The Combined Effect of Isolation and Fasciola hepatica Infection on the Life History Traits of Fossaria cubensis

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Journal of Invertebrate Pathology 78, 66–71 (2001)doi:10.1006/jipa.2001.5044, available online at http://www.idealibrary.com on

The Combined Effect of Isolation and Fasciola hepatica Infectionon the Life History Traits of Fossaria cubensis

Alfredo Gutierrez, Mary Yong, Lin Wong, and Jorge SanchezLaboratorio de Malacologıa, IPK, Apartado 601, Marianao 13, La Habana, Cuba

Received December 14, 2000; accepted July 9, 2001

Life history traits of Fossaria cubensis were com-pared between isolated and paired snails after infec-tion with three miracidia of Fasciola hepatica. Fourexperimental groups were tested: isolated–unexposed,paired–unexposed, isolated–infected, and paired–in-fected. A repeated-measures ANOVA showed statisti-cally significant interactions among isolation, infec-tion, and age effects for shell size, number of eggmasses per snail, number of eggs per snail, and num-ber of viable eggs per snail. Isolated–unexposed snailsexhibited the higher values of these variables andthose of survival and finite and intrinsic rates of nat-ural increase. Infection stimulated shell growth dur-ing the prepatent period, but differences were presentonly in paired snails since isolation causes a similareffect. Reproduction, in terms of the number of eggmasses per snail and the number of eggs per mass persnail, decreases in the presence of parasitic infection,whereas isolation stimulates it. These effects were ob-served from early stages of infection. © 2001 Academic Press

Key Words: Fossaria cubensis; Fasciola hepatica;Lymnaeidae; Trematoda; Digenea; life tables; repro-duction; growth; isolation; parasitism; snail; life his-tory traits; infection; mating system; net reproductionrate; mean generation time; intrinsic rate of increase;finite rate of increase; survival; fecundity; host–para-site interaction.

INTRODUCTION

Lymnaeid snails act as intermediate hosts for thedigenean trematode Fasciola hepatica. This cosmopol-itan parasite, known as the liver fluke, causes botheconomic and sanitary problems since it affects bovineand ovine livestock and people. Reports state that hu-man fasciolosis has increased during recent years, with2.4 million people infected worldwide and more than180 million at risk of infection (WHO, 1998)

Fossaria cubensis is the main intermediate snailost of F. hepatica in Cuba. Adult specimens attain an

approximate size of 13 mm and can live for about 4 to
6 months (Ferrer et al., 1989; Sanchez et al., 1995;
660022-2011/01 $35.00Copyright © 2001 by Academic PressAll rights of reproduction in any form reserved.

Gutierrez et al., 2000). This amphibious snail is highlyadaptable to changing environmental conditions; it in-habits different kinds of habitats such as temporaryponds, irrigation channels, lakes, rivers, streams, etc.and is able to resist long periods of desiccation, restor-ing its population as soon as appropriate environmen-tal conditions become available.

Previous studies have shown that F. cubensis ex-posed to F. hepatica exhibit a decreased survival andfecundity and a greater shell growth (Gutierrez et al.,2000). However, contrary to what has been foundfor other trematode–gastropod systems (Andersonand Crombie, 1984; Minchella, 1985; Woolhouse andChandiwana, 1989), there is no evidence of parasiticcastration in snails that have been exposed to the par-asite during juvenile–early adult stages; instead, theyare still able to reproduce during the prepatent periodof infection.

Other investigations have been conducted to deter-mine the influence of isolation on the life history traitsof some lymnaeids (Pseudosuccinea columella andLymnaea elodes among others), indicating that snailsraised in isolation show a greater shell growth andfecundity than grouped snails (de Witt and Sloan,1958; Florin et al., 2000). However, for other gastro-pods, a reduction in fecundity of isolated snails hasbeen reported, as is the case in Biomphalaria glabrata(Vianey-Liaud, 1976).

The present study focuses on the influence that iso-lation and F. hepatica miracidial infection cause on thelife history traits of F. cubensis.

MATERIALS AND METHODS

All snails in the study were selected from F. cubensisstocks maintained at the Laboratory of Malacology,Institute “Pedro Kouri” (IPK). Egg masses were ob-tained from adult cultures and placed in petri dishes(Pyrex, 43.33 cm3 of volume) until hatching occurred. Atotal of 80 newly hatched snails were selected andseparated into four experimental groups: isolated–
infected, isolated–unexposed, paired–infected, and

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67ISOLATION AND PARASITIC INFECTION OF Fossaria cubensis

paired–unexposed. Except for the group infected–isolated, in which a total of 17 snails became infected,all groups comprised 20 snails. The infection protocolwas performed as described below.

