Experimental Parasitology 114 (2006) 77–83

www.elsevier.com/locate/yexpr

EVect of the exposure to Fasciola hepatica (Trematoda: Digenea) on life history traits of Lymnaea cousini and Lymnaea columella

(Gastropoda: Lymnaeidae)

Laura Salazar ¤, Victoria E. Estrada, Luz E. Velásquez

Programa de Estudio y Control de Enfermedades Tropicales—PECET, Universidad de Antioquia, Colombia

Received 18 November 2005; received in revised form 10 February 2006; accepted 15 February 2006Available online 27 March 2006

Abstract

The snails Lymnaea columella and Lymnaea cousini have both been reported as intermediate hosts of Fasciola hepatica in Colombia.The eVect of the exposure to the parasite on survival, fecundity and size of these snails was evaluated by means of experimental infectionsand the life history traits of control and exposed groups were compared. Infection rates were 82.2 and 34% for L. columella and L. cousini,respectively. A reduction in Wtness was observed in both species when exposed to the parasite: fecundity alone was reduced in L. columellawhereas in L. cousini there was also a decline in survival rate. Unlike other studies, increased size was not observed in either species. Onthe contrary, a reduction in growth rate was observed in L. columella.© 2006 Elsevier Inc. All rights reserved.

Index Descriptors and Abbreviations: Fasciola hepatica; Trematode; Lymnaea columella; Lymnaea cousini; Life history traits; Life tables; Experimentalinfection

1. Introduction

The liver Xuke Fasciola hepatica is a digenean trematodewith worldwide distribution that requires snails of the fam-ily Lymnaeidae as intermediate hosts to complete its lifecycle.This parasite is responsible for fasciolosis or hepaticdistomatosis, a disease of cattle and sheep that also aVectsman, albeit less frequently (OMS, 1995).

In Colombia three species of Lymnaea have beenrecorded; Lymnaea ubaquensis (Piaget, 1914) from theLaguna de Ubaque, in the department of Cundinamarca;Lymnaea bogotensis (Pilsbry, 1935) from the Sabana deBogotá (both species are considered by Hubendick (1951))as identical with Lymnaea cousini; and Lymnaea columella(Say, 1817), widely distributed in the departments of Antio-quia (Unpublished data), Cundinamarca, Meta, Nariño,

* Corresponding author. Fax: +57 4 2106511.E-mail address: lauraalazar@yahoo.com (L. Salazar).

0014-4894/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.exppara.2006.02.013

Valle del Cauca (Malek and Congswell, 1980), and Tolima(Gómez, 1990). Of these, L. bogotensis (Brumpt et al., 1940;Muñoz-Rivas, 1953) and L. columella (Gómez, 1990; Malekand Congswell, 1980) have been incriminated as intermedi-ate hosts of Fasciola hepatica. Trematode-snail interactionshave been studied using diVerent approaches, which haveshown that the physiology and metabolism of the hosts canbe aVected by larval development of F. hepatica withinthem. The main traits altered by the presence of the parasiteare reproduction, survival and growth rate; host survivalbeing the most commonly reported of these (Gutiérrezet al., 2002; Loker, 1979; Sorensen and Minchella, 1998).Mortality due to parasitism is most severe at the moment ofcercarial shedding (Cohen, 1977; Woolhouse, 1989), partic-ularly in Lymnaea spp. (Sorensen and Minchella, 2001).

The eVect on fecundity has also been described in previ-ous studies, where infection with the trematode leads to thereduction or complete inhibition of reproductive activity(Jong-Brink, 1990; Loker, 1979; Sorensen and Minchella,1998, 2001). The latter phenomenon, known as parasitic

78 L. Salazar et al. / Experimental Parasitology 114 (2006) 77–83

castration, is assumed to beneWt the parasite since it liber-ates nutrients and space inside the snail (Thompson, 1997).Given that the reproductive tissue is not essential for hostsurvival, the parasite can use the energetic resources ofthese tissues without being aVected directly (Sorensen andMinchella, 2001).

