Phenetic studies on r andomly amplified polymor phic DNA-polymer ase
chain r eaction-var iability of four geogr aphical populations of
Lutzomyia whitmani
( Diptera: Psychodidae) in Brazil
Estudos fenéticos de variabilidade de polimorfismos de DNA amplificados ao acaso pela
reação em cadeia da polimerase em quatro populações geográficas
de
Lutzomyia
whitmani
( Diptera: Psychodidade) no Brasil
Car ina Mar go nar i de So uza1
, Co nsuelo Lato r r e Fo r tes- Dias2
, Pedr o Mar co s Linar di3
and Edelber to Santo s Dias1
ABSTRACT
Pre vio u s e va lu a tio n o f the ge n e tic va ria b ility o f fo u r b io ge o gra phic a l po pu la tio n s o f Lutzo myia whitmani fro m k n o wn fo c i o f c u ta n e o u s le i sh m a n i a si s i n Bra zi l d e m o n stra te d two m a i n sp a ti a l c lu ste rs: Co rte d e Pe d ra - BA, Ilh é u s- BA a n d Se rra d e Ba turité - CE in the first c luste r, a nd Ma rtinho Ca m po s- MG in the se c o nd. Furthe r a na lysis sho we d a high de gre e o f ho m o ge ne ity i n Co rte d e Pe d ra p o p u la ti o n b u t n o t i n th e o th e rs, wh i c h p re se n te d a si gn i f i c a n t p e rc e n ta ge o f sp e c i m e n s d i sp la c e d f ro m the ir phe n o n o f o rigin ( disc re pa n t in dividu a ls) . In the pre se n t wo rk we a n a lyze d the fre q u e n c ie s o f a sso c ia tio n c o e ffic ie n ts i n th e m a tri x e s o f si m i la ri ty p e r p o p u la ti o n o f Lutzo myia whitmani f ro m b o th se x e s a n d th e ge n e ra l p h e n o gra m s o b ta i n e d , i n a m o re de ta i le d stu dy o f tho se di sc re pa n t spe c i m e n s. Po pu la ti o n a l sta b i li ty wa s o b se rve d f o r Co rte de Pe dra po pu la ti o n , whe re a s the thre e re m a in in g po pu la tio n s sho we d va ryin g de gre e s o f he te ro ge n e ity a n d diffe re n t displa c e m e n ts a c c o rdin g to se x. Ou r re su lts stro n gly su gge ste d the e xi ste n c e o f a ge n e ti c f lo w b e twe e n the li n e a ge s No rth- So u th/No rth- Ea st a n d Ilhé u s/ Se rra d o Ba tu ri té o f Lutzo myia whitmani.
Ke y-words:Lutzo myia whitmani. Sa n d f ly. Ge n e ti c va ri a b i li ty. RAPD- PCR.
RESUMO
Um a a va li a ç ã o p ré vi a d a va ri a b i li d a d e ge n é ti c a d e q u a tro p o p u la ç õ e s b i o ge o grá f i c a s d e Lutzo myia whitmani o ri u n d a s d e fo c u s c o n he c ido s de le ishm a n io se c u tâ n e a n o Bra sil, e vide n c io u 2 a gru pa m e n to s e spa c ia is prin c ipa is: Co rte de Pe dra ( BA) , Ilh é u s ( BA) e Se rra d e Ba tu ri té ( CE) n o p ri m e i ro gru p o , e Ma rti n h o Ca m p o s ( MG) e m u m se gu n d o . O a p ro f u n d a m e n to d a a n á li se a c u so u u m a lto gra u d e h o m o ge n e i d a d e n a p o p u la ç ã o d e Co rte d e Pe d ra m a s n ã o n a s o u tra s, n a s q u a i s u m a p o rc e n ta ge m si gn i f i c a ti va d e e sp é c i m e n s d e slo c o u - se d o se u f e n o d e o ri ge m ( i n d i ví d u o s d i sc re p a n te s) . Ne ste tra b a lh o a n a lisa m o s a s fre q ü ê n c ia s do s c o e fic ie n te s de a sso c ia ç ã o n a s m a trize s de sim ila rida de po r po pu la ç ã o de Lutzomyia whitmani,
de a m b o s o s se xo s, e o fe n o gra m a ge ra l o b tido , e m u m e stu do m a is de ta lha do da q u e le s e spé c im e n s disc re pa n te s. Pa ra Co rte d e Pe d ra f o i o b se rva d a e sta b i li d a d e p o p u la c i o n a l, e n q u a n to a s o u tra s trê s p o p u la ç õ e s re sta n te s m o stra ra m gra u s d e h e te ro ge n e i d a d e va ri á ve i s e d e slo c a m e n to s d i sti n to s, d e a c o rd o c o m o se x o d o s i n d i ví d u o s. No sso s re su lta d o s su ge re m f o rte m e n te a e x i stê n c i a d e u m f lu x o ge n é ti c o e n tre a s li n h a ge n s No rte - Su l/No rte - Le ste e Ilh é u s/Se rra d o Ba tu ri té d e
Lutzo myia whitmani.
