J. Parasitol., 94(2), 2008, pp. 314–325 ᭧ American Society of Parasitologists 2008

LIFE HISTORY COST OF TREMATODE INFECTION IN ANCEPS USING MARK–RECAPTURE IN CHARLIE’S POND

N. J. Negovetich* and G. W. Esch Department of Biology, Wake Forest University, Winston-Salem, North Carolina 27109. e-mail: [email protected]

ABSTRACT: Parasitism has the potential to affect key life history traits of an infected host. Perhaps the most studied interactions are in snail–trematode systems, where infection can result in altered growth rates, survival, and/or fecundity of the individual. Positive correlations between host size and parasite prevalence are often attributed to changes in growth rates or mortality, which have been observed in the laboratory. Extending lab-based conclusions to the natural setting is problematic, especially when environmental conditions differ between the laboratory and the field. The present study uses reproduction experiments and mark– recapture methods to directly measure key life history traits of the pulmonate snail in Charlie’s Pond. Based on previous laboratory and field experiments on H. anceps, we predict a significant reduction in fecundity, but not growth rate or survival, of infected snails. Individual capture histories were analyzed with multistate models to obtain estimates of survival and infection probabilities throughout the year. Recaptured individuals were used to calculate specific growth rates. Trematode infection resulted in complete castration of the host. However, neither survival nor growth rates were found to differ between infected and uninfected individuals. The probability of infection exhibited seasonal variation, but it did not vary with size of the snail. These results suggest that the correlation between host size and trematode prevalence is not due to differential mortality or changes in growth rates. Instead, the infection accumulates in large snails via the growth of smaller, infected individuals.

Trematode larvae commonly castrate their molluscan hosts. been observed from infections by both sporocysts (Makanga, The mechanism of castration depends on the specific larval 1981; Minchella and Loverde, 1981; Zakikhani and Rau, 1999) stage infecting the host. For example, rediae mechanically cas- and rediae (Dreyfuss et al., 1999; Sandland and Minchella, trate the host by consuming host tissue, including the high- 2003). Physical damage caused by the parasite during feeding energy fat of the gonads. Sporocysts, lacking a mouth and phar- and cercariae release, and the energetic cost of an immune re- ynx, likely castrate the host via chemical inhibition. In partic- sponse may contribute to, if not cause, the increase in mortality. ular, Trichobilharzia ocellata stimulates the release of schisto- Reinfection by cercariae has also been implicated in decreasing somin, which inhibits oviposition by Lymnaea stagnalis (De survival of the snail host (Kuris and Warren, 1980; Sandland Jong-Brink, 1995). Castration could be complete, as in the Hel- and Minchella, 2003). isoma anceps–Halipegus occidualis system (Crews and Esch, The difficulties in estimating the true cost of parasitism arise 1986), or partial, as in the mudsnail Hydrobia ventrosa (Kube when extending the results derived from laboratory experiments et al., 2006). The individual cost of castration includes the loss to observations in the field. For example, lab-reared Lymnaea elo- of future reproduction. Fecundity compensation has been pro- des grew faster and laid more eggs than snails raised in the field posed as a mechanism by which the host could potentially off- (Eisenberg, 1966, 1970). If food was supplied in the field, then set this negative effect (Minchella, 1985; Sorensen and Min- both growth and fecundity increased. Likewise, H. anceps in Char- chella, 1998). Compensation has been recorded in the T. ocel- lie’s Pond are protein deprived, and they exhibit lower fecundity lata–L. stagnalis, Schistosoma mansoni–Biomphalaria glabra- and growth rates than snails raised in the laboratory (Keas and ta, and S. mansoni–Biomphalaria pfeifferi systems (Sorensen Esch, 1997). To accurately assess the impact of parasitism, studies and Minchella, 2001), and it occurs by increasing reproductive should be performed under natural field conditions. effort, i.e., oviposition or maturation, before castration. Mark–recapture sampling protocols provide the data required Many researchers have noted a positive correlation between to estimate survival in the field. Moreover, recent advances prevalence of infection and size of the snail host. This has led have expanded the scope of analysis, such that simple estimates some to question whether growth schedules vary between in- of survival and population size are becoming secondary to more fected and uninfected hosts (Baudoin, 1975; Fernandez and complex questions. One advance is the development of multi- Esch, 1991a; Sorensen and Minchella, 2001). Common re- state models, which permit the estimation of transition proba- sponses to infection include gigantism (ϭthe rapid increase in bilities between states (Hestbeck et al., 1991; Lebreton et al., size after infection) and stunting. The growth response by a 1992; Brownie et al., 1993; Nichols et al., 1994; Nichols and snail may depend on when it was infected. For example, if Kaiser, 1999; Lebreton and Pradel, 2002; White et al., 2006). infected before reproductive maturity, B. glabrata experiences For example, many researchers have used multistate models to rapid growth until maturity, and then it grows less rapidly than estimate the cost of breeding on survival rates (White et al., uninfected individuals (Gerard and Theron, 1997). 2006), whereas others have calculated dispersal rates between Recently, trematode infections have been shown to increase various habitats (Hestbeck et al., 1991). Application of mark– mortality of the snail host. Negative effects on survival have recapture protocols to snail populations has been limited to a few studies (O’Keeffe, 1985; Woolhouse, 1988; Goater et al., 1989; Woolhouse and Chandiwana, 1990; Fernandez and Esch, Received 19 March 2007; revised 7 August 2007; accepted 8 August 2007. 1991a; Chlyeh et al., 2002, 2003). One reason for the lack of * Present address: St. Jude Children’s Research Hospital, Infectious Dis- interest may be the low recapture rate of aquatic snails, as a ease, 332 N. Lauderdale Street, MS #330, Memphis, Tennessee 38105. consequence of their vagility. In Charlie’s Pond, 97% of H.

