Experimental Parasitology 105 (2003) 211–218

www.elsevier.com/locate/yexpr

Fasciola hepatica: identification of molecular markersfor resistant and susceptible Pseudosuccinea columella snail hostsq

Alfredo Guti�errez,a Jean-Pierre Pointier,b Jorge Fraga,a Edouard Jobet,b Sylvain Modat,b

R.T. P�erez,c Mary Yong,a J. Sanchez,a Eric S. Loker,d and Andr�e Th�eronb,*

a Laboratorio de Malacolog�ıa, Instituto ‘‘Pedro Kouri’’, IPK. Apartado 601, Marianao 13, Ciudad Habana, Cubab UMR CNRS-UP 5555, EPHE, Parasitologie Fonctionnelle et Evolutive, CBETM Universit�e 52, Av. Paul Alduy. 66860 Perpignan Cedex, France

c Facultad de Biolog�ıa, Universidad de la Habana, Cubad Department of Biology, University of New Mexico, Albuquerque, NM, USA

Received 8 April 2003; received in revised form 5 November 2003; accepted 11 December 2003

Abstract

Protein electrophoresis, RAPD-PCR and nuclear rDNA ITS sequencing were performed to search for genetic differences between

Pseudosuccinea columella snails susceptible and resistant to Fasciola hepatica infection. Of the 21 enzymatic loci analyzed in both

populations, none of them exhibited neither within- or between-group variation. Such an absence of enzyme polymorphism support

the hypothesis of selfing as the ‘‘prevalent’’ mating system for this hermaphroditic species. Conversely, the RAPD profiles displayed

clear differences between susceptible and resistant isolates for 17 of the 26 primers tested while no within-group variation was

detected. rDNA ITS sequence analysis from snails of each isolates showed only two bases that differed between groups accounting

for a 0.17% of variation confirming that suceptible and resistant snails belong to the same species. This is the first time that a genetic

variation using RAPD markers is demonstrated between susceptible and resistant lymnaeid snails vis-a-vis of F. hepatica infection in

absence of experimental selection.

� 2003 Elsevier Inc. All rights reserved.

Index Descriptors and Abbreviations: DNA, desoxyribonucleic acid; rDNA, ribosomal DNA; ITS, internal transcribed spacer; PCR, polymerase

chain reaction; RAPD, random amplified polymorphic DNA; S, similitude coefficient

Keywords: Fasciola hepatica; Lymnaeidae; Pseudosuccinea columella; Susceptibility; Resistance; Markers

1. Introduction

Fasciola hepatica is widely distributed in temperateand subtropical areas around the world, infecting nu-

merous species of mammals as definitive hosts including

human (Chen and Mott, 1990; Mas-Coma et al., 1999)

and various species of snails of the family Lymnaeidae as

intermediate hosts (Mas-Coma and Bargues, 1997). In

the island of Cuba, two lymnaeid snails, Fossaria cub-

ensis and Pseudosuccinea columella are involved in the

life cycle of F. hepatica and display experimentally a high

qThe sequence data reported herein have been deposited in

GenBank under Accession No. AY186751 (Pseudosuccinea columella).* Corresponding author. Fax: +33-4-68-66-22-81.

E-mail address: theron@univ-perp.fr (A. Th�eron).

0014-4894/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.exppara.2003.12.006

level of susceptibility to this parasite (Guti�errez et al.,

2000). However, we demonstrated previously that a

population of P. columella, sampled in the outskirts ofLa Palma municipality, province of Pinar del Rio, was

not susceptible to this parasite (Guti�errez et al., 2002,

2003). After repeated attempts to induce infection in

these snails, using high miracidial doses (more than 15

miracidia per snail), no evidence of intramolluscan stages

of F. hepatica was observed in any of the snails exposed

and many of them died a few weeks after exposure.