Palavr as-chave s:Lutzo myia whitmani. Fle b o to m í n e o . Va ri a b i li da de ge n é ti c a . Re a ç ã o e m c a de i a da po li m e ra se .
1 . Lab o r ató r io de Le ishmanio se s do Ce ntr o de Pe sq uisas Re né Rac ho u da Fundaç ão Oswaldo Cr uz, B e lo Ho r izo nte , MG; 2 . Ce ntr o de Pe sq uisa e De se nvo lvime nto da Fundaç ão Eze q uie l Dias, B e lo Ho r izo nte , MG; 3 . De par tame nto de Par asito lo gia da Unive r sidade Fe de r al de Minas Ge r ais, B e lo Ho r izo nte , MG, B r asil ( CNPq fe llo w) Wo r k suppo r te d b y: FIOCRUZ ( 0 2 5 0 . 2 5 0 . 2 0 0 ) ; FAPEMIG ( CB B 1 2 2 3 /9 5 ) ; CNPq ( 5 2 1 7 1 2 /9 3 -9 )
Ad d r e s s t o : D r. E d e l b e r to S a n to s D i a s . La b o r a tó r i o d e Le i s h m a n i o s e s /Ce n tr o d e P e s q u i s a s R e n é R a c h o u /FI OCR UZ, Av. Au gu s to d e Li m a 1 7 1 5 , 3 0 1 9 0 - 0 0 2 B e l o Ho r izo n te , MG.
Te l: 5 5 3 1 3 2 9 5 - 3 5 6 6 ; Fax: 5 5 3 1 3 2 9 5 - 3 1 1 5 . e - mail: e de l@ c pq r r. fio c r uz. b r
Lu tzo m yi a wh i tm a n i ( Antune s & Co utinho , 1 9 3 9 ) is a phle b o to mine sand fly in the sub ge nus Nysso m yi a that has b e e n pr e vale nt at B r azilian fo c i o f c utane o us le ishmaniasis in the state o f Minas Ge r ais1 3 1 5, B ahia4 1 4 and Ce ar á3 1 7 1 8
. It has
b e e n inc r iminate d as a ve c to r o f human Ame r ic an c utane o us le ishmaniasis, fo llo wing the finding o f spe c ime ns natur ally infe c te d with Le i sh m a n i a par asite s fr o m the b ra zi li e n si s
c o m ple x4 1 0, asso c iate d with hum an c ase s o f the dise ase .
Furthermore, a high anthropophilic behavior has been desc ribed fo r this sand fly spe c ie s3 8 9 1 6.