It has been proposed that the host might reallocate theenergy destined for reproduction to compensate forreduced fecundity. One of the forms of reallocation mostfrequently cited is increased growth, known as gigantism(Gerard and Theron, 1997; Gorbushin, 2000; Sorensen andMinchella, 2001; Wilson and Denison, 1980). Despite thefact that “fecundity compensation” has been documentedin diVerent studies, the ecological signiWcance and relationto gigantism remains debatable, given that in some studiesreduction in growth has been observed (Gerard andTheron, 1997; Loker, 1979; Smith, 1984).

In this study, we examined the eVects of the exposure toF. hepatica on life history traits (survival, reproduction, andsize) of L. cousini and L. columella.

2. Materials and methods

2.1. Parasite and snails

The wild snails used in this study were collected in fresh-water ecosystems in two localities and transported to thelaboratory.

(1) Lymnaea columella was collected in Llanogrande, afocus of F. hepatica situated to the SE of the urbancentre of the municipality of Rionegro (Antioquia), at06°09� N and 75°23� W.

(2) Lymnaea cousini was collected in Vereda la Toibitain Paipa (department of Boyacá) at 5°47� N and73°06� W.

The study was based on 150 Wrst generation (F1) individ-uals of each species reared in the laboratory. These weredistributed in aquaria (25 snails per aquarium) each con-taining 700 ml of continuously aerated, dechlorinatedWltered water at 21 °C, and a sterile mixture of red clay andagricultural chalk. The snails were provided with lettuce asfood and water was changed three times per week.

Eggs of F. hepatica were obtained from adult parasitesextracted from the livers of infected cattle donated by theCity of Medellín slaughterhouse. Eggs were stored in dis-tilled water at 4 °C for 30 days, then incubated at 25 °C fora further 2 weeks before hatching was stimulated using alight source (Malek, 1985).

2.2. Infection of snails

One hundred 2-week-old snails from each species, ofmean length sizes 1.4 mm (SD 0.25) and 2.4 mm (SD 0.31)for L. columella and L. cousini, respectively, were placed inindividual wells of 24-well cell culture plates. Each well con-

tained 1 ml of dechlorinated water, one snail and threemiracidia of F. hepatica. The preparation was maintainedfor 6 h to ensure contact of the miracidium with the snail.After exposure, all snails were removed to the aquaria.Infection in these snails was conWrmed four weeks afterexposure by checking for the presence of rediae under a ste-reomicroscope. The 50 remaining snails of each specieswere used as controls, submitted to the same procedure butwithout the addition of miracidia.

2.3. Life tables and statistical treatment

The week during which snails were infected was consid-ered to be tD 0 for the life tables. Surviving adults, numberof embryos and egg masses were counted weekly. Further-more, for each species the length of the shell was measuredfor 30 individuals chosen at random from the exposed andcontrol groups. All the individuals were measured when thenumber of snails in a group fell below 30. Data obtained forsurvival and fecundity were used to estimate both life his-tory traits: age-dependent (survival probability, fecundityrate, number of embryos per egg mass, and number of eggmasses per snail) and age-independent (net reproductiverate (Ro), mean generation time (T), intrinsic rate of naturalgrowth (r), and Wnite natural growth rate (�)) (Margalef,1986).

Survival of the snails was described using Kaplan–Meiercurves and the groups compared by Log rank test. Thenumbers of embryos per mass, egg masses per snail andembryos per snail were compared between the control andthe exposed groups using Mann–Whitney U test. DiVer-ences were considered to be statistically signiWcant atP < 0.05. The mean shell sizes of the two groups were com-pared by a repeated measures ANOVA, which evaluated:the eVect of the exposure as diVerences in growth ratebetween groups, the eVect of time, as diVerences in size fromone week to the next; and the interaction between time andtreatment.