Studies of Richards (1973, 1975, 1984) and Richardsand Merritt (1972) have demonstrated that snail sus-

ceptibility/resistance to schistosome infection is con-

trolled by genetic factors and genetic variability within

snail and parasite populations may affect parasite

transmission in endemic areas (Sire et al., 2001; Vidigal

212 A. Guti�errez et al. / Experimental Parasitology 105 (2003) 211–218

et al., 1994; Vieira et al., 1991). The present study wasdesigned to differentiate between susceptible and resis-

tant P. columella to F. hepatica infection based on en-

zyme electrophoresis and random amplification of

polymorphic DNA PCR-based technology. Both tech-

niques have been employed before to detect genetic

variations between susceptible and resistant laboratory

selected Biomphalaria snails to schistosome infection

(Abdel-Hamid et al., 1999; Knight et al., 1999; Larsonet al., 1996; Michelson and Dubois, 1981; Mulvey and

Woodruff, 1985). We also sequenced the nuclear rDNA

ITS region in order to detect genetic variation which

might separate the resistant isolate as a different species.

The knowledge about the molecular basis of snail–

trematode interactions and the identification of resis-

tance genes in the snail (Knight et al., 2000; Rollinson

et al., 1998) is of particular interest for a better under-standing of parasitic disease epidemiology and control.

2. Material and methods

2.1. Snails

All snails used in this study were obtained from cul-tures established from isolates of natural populations of

P. columella from Cuba. Susceptible snail cultures were

established with parental snails sampled from two field

populations, one located south (Parque Lenin) and the

other located west (Punta Brava) of Havana City. The

resistant snail culture was established with snails origi-

nating from La Palma population, located north of Pinar

del R�io province. Resistance was histologically demon-strated by the presence of amebocytes surrounding the

miracidium 24 h after infection (Guti�errez et al., 2003).

2.2. Enzyme analysis

Protein extracts: a total of 20 snails (both susceptible

and resistant) were used for the enzyme electrophoresis

Table 1

Number of loci found for 11 isoenzymes tested for the characterization of s

Protein

Code Abbreviation Na

EC 1.15.1.1 SOD Su

EC 3.2.1.1 AMY a-AEC 1.1.1.37 MDH M

EC 4.2.1.2 FUM Fu

EC 1.1.1.204 XDH Xa

EC 2.6.1.1 AAT As

EC 3.1.3.1 AKP Al

EC 3.1.1.1 EST Es

EC 3.1.3.2 ACP Ac

EC 5.3.1.8 MPI M

EC 3.1.4.47 GPI Gl

analysis (10 snails each group). Snails were removedfrom their shells and the soft parts were homogenized in

distilled water and stored at )70 �C until they were to be

used for the electrophoresis. Protein extracts of indi-

vidual snails were run on 10% starch gels for 4 h. After

completion of electrophoresis, bands were revealed by

specific enzymatic staining. Details of the methodology

for protein electrophoresis and enzymatic staining can

be found in Pasteur et al. (1987). Eleven enzymaticsystems were tested and the analysis revealed the pres-

ence of 21 putative loci (Table 1).

2.3. Random amplification of polymorphic DNA analysis

The foot of 14 ethanol-preserved snails from sus-

ceptible and resistant isolate was removed avoiding the

pigmented area and a portion of each foot was takenfor the analysis. Genomic DNA was extracted follow-

ing the methodology of Winnepenninckx et al. (1993)

modified by Bargues and Mas-Coma (1997): foot

samples were put in 400 ll of lysis buffer (10mM Tris–

HCl, pH 8.0; 100mM EDTA; 100mM NaCl; 1% so-

dium dodecyl sulfate [SDS]) containing 500 ll/ml of

proteinase K and digested for 4 h at 55 �C with manual

shaking every 15min. The extraction took place inthree steps: (i) an equal volume (400 ll) phenol was

added; (ii) the aqueous phase obtained (about 400 ll)was mixed with 200 ll phenol and 200 ll chloroform/

isoamyl alcohol (24/1); and (iii) the aqueous phase

obtained (about 400 ll) was mixed with an equal vol-

ume of chloroform/isoamil alcohol (24/1). Each step

was followed by a slow shaking and centrifugation for

15min at 14,000 rpm at room temperature. The aque-ous phase finally obtained was precipitated with 1/10

volume of 3M sodium acetate and 2.5 volumes of

chilled absolute ethanol, then refrigerated for 10min at

)20 �C. The DNA was centrifuged for 30min at

14,000 rpm at 4 �C and the pellet washed with 1ml of

70% ethanol then air dried before resuspending in

110 ll of ultra-pure water.

