanding this complex. It is not clear how thesefindingsrelate to those presentedhere becausein most cases voucherspecimens are not availablefor comparison. Kreutzer et al. (1976) found three chromosomallydifferentiatedpopulations,“C” in ColombiaandVenezuelaandtwoothers,B 1andB,, ranging from Rio de Janeiroto Belemandjust northof Ilha de Marajo. We do not believethat we have yet sampledtheir population“C”. Steineret al. ( 1982), usingisozymeanalysis,foundthree“geneticallydifferentiated’populationsat sitesfrom S%oPauloto just west of Marajo Islandbasedon 12 allozymeloci and chromosomeanalysis. Accordingto their map, species“a” and “b” were
found near the coastand “b” wasalso found inlandbut, accordingto their Table II, “a” wasalso foundwith “b” inlandat Logoa(sic,Lagoa?)Feia. An additionaldiscrepancyfailed to showspecies “b’ at Araraquara,sympatricwith their species “c”. Thesepossiblycorrespond to “B,” and “B,” of Steineret al. (1982). However, we were not able to locatepublishedinformationon chromosomescited in their analysis. In a studyof 10 populationsRosa-Freitaset al (1990) found no morphologicaldifferences,exceptfor An. (Ivys.) deaneorum, but throughisoenzymeanalysisconcludedthattherewerefive “groups”in Brazil and Argentina. Narang et al. (1993) comparedfour populationsin the AlbitarsisComplex in Brazil
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TABLE II Summary of Randon Amplified Polymorphic DNA markers used to identity species of the Albitarsis Complex Primer B16 B05 DO1
co7 B02 A01
Cl9
Cl6 Cl5
Bll Al2 A08
Approximate kbp 1.75 1.74 2.2-2.3 0.96 0.80 0.55
A
B
C
D
+Q + +
_ + _ _
_ + i+ +* _ _ +* +* +* +* +* +* _ _ + _ _ -
_ + +* + _ _ _ _ + -
_
0.38 1.88 2.03 1.41 1.22 0.85 1.30 1.17 0.88 0.99
+a + -
+* +* + -
-
1.41 1.24 1.oo 0.86 1.78 1.26 1.29 1.09 1.09
-
+* _ +* _ + _ _ -
+a + -
+* _ +* +* +* +*
a: speciesspecific markers; +: marker in common to more than one species; -: marker not present species
Cruz, Rio de Janeiro, Vol. 90(6),
nov./dec. 1995
731
using 18 allozymeloci and mitochondrialDNA restrictionfragmentlengthpolymorphisms.They reportedthat two morphologicallydistinct taxa existed,An. (Nys.) deaneorum and An. (Nys.) marajoara, the lattermadeup of 2 or 3 biochemically recognizableforms. One of the formsofAn. (Nys.) marajoara was sympatricwith An. (Nys.) deaneorum at CostaMarques,Stateof Rondonia, and the other one or two forms of An. (ik’ys.) marajoara, depending on interpretation, werefrom Iguape,Stateof S%oPaulo,and from its type localityof Marajo Island,Stateof Par& The Iguape specimens are the sameasthoseusedby usin the presentstudy(speciesC) and‘mayrepresenta speciesresponsible for a malaria outbreakin Iguape reportedby Schiavi(1945). As reportedherethe two speciesfrom CostaMarques(C, D) are probably An. (Nys.) marajoara and An. (Nys.) deaneaorum.
RAPD-PCRhasprovento be effectivein separatingcrypticAnopheles speciesandfor identifying u&nown specimens.It hasthe potential-for useas a routinediagnostictool for identification of Anopheles siblingspecies,but becauseof the sensitivityof RAPD-PCRto changingexperimental conditions, careshouldbetakenin its use(Williamset al. 1993,Black 1993). It hasthe advantage of relatively low initial investmentof time for determining species-specific markers when comparedto designingand testingcustom-made species-specificP.CRprimers basedon known nucleotidesequences.