314 NEGOVETICH AND ESCH—COST OF INFECTION IN A SNAIL HOST 315

anceps were collected within 1 m of the original release site class. Analysis of variance was performed in JMP 4.0.4 (SAS Institute, (Fernandez and Esch, 1991b), compared with the 28% of Physa Cary, North Carolina). If the variances of SGRs were not equal, then a Kruskal–Wallis (KW) test was performed. gyrina that moved more than 1.5 m away from the site of re- The mark–recapture protocol resulted in individual capture histories lease in just 2 wk (Snyder and Esch, 1993). Because of its low that span the entire collecting period. The capture histories consisted of vagility, H. anceps is a suitable candidate for an extensive an informational series of 0, A, and B. Each value holds unique infor- mark–recapture study to measure both growth rates and surviv- mation. Zero indicates that the snail was not captured during a specific al in the field. capture attempt. The letters indicate that the snail was captured and uninfected (A), or captured and infected (B). Although 11 trematode The goal of the present study was to determine how trema- were identified in 2006 alone, the prevalence, and thus sample tode infections affect the life history traits of H. anceps as mea- size, for most of these infections was too small to adequately estimate sured in a natural setting. The traits being investigated include their effect on survival. Therefore, trematode infections were pooled survival, fecundity, and growth rates. Mark–recapture methods into a single infection category. Capture histories grouped by size at initial capture were analyzed with multistate models in Program MARK were used, and the capture histories were then analyzed using ⌽ ␳ (White and Burnham, 1999). The general model tested was s·i·t s·t multistate models. Based on field (Fernandez and Esch, 1991a) ⌿ ⌽ ⌿ s·i·t. In this model, survival ( ) and infection ( ) probabilities vary as and laboratory estimates (Keas and Esch, 1997), we hypothe- a function of site (s), time (t), and infection status (i). We have no reason size that infection will not alter the observed growth rates or to suspect that infection status affects recapture probability (␳); thus, ␳ survival probabilities of H. anceps. This investigation will also the general model allows to vary only with s and t. Akaike’s infor- mation criterion (AIC) was used for model selection and ranking (An- produce estimates for the probability of infection, which is pre- derson and Burnham, 1999a, 1999b). Parametric bootstrap procedures dicted to vary seasonally. were used to estimate cˆ, which is a measure of lack-of-fit (Cooch and White, 2006). This method produced simulated capture histories using MATERIALS AND METHODS parameter estimates from the general model. These capture histories were then used to obtain simulated parameter estimates, including de- Helisoma anceps were collected from 2 sites in Charlie’s Pond, viance of the model. A new set of capture histories was created for each Stokes County, North Carolina. Charlie’s Pond is approximately 1 ha, iteration. One thousand iterations were performed for each size class, and it has been described previously (Crews and Esch, 1986; Esch et and the model deviances were saved. The deviance from the general al., 1997). Beginning in May 2005, snails were collected by random model was divided by the mean deviance from the simulated models to sampling with a dip net for 1 hr each week. Weekly collecting sites obtain cˆ (Cooch and White, 2006). AIC was divided by cˆ to obtain a alternated between the northwest cove (NW) and east side (ES) of Char- quasi-likelihood adjust AIC (QAIC), which corrects for excess variation lie’s Pond. No collections were made from mid-November 2005 through in the models (Cooch and White, 2006). Models with small QAIC are February 2006. The collection regime resumed in March 2006 and con- preferred to those with large QAIC. More specifically, differences in cluded in October 2006. QAIC of Ͼ7 strongly suggest a real difference between the models Snails were transported to the laboratory where they were individu- being compared (Anderson and Burnham, 1999a, 1999b; Cooch and ally isolated in 55-mm plastic jars. The shell length of each snail was White, 2006). measured to the