3. Results

Infection rates of L. columella and L. cousini at 4 weekswere 82.8 and 34.0%, respectively.

3.1. Survival

Snails in the control groups survived longer in bothcases: the longest-lived L. columella survived for 21 weekspost-exposure (WPE), while the last member of theexposed group died after 19 WPE. In L. cousini the maxi-mum survival times in the control and exposed groupswere 40 and 33 WPE, respectively. The survival curves forboth Lymnaea species are presented in Fig. 1. No signiW-cant diVerences were found between the control andexposed groups in L. columella. Individuals of bothgroups began to die in the fourth week and the declinecontinued at a similar rate, although the survival of

L. Salazar et al. / Experimental Parasitology 114 (2006) 77–83 79

controls always exceeded that of exposed snails (Fig. 1).Mean survival times for control and exposed snails were10 and 9 weeks, respectively.

In L. cousini signiWcant diVerences were found in sur-vival between control and exposed groups (P < 0.001). Theexposed group showed a pronounced decline from 13 WPEonwards, while numbers of the controls fell drastically after25 WPE. Mean survival times for the control and exposedgroups were 23 and 20 weeks, respectively.

3.2. Fecundity

Both control and exposed L. columella snails began tooviposit at 3 WPE, while those of L. cousini laid eggs fromweek 4 WPE onwards. However, signiWcant diVerenceswere found between control and exposed L. columella withrespect to the number of embryos per egg mass (P < 0.001),egg masses per snail (P < 0.05), and embryos per snail(P < 0.001) (Fig. 2). The most pronounced diVerencebetween the two groups occurred in 16 WPE, except for thenumber of embryos per egg mass. Here, the greatest dis-crepancy was in 18 WPE. In this species there was a cleardiVerence between the egg masses of the controls andexposed snails: many in the latter lacked embryos, some-thing that was not observed in L. cousini. SigniWcant diVer-ences were also found between the groups in L. cousini withregard to the number of embryos (P < 0.05) and egg massesper snail (P < 0.01). The most pronounced discrepanciesoccurred in 10 and 19 WPE. No signiWcant diVerences werefound between the groups with regard to the number ofembryos per egg mass (Fig. 3).

Fig. 1. Kaplan-Meier curves of Lymnaea columella and L. cousini. Survivalproportion of control (solid lines) and exposed snails (dashed lines) toFasciola hepatica.

3.3. Growth

The growth curves of both species are shown in Fig. 4.These were developed by only taking into account dataobtained until the week when the means reached a constantvalue. SigniWcant diVerences were found between the groupsfor L. columella (P < 0.001), presenting eVects for time(P < 0.001) and interaction between the treatment and timefactors (P < 0.001). Both the control and exposed snails pre-sented an accelerated growth rate up to 6 WPE, when theyhad attained 60 and 80% of their Wnal sizes, respectively. Thedivergence between growth rates began from 2 WPEonwards. No signiWcant diVerences were found between thetwo groups in L. cousini. There was no demonstrable eVect oftime or interaction between the group and time factors. At 10WPE the control and exposed snails had reached 82 and 75%of their Wnal sizes, respectively.

3.4. Life table

Although higher values were obtained for all parametersin the controls of both species (Table 1), the diVerenceswere greater in L. columella than in L. cousini. The parame-ter that showed greatest divergence was the net reproduc-tive rate (Ro), where the value in the control group wasalmost double that of the exposed group in L. cousini andwas nearly four times greater in L. columella.

4. Discussion

The infection rate of L. columella (82.8%) was compara-ble those reported in the literature for this species, such asvalues of 90 and 93% found by Gutiérrez et al. (2000).Other studies performed in our laboratory have providedrates of 87.5 and 44.1%, based on individual and groupinfections, respectively.