Several studies have suggested that L. whi tm a n i is, in fac t, a c o mplex o f spec ies. Phylo genetic analysis o f mo r pho metr ic , bioc hemic al and molec ular c harac ters of L. whitm a ni indic ated that the r e ar e , at le ast, thr e e to fo ur distinc t mito c ho ndr ial lineages in B razil1 1 1 9 2 0. The first lineage is an Amazo nian fo rm,
str ic tly silvatic , de sc r ib e d fr o m the no r the ast and so uthe ast r e gio ns o f the Amazo n r ive r, state o f Par á. A se c o nd fo r m was fo und in the no r the aste r n Atlantic Fo r e st zo ne , inc luding the spe c ie s type lo c ality, distinguishab le fr o m the thir d gr o up o f haplotypes, whic h is loc ated in the drier interior of Brazil ( north-south group) . A fourth lineage has been more rec ently desc ribed in the state o f Ro ndô nia, b ase d o n phylo ge ne tic studie s o f mito c ho ndr ial DNA. An o ve r all analysis sho we d that tho se line age s fr o m fo r e ste d r e gio ns ( Ro ndô nia, Amazo nian and No r the aste r n) fo r m a c lade to tally se par ate d fr o m the no r th-south lineage.
In a pr e vio us wo r k , we analyze d the ge ne tic var iab ility o f fo ur ge o gr aphic al po pulatio ns o f L. wh i tm a n i in B r azil b y r a n do m ly a m plifie d po lym o r ph ic DNA- po lym e r a s e c h a in r e ac tio n1 2. The gr e at maj o r ity o f individuals fitte d into the
inte r po pulatio nal phe no gr ams ac c o r ding to the ir r e gio n o f origin. However, a small disc repant group displac ed from their r e gio n o f o r igin was no te d. The o b j e c tive o f the c ur r e nt wo r k was to fo c us o n tho se spe c ime ns unlink e d to the phe no n o f origin.
MATERIAL AND METHODS
Phlebotomine. Sand flies were captured at night by Shannon24
and CDC2 6 traps at the Brazilian munic ipalities of Corte de Pedra,
Bahia ( 1 3 ° 2 6 ’2 3 ”S, 3 9 ° 3 9 ’3 6 ”W) ; Ilhéus, Bahia ( 1 4 ° 4 7 ’2 ”S, 3 9 ° 0 2 ’5 8 ”W) ; Martinho Campos, Minas Gerais ( 1 9 ° 1 9 ’5 4 ”S, 4 5 ° 1 4 ’1 3 ”W) and Se r r a de B atur ité , Ce ar á ( 0 4 ° 1 9 ’4 3 ”S, 3 8 ° 5 3 ’0 5 ”W) . The traps were mounted at 5 :0 0 p.m. and taken down at 7 :0 0 a.m. the next day, for 5 -7 consecutive days per region. In the laboratory, female sand flies were fed on non-infec ted hamsters (Me so crice tus a ura tus) . After 2 4 h they were isolated into individual containers until oviposition1 4 after which they were
mounted on microscope slides in Berlese medium2 7 and identified
according to Young and Duncan30. Eggs from 5 0 0 females identified
as L. whitm a ni were c ombined, transferred to new c ontainers and inc ubated in a humid c hamber at 2 7 ºC until ec losion. We o b taine d apr o ximate ly 2 0 0 spe c ime ns o f L. wh i tm a n i pe r population in F1 , from which 5 0 males and 5 0 females were frozen and stored in liquid nitrogen for later use.
