ORIGINAL PAPER

Alfredo Gutierrez Æ Mary Yong Æ Gloria Perera

Jorge Sanchez Æ Andre Theron

Fasciola hepatica (Trematoda: Digenea): its effect on the life historytraits of Pseudosuccinea columella (Gasteropoda: Lymnaeidae),an uncommon interaction

Received: 18 January 2002 /Accepted: 30 January 2002 / Published online: 16 March 2002� Springer-Verlag 2002

Abstract An uncommon snail-trematode interactionpattern was found for the Pseudosuccinea columella –Fasciola hepatica model under laboratory conditions.Week-old juveniles from two isolates showed very highrates of infection (90.0% and 93.3%) after exposure tofive miracidia. They also showed differences in their lifehistory traits when compared to a control group.However, they did not exhibit the increase in size andreduction/cessation in host fecundity which is usuallyobserved in most of the other related snail-trematodesystems. In contrast, infected juvenile P. columellashowed increased egg laying after the onset of cercarialemission and there was no effect on growth. A thirdisolate of P. columella was found to be refractory tomiracidial infection. This constitutes the first report ofnon-susceptibility to F. hepatica in a lymnaeid specieswhich is usually susceptible. These non-susceptible snailsexhibited lower fecundity and survival compared to non-exposed susceptible snails under identical laboratoryconditions. The differences observed in terms of life spanand reproduction could result from the cost of resistancefor individuals from this isolate.

Introduction

Lymnaeid snails act as intermediate hosts for the trem-atode Fasciola hepatica, Linnaeus 1758. This parasite

causes fascioliasis, which affects domestic animals aswell as people from various countries (Malek 1985;Mas-Coma 1998; Mas-Coma et al. 1999), and is there-fore important from the economic and public healthstandpoints.

Numerous studies have been conducted on the in-teraction between freshwater snails and trematodes.Although dissimilarities can be found in the differentsystems, most digenetic parasites affect the physiologyand (neuro)endocrinology of the snail host, inducingabnormal body growth as well as inhibiting the devel-opment of the male and female reproductive organs andresulting in either parasitic castration or a considerablereduction in snail fecundity (Wilson and Denison 1980;Jong-Brink 1990; Hordijk et al. 1991; Gerard andTheron 1997). It is also known that survival is generallyreduced in infected snails and that most die a few weeksafter the beginning of cercarial emission (Woolhouse1989; De Kock 1993).

The freshwater snail Pseudosuccinea columella (Say,1817) is the largest lymnaeid species found in Cuba, withaverage adult specimens measuring 14–16 mm in shellheight. It generally occurs in permanent water bodiessuch as rivers, ponds and lakes. This species is distrib-uted in North, Central and South America, and has alsobeen introduced into some areas of Europe, Africa, Asiaand Australia (Malek 1985). In Cuba it has only beenfound in the western and central provinces. Mauri(1981) reported this species as an intermediate host ofF. hepatica in Cuba, in addition to Fossaria cubensis(Pfeiffer, 1839) which had been previously reported. Sofar, no information is available on the snail-trematodeinteractions for the P. columella – F. hepatica system.However, data for the Fos. cubensis – F. hepatica systemwas recently published (Gutierrez et al. 2000). In thispaper, we analyse the influence of F. hepatica develop-ment on some of the life history traits of three Cubanisolates of P. columella, one of which is refractory to theparasite infection. Such data are of importance for abetter understanding of host-parasite interactions whichin turn might help in controlling this disease.

Parasitol Res (2002) 88: 535–539DOI 10.1007/s00436-002-0625-4

A. Gutierrez Æ M. Yong Æ G. Perera Æ J. SanchezLaboratorio de Malacologıa,Instituto ‘‘Pedro Kourı’’(IPK),Apartado 601, Marianao 13, Havana, Cuba

A. Theron (&)Laboratoire de Biologie Animale,UMR 5555 CNRS-UP, Centre de Biologie etd’Ecologie Tropicale et Mediterraneenne,Universite. 52, Av. de Villeneuve,66860 Perpignan Cedex, FranceE-mail: theron@univ-perp.frFax: +33-4-66682281

Materials and methods

Snail and parasite origin

The snails used in this study were laboratory isolates that originallycame from the following three natural populations: (1) ParqueLenin, an artificial lake which is part of the recreational park of thesame name located south of Havana; (2) Punta Brava, the sectionof the river that crosses the town of Punta Brava located west ofHavana; and (3) La Palma, a creek in the municipality of Pinar delRio Province. F. hepatica eggs were obtained from cow livers at aslaughterhouse in Havana, which receives bovine livestock fromgovernment dairies from throughout the country.