The only published report of experimental infection forL. cousini is from the work of Muñoz-Rivas (1953) carriedout on L. bogotensis, which may be a synonym of theformer species (Hubendick, 1951). The highest value in thisreport was 0.6% (Muñoz-Rivas, 1953), much lower than theprevalence of 34.0% obtained in the present study. Otherstudies performed in our laboratory have produced infec-tion rates between 39 and 60%.

In this study, the exposure to F. hepatica did not signiW-cantly aVect survival of L. columella, in contrast to Wndingsfor other species of molluscs (Cohen, 1977; Gutiérrez et al.,2002; Loker, 1979; Woolhouse, 1989). This can be explainedsince this species has a short lifespan (mean 9–11 weeks) rela-tive to the occurrence of cercarial shedding (7 WPE).

The mean lifespan of L. cousini is much longer (mean20–23 weeks). This allows the parasite to remain longerwithin the snail and generate more pronounced changes insurvival, due to the greater production of rediae.

The control and exposed groups of L. columella diVeredwith respect to fecundity, through a reduction in the num-ber of embryos per egg mass and egg masses per snail, the

80 L. Salazar et al. / Experimental Parasitology 114 (2006) 77–83

former being the most marked (Figs. 2B and C). On theother hand, the diVerences in fecundity between the groupsin L. cousini were due solely to reduction of the number ofegg masses per snail. The egg masses found in the exposedgroup of L. cousini may have been laid by uninfected snails,given that the overall number produced was approximatelythe same as that expected from the non-infected specimensfound within this group.

Based on our results, the exposure to F. hepatica appears toexert a greater eVect on the reproduction of L. cousini than

that of L. columella. The latter presented eggs with few or noembryos but in L. cousini, egg mass production ceased almostcompletely in infected snails. In laboratory studies involvingisolation of infected individuals, only 10 egg masses were laidby 32 individuals in four weeks (data not shown). This agreeswith other studies, where parasites castrated snails or reducedtheir reproduction considerably (Baudoin, 1975; Jong-Brink,1990; Wilson and Denison, 1980; Woolhouse, 1989), especiallywhen infection occurred during the early stages of develop-ment (Gerard and Theron, 1997; Loker, 1979).

Fig. 2. Fecundity of Lymnaea columella: (A) number of embryos per mass, (B) number of egg mass per snail, (C) number of embryos per snail of control(clear bars) and exposed to Fasciola hepatica (dark bars).

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L. Salazar et al. / Experimental Parasitology 114 (2006) 77–83 81

Such eVects on fecundity and survival of the host resultingfrom parasite exposure should result in drastic reduction ofhost populations. However, natural prevalences of F. hepat-ica infection are very low among lymnaeids in Colombia.

Values of only 0.65–12.5% were recorded for L. colu-mella in the locality from which specimens were obtainedfor the present study (Unpublished results) while infectionof L. bogotensis was only 0.07–1.64% in lakes of the Cundi-namarca–Boyaca plateau (Brumpt et al., 1940; Muñoz-Rivas, 1952, 1953).

Studies of the eVect of the parasite on growth of the snailhave produced contrasting results. In the present study, weobserved that exposure to F. hepatica reduced the growth

rate of L. columella throughout the life of the snail (eVect oftime). No such eVect was found for L. cousini, perhapsbecause the infection rate was low and any diVerences wereimperceptible. Neither was any temporal eVect observed inthis species, probably due to the high variability of theweekly means and the fact that snails of this species attaintheir full size early in their lifespan (Fig. 4).

A recent hypothesis to explain the parasite–host rela-tionship proposes that changes in life history traits of thehost as a response to parasitism are driven principally byenvironmental eVects (Sorensen and Minchella, 2001). Thusgigantism, as compensation for reduced fecundity, may beobserved most commonly in populations from less stable

Fig. 3. Fecundity of Lymnaea cousini: (A) number of embryos per mass, (B) number of egg mass per snail, (C) number of embryos per snail of control(clear bars) and exposed to Fasciola hepatica (dark bars).

