3 5 1
Revista da Sociedade Br asileir a de Medicina Tr opical 3 7 ( 4 ) :3 5 1 -3 5 3 , jul-ago, 2 0 0 4
Polymer ase chain r eaction and r estr iction fr agment length polymor phism
analysis of the ITS2 region for differ atiation of Brazilian
Biomphalaria
inter mediate hosts of
the
Schistosom a m an son i
Reação em cadeia da polimerase e polimorfismo de tamanho de fragmento de restrição
da
região do ITS2 para a diferenciação dos moluscos brasileiros do gênero Bio m pha la ria
hospedeiros intermediários do Schisto so m a m a nso ni
Teofânia Heloísa Dutr a Amor im Vidigal
1 , 2, Kelly Gr ace Magalhães
1and Omar dos Santos Car valho
1ABSTRACT
We se q u e n c e d th e i n te rn a l tra n sc ri b e d sp a c e r 2 o f th e ri b o so m a l DNA ( ITS2 - DNAr) f ro m th e th re e
Sc histo so ma manso ni
i n te rm e d i a te h o sts i n Bra zi l:
B io mphalar ia glab r ata
,
B io mphalar ia te nago phila
a n d
B io mphalar ia str amine a
. An a lysi s o f a
re stri c ti o n m a p f ro m th o se se q u e n c e s a llo we d u s to se le c t p u ta ti ve re stri c ti o n e n zym e s a b le to i d e n ti f y th e sn a i l sp e c i e s
u n d e r stu d y. Fo u r re stri c ti o n e n zym e s we re u se d a n d Hp a II p ro vi d e d si m p le sp e c i e s- sp e c i f i c p ro f i le s e a si ly vi su a li ze d i n
p o lya c ryla m i d e ge ls. Th e u se o f ITS2 i s a d va n ta ge o u s a s i t p ro vi d e s a sm a ll f ra gm e n t o f 4 6 0 b p wh i c h m a y b e e a si ly
a m p li f i e d b y PCR. In th e c u rre n t wo rk , we sh o we d th a t th e a m p li f i c a ti o n o f ITS2 - DNAr to ge th e r wi th Hp a II e n zym e
re stri c ti o n i s a n a u x i li a ry m o le c u la r to o l f o r th e m o rp h o lo gi c a l i d e n ti f i c a ti o n o f su c h sn a i ls a s we ll a s f o r ta x o n o m i c
a n d p h ylo ge n e ti c stu d i e s o f n e o tro p i c a l p la n o rb i d s.
Ke y- wo r ds:
B io mphalar ia glab r ata
.
B io mphalar ia te nago phila
.
B io mphalar ia str amine a. Po lyme r ase c hain r e ac tio n. Inte r nal
tr ansc r ib e d spac e r 2 .
RESUMO
O se q u e n c ia m e n to da re giã o e spa ç a do ra tra n sc rita in te rn a 2 do DNA rib o sso m a l ( ITS2- DNAr) da s e spé c ie s b ra sile ira s
gê n e ro
B iomphalaria
(
B . glabrata
,
B . tenagophila
a n d
B . straminea
) ho spe de ira s in te rm e diá ria s do
Sc histosoma mansoni
n o
Bra sil, pe rm itiu a a n á lise do s sítio s de re striç ã o pre se n te s n e sta s se q ü ê n c ia s. A a n á lise do m a pa de re striç ã o o b tido de ssa s
se q ü ê n c ia s n o s pe rm itiu se le c io n a r e n zim a s m a is pro m isso ra s q u e ge ra sse m pe rfis de re striç ã o c a pa ze s de ide n tific a r
e ssa s e spé c ie s. Fo ra m te sta da s 4 e n zim a s e a e n zim a Hpa II fo i se le c io n a da po r pro du zir pe rfis e spé c ie e spe c ífic o s de fá c il
visu a liza ç ã o e m ge l de po lia c rila m ida . A u tiliza ç ã o da re giã o ITS2 te m c o m o va n ta ge n s a o b te n ç ã o de u m fra gm e n to
pe q u e n o de 460b p, o q u a l po de se r fa c ilm e n te a m plific a do po r PCR. Ne ste tra b a lho , n o s de m o n stra m o s q u e a a m plific a ç ã o
da re giã o ITS2- DNAr e a re striç ã o de ste c o m a e nzim a Hpa II é um a fe rra m e nta m o le c ula r a uxilia r a ide ntific a ç ã o m o rfo ló gic a
de sse s m o lu sc o s, b e m c o m o pa ra e stu do s ta xo n ô m ic o s e filo ge n é tic o s de pla n o rb íde o s n e o tro pic a is.