Snail culture and infection

All of the snails used in this study were laboratory reared to thethird generation from field-collected specimens. A total of 180newly born individuals (60 per isolate) were placed in Petridishes with 88 ml water, using four dishes per population(15 snails/dish).

Eggs of F. hepatica were transported to the laboratory andimmediately incubated at 36�C. Hatching was induced by lightstimulation 15 days later. Week-old snails (30 snails per isolate)were individually exposed to five F. hepatica miracidia in a 96-wellsmicrotitre plate. All of the wells to be used were filled with de-chlorinated tap water and each snail was placed together with themiracidia in each well. All wells were covered with glass to keep thesnails from escaping. After 3 h exposure, all snails were removedfrom the wells and immediately deposited in culture medium(Sanchez et al. 1995). Infection was confirmed 2 weeks post expo-sure by checking for the presence of redia in exposed snails under astereoscopic microscope. In the Parque Lenin and Punta Bravaisolates, only infected snails (n=25 and 27, respectively) weresubsequently used. In the La Palma population, no infection wasfound. This isolate has been exposed several times to a high mi-racidial dose and neither F. hepatica larval stages nor any cercarialshedding have ever been observed at any time post exposure (un-published data). Therefore, all exposed snails from La Palma wereused for comparison with susceptible (Parque Lenin and PuntaBrava) isolates. The remaining 30 snails from each group, includingLa Palma, were used as controls. Infected, exposed and controlsnails were then maintained using the methods described bySanchez et al. (1995).

Life-table computations and comparisons

The week during which the snails were born was considered to betime zero for the experiment. Measurements of the shell size, countsof live and dead individuals and the numbers of eggs and eggmasses were made on a weekly basis. The following age-dependentand age-independent life-history traits were determined (Stearns1992): (1) age-dependent life tables: survivorship probability, fe-cundity rate, number of eggs per mass, number of egg masses persnail and growth curve; (2) age-independent life tables: net repro-ductive rate (Ro), mean generation time (T), intrinsic rate of nat-ural increase (r) and finite rate of natural increase (k). Allparameters were calculated using software specifically developedfor this purpose.

The mean values of the shell size, the number of eggs per massand the number of masses per individual were compared betweenthe exposed and control groups of each population at weeks 3, 14and 19. These weeks were selected for comparisons on the basis ofearly, middle and late snail developmental age, respectively.Statistical comparisons between infected and control snails wereperformed using a Student’s t-test for the shell size and a Mann-Whitney U-test for both the number of eggs per mass andthe number of egg masses per snail. Differences were consideredstatistically significant at P<0.05.

Results

Individuals from the Parque Lenin and Punta Bravaisolates displayed very high infection rates (90.0% and93.3%, respectively) which were greater than the 62%typical for Fos. cubensis under similar laboratory con-ditions (Gutierrez et al. 2000).

In contrast, exposed snails from the La Palma isolateshowed no sign of infection, and were considered as non-susceptible to F. hepatica. The age-dependent life historytraits of infected, exposed and control individuals for thethree groups are plotted in Fig. 1 (survival and growthdata) and Fig. 2 (fecundity data).

The survival curves of the three groups showed verydistinct differences between the control and exposedsnails from the beginning of the experiment (Fig. 1A).Infected snails from Parque Lenin and Punta Bravadisplayed a rapid decline in survival at week 7, corre-sponding to the onset of cercarial shedding, whereasexposed snails from La Palma exhibited a step-likecurve, with more pronounced descents during the juve-nile period. Snails from La Palma had the longest lifespan (32 and 31 weeks for control and exposed groupsrespectively) followed by Parque Lenin (29 and 19 weeksrespectively) and Punta Brava (19 and 20 weeks re-spectively). The control group of the last isolate suffereda great reduction in number during week 19, when forunknown reasons all of the remaining snails died. Beforethis, all of the parameters of both the infected andcontrol groups were very similar to those of ParqueLenin.