TABLE III Diagnostic primers used for identification of Albitarsis Compelex species and comparison to Wilkerson (1995) results Species A
B
C
Primer
Appox. kbp
Wilkerson (1995)
Present study
B05
1.74 1.75
121/121 (100%)
8/8 (100%)
B16
81/82 (98.8%)
818 (100%)
A01
2.03
1181118 (100%)
818 (100%)
co7
0.38 1.88
32/32 ( 100%)
B02
35135 (100%)
40141 (97.5%) 38/4 1 (92.6%)
Cl5
1.00
15/15 (100%)
28/41 (68.3%)
1.41
1505 (100%)
41/41 (100%)
Cl6
0.99
0.55
63163 ( 100%) 42/42 ( 100%)
66166 ( 100%)
DO1 A01
1.41
59/61 (96.7%)
9!/108 (84.3%)
1.22 0.85
58/61 (95.1%) 59/61 (96.7%)
100/108 (92.3%)
Cl9
D
Al2 DO1
29/29 (100%) 86/l 08 (79.6%)
1.17
58/58 (100%)
84/84 (100%)
0.88
58/58 (100%)
84/84 (100%)
1.29
9/9 (100%)
1.09
919 (100%)
26/26 (100%) 26i26 (100%)
0.96
9/9 (100%)
26126 (100%)
732
Identification
of Albitarsis
Complex
by RAPD-PCR
ACKNOWLEDGMENTS To the personnelof the U.S. Army Medical Research Unit, Rio de Janeiro, Brazil, for collecting, rearing and curatingmany of the specimensusedin this study. They are: Eric G Milstrey, Maria de Fatima Barros, Jose Rodrigues Vargas and Temizio Rodrigues Pereia. Also in Brazil, Agostinho Marques Cruz, FundacBoNational da Saude, Brasilia, provided invaluable administrative support as did Jorge Arias, Pan American Health Organization, Brasilia. To Soma Mitra for her conscientious and careful DNA analyses. To Jan Conn and Phil Lounibos(supportedby NIH grantAI-3 1034) who kindly provided specimens from Venezuela. REFERENCES Black WC III 1993. PCR with arbitrary primers: approach with care. Insect Mol Biol2: 1-6. Black WC III, Duteau NM, Puterka GJ, Nechols JR, Pettorini JN 1992. Use of the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) to detect DNA polymorphisms in aphids (Homoptera: Aphididae). Bull Ent Res 82: 151-159. Bowditch BM, Albright DG, Williams JGK, Braun MJ 1993. The use of RAPD markers in comparative genome studies. Meth Enzvmol224: 294-309. Chapco W, Ashton NW, Martel RKB, Antonishyn N 1992. A feasibility study of the use of random amplified polymorphic DNA in the population genetics and systematicsof grasshoppers. Genome 35: 569-574. Galvgo ALA, Damesceno RG 1942. Sobre urn novo anofelino da Ilha de Marajo, Anopheles (nyssorhynchus)marajoara. Folia Clin Biol, SZo Paul0 14: 60-66. Galvgo ALA, Damesceno RG 1944. Observacoessobre anofelinos do complexo albitarsis (Diptera Culicidae). Ann Fat Med, SZo Paul0 20: 73-87. Gawel NJ, Bartlett AC 1993. Characterization of differences between whiteflies using RAPD-PCR. Insect Mol Biol2: 33-38. Hadrys H, Balick M, Schierwater B 1992. Apphcations of random amplified polymorphic DNA (RAPD) in molecular ecology. Mol Ecol I: 55-63. Kambhampati S, Black WC IV, Rai KS 1992. Random amplified polymorphic DNA of mosquito species and populations (Diptera: Culicidae): Techniques, statisticalanalysis,and applications.JMed Entomol 29: 939-945. Klein TA, Lima JB 1990. Seasonal distribution and biting patterns of Anopheles mosquitoes in Costa Marques, Rondonia, Brazil. J Amer Mosq Cont Assoc 6: 700-707. Kreutzer RD, Kitzmiller JB, Rabbani MG 1976. Cytogenetically distinguishable sympatric and allopatric populationsof the mosquitoAnophelesalbitarsis. Acta Amaz 6: 473-481. Linthicum KJ 1988. A revision of the ArgyritarsisSection of the subgenusNyssorhynchusof Anopheles (Diptera: Culicidae). Mosq Syst 20: 98-271. Lynch-Arribalzaga F 1878. Description de tres nuevos Culicidae de Buenos Aires. Nat Argent I: 149-152. Narang SK, Klein TA, Perera OP, Lima JB, Tang AT
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