nearest 0.05 mm, and the jars were examined for pres- The POPAN algorithm of Program MARK was used to estimate re- ence of cercariae. Each snail was marked with colored enamel paint to cruitment (births ϩ immigration) and population size. This method signify month, with a letter indicating year and site, and a number. The models a super population N that enters the local population with a combination of color, letter, and number represented a unique identifier probability of bi. The product of the probability bi and the super pop- so that a set of individual capture histories could be obtained for each ulation is the net number of new recruits from either immigration or site within the pond. Snails were returned to their site of capture the ˆ ϭ ˆ births during monthly transition i (Bi N·bi). Population size (Ni) dur- following week. ing month i is derived from estimates of survival, population size, and Fecundity was directly measured in the pond using 2 reproduction recruitment in the previous month (Cooch and White, 2006). platforms. Each platform holds 24 plastic jars (Negovetich, 2003); they were placed in the pond 1 wk before the addition of snails. From May to October 2005, reproduction was measured monthly at NW and ES. RESULTS In 2006, bimonthly measurements were made from March to October. To measure fecundity, 24 snails were collected from NW or ES. A In total, 910 H. anceps were placed in the reproduction plat- concerted effort was made to collect snails of various sizes. A single snail was placed in each jar, and the platform was submerged to a depth forms. Because fecundity varies from year to year (Negovetich of 5 cm. The following week, the jars were transported to the lab, and and Esch, 2007), the effect of infection status was assessed by the number of eggs and egg masses was recorded. The snails were comparing fecundity of only those snails that were isolated in isolated, measured, and marked in the same manner as individuals that 2006. The analysis included 674 snails, 34 were infected with were not placed in the reproduction platform. Fecundity was normalized to eggs/week for each snail, and mean values from infected and unin- H. occidualis (rediae), and 27 with miscellaneous trematode fected individuals were compared with nonparametric tests of signifi- species (mostly sporocysts). Infection resulted in a significant cance. Randomization tests (10,000 iterations) were performed to de- reduction in egg output (KW: ␹2 ϭ 48.3, 2 df, P Ͻ 0.0001). termine whether fecundity differs between ES and NW for each month Uninfected snails laid an average Ϯ SE of 13.46 Ϯ 0.68 eggs/ (Manly, 1991). Ϯ Ϯ Differences in mean size of uninfected and infected individuals were wk compared with 0.56 0.32 and 0.74 0.44 eggs/wk for investigated. To correct for seasonal variation, an adjusted shell length H. occidualis-infected and other infected snails, respectively. was calculated for each snail. This was accomplished by extracting the Restricting analysis to uninfected snails reveals that monthly residuals from a linear regression of size as a function of year and mean fecundity decreased throughout the year for both loca- month (Sokal and Rohlf, 1995). The seasonally corrected sizes of un- infected, H. occidualis-infected, and non–H. occidualis-infected snails tions within the pond (Fig. 1). Randomization tests detected a from NW and ES were compared with nonparametric tests. significant difference in March (P Ͻ 0.0001), May (P Ͻ Specific growth rates (SGRs) were calculated for each recaptured 0.0001), June (P Ͻ 0.0003), and August (P Ͻ 0.0001). Analysis ϭ Ϫ individual (SGR [S2 S1]/D, where S1 is size at initial capture, S2 with POPAN strongly suggests variation in both site and time is size at recapture, and D is the number of d between capture). Snails for the probability to enter the population (AIC ϭ 7,175.8, mod- were classified as infected if they were shedding cercariae when initially captured or at recapture. Differences between sites and infection status el likelihood ϭ 1.00, deviance ϭ 7,036.1). For monthly tran- were investigated by comparing average SGR for each month and size sitions in 2006, recruitment was higher at NW than ES; yet, the 316 THE JOURNAL OF PARASITOLOGY, VOL. 94, NO. 2, APRIL 2008