F. hepatica eggs were collected from infected bovineliver in a slaughter house that processes livestock fromdifferent localities of Cuba. The lot used in this exper-iment was received from a cattle-rearing station inSancti Spiritus Province, Cuba central region. Theeggs were kept in darkness for 15 days and after thattime hatching was induced by light exposure. Eachsnail to be infected (those from the groups infected–isolated and paired–infected) was exposed to threemiracidia of F. hepatica for 3 h in a 96-well microtiterplate (one snail per well). Snails were 1 week old (meanshell height 5 1.5 mm) at the time of infection. Infec-tion was confirmed by the presence of rediae 2 weeksafter exposure by examination under stereoscope(Olympus JM Optical Co. Ltd).

Infected and unexposed snails were separated intothe above-mentioned groups and raised in petri dishes(as described above). Snails from the groups infected–isolated and isolated–unexposed were raised in isola-tion (one snail/dish), whereas those from the groupspaired–infected and paired–unexposed were raised inpairs (two snails/dish). All groups were maintained inthe laboratory until the last snail died. The methodol-ogy described by Sanchez et al. (1995) was used forsnail culture. Calcium-enriched mud was used as sub-strate, a complex of four algae was used as food source,the mean temperature during the study period was25 6 0.19°C, and the photoperiod was 12 h/day with

uorescent lamp artificial light. The week zero wasonsidered that when all snails for the experimentmerged from the eggs.Every week snails were moved into new dishes. In all

roups there was always a residue of algae and no snailas seen attached to the dish cover, indicating that the

onditions within the dishes were not limiting eitheror isolated or for paired snails. During the process ofoving snails into new dishes, the shell size was mea-

ured with a caliper to the nearest 0.1 mm. The num-er of living and dead snails, the number of eggasses, and the number of eggs in each mass were also

ecorded. Counts of the number of eggs per mass wereerformed under a stereoscope (described above).The following life table parameters were determinedith the software TABVID v3.0 (Laboratory of Mala-

ology, IPK): age-dependent life tables [survivorshiprobability (lx), defined as the number of individualshat arrived alive at age x; fecundity rate (mx), defineds the number of viable eggs laid by an average indi-idual during age x; average shell size in millimeters;umber of egg masses per snail; and number of eggser mass] and age-independent life tables (see Stearns1992) for parameter description) [net reproduction
ate (Ro); mean generation time (T); intrinsic rate of
atural increase (r); and finite rate of natural increasel)].

A repeated-measures ANOVA was designed to com-pare the shell size, the number of eggs per mass, thenumber of eggs per snail, and the number of viableeggs per snail in the four groups (factors). The age wasincluded in the design as the repeated-measures factorto determine the time at which differences betweengroups were significant. A Tukey HSD test for multiplecomparisons of means (Hays, 1988) was performed totest the significance of interactions reported by therepeated-measures ANOVA.

RESULTS

The four experimental groups showed different lifehistory patterns for the parameters considered. Theage-dependent life history traits exhibited between-group variations (Figs. 1 and 2).

Survival curves (Fig. 1A) were very different be-tween isolated and paired snails of the unexposedgroups (isolated–unexposed and paired–unexposed).Isolated–unexposed snails exhibited the highest sur-vival rates at the beginning of their life and starteddying at week 15, whereas paired–unexposed snailsshowed a high mortality at week 3 and onward. Bothisolated and paired snails of the infected groups (in-fected–isolated and paired–infected) showed interme-diate values of the survival probability, and the shapesof the curves were very similar; paired snails starteddying at week 8, whereas isolated snails started dyingat week 10.

Snails of the group isolated–unexposed showed thehighest shell size at the end of the experiment. How-ever, the growth rate was higher in snails of the groupinfected–isolated. Those of the group paired–infectedalso showed greater shell sizes and growth rates thanthose of the group paired–unexposed. Significant inter-actions were found for the shell size in all groups (Ta-ble 1), indicating that snails raised under certain con-ditions of infection and isolation exhibited higher val-ues of this variable than others at a specific time.Snails of the group isolated–unexposed were signifi-cantly different from those of the infected groups (in-fected–isolated and paired–infected) only at week 8and onward. The group paired–unexposed exhibitedthe lowest values of the shell size at all ages, thedifferences from the other groups being significantfrom the second week of life.