The growth curves did not exhibit significant differ-ences between the exposed and control snails from anyof the three isolates (Fig. 1B) (P>0.05). Only slightlyhigher values were observed in the infected-exposedgroups during the first 8 weeks of life.

The fecundity rates also showed a notable variationbetween the infected and control groups from theParque Lenin and Punta Brava isolates (Fig. 2A). Thehighest values were attained by the infected groups inboth cases. These differences were more pronouncedduring the period of cercarial shedding (patent period),with peaks at weeks 17 and 13 for Parque Lenin andPunta Brava, respectively. Differences between theexposed and control snails from the La Palma non-susceptible strain were not as high as those fromParque Lenin and Punta Brava. In addition, the resis-tant isolate showed much lower values than thesusceptible groups.

Significant differences between the infected and con-trol snails from Parque Lenin and Punta Brava were alsoobserved for the number of eggs per egg mass (P<0.05,Mann-Whitney U-test), with the highest values beingattained by controls for most of their lifespan (Fig. 2B).For the La Palma isolate, histograms for exposed andcontrol groups appeared similar in shape and magnitude(Fig. 2B). This strain also exhibited lower values thanthe control snails of the other two isolates.

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The number of egg masses per individual also showedsignificant differences (Fig. 2C) between the infected andcontrol snails (P<0.01, Mann-Whitney U-test) for thetwo susceptible isolates, Parque Lenin and Punta Brava.The number of egg masses laid by infected susceptiblesnails during the patent period of infection was twicethat of the controls of the same age. Non-susceptiblesnails from the La Palma population showed no differ-ences between the control and exposed groups, theirvalues being comparable only to the controls from theother two populations before week 20 (Fig. 2B).

The age-independent life history traits also mani-fested between-group variation for each isolate, butsome parameters showed greater differences than others(Table 1). Values of Ro and T were higher for the con-trols than for the infected/exposed individuals from allisolates. These traits displayed more variation betweencontrols and infected snails from Parque Lenin andPunta Brava than between the control and exposedsnails from La Palma. The k and r values also exhibiteddifferences between groups in all populations, but in thiscase, the relationship was the inverse with the values forinfected snails being higher than those for controls.Exposed snails from La Palma showed intermediatevalues for these traits.

Discussion

Numerous studies have documented the influence thattrematode parasites exert on their intermediate snailhosts (reviewed by Jong-Brink 1990; Hurd 1990;Beckage 1993). The potential differences observed on thegrowth and reproduction of infected snails (i.e. no effect,

stimulation, reduction or cessation) are mainly linkedwith the diversity of the host-parasite combinations usedand, sometimes, depend on the age/size of the snails atinfection (Gerard and Theron 1997). For the majority ofthe lymnaeid snail-parasite combinations studied, dige-netic trematodes such as Schistosomatium douthitti,Trichobilharzia ocellata or F. hepatica induce a strongreduction (and in some cases a complete cessation) offecundity (Loker 1979; Sluiters et al. 1980; Wilson andDenison 1980; Dreyfuss et al. 1999) and they alsostimulate the body growth during the prepatent period(McClelland and Bourns 1969; Wilson and Denison1980; Joosse and Van Elk 1986).

This was also the pattern observed for the Fos.cubensis – F. hepatica system in which juvenile infectedsnails grow faster and reach larger sizes than the con-trols, although fecundity was reduced (Gutierrez et al.2000). On the other hand, individuals infected duringtheir adult phase did not show an enhanced growth atany time of infection, their egg production being in-creased during the prepatent period and decreased andstopped at some point during the patent period(Gutierrez et al. 2000). Wilson and Denison (1980)showed that F. hepatica castrated and stimulated shellgrowth in Lymnaea truncatula. Later, for the same host-parasite system, Dreyfuss et al. (1999) found that cer-caria shedding snails exposed to one miracidiumresumed reproduction after week 8 post-infection, butthat the oviposition of the infected snails was never asintense as that of the controls. In addition, snailsexposed to two or three miracidia stopped oviposition atweeks 4 and 3 respectively, with no further egg-layingactivity. The observed restoration of reproduction in thesnails exposed to a low miracidial dose was attributed to

Fig. 1 Pseudosuccinea columel-la/Fasciola hepatica: A survivaland B growth curves (±SD) ofcontrol (solid lines), infected(dashed line, Parque Lenin andPunta Brava) and exposedresistant (dashed line, La Palma)snails

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a lower parasite burden, which enabled the reconstitu-tion of the gonadal epithelium after a phase of necrosis.