FIGURE 1. Mean fecundity (ϮSE) of uninfected snails from ES and NW of Charlie’s Pond in 2006. Data points with an asterisk indicate site differences in fecundity, as determined by randomization tests. population in ES experienced less fluctuation in snail recruit- sizes were significantly different between uninfected, H. occi- ment than NW (Fig. 2). dualis-infected, and miscellaneous infected snails (KW: ␹2 ϭ The mark–recapture experiment resulted in a total of 4,850 153.1, 2 df, P Ͻ 0.0001). Snails infected with H. occidualis (x¯ individual capture histories. Of these, 1,114 capture histories ϭ 9.72 Ϯ 0.05 mm [ϮSE]) were larger than snails infected with represented individuals that were recaptured at least once (Table other trematode species (x¯ ϭ 9.61 Ϯ 0.10 mm), and these were I). Only a single snail migrated across the pond, and this indi- larger than uninfected snails (x¯ ϭ 9.07 Ϯ 0.02 mm; Steel-type vidual was excluded from analysis. Seasonally corrected mean MC: P Ͻ 0.01 for all comparisons).

FIGURE 2. Number of new recruits (ϮSE) estimated with POPAN in 2006 for ES and NW of Charlie’s Pond. Recruitment is measured as the increase in population size from 1 mo to the next after accounting for mortality, and entrance via birth and immigration. NEGOVETICH AND ESCH—COST OF INFECTION IN A SNAIL HOST 317