Pa la vr a s - c ha ve s :
B io m pha la r ia gla b r a ta
.
B io m pha la r ia te na go phila
.
B io m pha la r ia s tr a m ine a .
Re a ç ã o e m c a d e i a d a
p o li m e ra se
.
Re gi ã o e sp a ç a d o ra tra n sc ri ta i n te rn a 2
.
COMUNICAÇÃO/COMMUNICATION
1 . 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, B r asil. 2 . De par tame nto de Zo o lo gia do Instituto de Ciê nc ias B io ló gic as da Unive r sidade Fe de r al de Minas Ge r ais, B e lo Ho r izo nte , MG, B r asil
Wo r k par tially suppo r te d b y CAPES, FAPEMIG and PIB IC/FIOCRUZ
Addr e ss to: Dr. Omar do s Santo s Car valho . CPq RR/FIOCRUZ. Av. Augusto de Lima 1 7 1 5 , 3 0 1 9 0 - 0 0 2 B e lo Ho r izo nte , MG, B r asil. Fax: 5 5 3 1 3 2 9 5 - 3 1 1 5 .
3 5 2
Vidiga l THDA e t al
Among the 1 0 Brazilian snail spec ies and one subspec ies of
th e ge n u s
B i o m p h a la r i a
d e s c r i b e d u p to th e p r e s e n t,
B . gla b ra ta
1 7,
B. te na go phila
8,
B. stra m ine a
5,
B. te na go phila
gu a i b e n si s
1 3B. p e re gri n a
8, B. k u h n i a n a
3, B. sc h ra m m i
4,
B . a m a z o n i c a
9, B . o l i g o z a
1 0, B . i n t e r m e d i a
1 6a n d
B . o ccide nta lis
1 2only the first three spec ies have been found
naturally infec ted by
Schisto so m a m a nso ni
1 4.
Th e c o r r e c t ide n tific a tio n o f
Bi o m p h a la ri a
s n a ils is
c omplic ated due to the high intra-spec ific variation in anatomic al
and morphologic al c harac ters or great similarity among some
spe c ie s
9 1 1 1 5. The a va ila b ility o f m e tho do lo gie s b a se d o n
molec ular analysis has enabled the ac c ess to more c onsistent
information on
Bio m pha la ria
populational struc ture among
Planorbidae. Molec ular taxonomy has been able to solve several
problems c onsidered insoluble so far by traditional morphology.
The po lymerase c hain reac tio n and restric tio n fragment
le ngth po lymo r phism ( PCR- RFLP) analysis o f the inte r nal
tr a n s c r ib e d s p a c e r ( I TS) r e gio n o f th e r DNA ( 1 3 0 0 p b
appr o ximate ly) and a par t o f COI r e gio n o f mito c ho ndr ial
DNA ( mit-COI – 7 8 0 bp appro ximately) have been used fo r
identific ation of several
Bio m pha la ria
spec ies from Brazil and
some regions of South Americ a
1 2 1 8 1 9 2 1 2 2. Restric tion enzymes
were randomly selec ted due to the lac k of available sequenc es
from
Bio m pha la ria
ITS region. But for part of the mit-COI region,
analysis of a restric tion map available in a data base allowed us
to selec t partic ular enzymes to be tested and used in PCR-RFLP.
In the present work, we report the use of this methodology for
spe c ific ide ntific atio n o f fie ld po pulatio ns o f
B. gla b ra ta ,
B. stra m i n e a
and
B. te n a go phi la
fr o m
diffe r e nt B r azilian
localities using restriction profiles provided by the ITS2 region.