DNA e x tr a ctio n. Fifte e n spe c ime ns o f L. wh i tm a n i o f eac h sex and population were randomly selec ted from the frozen sto c k . Eac h spe c ime n was mac e r ate d in a mic r o tub e using a plastic pe stle , fo llo we d b y additio n o f 5 0
µ
l o f lysis b uffe r ( 1 0 0 mM TRIS-HCl, 1 0 0 mM NaCl, 2 5 mM EDTA, 0 .5 % SDS, pH 8 .0 ) and mac erated again. Geno mic DNA extrac tio n was perfo rmed as desc ribed by Ausubel et al2.. The final pellet was ressuspended
in 2 0
µ
l o f TE 1 X. Te n mic r o lite r s we r e use d fo r e stimatio n o f DNA c o nc e ntr atio n and pur ity b y o ptic al r e adings at 2 8 0 and 2 6 0 nm in a spe c tr o pho to m e te r a nd the n disc a r de d. The r emaining 1 0µ
l wer e sto r ed at 4 ºC until use.DNA amplificatio n. DNA was amplified by RAPD-PCR2 8 2 9
in a thermocycler ( Perkin-Elmer 2 4 0 0 ) in a total reaction volume of 6 0
µ
l. The reac tion mixture was prepared as follows: 1 2µ
l of PCR b uffe r ( 1 0 0 mM TRIS-HCl, 5 0 0 mM KCl, 2 5 mM MgCl2, pH
8 . 3 ) , 9 . 6
µ
l dNTPs ( 1 0 m M e ac h) , 8µ
l o f a r ando m pr im e r ( 7 0 ng) and 1 8 .4µ
l o f ultr apur e wate r. Six mic r o lite r s o f DNA ( 2 ng) were added per reac tion and the mic rotubes inc ubated at 9 4 º C fo r 1 m in . Afte r a dditio n o f 6µ
l ( 1 . 5 U) o f dilute d Ta q po lyme r ase ( Pe r k in Elme r ) , the amplific atio n r e ac tio n was perfo rmed fo r 4 5 c yc les o f 3 0 s at 9 4 ºC, 3 0 s at 3 4 ºC and 1 min at 7 2 ºC. The r ando m pr ime r s we r e : OPG0 9 ( CTGACGTCAC) , OP G1 2 ( CAGCT CACGA) , 3 3 0 2 ( CT GAT GCTAC) o r 3 3 0 3 ( TCACGATGCA) . A ne gative ( no DNA) and a po sitive ( lamb da DNA) c o ntr o l wer e used in all amplific atio ns.DNA PAGE. Twelve mic roliters of the amplific ation produc ts were analyzed by 4 % PAGE ( 1 0 x 8 .5 x 1 .5 c m) in TB E buffer for 1 0 min at 5 0 V fo llo we d b y 9 0 min at 8 0 V, and de ve lo pe d b y silve r staining5 2 3.
φ
X1 7 4 RF DNA/Ha eIII fr agme nts and 1 Kbladde r ( Gib c o -B RL) we r e use d as size mar k e r s. Ge ls we r e do c ume nte d b y a Po lar o id Came r a ( Ko dak ) o r an Eagle Eye System ( Stratagene, La Jolla, US) .
Da ta a na lysis.The PAGE fr agme nts se le c te d fo r analysis we r e the mo st c o nsiste ntly visib le in all ge ls. The pr e se nc e o r absenc e of a given gel band was c odified as 1 or 0 , respec tively, and use d in the c o nstr uc tio n o f a se r ie s o f matr ixe s taxo n/ c har ac ter. Asso c iatio n c o effic ients wer e deter mined fo llo wing Dic e7 and the phe no gr ams we r e c o nstr uc te d b y unwe ighte d
pair-group method analysis ( UPGMA)2 5 through the NTSYS - pc
ve r sio n 2 .0 so ftwar e2 2.
RESULTS
Despite the high variability of assoc iation c oeffic ients in the po pulatio ns, the gr e at maj o r ity o f spe c ime ns was gr o upe d ac c ording to their region of origin, in the interpopulational analysis per sex ( Figures 2 and 3 ) . However, a disc repant group was c learly formed by a limited number of spec imens. For the females, it was c omposed of spec imens numbered 3 0 1 , 3 2 2 and 3 2 5 from Serra de B aturité and 1 8 , 1 9 , 2 0 , 2 2 , and 2 3 from Martinho Campos ( Figure 2 ) . Another disc repant group was observed for the males, c omprising spec imens 3 5 8 from Serra de B aturité, 2 7 5 and 2 8 0 from Ilhéus ( B A) and 5 2 and 8 1 from Martinho Campos ( Figure 3 ) . Analysis of the displac ement of similarities for the disc repant groups is summarized in Table 1 .