TABLE I. Percentage of capture histories representing the number of throughout the year, but NW snails lay more eggs/week until times an individual was captured for ES, NW, and the entire Charlie’s cohort turnover in July (Fig. 1). In March, the difference in pond. Snails captured once were marked and never recaptured. The total number of capture histories for each site and for the Charlie’s Pond is reproductive output is most likely related to emergence of snails also included. from the substratum as the water warms after winter. Specifi- cally, more snails per hour were collected in NW than ES dur- Times captured ES NW Pond ing March, which suggests that snails become active earlier in the year at NW than in ES. May was the second month that 1 77.54 76.60 77.03 produced a difference in fecundity. In this month, snails Ͼ9 2 20.49 20.08 20.27 mm were more fecund, and closer inspection reveals that re- 3 1.84 3.13 2.54 productive activity is responsible. The percentage of reproduc- 4 0.09 0.11 0.10 5 0.04 0.08 0.06 tively active snails differed for the 9-mm (93% in NW vs. 0% Ͼ Total no. of capture histories 2,226 2,624 4,850 in ES) and 10-mm size classes (96% in NW vs. 43% in ES). When accounting for reproductive activity, snails Ͼ10 mm re- main more fecund at NW compared with ES. Reproduction is depressed when a snail approaches death (Herrmann and Har- SGR did not differ by site or infection status for almost all man, 1975). Based on size frequency histograms (Fig. 7), cohort size classes examined (Fig. 3). The single exception when com- turnover at ES is apparent by June, which suggests that those paring the sites was for the 7-mm size class. SGR for NW was snails were near death in May. Moreover, the NW decrease of significantly higher than ES (analysis of variance [ANOVA]: fecundity in July corresponded to turnover at that location, F ϭ 6.9, P Ͻ 0.01). Upon closer inspection, the difference 1,131 which occurred nearly a month after ES. in SGR persisted for uninfected (ANOVA: F ϭ 7.9, P Ͻ 1,108 Alternative explanations for a difference in fecundity be- 0.006), but not infected (ANOVA: F ϭ 0.00003, P Ͼ 0.05), 1,21 tween the sites include local productivity and snail size. First, individuals. Seasonally, SGR fluctuated little from March to large snails have the capacity to lay more eggs than smaller May, increased to a peak in August, and then decreased until individuals (Brown, 1985; Brown et al., 1985; Lazaridou-Dim- winter (Fig. 4). Uninfected individuals exhibited a higher SGR itriadou et al., 1998). The NW snails placed in the reproduction in August (KW: ␹2 ϭ 20.0, 1 df, P Ͻ 0.0001). Comparing the platforms were larger than ES snails in May–July. However, sites, significant differences in SGR were observed in August fecundity at the 2 sites was similar in July, which diminishes (ANOVA: F ϭ 6.1, P Ͻ 0.015) and September (KW: ␹2 ϭ 1,134 the likelihood that snail size is the primary cause of the fecun- 19.2, 1 df, P Ͻ 0.0001). When examining infection status with- dity differences. Second, reproduction is depressed when food in these months, SGR was not different in August for both supplies are limiting (Brown et al., 1985; Byrne et al., 1989; uninfected (ANOVA: F ϭ 3.5, P Ͼ 0.05) and infected in- 1,98 Lam and Calow, 1989; Wayne, 2001). NW contains more emer- dividuals (ANOVA: F ϭ 0.74, P Ͼ 0.05). However, in Sep- 1,34 gent vegetation and leaf litter, surfaces on which periphyton can tember, NW growth rates were significantly higher only for the grow. If productivity is contributing to the differences in fe- uninfected snails (KW: ␹2 ϭ 13.3, 1 df, P Ͻ 0.0003). cundity, then NW snails of a specific size class should be con- The effect of infection on survival was assessed by fitting sistently more fecund than ES snails of the same size class various models to the set of capture histories from the mark– during a particular month. This was only observed in March recapture experiment. To avoid complications of initial size and for the 8- and 10-mm size classes, and in May for the 9- and growth of the snails, analyses were performed by size class. For 10-mm size classes. The lack of consistency from 1 mo to the example, the capture histories of all snails that were 6–6.95 mm next implies that productivity is not the only factor affecting at initial capture were analyzed independently from the remain- egg production in the Pond. Instead, it may be contributing to ing size classes. After fitting the general model and correcting the observed differences between the 2 sites. for overdispersion of the errors, the most likely model that fits Infection severely decreased egg production in H. anceps. the data was ⌽ ␳ ⌿ (Table II). For the 10-mm size class, t t i·t For snails infected with rediae of H. occidualis, the mechanism there is some support for a model that allows survival to vary is mechanical castration by direct consumption of the gonads with site and time; yet, the best model was 2.5 times more likely (Crews and Esch, 1986). Of the 27 snails infected with a trem- than this second best model. Survival from 1 mo to the next atode species other than H. occidualis, 22 individuals were varied seasonally (Fig. 5). Small snails suffered from higher shedding 1 of 2 unknown spirorchiid cercariae. Within the snail mortality (ϭlow survival probabilities) than larger snails, ex- host, the 2 spirorchiids have sporocyst larval stages. Unlike H. cept in the period during cohort turnover. occidualis, sporocysts have been known to chemically castrate Recruitment of trematode infections is reflected in the prob- the host (Cheng et al., 1973; De Jong-Brink et al., 1991; Wayne, ability of being infected (Fig. 6). Early in the year, infection 2001). The size at first reproduction was 6 mm, and given that probabilities remained under 0.2 for all size classes. Peak in- all but 1 snail were larger than 7 mm, it is reasonable to assume fection probabilities occurred from June to August, which cor- that the unknown spirorchiids also castrate H. anceps in Char- responds to a large recruitment of trematode infections and new lie’s Pond. In fact, 19 of 22 spirorchiid-infected snails did not snails at this time. The risk of infection then returned to levels lay eggs, and the 3 snails that were reproductively active pro- previously observed during spring. duced no more than 10 eggs within a week. In some snail– trematode systems, snails often exhibit fecundity compensation DISCUSSION (Sorensen and Minchella, 2001). Specifically, some species in- When comparing fecundity, prominent differences exist be- crease their reproductive activity between the time of initial tween ES and NW. In both locations, fecundity decreases exposure to the egg or miracidium, and patency. Fecundity 318 THE JOURNAL OF PARASITOLOGY, VOL. 94, NO. 2, APRIL 2008