The choice of such region was due to two specific reasons: 1 ) on
account of the size of the generated fragment for
Bio m pha la ria
,
afte r PCR amplific atio n ( appr o ximate ly 4 6 0 b p) . This is a
considerably small product when compared with the size of
ITS-rDNA and the COI region from this genus, enabling an easier
amplification with no need of a high quality DNA; 2 ) this region
proved to be appropriate as it had been sequenced and analyzed
in phylogenetic studies of Brazilian
Bio m pha la ria
species
2 0.
Ten spec imens of eac h population were killed and fixed.
B e fo r e fixing the spe c ime ns, a fr agme nt o f the ir fo o t was
removed for subsequent DNA extrac tion. Fixed spec imens were
ide n tifie d b y m e a n s o f c o m pa r a tive m o r ph o lo gy o f th e
reproduc tive organs and shells
1 1 1 2 1 3 1 5.
Total DNA was extrac ted
fro m the fo o t o f eac h snail using the Wizard Geno mic DNA
Purific ation Kit ( Promega)
2 1. The ITS2 region was amplified
using the primers ITS2 F ( 5
’-CGTCCGTCTGAGGGTCGGTTTGC-3 )
2 0and ETTS1 ( 5
’-TGCTTAAGTTCAGCGGGT-3 ’)
7anc hored in
the c onserved extremities of the 5 .8 S and 2 8 S ribosomal genes,
respec tively. The PCR amplific ation c onditions were the same
used by Vidigal et al
1 9, exc ept for the annealing temperature,
whic h was 6 0 ° C.
The PCR amplific ation of
Bio m pha la ria
ITS2 region, from
four spec imens of eac h spec ies under study, resulted in a produc t
of approximately 4 6 0 bp. Theses produc ts were digested using
the following restric tion enzymes:
Ta q
I and
Mb o
I
( Invitrogen,
Life Sc ienc e)
, Rsa
I and
Hpa
II ( Promega Co, USA) . These enzymes
were selec ted based on the restric tion map analysis using the
program Webc utter version 2 .0 , ( www.firstmarket.c om/c utter/
c ut2 .html) of the ITS2 sequenc es available in the Genbank
2 0. –
The ac c e ss numb e r s use d in o ur study we r e :
B. gla b ra ta ,
AF1 9 8 6 5 9 , AF1 9 8 6 6 0 , AF1 9 8 6 6 1 , AF1 9 8 6 6 2 ;
B. te na go phila
AF1 9 8 6 5 4 , AF1 9 8 6 5 5 , AF1 9 8 6 5 6 ;
B. stra m i n e a
AF1 9 8 6 6 8 ,
AF1 9 8 6 6 9 , AF1 9 8 6 7 0 , AF1 9 8 6 7 1 , AF1 9 8 6 7 2 .
Afterwards, the fragments were visualized in 6 % silver-stained
po lya c r yla m ide ge ls . Dige s tio n a n d R FLP a n a lys is we r e
performed
1 8and the gels photographed with a Mavic a digital
c amera ( Sony) .
The profiles obtained with
Ta q I
and
Rsa
I did not allow us to
distinguish between the three spec ies due to the high similarity
among RFLP profiles ( data not shown) . The most promising
profiles were those produc ed by
Mb o I
and
Ha p
II, and the best
result was obtained with
Ha p
II ( Figure 1 ) , whic h provided a
simple profile of four fragments for
B. gla b ra ta
, ( 2 0 0 , 1 3 0 , 9 0
and 7 0 bp) ,
B. te na go phila
( 2 0 0 , 1 2 0 , 9 0 and 6 0 bp) and two
fragments fo r
B. stra m in e a
( 3 0 0 and 1 8 0 bp) . Altho ugh
B.
gla b ra ta
and
B. te na go phila
share the fragments of 2 0 0 and
9 0 bp ( Figure 1 ) , they c ould be separated by other two
non-shared fragments: Bg1 ( 1 3 0 bp) and Bg2 ( 7 0 bp) for
B. gla b ra ta
;
Bt1 ( 1 2 0 bp) and Bt2 ( 7 0 bp) for
B. te na go phila
. The restric tion
pro file fo r
B. stra m i n e a
c o mprised: B s1 ( 3 0 0 bp) and B s2
( 1 8 0 bp) ( Figure 1 ) . Reproduc ibility of the generated profiles
with
Ha p
II was supported by the use of spec imens originated
from different loc alities in Brazil ( Figure 1 ) .
