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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 92, Num. 5, 1997, pp. 661-662
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 92(5),
September/October 1997, pp. 661-662
A laboratory-based approach to biological control of snails
Fred A Lewis^+, Matty Knight, Charles S Richards
Biomedical Research Institute, 12111 Parklawn Drive, Rockville, MD
20852, USA
^+Corresponding author. Fax: 301-770-4756
Received 16 April 1997; Accepted 30 June 1997
Code Number:OC97124
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Development of Schistosoma mansoni in the intermediate host
Biomphalaria glabrata is influenced by a number of parasite and
snail genes. Understanding the genetics involved in this complex
host/parasite relationship may lead to an often discussed approach of
introducing resistant B. glabrata into the field as a means of
biological control for the parasite. For the snail, juvenile susceptibility
to the parasite is controlled by at least four genes, whereas one gene
seems to be responsible for adult nonsusceptibility. Obtaining DNA from F2
progeny snails from crosses between parasite-resistant and-susceptible
snails, we have searched for molecular markers that show linkage to either
the resistant or susceptible phenotype. Both restriction fragment length
polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) approaches
have been used. To date, using a variety of snail and heterologous species
probes, no RFLP marker has been found that segregates with either the
resistant or susceptible phenotype in F2 progeny snails. More promising
results however have been found with the RAPD approach, where a 1.3 kb
marker appears in nearly all resistant progeny, and a 1.1 kb marker appears
in all susceptible progeny.
Key words: snails - genetic susceptibility - biological control -
schistosomiasis - Biomphalaria glabrata - Schistosoma mansoni
- resistance - susceptibility
Infection of the planorbid snail Biomphalaria glabrata by the
helminth parasite Schistosoma mansoni depends on genes from both
snail and parasite (Richards & Shade 1987). A number of laboratory-derived
stocks of B. glabrata have been used to sort out this complex
genetic relationship. The use of snails possessing different susceptibility
phenotypes, and progeny from their crosses, might enable investigators to
determine molecular factors important in these phenotypes. To develop the
snail populations for study, use of pigmentation markers has been essential
for verifying that progeny snails in question result from cross-, and not
by self-, fertilization (Richards et al. 1992). This report describes
attempts to determine molecular markers that segregate with either
susceptibility or resistance to the parasite in F2 progeny from crosses
between B. glabrata snails which are either susceptible or resistant
to S. mansoni. The techniques used for this study encompassed both
restriction fragment length polymorphism (RFLP) and random amplified
polymorphic DNA (RAPD) approaches.
MATERIALS AND METHODS
Snails - Crosses were made from M-line susceptible (Newton 1953)
and BS-90 resistant B. glabrata snails (Paraense & Correa 1963).
Infection phenotypes of progeny F2 snail cohorts was assessed by exposing
5-8 mm dia. snails to S. mansoni miracidia. Snails were examined
weekly and scored as positive either by establishment of primary or
secondary sporocysts, or by shedding of cercariae 4-5 weeks after exposure.
Molecular analysis - DNA was isolated from the snails by standard
procedures (Knight et al. 1991). The quality of DNA was determined by
horizontal flat-bed gel electrophoresis on 0.6% agarose gels.
For RFLP analysis, genomic DNA was digested with a number of restriction
enzymes, and the fragments separated by flat-bed electrophoresis through a
0.6% agarose gel, then transferred to either a nylon or nitrocellulose
membrane according to the technique of Southern (Southern 1975).
Hybridization with radioisotopically-labelled probe pSM389 (McCutchan et
al. 1984), or several homologous probes (unpublished) was followed by
washing, then autoradiography on Kodak X-Omat film with intensifying
screens at -70 C.
For the RAPD-polymerase chain reaction, conditions used were those
described by Williams et al. (1990). A series of 20 arbitrary
oligonucleotide primers were used (Operon Technologies, Inc, Alameda, CA,
USA), and amplifications carried out with Taq DNA polymerase. Amplified
products were initially screened by ethidium bromide staining of gels, and
more sensitive resolution of fragments by silver staining of 4%
polyacrylamide gels.
