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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 97, Num. 7, 2002, pp. 997-1004
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Mem Inst Oswaldo Cruz, Rio de
Janeiro, Vol. 97(7), October
2002, pp. 997-1004
Morphological and
Polymerase Chain Reaction-Restriction Fragment Lenght Polymorphism Characterization
of Biomphalaria kuhniana and Biomphalaria amazonica from Colombia
Luz E Velásquez*, Roberta
L Caldeira, Victoria Estrada*/**, Omar S Carvalho/+
Centro de Pesquisas René Rachou-Fiocruz,
Av. Augusto de Lima 1715, 30190-002 Belo Horizonte, MG, Brasil *Laboratorio
de Malacología Medica, Universidad de Antioquia, Colombia **Grupo de
Sistemática Molecular, Universidad Nacional de Colombia, Medellín,
Colombia
+Corresponding author. Fax: + 55-31-3295.3115.
E-mail: omar@cpqrr.fiocruz.br
Work partially
supported by Fapemig.
Receibed 11 April 2002
Accepted 12 June 2002
Code Number: oc02224
In Colombia, five Biomphalaria
planorbid species are known: B. kuhniana, B. straminea, B. peregrina, B.
canonica and B. oligoza(var. B. philippiana). Among them, B.
straminea is intermediate host of Schistosoma mansoni and B.
peregrina has been found to be experimentally susceptible to this parasite.
B. straminea is commonly confused with B. kuhniana and they have been
clustered together with B. intermedia in the complex named B. straminea.
The difficulties involved in the specific identification, based on morphological
data, have motivated the use of new techniques as auxiliary tools in cases of
inconclusive morphological identification of such planorbid. In the present
study, five Biomphalaria populations from the Colombian Amazon region
and from Interandian Valleys were morphologically identified and characterized
by polymerase chain reaction-restriction fragment lenght polymorphism directed
at the internal transcribed spacer region of the rRNA gene, followed by digestion
of the generated fragment with restriction enzymes (DdeI, AluI,
RsaI, MvaI and HaeIII). Known profiles of the Brazilian
species B. straminea, B. peregrina, B. kuhniana, B. intermedia and B.
amazonica, besides B. kuhniana from Colombia, were used for comparison.
The five populations under study were morphologically and molecularly identified
as B. kuhniana and B. amazonica.
Key words: Biomphalaria kuhniana
- Biomphalaria amazonica - snails - polymerase chain reaction - restriction
fragment length polymorphism - Colombia
The Colombian snail fauna of the
genus Biomphalaria is represented thus far by five species: B. kuhniana
(DeJong et al. 2001), B. peregrina (Malek 1985), B. straminea
(Barbosa 1968), B. canonica and B. oligoza var. B. philippiana
(Uribe 1950). Among them, B. straminea is intermediate host
of Schistosoma mansoni, being one of the main species responsible for
schistosomiasis transmission in many localities of the Northeastern region of
Brazil. This species was described by Dunker (1848) and according with Paraense
(1963) "The type locality was vaguely mentioned as South America, where
several species answering to that description are known to occur" and "Martens
(1873) restricted the type locality of P. stramineus to Venezuela (Lagunilla
and Caracas), and also referred it to the State of Ceará, Brazil".
Being experimentally susceptible to S. mansoni, the species B. peregrina
is regarded as a potential host of the trematode (Paraense & Corrêa
1973). Concerning the epidemiological importance of B. straminea for
schistosomiasis, this species is commonly confused with B. kuhniana and
B. intermedia. For this reason, these three species were clustered into
a group named B. straminea complex (Paraense 1988). The difficulties
involved in specific identification based on morphological characters have motivated
the use of more modern techniques such as molecular biology.
The type locality of B. kuhniana
(Clessin, 1883) is Paramaribo, Surinam but it is also found in Cayenne, French
Guyana (Floch & Fauran 1954a,b, Floch & Lajudie 1945), Venezuela (Baker
1930), Tucuruí, Pará, Brazil (Paraense 1988), and Panama (Paraense
- pers. commun. 1998). Regarding susceptibility, Floch and Fauran (1954b) showed
that B. kuhniana is resistant to S. mansoni infection.
The type locality of B. amazonica
Paraense, 1966, is Manaus, Amazonas, Brazil. To date, its distribution is restricted
to the Brazilian states of Acre, Amazonas, Rondônia (Paraense 1983), Mato
Grosso (Paraense 1983), Mato Grosso do Sul (Dorval & Silva 1990) and Bolivia
(Pontier et al. 2002). Experiments on susceptibility showed that such species
is a potential host of S. mansoni (Corrêa & Paraense 1971,
Paraense & Corrêa 1985). Vidigal et al. (2000a) characterized this
snail by polymerase chain reaction-restriction fragment lenght polymorphism
(PCR-RFLP) with the enzyme DdeI and detected three species-specific profiles
in specimens from the same or different localities.