The fecundity rate (mx, number of viable eggs persnail) (Fig. 1C) was also different among experimentalgroups, the group isolated–unexposed showing thehighest values with a reproductive peak during week 7.Snails from the group paired–unexposed followed thegroup isolated–unexposed in decreasing order of fecun-dity rate, but with much lower values, showing no
definite reproductive peak. Reproduction as measured

68 GUTIERREZ ET AL.

by this variable was very poor in infected snails (in-fected–isolated and paired–infected), the lowest fecun-dity being observed in paired snails. Reproduction asmeasured by the number of egg masses per snail andthe number of eggs per snail was very similar to mx.Significant interactions were found for the three vari-ables only in those comparisons involving the group

FIG. 1. Age-dependent life history traits of four experimentalgroups of snails. (A) Survival probability; (B) shell growth rate; (C)fecundity rate. Vertical bars represent standard deviations.

isolated–unexposed from week 3 to 10 (Table 1). There

were no significant differences in reproduction amongthe other groups for any of the three reproductive vari-ables considered herein.

The age-independent life history traits (Table 2)showed that snails of the group isolated–unexposedattained the highest population growth rates followedin order of magnitude by the groups paired–unex-posed, infected–isolated, and paired–infected. Allgroups started reproducing in the second week of lifeand slight differences were apparent in regard to theage at last reproduction, the longest reproductive pe-riod being for the group isolated–unexposed and theshortest being for the group paired–unexposed. Vari-able T was greater in the group paired–infected, whichalso showed the lowest values of Ro, r, and l.

DISCUSSION

The results clearly show the effects exerted by isola-tion and F. hepatica infection on the intermediate

FIG. 2. Age-dependent life history traits of four experimentalgroups of snails. (A) Number of egg masses per snail; (B) number of
eggs per snail. Vertical bars represent standard deviations.

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host’s life history traits. It seems evident that a similarstimulatory effect is caused by both events on shellgrowth, but the consequences of isolation are morepronounced than those of infection. On the other hand,reproduction and survival seem to be negatively af-fected by infection and stimulated by isolation. Wefound no explanation for the unexpected mortality ob-served at relatively early ages in paired unexposedsnails. This was probably caused by an unpredictedaccidental factor, since trematode infection has beenreported to negatively affect snail survival (see Gerardand Theron, 1997), as was observed between infectedand unexposed isolated snails in the current experi-ment.

All snails raised in isolation exhibited higher valuesof shell size and fecundity rate than those raised in the

TABResults of the Repeated-Measures ANOVA and the Tuk

Age-Dependent Life Tables, between Fou

Trait Comparison

Size infected–isolated vs isolated–uF 5 8.10; P , 0.001 infected–isolated vs paired–in

infected–isolated vs paired–unisolated–unexposed vs paired–isolated–unexposed vs paired–paired–infected vs paired–une

Egg masses/snail infected–isolated vs isolated–uF 5 13.13; P , 0.001 infected–isolated vs paired–in


Eggs/snail infected–isolated vs isolated–uF 5 21.40; P , 0.001 infected–isolated vs paired–in


Viable eggs/snail infected–isolated vs isolated–uF 5 19.44; P , 0.001 infected–isolated vs paired–in


Note. Infected and unexposed snails correspond to snails infectedpaired snails correspond to snails reared in complete isolation and i

TABAge-Independent Life Tables Obtained from F

Group Ro T

infected–isolated 97.76 5.80isolated–unexposed 2181.60 7.13paired–infected 15.00 7.34paired–unexposed 239.70 3.62

Note. Group legend as in Table 1. Ro, net reproduction rate; T, mea
.F.R., age at first reproduction; A.L.R., age at last reproduction.
presence of a partner, no matter the infection condi-tion. However, the reproductive differences are signif-icant only in comparisons between the group isolated–unexposed and any other group, which supports thatboth infection and pairing act against fecundity. Dif-ferences in shell size are significant in comparisonsamong all groups, but as infection stimulates growth,infected snails do not differ from unexposed isolatedsnails during the first 8 weeks of life, indicating thatshell growth is similarly affected by both events duringthe juvenile stage.

The effect of trematode infection on the intermediatesnail hosts has been widely studied. The observed re-sponses of snails to miracidial infection are to someextent divergent as trematodes can cause parasitic cas-tration, reduce or increase the host fecundity in differ-

1HSD Test for Multiple Comparisons of Means of Five

xperimental Groups of Fossaria cubensis

Significant differences P

xposed From week 8 onward ,0.001ted From week 2 to 4 ,0.001posed From week 2 onward ,0.001ected From week 8 onward ,0.001exposed From week 2 onward ,0.001osed From week 2 to 8 ,0.05xposed From week 3 to 10 ,0.001ted None .0.05posed None .0.05ected From week 2 onward ,0.05exposed From week 3 onward ,0.001osed On week 5 ,0.01xposed From week 3 to 10 ,0.001ted None .0.05posed None .0.05ected From week 3 to 10 ,0.001exposed From week 3 to 10 ,0.001osed None .0.05xposed From week 3 to 10 ,0.001ted None .0.05posed None .0.05ected From week 3 to 10 ,0.001exposed From week 4 to 10 ,0.001osed On week 5 ,0.001

ith and unexposed to Fasciola hepatica, respectively; isolated andairs, respectively.