DISCUSSION
The histo gr am pr o file s po inte d to a var iab le r ange o f assoc iation c oeffic ients for the populations under study ( Figure 1 ) . Mo dal disc o ntinuity was a c o mmo n po int, indic ating the existenc e o f a high level o f intrapo pulatio nal genetic variability a s p r e vio us ly de s c r ib e d1 2. I n tr a p o p ula tio n a l va r ia b ility
asso c iated with a lo w value o f the highest frequenc y c o effic ient ( 0 .5 6 ) distinguishe d Mar tinho Campo s po pulatio n fr o m the o th e r s . Th e p e r s i s te n t di s c o n ti n u i ty i n th e a s s o c i a ti o n c oeffic ients ( 0 .6 5 ) in the histograms of Corte de Pedra, Ilhéus, and Se r r a de B atur ité might r e pr e se nt an aspe c t o f similar ity between these three populations, if one c onsiders the positioning o f this disc o ntinuity in the same mo de.
The phe no n line s ( e quivale nt to the me an o f similar itie s amo ng all the spe c ime ns analyze d) in the inte r po pulatio nal phenograms of males and females ( Figures 2 and 3 ) denote the gr e ate r ho mo ge ne ity o f Co r te de Pe dr a and Se r r a de B atur ité po pulatio ns. All the individuals in the se po pulatio ns we r e grouped ac c ording to their region of origin. Females from Ilhéus fo r me d a phe no n inc luding the disc r e pant gr o up ( Figur e 2 )
0 5 10 15 20 25 30 35 40
0.48 0.52 0.56 0.60 0.64 0.68 0.72 0.76 0.8 0.84
CP 0 5 10 15 20 25 30 35 40 0. 39 0. 43 0. 47 0. 51 0. 55 0. 59 0. 63 0. 67 0. 71 0. 75 MC 0 5 10 15 20 25 30 35 40 45 50 0. 52 0. 55 0. 58 0. 61 0. 64 0. 67 0. 70 0. 73 0. 76 0. 79 0. 82 0. 85 IL
Frequency
Coefficient of association SB 0 5 10 15 20 25 30 35 40
0.53 0.56 0.59 0.62 0.65 0.68 0.71 0.74 0.77 0.80 0.83 CB
Fi gu re 1 - Fre q u e n c i e s o f a sso c i a ti o n c o e f f i c i e n ts i n th e m a tri x e s o f si m i la ri ty p e r p o p u la ti o n o f
Lutzo myia whitmani f ro m b o th se x e s. Ori gi n o f p o p u la ti o n s- CP: Co rte d e Pe d ra /MG, IL: Ilh é u s/BA,
MC: Ma rti n h o Ca m p o s/ MG a n d SB: Se rra d e Ba tu ri té / CE.
CP-F106 CP-F110 CP-F112 CP-F109 CP-F133 CP-F111 CP-F107 CP-F114 CP-F118 CP-F119 CP-F120 CP-F129 CP-F137 CP-F122 CP-F127 IL-F202 IL-F225 IL-F208 IL-F209 IL-F242 IL-F204 IL-F228 IL-F226 IL-F239 IL-F231 IL-F235 IL-F240 IL-F230 IL-F241 SB-F301 SB-F322 SB-F325 MC-F20 MC-F18 MC-F23 MC-F22 SB-F308 SB-F323 SB-F313 SB-F320 SB-F315 SB-F316 SB-F324 SB-F329 SB-F326 SB-F327 SB-F330 SB-F328 MC-F21 MC-F24 MC-F26 MC-F28 MC-F27 MC-F31 MC-F41 MC-F33 MC-F30 MC-F34
0.54 0.62 0.69 0.76 0.84
Coefficient of association Deviation= 0.004 IL-F238
MC-F19
Figure 2 - Ge ne ra l phe no gra m fo r fe m a le s o f Lutzomyia whitmani ge ne ra te d b y NT-SYS so ftwa re . Ea ch spe cim e n wa s ide ntifie d b y the initia ls o f the po pula tio n ( CP: Co rte de Pe dra /MG; IL: Ilhé us/BA; MC: Ma rtinho Ca m po s/MG; SB: Se rra de Ba turité / CE) , se x ( F fo r fe m a le ) a nd a thre e digits num b e r. The ve rtica l line re pre se nts the a ve ra ge le ve l o f sim ila rity b e twe e n a ll the pa irs o f OTU’s a na lyze d ( phe no n line ) .