FIGURE 3. Mean specific growth rate (ϮSE) for the different size classes of snails, examined by (A) infection status and (B) site (ES and NW). Pairs of bars marked with an asterisk were significantly different at the P Յ 0.05 level. Only 1 infected snail from the 5-mm size class was recaptured, and that value is excluded from the graph. compensation may be occurring in the Pond, but we feel that not imply a difference in growth rate. Much work has been it is unlikely given that increases in egg production were not done on the influence of trematode infections on the growth observed in lab studies on H. anceps (Keas and Esch, 1997). rate of snails. In all of the field studies at Charlie’s Pond, H. Infected snails are larger than uninfected snails, but this does occidualis-infected H. anceps were larger than uninfected in- NEGOVETICH AND ESCH—COST OF INFECTION IN A SNAIL HOST 319

FIGURE 4. Mean specific growth rate (ϮSE) throughout the year, examined by (A) infection status and (B) site (ES and NW) of the uninfected snails. Pairs of bars marked with an asterisk indicate a significant difference at the P Յ 0.05 level. dividuals (Crews and Esch, 1986; Fernandez and Esch, 1991c; between host size and parasite prevalence, i.e., unequal catch- Williams and Esch, 1991; Negovetich, 2003). Similar associa- ability, differential mortality, variation in susceptibility, and tions between prevalence and snail size occur in 68% of marine changes in growth rates (Baudoin, 1975). and 73% of freshwater gastropods (Sorensen and Minchella, Catchability describes the likelihood of collecting snails giv- 2001). Four possibilities can explain the positive correlation en their infection status. In some systems, the parasite alters the 320 THE JOURNAL OF PARASITOLOGY, VOL. 94, NO. 2, APRIL 2008

TABLE II. Summary of the various multistate models tested in Program MARK. Analyses were performed in groups based on the size class of snails at initial capture. Number of parameters and the dispersion parameter (cˆ) were adjusted to generate the QAIC, the change in QAIC (⌬QAIC), and the quasi-likelihood deviance (QDeviance). Survival (⌽), catchability (␳), and infection transitions (⌿) were allowed to vary by site, infection status (inf), and time.

Model QAIC ⌬QAIC Parameters QDeviance

6mm(cˆ ϭ 2.051) ⌽ ␳ ⌿ time time inf*time 339.6 0 55 58.6 ⌽ ␳ ⌿ site*time time inf*time 363.3 23.7 69 44.6 ⌽ ␳ ⌿ inf*time time inf*time 373.5 33.9 69 54.7 ⌽ ␳ ⌿ site*inf*time time inf*time 441.4 101.8 97 38.2 ⌽ ␳ ⌿ site*inf*time site*time site*inf*time 588.0 248.4 138 33.6 7mm(cˆ ϭ 1.015) ⌽ ␳ ⌿ time time inf*time 788.2 0 55 177.3 ⌽ ␳ ⌿ site*time time inf*time 808.7 20.5 69 163.7 ⌽ ␳ ⌿ inf*time time inf*time 809.7 21.5 69 162.6 ⌽ ␳ ⌿ site*inf*time time inf*time 872.2 84.0 97 149.8 ⌽ ␳ ⌿ site*inf*time site*time site*inf*time 979.4 191.2 138 128.0 8mm(cˆ ϭ 1.318) ⌽ ␳ ⌿ time time inf*time 1,185.4 0 55 247.0 ⌽ ␳ ⌿ site*time time inf*time 1,205.2 19.8 69 235.0 ⌽ ␳ ⌿ inf*time time inf*time 1,207.1 21.7 69 236.9 ⌽ ␳ ⌿ site*inf*time time inf*time 1,254.7 69.3 97 217.9 ⌽ ␳ ⌿ site*inf*time site*time site*inf*time 1,332.3 146.9 138 190.3 9mm(cˆ ϭ 1.359) ⌽ ␳ ⌿ time time inf*time 1,562.1 0 55 321.5 ⌽ ␳ ⌿ site*time time inf*time 1,570.9 8.8 69 299.5 ⌽ ␳ ⌿ inf*time time inf*time 1,580.1 18.0 69 308.7 ⌽ ␳ ⌿ site*inf*time time inf*time 1,617.9 55.8 97 283.0 ⌽ ␳ ⌿ site*inf*time site*time site*inf*time 1,699.1 137.0 138 265.9 10 mm (cˆ ϭ 1.182) ⌽ ␳ ⌿ time time inf*time 1,561.6 0 55 350.4 ⌽ ␳ ⌿ site*time time inf*time 1,563.5 1.9 69 321.0 ⌽ ␳ ⌿ inf*time time inf*time 1,584.3 22.7 69 341.9 ⌽ ␳ ⌿ site*inf*time time inf*time 1,611.9 50.3 97 304.4 ⌽ ␳ ⌿ site*inf*time site*time site*inf*time 1,703.7 142.1 138 294.4 11 mm (cˆ ϭ 1.343) ⌽ ␳ ⌿ time time inf*time 690.8 0 55 173.7 ⌽ ␳ ⌿ site*time time inf*time 716.3 25.5 69 163.1 ⌽ ␳ ⌿ inf*time time inf*time 719.7 28.9 69 166.5 ⌽ ␳ ⌿ site*inf*time time inf*time 783.5 92.7 97 151.0 ⌽ ␳ ⌿ site*inf*time site*time site*inf*time 905.9 215.1 138 136.3