usceptible and resistant P. columella

me Number of loci

peroxide dismutase 2

mylase 2

alate deshydrogenase 2

marase 1

nthine dehydrogenase 1

partate amino trasferase 1

kaline phosphatase 1

terases 8

id phosphatase 1

annose phosphate isomerase 1

ycosylphosphatidyl inositol 1

A. Guti�errez et al. / Experimental Parasitology 105 (2003) 211–218 213

Twenty six oligonucleotides (Kits A, B and G, Op-eron Technologies, California, USA), were used for the

amplification of random DNA markers to reveal genetic

diversity between individual snails. RAPD reagents and

concentrations were optimized in preliminary experi-

ments (data not shown). DNA amplification was per-

formed in a final volume of 25 ll containing 2.5 ll of10� PCR buffer (100mM Tris–HCl pH 8.3, 15mM

MgCl2, 500mM KCl, and 0.01% gelatin) (Boehringer–Mannheim, Germany), 200 lM each dNTP (Boehrin-

ger–Mannheim, Germany), 5 pmol single primer, 2.5U

Taq DNA polymerase (Boehringer–Mannheim, Ger-

many) and 10 ng template DNA. Negative controls for

each of the 26 primers used contained all of the above

components except sterile distilled water in place of

template DNA. The amplification profile consisted of an

initial denaturation step at 94 �C for 5min followed by45 repetitions of 1min at 94 �C, 1min at 36 �C, and

2min at 72 �C. In the final cycle, the extension step was

for 5min PCR products were analyzed by electropho-

resis in 1.2% agarose gels in TBE buffer containing

ethidium bromide (0.5mg/ml) and visualized using a UV

transilluminator.

A similitude coefficient (S) between groups was calcu-

lated for each primer using the following formula (Dice,1945): S ¼ 2a=ð2aþ bþ cÞ where a represents the num-

ber of bands shared by both groups, b represents the

number of marker bands specific for susceptible snails,

and c represents the number of marker bands specific for

resistant snails. This coefficient was useful to measure the

degree of between-group differentiation among primers.

2.4. rDNA sequencing and analysis

The foot of 13 ethanol-preserved snails of each isolate

was taken for the analysis. Three foot samples of each

Table 2

Size (in base pairs) of the RAPD markers obtained for the characterization

Primer Sequence Suscepti

OPA-01 CAGGCCCTTC 1380

OPA-02 TGCCGAGCTG 250

OPA-04 AATCGGGCTG 480

OPA-06 GGTCCCTGAC 1100

OPA-07 GAAACGGGTG 710, 650

OPA-08 GTGACGTAGG 530

OPA-09 GGGTAACGCC 697, 730

OPA-16 AGCCAGCGAA —

OPA-17 GACCGCTTGT 1489

OPA-18 AGGTGACCGT 697, 740

OPA-19 CAAACGTCGG 421

OPA-20 GTTGCGATCC 1000

OPB-08 GTCCACACGG 500

OPG-05 CTGAGACGGA 350, 450

OPG-10 AGGGCCGTCT —

OPG-17 ACGACCGACA 400, 590

OPG-19 GTCAGGGCAA 240

S, similitude coefficient.

isolate were pooled together for a first sequencing trial,the remaining 10 foot samples were used individually in

a second trial. Genomic DNA extraction protocol was

identical as previously described for the RAPD analysis.