Fi gu r e 1 - Si lve r- s ta i n e d 6 % p o lya c r yla m i d e ge l s h o w i n g th e
re stri c ti o n p ro f i le s o b ta i n e d wi th e n zym e
HpaII o f th e ITS2 re gi o n
o f DNA e x tra c te d f ro m
B io mphalar iasp e c i e s i n te rm e d i a te h o sts o f
th e
S. manso nii n th e Bra zi l. La n e 1 :
B . glab r ataf ro m To u ro s
( Sta te
o f Ri o Gra n d e d o No rte ) ; La n e 2 :
B . glab r ataf ro m Cu ru ru p u ( Sta te
o f Ma ra n h ã o ) ; La n e 3 :
B . glab r ataf ro m Ja c o b i n a ( Sta te o f Ba h i a ) ;
La n e 4 :
B . glab r ataf ro m Sa b a rá ( Sta te o f Mi n a s Ge ra i s) ; La n e 5 :
B . te nago philaf ro m Ve sp a si a n o ( Sta te o f Mi n a s Ge ra i s) ; La n e 6 :
B . te nago philaf ro m Fo rm o sa ( Sta te o f Go i á s) ; La n e 7 :
B . te nago philaf ro m Flo ri a n ó p o li s ( Sta te o f Sa n ta Ca ta ri n a ) ; La n e 8 :
B . te nago philaf ro m Im b é ( Sta te o f Ri o Gra n d e d o Su l) ; La n e 9 :
B . str amine af ro m
Pi c o s ( Sta te o f Pi a u í ) ; La n e 1 0 :
B . str amine af ro m Ja c o b i n a ( Sta te
o f Ba h i a ) ; La n e 1 1 :
B . str amine af ro m Ju i z d e Fo ra ( Sta te o f
Mi n a s
Ge ra i s) ; La n e 1 2 :
B . str amine af ro m Gu a í ra ( Sta te o f Pa ra n á ) . Th e
a rro ws a n d a b b re vi a ti o n s i n d i c a te sp e c i e s sp e c i f i c f ra gm e n ts.
B . glab r ata: Bg1 - 1 3 0 b p a n d Bg2 - 7 0 b p ;
B . te nago phila, Bt1 - 1 2 0 b p a n d
Bt2 - 7 0 b p a n d
B . str amine a, Bs1 3 0 0 b p a n d Bs2 1 8 0 b p . Mo le c u la r
si ze m a rk e rs a re sh o w n o n th e le f t o f e a c h ge l.
B. Glabrata B. tenagophila B. straminea
1 2 3 4 5 6 7 8 9 1 0 1 1 1 2
6 0 3
3 1 0
1 9 4 Bg1
1 1 8
Bg27 2
Bg1
3 5 3
Revista da Sociedade Br asileir a de Medicina Tr opical 3 7 ( 4 ) :3 5 1 -3 5 3 , jul-ago, 2 0 0 4
These results demonstrated that PCR-RFLP of the ITS2 region,
using
Hpa
II restriction enzyme, is an important tool to distinguish
among
B. glabrata
,
B. stram inea
and
B. tenagophila
species. Such
data is in accordance with those produced for ITS and COI regions,
through the same technique
19 22and it also corroborates classical
morphological taxonomy.
In general success of the amplification using degraded DNA is
difficult to achieve and severely restricted in target size ( degraded
DNA results in amplific atio n o f relatively small fragments)
6.
Regarding this aspect, we believe that such methodology may be
used in studies, in whic h degraded DNA is rec o vered fro m
improperly conserved material ( low molecular weight < 5 0 0 bp) .
Thus, the fragment of 4 6 0 bp correspondent to the ITS2 region
may be more easily amplified by PCR than a region of approximately
1 3 0 0 bp ( approximate size of ITS-rDNA from Planorbidae) , due
to the need for a more conserved or high quality DNA.
ACKNOWLEDGEMENTS
To Dr a Liana K. Passo s fr o m Ce ntr o de Pe squias Re né
Ra c ho u/FI OCRUZ who pr o vide d us with va lua b le c r itic a l
c o mme nts.