behavior of the host so that infected individuals are more likely infection, which seems more common with rediae stages than to be consumed by the next host in the parasite’s life cycle with sporocysts (Sorensen and Minchella, 2001). If younger (Poulin, 2007). In the context of Baudoin (1975), the parasite snails are more likely to die after infection, then the size dis- may have a size-dependent effect on behavior, such that large, tribution of infected snails will be skewed, with a greater quan- infected individuals will be easier to catch than small, infected tity of infected snails tending to larger sizes. The second situ- individuals. Thus, prevalence would be positively correlated ation is a reduction of mortality of infected individuals. Spe- with size of the snail. In Charlie’s Pond, no evidence exists that cifically, if infected snails live longer than uninfected snails, would suggest that infected snails are easier to collect than un- and size is positively correlated with age, then the infected pop- infected snails. Furthermore, analysis of capture histories indi- ulation will be larger, on average, than the uninfected popula- cates that models with capture probabilities that vary with in- tion. In the current study, differential mortality is not apparent. fection status do not fit the data better than the general model None of the most parsimonious models included infection status (data not shown). Thus, catchability of H. anceps is not influ- as a determinant of survival (Table II). Prepatent infections enced by infection status. could have increased mortality, especially in small snails. This There are 2 situations where differential mortality will pro- increase in mortality would not be detected because the sam- duce a size difference between uninfected and infected individ- pling methods only permitted the identification of patent infec- uals. The first is an increase in mortality of young snails after tions. However, laboratory studies have also failed to reveal a NEGOVETICH AND ESCH—COST OF INFECTION IN A SNAIL HOST 321

FIGURE 5. Probability to survive (⌽) during the monthly transitions for the 6 size classes of snails. significant difference in survival (Keas and Esch, 1997); so, it infected at a smaller size and grew into the size class. In the seems that mortality of H. anceps in the field is not influenced absence of increased mortality, a plot of prevalence versus size by infection status. class will produce a straight line with positive slope, where the A positive correlation between prevalence and snail size may slope is equal to the probability of infection. Alternatively, the have arisen from differences in susceptibility. Two situations probability of infection could increase with size, in which case would produce the same observation, i.e., equal probability of prevalence will exhibit a curvilinear increase as a function of infection across size classes, or increases of infection probabil- size class. The highest prevalence, regardless of trematode spe- ities with size (Baudoin, 1975). If the probability of infection cies, was observed in June (20%), and it remained above 16% is constant across size classes, then infections will accumulate in July and August. To maintain the level of prevalence during in larger snails. For example, the 9-mm size class includes new- cohort turnover, the probability of infection must increase so ly infected 9-mm snails, in addition to individuals that were that young, newly infected individuals replace the dying, in- 322 THE JOURNAL OF PARASITOLOGY, VOL. 94, NO. 2, APRIL 2008