The polymerase chain reaction (PCR) was used to

amplify the rDNA fragment corresponding to the 30 re-gion of the 18S, ITS-1, 5.8S, ITS-2, and the 50 region of

the 28S contained between the forward primer Pc ITS f

50-GTCGTAACAAGGTTTCCGTAG-30 (obtained byaligning 10 sequences of the 18S, found on GenBank,

from 10 snails using the software CLUSTALW) and the

reverse primer Pc ITS r 50-TTCTATGCTTAAATTC

AGGGG-30 (Bargues et al., 2001). PCR were carried out

using 3 ll of genomic DNA for a total reaction volume

of 40 ll. After cloning and sequencing the resulting long

products (containing 1170 bp), other primers were de-

signed in order to amplify and sequence their centralpart where only about 400 bp had been clearly se-

quenced on a first trial at the 50 and 30 regions: Pc ITS r1

50-CGATATCGGTGGAAGCA-30 (position 289) and

Pc ITS f1 50-CAACACGCCTCTCGAGA-30 (position810). Each PCR was programmed for 35 cycles with the

following stages: (i) 95 �C for 3min, (ii) 35 cycles at

95 �C for 1min, 55 �C for 1min, and 72 �C for 2min, and

(iii) 72 �C for 5min to complete extension. The productsobtained were viewed on an ethidium bromide stained

1.5% agarose gel. The amplified products were cloned

using the ‘‘TOPO TA cloning kit’’ (Invitrogen) and se-

quenced using the ‘‘Big Dye Terminator’’ sequencing kit

(Perking–Elmer, Applied Biosystems) which uses the

method described by Sanger et al. (1977). The reaction

products were separated by 6% polyacrylamide gel

electrophoresis using an automatic sequencer ‘‘373ADNA sequencer’’ (Applied Biosystems) and DNA se-

quences were aligned using the software ‘‘Sequencher

3.1.1’’ (Gene Codes).

of susceptible and resistant P. columella

ble snails Resistant snails S

150, 1490 0.5

— 0.75

421 0.6

2300 0.5

510, 810 0.43

600 0.57

710, 820 0.43

1037 0.83

— 0.83

718 0.57

380, 500 0.5

970 0.66

400, 505 0.25

, 780 225, 400 0.36

710 0.83

, 600 500, 605,870 0.22

— 0.75

Fig. 1. RAPD profiles produced from P. columella susceptible (Parque Lenin and Punta Brava) and resistant (La Palma) to F. hepatica using primers

OPB-8 (A), OPG-5 (B), and OPG-17 (C).

214 A. Guti�errez et al. / Experimental Parasitology 105 (2003) 211–218

A. Guti�errez et al. / Experimental Parasitology 105 (2003) 211–218 215

3. Results

3.1. Protein electrophoresis

The number of loci for each of the 11 isoenzyme

systems tested is shown in Table 1. Neither within- nor

between-group polymorphism was obtained for any of

the 21 loci revealed in both susceptible and resistant

snails. A high allele fixation was observed for all systemsat every locus. The highest number of loci (8) was found

for the esterase system. The remaining enzymes showed

either one locus or two loci.

3.2. RAPD-PCR analysis for the identification of markers

Of the 26 primers tested, 17 showed notable differ-

ences between resistant and susceptible snails. Allprimers together produced a total of 140 bands and 44

bands were specifically obtained in one group or the

other. Twenty-two bands were found to be markers

for susceptible snails and 22 bands for resistant snails.

Molecular weights of marker bands are shown in

Table 2. Fig. 1, shows the RAPD profiles of three of the

primers (OPB-08, OPG-05, and OPG-17) that produced

the highest between-group differences (e.g., similitudecoefficient of 0.25, 0.36, and 0.22, respectively).

Using primers OPB-8, OPG-5 (Fig. 1), OPG-10,

OPG-16, and OPG-19, no difference was observed be-

tween the two susceptible groups of P. columella (Par-

que Lenin and Punta Brava).

Fig. 2. rDNA sequence of the region comprising the 30 portion of the 18S, the

resistant and susceptible P. columella. X at the position 555 corresponds to ad

725 corresponds to cytosine for susceptible and thymine for resistant snails. H

and Pc ITS r1 used for reamplify the central region.