evidenc ing the similarity between these insec ts. Inc o rpo ratio n o f the disc r e pant gr o up was no t o b se r ve d fo r the male s fr o m the same region ( Figure 3 ) . Spreading of disc repant individuals from Martinho Campos population in distinc t groups suggested a no n-unifo r mity in this po pulatio n fo r b o th se xe s.
Fur ther analysis o f the displac ement o f similar ities fo r the disc r e pant gr o ups c o nfir me d that Co r te de Pe dr a is the mo st unifo r m po pulatio n with 1 0 0 % o f fe male s and male s gr o upe d
in a single pheno n ( Table 1 ) . The high level o f ho mo geneity in both sexes suggests a populational stability and perhaps a genetic iso latio n o f this r e gio n. This hypo the sis is r e info r c e d b y the overall analysis of disc repant individuals from the other regions with the Co rte de Pedra pheno n was never o bserved. The three populations remaining showed varying degree of heterogeneity and diffe r e nt displac e me nts fo r fe male s o r male s. In the fir st c ase, 9 3 % of the spec imens from Ilhéus formed a phenon while 7 % suffered a displac ement to the Serra de B aturité population. Martinho Campos displayed the highest heterogeneity with only 6 7 % o f s pe c im e n s gr o upe d in a c h a r a c te r is tic ph e n o n . Disc r e pant individuals ( 3 3 % ) we r e displac e d c o nsiste ntly to war ds Ilhé us gr o up. A similar displac e me nt was o b se r ve d fo r disc repant females fro m Serra de B aturité. Differently fro m previously reported6 1 9. Ilhéus and Serra de B aturité populations
were not grouped into the same phenon but c lose to eac h other1 2.
Ta b le 1- Pe rc e nta ge o f spe c im e ns o f L. whitmani link e d in the sa m e phe no gra m a nd displa c e m e nt a na lysis o f the disc re pa nt gro up. CP: Co rte de Pe dra / BA, MC: Ma rtinho Ca m po s/ MG, IL: Ilhé us/ BA a nd SB: Se rra de Ba turité / CE, F- fe m a le s, M- m a le s.
Software Loc ality Total number of Individuals in Loc ality of displac ement
individuals analyzed Linked individuals the disc repant group of the disc repant group
F M F M F M F M
no % no % no % no %
CP 1 5 1 5 1 5 1 0 0 .0 1 5 1 0 0 .0 0 0 0 0 -
-NTSYS IL 1 5 1 5 1 4 9 3 .0 1 3 8 7 .0 1 7 .0 2 1 3 .0 SB SB
MC 1 5 1 5 1 0 6 7 .0 1 3 8 7 .0 5 3 3 .0 2 1 3 .0 IL SB
SB 1 5 1 5 1 2 8 0 .0 1 4 9 3 .0 3 2 0 .0 1 7 .0 IL SB
N
S L W
Capricórnio
Tropic Equador
Baturité - CE
Corte de Pedra -BA
Ilhéus - BA
Martinho Campos -MG
1
2
3
4
CP-M154 CP-M168 CP-M171 CP-M157 CP-M162 CP-M170 CP-M184 CP-M188 CP-M174 CP-M175 CP-M179 CP-M181 CP-M190 CP-M176 CP-M189
IL-M253 IL-M259 IL-M271 IL-M260 IL-M263 IL-M277 IL-M270 IL-M274 IL-M272 IL-M279 IL-M264 IL-M276 IL-M268 SB-M358 IL-M275 IL-M280 MC-M52 MC-M81
SB-M357 SB-M369 SB-M387 SB-M379 SB-M380 SB-M375 SB-M371 SB-M372 SB-M384 SB-M373 SB-M374 SB-M368 SB-M370 SB-M383 MC-M73 MC-M74 MC-M54 MC-M67 MC-M79 MC-M70 MC-M78 MC-M75 MC-M89 MC-M97 MC-M91 MC-M96 MC-M100
0.53 0.61 0.69 0.78 0.86