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82 L. Salazar et al. / Experimental Parasitology 114 (2006) 77–83

habitats, since the energy destined for reproduction in suchareas is greater compared to that for maintenance. Thus,reallocation of energy results in increased size. Conversely,stunting is thought to occur in environments favoring sta-ble population sizes, where energy invested for maintenanceis greater than that for reproduction and therefore its real-location is not reXected in increased size (Sorensen andMinchella, 2001). Based on this and that the exposed indi-viduals of L. columella presented a reduced growth rate, itmay be surmised that the snails used in our study were from

Table 1Independent age parameters for L. columella and L. cousini

Ro, net reproduction rate; T, mean generation time; �, Wnite rate of natureincrease; r, intrinsic rate of natural increase.

Species Group Ro T r �

Lymnaea columella Control 708.40 10.93 0.60 1.82Exposed 151.30 10.88 0.46 1.57

Lymnaea cousini Control 345.66 15.60 0.37 1.45Exposed 147.63 14.33 0.35 1.42

a stable population that has been exposed to the parasitelong enough for changes in reproduction to produceimproved defense mechanisms rather than reallocatedenergy for maintenance. The latter could explain why nosigniWcant diVerences in survival were found between theexposed snails and controls of this species.

By contrast no conclusions can be made for L. cousiniregarding stability of the original population, since no signiW-cant diVerences were found between the growth rates of thetwo groups. Since this species did show diVerences both insurvival and fecundity, it is likely that the studied populationhas never been exposed to F. hepatica, so that there was noeYcient reallocation of energy in response to infection.

Based on life tables, the age-independent traits in theexposed groups of both lymnaeid species were smaller. Thisindicates a reduction in snail Wtness because of parasiteexposure, through its eVect on survival, fecundity, or both.

Our study shows clear diVerences in the response ofL. cousini and L. columella exposed to F. hepatica: in thelatter snail species infestation rates are high and cercarial

Fig. 4. Growth curves (Mean § SD) of L. columella (A) and L. cousini (B): controls (�) and exposed snails to F. hepatica (�).

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L. Salazar et al. / Experimental Parasitology 114 (2006) 77–83 83

shedding time is brief due to its short lifespan. In contrast,in L. cousini the prevalences are low but cercarial shed-ding is more prolonged since its lifespan is greater. Thus,in both cases, parasite exposure exerts a clear eVect on thelife history traits of the host, which translates into reducedWtness of the latter.

Acknowledgments

We wish to express our gratitude to Dr. Iván DaríoVélez director of PECET, for his constant support of mala-cology research and to the staV from the Malacology Labo-ratory at the PECET. We thank Bruce Alexander forEnglish corrections. This study was Wnanced by grant 1115-05-13664 from COLCIENCIAS, Colombia.

References

Baudoin, M., 1975. Host castration as parasitic strategy. Evolution 29,335–352.

Brumpt, E., Velásquez, J., Ucroz, H., Brumpt, L.C., 1940. Decouvete del’hote intermediarire Limnaea bogotensis Pilsbry, de la grande douveFasciola hepatica L., en Colombie. Annales de Parasitologie 6, 563–579.

Cohen, J., 1977. Mathematical models of schistosomiasis. Annual Reviewof Ecology and Systematic 8, 209–233.

Gerard, C., Theron, A., 1997. Age/size- and time-speciWc eVects of Schisto-soma mansoni on energy allocation patterns of its snail host Biomphala-ria glabrata. Oecologia 112, 447–452.

Gómez, T., 1990. Ciclo de vida de Fasciola hepatica (Linnaeus, 1758) eidentiWcación de su huésped intermediario en algunas zonas ganaderasdel departamento del Tolima. Revista de la Universidad del Tolima.Ciencia y Tecnología 5, 45–75.

Gorbushin, A., 2000. Comparative morphofunctional analysis of the gas-tropodtrematode interactions. Parazitologiia 34, 502–514.