Our results on P. columella revealed unusual effectsof a trematode on its intermediate snail host’s life

history traits. In spite of the fact that all snails wereinfected as juveniles (only 1 week post-hatching), indi-viduals of P. columella from susceptible populationsfailed to exhibit the enhanced growth reported forrelated species such as L. truncatula infected withF. hepatica (Hodasi 1972; Wilson and Denison 1980)and Lymnaea stagnalis infected with Trichobilharziaocellata (Joose and van Elk 1986). On the other hand,despite a relatively high miracidial dose (five miracidia),infected snails also kept laying eggs for more than10 weeks after the onset of cercarial shedding (paten-cy). During this time, the production of egg masses perinfected snail was even stimulated, which may com-pensate for the decreased survival and lower number ofeggs per mass compared to the controls. More studiesare needed in order to explain the physiological basis ofthe effects observed on the host reproduction and bodygrowth.

Fig. 2 Pseudosuccinea columel-la/Fasciola hepatica: A numberof eggs per snail, B number ofeggs per egg mass, C numberof egg masses/snail for suscepti-ble (Parque Lenin and PuntaBrava) and resistant (La Palma)isolates

Table 1 Age-independent life history parameters of Pseudosucci-nea columella snails infected with Fasciola hepatica (Parque Leninand Punta Brava), resistant exposed (La Palma) and non-exposedcontrols (all three isolates). Ro Net reproductive rate, T meangeneration time, r intrinsic rate of natural increase, k finite rate ofnatural increase

Isolate Group Ro T r k

Parque Lenin Control 265.33 14.34 0.59 1.80Infected 79.90 8.06 0.90 2.46

Punta Brava Control 154.03 11.95 0.50 1.65Infected 58.30 9.45 0.73 2.08

La Palma Control 160.90 12.15 0.55 1.74Resistant 110.90 10.77 0.64 1.89

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Because of the lower survival rates attained by in-fected snails during the prepatent period, Ro valueswere also lower than those of the controls. However,since T estimates for infected snails of this species weresignificantly reduced as a result of a shorter lifespan andan enhanced production of egg masses, values of r and kwere higher for this group than for controls. It can thenbe concluded that under the conditions used, infectedisolates of P. columella experienced a greater increase innumber as a consequence of an enhanced fecundity anda shorter T.

Another important result concerns the isolate fromLa Palma from which snails appear to be refractory tothe strain of F. hepatica used, since no infection wasachieved after exposing 30 individuals to five miracidiaeach. We have subsequently exposed a greater numberof snails (both collected from the field and laboratorymaintained stocks) to higher miracidial doses and nosign of the intramolluscan stages of F. hepatica has everbeen observed (unpublished data). Age-independent lifehistory parameters (Table 1) showed that the fitness ofresistant individuals from this population was lowercompared to susceptible snails under identical labora-tory conditions. The differences observed, mainly interms of lifespan and reproduction, could be a result ofthe cost of resistance found in individuals from thispopulation (Minchella and Loverde 1983; Coustau et al.2000).

Interestingly, this population comes from a zonewhere an outbreak of more than 80 cases of humanfascioliasis took place in 1996 (Espino et al. 1998). Inaddition, the province of Pinar del Rıo, where La Palmais located, has previously been reported to have thecountry’s highest occurrence of the parasite (Kourı1963). The possible origin of this population is unknownand studies to determine the genetic differences betweenthe susceptible and resistant snails of this species arecurrently in progress.

Acknowledgements We are indebted to Dr. J-P. Pointier (EPHE,University of Perpignan) and Dr. E. S. Loker (University of NewMexico) for their valuable comments on the manuscript and to LinWong for her technical assistance during the life table experiments.

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