Coefficient of association
Deviation=0.002
Scale 1:22 000 000 110 200 400Km
Fi gu re 3 - Ge n e ra l p h e n o gra m f o r m a le s o f Lutzo myia whitmani ge n e ra te d b y NT- SYS s o f tw a r e . Ea c h s p e c i m e n w a s i d e n ti f i e d b y th e i n i ti a ls o f th e p o p u la ti o n ( CP: Co rte d e Pe d ra /MG; IL: Ilh é u s/BA; MC: Ma rti n h o Ca m p o s/ MG; SB: Se rra d e Ba tu ri té /CE) , se x ( M f o r m a le ) a n d a th re e d i gi ts n u m b e r. Th e ve rti c a l li n e re p re se n ts th e a ve ra ge le ve l o f si m i la ri ty b e tw e e n a ll th e p a i rs o f OTU’s a n a lyze d ( p h e n o n li n e ) .
Fi gu re 4 - Ge o gra p h i c d i stri b u ti o n o f th e lo c a li ti e s u se d f o r sa n d f ly c a p tu re s i n Bra z i l, sc h e m a ti c re p re se n ta ti o n o f th e Lutzo m yia whitm ani
The dis c r e pa nt fe m a le s fr o m I lhé us po pula tio n gr o upe d c onsistently with Serra do B aturité ( CE) phenon and vic e-versa. The fac t is indic ative o f a high de gr e e o f similar ity b e twe e n these two po pulatio ns. The displac ements o bserved ( Martinho Cam po s and Se r r a de B atur ité to war ds Ilhé us and Ilhé us towards Serra do Baturité) suggest the existenc e of genetic flows b e twe e n the line age s no r th-so uth/no r th-e ast po pulatio ns and Ilhéus/Ser r a do B atur ité ( Figur e 4 ) .
Analysis of male populations revealed aspects similar to those discussed for females. The great majority of specimens were grouped accordingly to their region of origin ( Figure 3 ) . However, differently from the females, 13% of males from Martinho Campos were displaced towards Serra de Baturité instead of Ilhéus. It is most probable that Martinho Campos population belongs to the north-south lineage, which is extensively distributed in Brazil from the States of Piauí ( PI) to Paraná ( PR)2 0 2 1. It is probable that the genetic non-uniformity of
the lineage results from the existence of sympatric populations in those regions. As pointed out before, individuals with intermediate profiles could be explained by the occurrence of a gene flow.
I n c o n c lus io n , a n a lys is o f th e dis c r e pa n t s pe c im e n s o f
L. whitm a n i suggested some gene flow among the populations under study. We believe that further studies on the susc eptibility of L. whitm a ni from different regions to Le ishm a nia infec tion, asso c iate d with intr a- and inte r-line age s analyse s, will he lp to c la r ify o ur un de r s ta n din g o f s ym pa tr y, ge n e tic flo w a n d adaptability o f these po pulatio ns o f leishmaniasis vec to r s.
ACKNOWLEDGMENTS
To Ric ar do Andr ade B ar ata fo r te c hnic al he lp with the phle b o to mine b r e e ding and slide mo unting, to the Núc le o de Medic ina da Universidade do Ceará and to the Fundaç ão Nac ional de Saúde ( Coordenadorias Regionais B ahia and Ceará) for their assistanc e dur ing field wo r k.
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