Gutiérrez, A., Perera, G., Yong, M., Sanchez, J., Wong, L., 2000. Life-his-tory traits of Fossaria cubensis (Gastropoda: Lymnaeidae) underexperimental exposure to Fasciola hepatica (Trematoda: Digenea).Memorias do Instituto Oswaldo Cruz 95, 747–752.

Gutiérrez, A., Yong, M., Perera, G., Sanchez, J., Theron, A., 2002. Fasciolahepatica (Trematoda: Digenea): its eVect on the life history traitsPseudosuccinea columella (Gastropoda: Lymnaeidae) an uncommoninteraction. Parasitology Research 88, 535–539.

Hubendick, B., 1951. Recent Lymnaeidae; their variation, morphology,taxonomy, nomenclature, and distribution. Kungl Svenska Vetensk-apsakad Handl (4 ser) 3, 1–223.

Jong-Brink, M., 1990. How trematode parasites interfere with reproduc-tion of their intermediate hosts freshwater snails. Journal for Medicaland Applied Malacology 2, 101–133.

Loker, E.S., 1979. EVect of Schistosomatium douthitti infection on growth,survival, and reproduction of Lymnaea stagnalis. Experimental Parasit-olgy 24, 137–146.

Malek, E., Congswell, F.B., 1980. Lymnaea (Pseudosuccinea) columella inColombia. Nautilus 94, 112–114.

Malek, E., 1985. Snail hosts of schistosomiasis and other snail-transmitted dis-eases in tropical America: a manual. PAHO scientiWc publication N°, 478.

Margalef, R., 1986. Ecología. Ediciones Omega, Barcelona.Muñoz-Rivas, G., 1952. Coccidiosis y distomatosis humanas en Colombia.

Revista de la Facultad de Medicina de Bogotá 21, 47–58.Muñoz-Rivas, G., 1953. Fasciolosis experimental. Revista de la Academia

Colombiana de Ciencias Exactas, F´sicas y Naturales 9, 156–158.OMS, 1995. Lucha Contra las Trematodiasis de Transmisión Alimentaria.

Serie de informes técnicos 849 Ginebra.Piaget, J., 1914. Voyage D’Exploration ScientiWque en Colombie. Mémor-

ies de la Sociétè neuchâteloise des Sciences naturelles. Neuchâtel, Att-inger Frères 5, 266. pl 9.

Pilsbry, H.A., 1935. South American land and freshwater mollusks, IXColombian species. Proceedings of the National Academy of Sciencesof Philadelphia 87, 83–88.

Say, T., 1817. Description of seven species of American fresh water andland shells, not noticed in the system. Journal of the Academy of Natu-rals Sciences of Philadelphia 1, 217–237.

Smith, G., 1984. The relationship between the size of Lymnaea truncatulanaturally infected with Fasciola hepatica and the intensity and maturityof redial infection. Journal of Helmintology 58, 123–127.

Sorensen, R.E., Minchella, D.J., 1998. Parasite inXuences on host life-his-tory: Echinostoma revolutmu parasitism of Lymnaea elodes snails. Oec-ologia 115, 188–195.

Sorensen, R.E., Minchella, D.J., 2001. Snail-trematode life history interac-tions: past trends and future directions. Parasitology 123, S3–S18.

Thompson, S.N., 1997. Physiology and biochemistry of snail–larval trema-tode interactions. In: Fried, B., Graczyk, T.K. (Eds.), Advances inTrematode Biology. CRC Press, Boca Raton, Florida, pp. 149–196.

Wilson, R.A., Denison, J., 1980. The parasitic castration and gigantism ofLymnaea truncatula infected with the larval stages of Fasciola hepatica.Zeitschrift fur Parasitenkunde 61, 109–119.

Woolhouse, M.E., 1989. The eVect of Schistosome infection on the mortal-ity rates of Bulinus globosus and Biomphalaria pfeiVeri. Annals of Trop-ical Medicine and Parasitology 83, 137–141.