REFERENCES
1 . Ca lde ir a R L, Vidiga l THDA, Ma r tin e la L, Sim ps o n AJ G, Ca r va lh o OS. Ide ntific atio n o f Plano r b ids fr o m Ve ne zue la b y Po lyme r ase Chain Re ac tio n Amplific atio n and Re str ic tio n Fr agme nt Le ngth Po lymo r phism ( PCR-RFLP) . Me mó r ias do Instituto Oswaldo Cr uz 9 5 : 1 7 1 - 1 7 7 , 2 0 0 0 .
2 . Ca lde ir a RL, Vidiga l THDA, Pa ulin e lli ST, Sim ps o n AJ G, Ca r va lh o OS. Mo le c ular ide ntific atio n o f sim ilar spe c ie s o f the ge nus Bi o m p h a la ri a ( Mo llusc a: Plano r b idae ) de te r mine d b y PCR-RFLP. Me mó r ias do Instituto Oswaldo Cr uz 9 3 : 2 1 9 - 2 2 5 , 1 9 9 8 .
3 . Cle ssin S. Die Familie de r Limnae ide n, e tc . In : Mar tini, Che minitz ( e ds) Syste matisc he s Co nc hy - Cab ine t, Editio n 2 , vo l. 1 , pt. 1 7 . B aue r & Raspe , Nür nb e r g, 1 8 8 3 -1 8 8 4 .
4 . Cr o s s e H. De s c r i p ti o n d’ e s p é c e s n o u ve l l e s . J o u r n a l o f Co n c h yl i o l o gi e 1 2 : 1 5 2 - 1 5 4 , 1 8 6 4 .
5 . Dunk e r W. Diagno se s spe c ie r um no var um ge ne r is Pla n o rb i s c o lle c tio nis Cumingianae . Pr o c e e dings o f the Zo o lo gic al So c ie ty o f Lo ndo n 1 6 : 4 0 - 4 3 , 1 8 4 8 , 1 9 9 6 .
6 . Go le nb e r g EM, B ic k e l A, We ihs P Effe c t o f highly fr agme nte d DNA o n PCR. Nuc le ic Ac ids Re se ar c h 2 4 : 5 0 2 6 - 5 0 3 3 , 1 9 9 6 .
7 . Ka n e R A, R o l l i n s o n D R e p e ti ti ve s e q u e n c e s i n th e r i b o s o m a l DNA in te r n a l tr ansc r ib e d spac e r o f Sc h i sto so m a h a e m a to b i u m, Sc h i sto so m a
i n t e r c a la t u m a n d Sc h i s t o s o m a m a t t h e e i. Mo l e c u l a r B i o c h e m i c a l Par asito lo gy 6 3 : 1 5 3 - 1 5 6 . 1 9 9 4 .
8 . Or b igny A. Syno psis te r r e str ium e t fluviatilium mo llusc o r um, in suo pe r Am e r i c a n Me r i di o n a l e m i ti n e r e c o l l e c to r u m . Ma ga s i n de Zo o l o gi e Plano r b is 2 6 -2 8 , 1 8 3 5 .
9 . Par ae nse WL. Bi o m p h a la ri a a m a zo n i c a and B. c o u si n i, two ne w spe c ie s o f Ne o tr o pic al plano r b id mo llusk s. Re vista B r asile ir a de B io lo gia 2 6 :1 1 5 -1 2 6 , -1 9 6 6 .
1 0 . Par ae nse WL. Bi o m p h a la ri a o li go za N. N. fo r Tro p i c o rb i s p h i li p p i a n u s ( Dunk e r ) se nsu Luc e na. Re vista B r asile ir a de B io lo gia 3 4 : 3 7 9 -3 8 6 , 1 9 7 4 . 1 1 . Par ae nse WL. Estado atual da siste mátic a do s plano r b íde o s b r asile ir o s.