3.3. rDNA sequencing

The sequence of ribosomal DNA corresponding to

the 30 region of 18S, ITS-1, 5.8S, ITS-2, and the 50 regionof the 28S from pools of three resistant and three sus-

ceptible snails (Fig. 2) shows a difference of only two

bases from a total of 1170, accounting for a 0.17% of

variation between both isolates. These differences cor-

respond to position 555 (adenine for susceptible andguanine for resistant snails) and to position 725 (cyto-

sine for susceptible and thymine for resistant snails).

When comparing this sequence with those published in

GenBank the structure of this fragment may be de-

scribed as follows: bases 1–45, 30 portion of 18S; bases

46–565, ITS1; bases 566–722, 5.8S; bases 723–1122,

ITS2; bases 1123–1170, 50 portion of 28S. A G+C bias

can be observed in the sequence bases composition(57.95% of G+C vs. a 41.88% of A+T).

A second amplification and sequencing trial was

carried out on single rDNA extracted from 10 snails of

each isolate. This fragment comprised a region of about

500 bp containing the bases that had shown variation in

the first trial. Amplification was performed using the

primers Pc ITS f1 and Pc ITS r1 (see sequence in Fig. 2).

Results confirmed those two bases as the only geneticdifference between susceptible and resistant snails. It is

worth noting that one of those bases (position 555 of the

rDNA sequence) is located at the 30 portion of ITS1

whereas the other (position 725 of the same sequence) is

located at the 50 portion of ITS2.

ITS-1, the 5.8S, the ITS-2 and the 50 portion of the 28S of individuals of

enine for susceptible and guanine for resistant snails; Y at the position

ighlighted bases correspond to the sequences of the primers Pc ITS f1

216 A. Guti�errez et al. / Experimental Parasitology 105 (2003) 211–218

4. Discussion

Various biochemical and molecular techniques have

been used to search for genetic markers associated with

susceptible and resistant phenotypes of snails in relation

to digenetic trematode infection (Rollinson et al., 1998).

Most of the studies have concerned the Biomphalaria

glabrata/Schistosoma mansoni combination (see Knight

et al., 2000).From isoenzyme analysis, Mulvey and Woodruff

(1985) showed that biochemical differences existed be-

tween genetically defined resistant and susceptible

snails. Michelson and Dubois (1981), reported the

identification of an isoenzyme marker (AcP2), which

appeared to be associated with susceptibility and re-

fractory (dominant) state of laboratory strains of Bi-

omphalaria glabrata to S. mansoni. In the presentstudy, we were unable to detect biochemical variation

from the analysis of 11 enzymatic systems between and

within groups of P. columella resistant and susceptible

to F. hepatica infection. This absence of enzyme poly-

morphism and the presence of a high allele fixation

support the hypothesis of selfing as the ‘‘prevalent’’

mating system for this hermaphroditic species. Jab-

bour-Zahab et al. (1997) have already observed such amonomorphism within populations of F. cubensis and

L. truncatula, from different geographic sites. They

attributed this absence of variation to the possible role

of the mating system and the population dynamics, as

proposed previously for several monomorphic popula-

tions of Biomphalaria and Bulinus (Jarne, 1995). Con-

versely, the RAPD analysis performed in this study

demonstrated sharp differences between resistant andsusceptible P. columella. This indicates that, despite the

lack of within-group polymorphism observed, genetic

variability does exist at the DNA level between both

isolates. The genetic differences were obtained for 17

out of 26 primers tested and concerned 44 of the 140

bands scored. This is the first time that a genetic var-

iation is demonstrated between susceptible and resis-

tant lymnaeid snails vis-a-vis of F. hepatica infection inabsence of experimental selection. By using RAPD-

PCR analysis Larson et al. (1996) were able to differ-

entiate genetically defined lines of B. glabrata selected

for susceptibility and resistance to S. mansoni. Of the

20 primers they tested, 2 (OPA-1 and OPA-6) were

useful in distinguishing between the two snail lines. The

stable inheritance of these markers in individual prog-

eny was also studied. Using bulked segregant analysisand RAPD-PCR, Knight et al. (1999) identified two

other markers (OPM-04, 1.2 kb and OPZ-11, 1.0 kb)

from 205 primers tested that segregate with the adult

resistant phenotype of B. glabrata snails. RAPDs ge-

netic variation was also detected for Biomphalaria

tenagophila susceptible and resistant laboratory selected

snails (Abdel-Hamid et al., 1999).