The PCR-RFLP technique has been successfully
used in studies on Biomphalaria (Vidigal et al. 1998, 2000a, Caldeira
et al. 1998, 2000, Spatz et al. 1999), Oncomelania (Hope & McManus
1994) and Bulinus species (Stothardet al. 1996, Stothard & Rollinson
1997). This methodology was also used for molecular identification of Mammalia,
Coleoptera and Platyhelminthes (Clark et al. 2001, Dynes et al. 2001, Verkaar
et al. 2002) and for the distinction of cryptic species within the Trypanosoma
brucei group (Agbo et al. 2001). The technique is based on the amplification
of a particular genomic DNA region, followed by digestion of the generated fragment
with restriction enzymes. Considering the simplicity and easy execution of the
technique, in the current study we used PCR-RFLP and morphological identification
in order to characterize Biomphalaria snails from the Colombian Amazon
region and the Valles Interandinos.
MATERIALS AND METHODS
Snail populations - This
study was carried out using snail populations from five localities in Colombia
(Fig. 1), together with specimens
identified as B. peregrina, B. straminea, B. intermedia, B. amazonica
and B. kuhniana (Table) used
for comparison. All the snails obtained from the field were examined for the
presence of S. mansoni cercariae.
Morphological identification
- Ten specimens of each populationfrom Colombiawere killed, their feet removed
and conserved in ethanol. The remaining material was fixed and dissected for
morphology of the shell and reproductive organs as described by Deslandes (1951)
and Paraense (1975, 1976, 1988).
DNA extraction -Total DNA
was extracted from the feet of the snails by phenol-chloroform extraction and
ethanol precipitation (Vidigal et al. 1994).
PCR-RFLP analysis - The entire
ITS region (which includes the 5.8S rDNA gene together with the flanking ITS1
and ITS2 spacers) was amplified using the primers ETTS2 (5-TAACAAGGTTTCCGTAGGTGAA-3)
and ETTS1 (5-TGCTTAAGTTCAGCGGGT-3) (Kane & Rollinson 1994). PCR amplification
conditions were the same as used by Vidigal et al. (1998). Several enzymes employed
in our previous studies with Biomphalaria snails (Vidigal et al. 1998,
2000a, Caldeira et al. 1998, 2000) were used here: DdeI, AluI,
RsaI, MvaI and HaeIII. Digestion and RFLP analysis were performed
as described by Vidigal et al. (1998).
RESULTS
Morphological identification-
The snails were morphologically identified as B. amazonica (Fig.
2) and B. kuhniana (Figs 3-4).
The vaginal corrugation, so characteristic of the B. straminea complex,
show a difference among the B. kuhniana populations studied. Specifically,
in the snails from Llanogrande (located at 2,100 m altitude), minimal swellings
in the vaginal wall were found, in sharp contrast with the finding in snails
from Acacias (530 m altitude).
All populations showed to be negative
for S. mansoni cercariae. Some specimens of B. kuhniana from Acacias
were infected with unidentified trematode cercariae.
Restriction profile analysis
- DNA amplification with the ETTS1 and ETTS2 primers generated a fragment of
approximately 1,300 bp for all specimens. Fig.
5 shows the profiles obtained with the enzyme DdeI for Brazilian
populations of B. peregrina (lanes 1, 2), B. intermedia (lanes
3, 4), B. straminea (lanes 5, 6), B. kuhniana (lanes 7, 8), for
B. kuhniana populations from Venezuela (lanes 9, 10) compared with populations
from the Interandean Valleys (lanes 11 to 22). The four populations from the
Interandean Valleys showed species-specific profiles for B. kuhniana.
The enzymes AluI, RsaI, MvaI and HaeIII generated
species-specific profiles for the four B. kuhniana populations from the
InterAndean Valleys (data not shown).
Fig.
6 shows the profiles obtained with the enzyme DdeI for Brazilian
populations of B. amazonica (lanes 1 to 11; 16-17) compared with the
samples from Colombian Amazonia (lanes 12 to 15). The profiles of the latter
proved similar to each other and also to one of the three profiles of the Brazilian
B. amazonica.
DISCUSSION
We report here for the first time
the presence of B. amazonica in Colombia. The genitalia characters of
this species correspond to those described by Paraense (1966). It does not present
phenotypic plasticity but has well-defined morphological characters. On the
other hand, the molecular profile of these snails sometimes shows three variants
in the same locality (Vidigal et al. 2000a). Such intraspecific variation was
further studied by Vidigal et al. (2000b), when the ITS2 region of Brazilian
Biomphalaria snails was sequenced. Through PCR-RFLP analysis, using the
same snail samples, these investigators could observe, in all trees, a polymorphism
concerning the position of the three individuals (two from Amazonas and one
from Mato Grosso, Brazil). Dejong et al. (2001) observed an intraspecific variation
in two B. amazonica Brazilian snail populations (the same under study
by Vidigal et al. 2000a, b) and in another from Bolivia, using combined data
of the sequenced regions ITS1, ITS2 of rRNA gene and partial subunit 16S mitochondrial
of rRNA gene of 23 Biomphalaria species (16 Neotropicals and 7 Africans).