Ar q uivo s do Muse u Nac io nal do Rio de J ane ir o 5 5 : 1 0 5 - 1 2 8 , 1 9 7 5 . 1 2 . Pa r a e n s e WL. Bi o m p h a la ri a o c c i d e n ta li s s p. n . fr o m So uth Am e r ic a
( Mo llusc a B aso mmato pho r a Pulmo nata) . Me mó r ias do Instituto Oswaldo Cr uz 7 6 : 1 9 9 - 2 1 1 , 1 9 8 1 .
1 3 . Par ae nse WL. Bi o m p h a la ri a te n a go p h i la gu a i b e n si s ssp.n. fr o m so uthe r n B r azil and Ur uguay ( Pulmo nata: Plano r b idae ) . I. Mo r pho lo gy. Me mó r ias do Instituto Oswaldo Cr uz 7 9 : 4 6 5 - 4 6 9 , 1 9 8 4 .
1 4 . Par ae nse WL. Distr ib uiç ão do s c ar amuj o s no B r asil. In : Re is FA, Far ia I, Ka tz N ( e d s ) Mo d e r n o s c o n h e c i m e n to s s o b r e a e s q u i s to s s o m o s e mansô nic a. B ib lio tec a da Ac ademia Mineir a de Medic ina, p.1 1 7 -1 2 8 . 1 9 8 6 . 1 5 . Pa r a e n s e WL. B i o m p h a la r i a k u h n i a n a ( Cl e s s i n , 1 8 8 3 ) , p l a n o r b i d m o llus c fr o m So uth Ame r ic a. Me mó r ias do Instituto Oswaldo Cr uz 8 3 : 1 -1 2 , -1 9 8 8 .
1 6 . Par ae nse WL, De slande s N. Au stra lo rb i s i n te rm e d i u s sp. n. fr o m B r azil. Re vista B r asile ir a de B io lo gia 2 2 : 3 4 3 - 3 5 0 , 1 9 6 2 .
1 7 . Say T. Ac c o unt o f two ne w ge ne r a, and se ve r al ne w spe c ie s, o f fr e sh wate r and land she lls. J o ur nal o f the Ac ade my o f Natur al Sc ie nc e s Philade lphia 1 : 2 7 6 - 2 8 4 , 1 8 1 8 .
1 8 . Spatz L, Vidigal THDA, Calde ir a RL, Dias Ne to E, Cappa SMG, Car valho OS. Study o f Bi o m p h a la ri a te n a go p h i la te n a go p h i la, B. t. gu a i b e n si s a nd B. o c c i d e n ta li s b y po lyme r ase c hain r e ac tio n amplific atio n and r e str ic tio n e nzyme dige stio n o f the r ib o so mal RNA inte r ge nic spac e r r e gio ns. J o ur nal Mo llusc an Studie s 6 5 : 1 4 3 - 1 4 9 , 1 9 9 9 .
1 9 . Vidigal THDA, Calde ir a RL, Simpso n AJ , Car valho OS. Fur the r studie s o n the mo le c ular syste matic s o f Bi o m p h a la ri a snails fr o m B r azil. Me mó r ias do Instituto Oswaldo Cr uz 9 5 : 5 7 - 6 6 , 2 0 0 0 b .
2 0 . Vidigal THDA, Kissinge r J C, Calde ir a RL, Pir e s EC, Mo nte ir o E, Simpso n, AJ. Car valho OS. Phylo ge ne tic r e latio nships amo ng B r azilian Bi o m p h a la ri a spe c ie s ( Mo llusc a: Plano r b idae ) b ase d upo n analysis o f r ib o so mal ITS2 se q ue nc e s. Par asito lo gy 1 2 1 : 6 1 1 -6 2 0 , 2 0 0 0 a.
2 1 . Vidigal THDA, Mo ntr e so r LC, Simpso n AJG, Car valho OS. Po lyme r ase Chain Re ac tio n and Re str ic tio n Fr agme nt Le ngth Po lymo r phism o f Cyto c hr o me o x i da s e I u s e d fo r di ffe r e n ti a ti o n B r a zi l i a n B i o m p h a la r i a s p e c i e s in te r m e dia te h o s t o f Sc h i s to s o m a m a n s o n i. Me m ó r ia s do I n s tituto Oswaldo Cr uz 9 7 : 4 7 - 5 2 , 2 0 0 2 .