One question that may arise is whether resistant iso-lates belong or not to a different species other than P.

columella, given the genetic differences observed with the

RAPDs markers. Despite some phenotypic differences

that have been found between resistant and susceptible

isolates regarding shell morphometry, mantle pigmen-

tation pattern, and egg laying behavior (Guti�errez et al.,2003), no variation has been observed in the general

characteristics that define the species like for instance theshell width and body whorl shape and some specific traits

of the reproductive system (unpublished data). The

rDNA sequencing from snails of each isolate shows

clearly that only a little variation (two bases which ac-

count for 0.17% of the sequenced fragment) is present

between resistant and susceptible snails and this result

should confirm that both isolates correspond to the same

species. Vigo et al. (2000) and Bargues et al. (2001) haveconcluded that the ITS-2 (comprised in the sequenced

rDNA fragment in this study) is an useful marker for

resolving supraspecific, specific, and population rela-

tionships in Lymnaeidae, even in spite of the G+C bias.

Pseudosuccinea columella has been always reported as

highly susceptible to F. hepatica in different parts of the

world (Boray et al., 1985; Cruz-Reyes and Malek, 1987;

Dacal et al., 1988; Rondelaud and Barthe, 1987) and wehave successfully infected several Cuban isolates in our

laboratory for many years. The resistant snails from La

Palma locality are an exception. Regarding life-history

traits, uncommon relationships have been demonstrated

between susceptible infected Cuban specimens of P.

columella and F. hepatica (Guti�errez et al., 2002, 2003).

While F. hepatica is known to castrate its host and to

induce an increase of shell growth in L. truncatula

(Wilson and Denison, 1980), infected susceptible P.

columella showed an increased fecundity during the

patent period of infection and no increase of size (Gu-

ti�errez et al., 2002, 2003). On the other hand, exposed

resistant snails exhibit a reduced survival and fecundity

rates compared to non-exposed resistant controls and to

infected susceptible snails. Furthermore, non-exposed

resistant snails showed lower fecundity rates comparedto non-exposed susceptible P. columella. These observed

differences suggest a possible fitness and physiological

cost associated with resistance of P. columella to dige-

nean infection (Coustau et al., 2000; Minchella and

LoVerde, 1983).

The inheritance pattern of the specific markers iden-

tified for susceptibility and resistance of P. columella

remains to be established. However, mating experimentsmay not resolve this issue if selfing constitutes the al-

most exclusive mode of reproduction of this snail. Re-

cently Meunier et al. (2001) also demonstrated that L.

truncatula was almost exclusive self-crossing, since they

observed a single microsatellite multilocus genotype

among all the individuals sampled from a wide endemic

area of F. hepatica at the Bolivian Altiplano.

A. Guti�errez et al. / Experimental Parasitology 105 (2003) 211–218 217

The origin of the natural population of resistant P.columella is so far unknown and studies to determine the

nature and distribution of the resistant gene(s)

throughout the species range in Cuba are now necessary.

Subsequent studies will be directed toward a better un-

derstanding of the mechanisms involved in the recog-

nition and destruction of the parasite within the snail

(Coustau and Yoshino, 2000; Sapp and Loker, 2000) as

well as the isolation of genes or gene products involvedin the susceptibility or resistance of snails to this para-

sitic infection (Knight et al., 2000).

Acknowledgments

We are grateful to G. Espinosa, Faculty of Biology,

University of Havana, for her valuable help with RAPDtechnique and J.R. Pag�es, University of Perpignan for

his contribution with image management. We thank C.

Coustau from the CNRS and M.D. Bargues from the

University of Valencia (Spain) for reading the manu-

script and helpful comments. This work received finan-

cial support from the CNRS (France)/CITMA (Cuba)

12090-project and CORUS 02 736 087-project.

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