2r Experimental Groups of Fossaria cubensis

r l A.F.R. A.L.R.

1.28 3.61 1 162.12 8.39 1 170.86 2.37 1 141.79 6.04 1 10

eneration time; r, intrinsic rate of increase; l, finite rate of increase;

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70 GUTIERREZ ET AL.

ent stages of their life cycle, enhance, reduce, or notalter the host shell growth rate, and reduce the sur-vival of the host. The specific patterns of host responseto parasitic infection seems to depend on the trema-tode–snail system and on the time of the snail life cycleat which infection took place (Baudoin, 1975; Becker,1980; Hurd, 1990; Gerard and Theron, 1997; Zakikhaniand Rau, 1998).

Gutierrez et al. (2000) reported on the effect of expo-sure of a group of F. cubensis to F. hepatica miracidia,observing an increased growth and a reduced fecundityin exposed snails compared to those in controls. Thetrematode stimulation of the snail shell growth hasbeen reported to be a consequence of a host’s endocrinesystem manipulation to increase the intramolluscanavailable space (Jong-Brink, 1995). Recent observa-tions on the F. cubensis–F. hepatica system show thata trade-off between shell size increment and reproduc-tion occurs only in snails exposed during early stages oflife, infected snails exhibiting the highest correlations(Gutierrez et al., 2000). This could be the reason whydifferences in shell growth between isolated–unex-posed snails and infected snails in this study are sig-nificant only after week 8. It may be possible that theenergy normally allocated for reproduction is then re-directed to the shell growth in young adult snails.

The trematode effect on host reproduction has beeninvestigated for different systems, and in most casesfecundity is decreased, with parasitic castration some-times evoked in snails infected during early stages oflife. Several workers have given explanations for thisalteration of host reproduction. Their results suggestthat infection induces the hosts to produce (or changethe activity of) a molecule that interferes with theirown reproductive system, inhibiting the normal func-tioning of the albumen gland (Jong-Brink, 1995; Bai etal., 1997). The results of the present study show thatno parasitic castration seems to occur in F. cubensissnails infected with F. hepatica during early stages oflife. However, a strong reduction of the reproductivefunction is indeed visible in infected snails, and thismalfunction inevitably affects the population fitness,as infected adult snails showed lower values of thefinite and intrinsic rates of increase.

The influence of isolation in freshwater snails hasbeen also studied. Vianey-Liaud (1976) reported thatisolated Biomphalaria glabrata exhibited a reducedfecundity compared to that of paired snails, and theseresults were discussed in relation to the differences insperm storage location for each type of mating system(selfing and outcrossing). For lymnaeids, de Witt andSloan (1958) observed an increase in fecundity andshell growth in isolated Pseudosuccinea columella com-pared to paired snails and attributed the differences tosome unknown genetic mechanism. Florin et al. (2000)compared growth and fecundity between isolated and
grouped Lymnaea elodes, finding that isolated snails
also grew and reproduced at a higher rate. They spec-ulated about the possibility of an inhibition of growthand reproduction in grouped snails by the release ofsubstances into the water (i.e., excretory/secretoryproducts). However, we have observed in P. columellathat isolated snails raised in snail-conditioned watershow the same reproduction and growth patterns asthose of control isolated snails (data not published),which indicates that at least for this species the above-mentioned hypothesis is not valid. There may still beseveral explanations for this phenomenon: (1) an inhi-bition of reproduction and growth may occur betweensnails by direct contact, for instance during copulation,and (2) copulation may be an energy-costly event andsnails reproducing only by selfing may allocate moreenergy, otherwise expended during copulation, to re-production and growth. In any case, it seems that thereproductive strategy for isolated snails depends on thespecies, since different authors have reported contra-dictory results in regard to the effect of isolation (seereview in Vianey-Liaud, 1998). At least for some lym-naeids, including F. cubensis, it seems plausible thatisolated snails reproduce at a higher rate to quicklycolonize and expand their populations. As the numberof individuals increase thereafter, interactions be-tween them inhibit their fecundity rates as an intrinsicmechanism to regulate the population size, avoidingthe negative effects of crowding. Further experimentsare needed to provide more data to test these hypoth-eses.

ACKNOWLEDGMENTS

We are indebted to Dr. Gloria Perera for her valuable contribu-tions to the development of this work, to Dr. Jean P. Pointier and Dr.Andre Theron for their ideas and suggestions in the interpretationsof results, and to two anonymous reviewers for their helpful com-ments on the original manuscript.

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The Combined Effect of Isolation and Fasciola hepatica Infection on the Life History Traits of Fossaria cubensis