However, the Colombian specimens showed only one of the three profiles, reported
by Vidigal (2000a), which mirrors less polymorphism when compared with Brazilian
specimens. The phylogenetic relationship of B. amazonica, obtained through
sequencing analyses by Vidigal et al. (2000b) and DeJong et al. (2001), with
species of the same genus, from Neotropical and African regions, showed that
B. amazonica possesses high affinity with the species of the complex
B. straminea.
Among the populations of B. kuhniana
under study presented variability of the vaginal corrugation, with more marked
differences among the populations from Llanogrande, which showed very slight
swellings, and from Acacias, which exhibited conspicuous corrugation. Interestingly,
this kind of morphological variability was not detected at the intrapopulational
level in contrast with the finding reported in B. kuhniana from Tucuruí,
Brazil (Paraense 1988).
Owing to the morphological similarity
and short genetic distance between B. straminea and B. kuhniana
(Paraense 1988, Caldeira et al. 1998), these species are commonly confused by
health technicians who are not specialized in Malacology. Indeed, snails from
Venezuela identified as B. straminea were actually B. kuhniana
(Caldeira et al. 2000). Such misclassification may also have occurred in Colombia
since some populations of B. kuhniana, previously identified as B.
straminea (Velásquez & Vélez 1999), were molecularly characterized
as B. kuhniana in the present study.
Caldeira et al. (1998), by using
PCR-RFLP analysis of the ITS region of rRNA of B. straminea, B. intermedia,
B. kuhniana and B. peregrina observed the cluster of three groups,
which comprise: I) B. straminea and B. kuhniana; II) B. intermedia
and III) B. peregrina. Groups I and II are more closely related while
the third one showed to be a distant group. It was very clear the close relationship
between B. straminea and B. kuhniana, and despite the morphological
similarity of B. straminea and B. intermedia with B. peregrina,
it could not be included in the complex, which is supported by the morphological
information reported by Paraense (1988). Following this, Vidigal et al. (2000b),
through the sequencing of ITS2 of rRNA of the ten Brazilian Biomphalaria
species, confirmed that B. kuhniana (from Venezuela and Brazil) is more
closely related with B. straminea (from Brazil) than with B. intermedia
(from Brazil). Afterwards, Dejong et al. (2001) observed that, among the
23 Biomphalaria species studied, the most closely related group was that
formed by B. straminea (Brazil), B. kuhniana (Dominica, Colombia
and Venezuela) and B. intermedia (Paraguay). However, these authors remark
that when the region 16S was separately analyzed, two groups were then formed:
(1) B. straminea (Pará, Brazil) and B. kuhniana; (2) B.
straminea (São Paulo, Brazil) and B. intermedia. Only after
the analysis of the three DNA regions was it possible to observe the cluster
of a single group. These authorsspeculate about a possible hybridization among
these species, which could explain the close relationship among them. It is
also important to remark that for Venezuela, country considered the type-locality
of B. straminea, such species appear not to exist anymore, as recently
reported by Caldeira et al. (2000), perhaps suggesting a misclassi-fication
of the species, or, yet, a progressive substitution of B. straminea for
B. kuhniana.
Therefore, the correct identification
of these snails is of great importance since it allows the detection of species
in areas of schistosomiasis transmission, as well as in areas free of the disease,
which might become schistosomiasis foci, owing to the presence of natural or
experimentally susceptible species. Thus, the methodology using PCR-RFLP proved
to be effective for the characterization of Colombian Biomphalaria snails
since it was able to confirm the classical morphologic identification.
ACKNOWLEDGMENTS
To Dr W Lobato Paraense, Department
of Malacology, Instituto Oswaldo Cruz, Rio de Janeiro, for confirming the identification
of Biomphalaria snails from Colombia; to Dr Sandra Uribe Soto, Universidad
Nacional, Medellín for logistic support for the trip to Brazil; to Dr
Piedad Vitoria Daza and Ariel Rodriguez for collecting specimens in Meta; to
Mauricio Rodriguez for collecting and sending Colombian snail specimens and,
finally, to the biologist María Carmenza Hincapie for the drawings.
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Copyright 2002 Instituto Oswaldo
Cruz - Fiocruz
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