The genetic heritability of genotypically stable markers, as employed
with both molecular analyses, was determined with DNA from F2 progeny
snails resulting from crosses between resistant and susceptible parents.
RESULTS
Cohort snails from the adult population of F2 progeny snails, derived
from crosses between susceptible and resistant parents, developed a
resistant:susceptible phenotypic ratio of approximately 3:1. This ratio
would be expected if there were one gene involved in resistance to this
parasite strain and inherited in a Mendelian fashion.
When DNA from F2 progeny snails were subjected to RFLP analysis, most
alleles present were inherited in a co-dominant fashion. However, to date
no association with either resistance or susceptibility was noticed with
any of the markers used. This was the case regardless if either homologous
or heterologous probes were tested.
Experiments using the RAPD approach indicated that the majority of
primers tested showed no polymorphisms between the various snail lines
tested, or they were not reproducible among individual isolates from the
same line. Only 2 primers were initially useful for distinguishing snail
lines. Amplification with primer OPA-01 produced a major 180 bp marker in
resistant snails, and a 400 bp fragment in susceptible snails. With primer
OPA-06, a 600 bp marker was found only in resistant snails. A major
specific marker (1100 bp) in susceptible snails was amplified with OPA-06.
Further studies in the reproducibility of these major markers showed that
these may represent invariant structural genotypic differences between
snails of the two separate phenotypes (Larson et al. 1996).
Strain specific markers were inherited in a codominant fashion in the
majority of F2 progeny from crosses between susceptible and resistant
snails. From infectivity studies however there was no apparent linkage in
the inheritance of these markers to the parasite susceptibility phenotypes
of the progeny snails.
DISCUSSION
These studies summarize attempts to determine markers that segregate
according to susceptibility phenotypes among progeny snails derived from
crosses between resistant and susceptible parents. Although snail strain
differentiation can be accomplished by such techniques as RFLP and RAPD
analysis, to date we have not been able to show linkage of susceptibility
phenotypes with the RFLP approach, although some promising results have
been obtained with the RAPD method with several of the markers used.
Such approaches have considerable promise, not only in this type of
study, but in studies of genetic diversity of populations in the field. For
instance Langand et al. (1993) showed the usefulness of the RAPD approach
for evaluating genetic diversity within the genus Bulinus. A recent
report by Vidigal et al. (1994) showed similar genetic diversity, using a
comparable approach, among field isolates of B. glabrata in
Brazil.
The ability to determine resistance or susceptibility markers in field
populations would be an important component of studies to potential
biological control measures employing the use of refractory snails. The
genetic flow of markers within a population would have to be known before
the feasibility of this technique can be realized. Since many complex
issues determine susceptibility and transmission potential in any
population of snails, we are hopeful that approaches such as ours described
here can be useful for developing additional control measures for
schistosomiasis in the near future.
REFERENCES
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mansoni: Use of a cloned ribosomal RNA gene probe to detect restriction
fragment length polymorphisms in the intermediate host Biomphalaria
glabrata. Exp Parasitol 73: 285-294.
- Langand J, Barral V, Delay B, Jourdane J 1993. Detection of genetic
diversity within snail intermediate hosts of the genus Bulinus by
using random amplified polymorphic DNA markers (RAPDs). Acta Tropica
55: 205-215.
- Larson SE, Andersen PL, Miller AN, Cousin CE, Richards CS, Lewis FA,
Knight M 1996. Use of RAPD-PCR to differentiate genetically defined lines
of the intermediate host of Schistosoma mansoni, Biomphalaria
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- Paraense WL,Correa L 1963. Variation in susceptibility of populations
of Australorbis glabratus to a strain of Schistosoma mansoni.
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random amplified polymorphic DNA analysis. Exp Parasitol 79:
187-194.
- Williams JGK, Hanafey MK, Rafalski JA, Tingey SV 1990. DNA
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Meth Enzymol 218: 704-740.
Copyright 1997 Fundacao Oswaldo Cruz
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