FIGURE 6. Probability to become infected (⌿) during the monthly transitions for the 6 size classes of snails. Note the change in scale between 2 graphs. Transitions without a value indicate that Program MARK was unable to estimate the parameter. fected snails (Fig. 6). This corresponds with the timing of peak The final explanation for the correlation between prevalence recruitment and maturation of H. occidualis in the green frogs and size involves growth rates. Sorensen and Minchella (2001) (Goater, 1989; Wetzel and Esch, 1996). There is little evidence reviewed the literature on snail–trematode life history interac- to suggest that infection probability varies with size class. With tions. They found that enhanced growth rates of infected snails the exception of the July–August and August–September tran- occur more frequently in freshwater systems, whereas stunting sitions, the 3 largest size classes differ by no more than 12%, is more common in marine systems. Trematode infections can and the SE of the estimates overlap, suggesting that infection have stage-specific effects on the host, such that infected ju- probabilities are not significantly different. The SE of the 3 veniles might grow faster than uninfected juveniles, whereas smallest size classes also overlap. Thus, the correlation between infected adults may grow more slowly. Most work has occurred shell length and prevalence is probably caused by accumulation in the laboratory, including experiments on H. anceps (Keas of infected individuals in the larger size classes. and Esch, 1997). Fernandez and Esch (1991c) suggested that NEGOVETICH AND ESCH—COST OF INFECTION IN A SNAIL HOST 323

FIGURE 7. Histogram of shell size (millimeters) for snails collected from ES and NW of Charlie’s Pond throughout the year. the disparity of conclusions between the field and lab experi- uals, the parasite liberates energy for the host that was previ- ments may be due to the abundance of food and constant en- ously allocated to reproduction. In response, the host exhibits vironmental conditions in the laboratory. Specifically, the cost faster growth rates. Alternatively, gigantism could result as a of parasitic infection on growth rates could be strengthened or response by the host to increase fitness before castration (Min- diminished depending on the environmental conditions. chella, 1985; Sorensen and Minchella, 1998). Specifically, once Growth rates vary by size class and month. Specifically, infected, the host attains reproductive size and oviposits before small snails grow faster than large snails (Fig. 3), and SGR castration. Increased growth rates may also delay castration be- peaks in August (Fig. 4). The dependence of size on growth cause large hosts maintain viable gonadal tissue longer than rate is well documented for many species, including H. anceps smaller snails. In both hypotheses, life history tradeoffs mini- (Herrmann and Harman, 1975; Fernandez and Esch, 1991a). mize the effect of infection. At Charlie’s Pond, trade-offs in the The 7-mm size class produced a significant difference of SGR life history traits of H. anceps were not detected. Although the between NW and ES. However, 71% of the uninfected NW trematode infections castrated the snail host, neither growth rate snails were initially captured in August–September, the 2 mo nor survival probability was altered in comparison with unin- with the fastest growth rate, compared with 31% of the ES fected individuals. snails. In contrast, 75% of the infected snails for each site were Previous studies have examined key life history traits of H. collected from July to September. Thus, the observed difference anceps from Charlie’s Pond in the field (Crews and Esch, 1986; in SGR for the 7-mm size class was primarily influenced by Fernandez and Esch, 1991a; Negovetich, 2003) and in the lab seasonal changes in growth rate, and not by variation between (Keas and Esch, 1997). The current study expands on the pre- the 2 locations within Charlie’s Pond. Similarly, the significant vious research by comparing 2 locations within Charlie’s Pond difference in September between the sites is most likely due to and directly estimating life history traits with a mark–recapture the population structure in that month. Snails Ͻ8 mm repre- experiment. A decrease in fecundity was the only individual sented 20% of the ES population and 42% of the NW popu- cost associated with trematode infection. Castration will nega- lation. Seasonal variation and size class effects suggest that tively impact the snail host by decreasing the population growth SGR does not differ by site. Therefore, site was ignored when rate and reducing the number of genotypes that individual hosts examining the effect of infection on SGR. pass on to the next generation. Although individual responses August was the only month when SGR of uninfected and to offset castration were not detected, the population may have infected individuals were significantly different. The distribu- responded with changes in the life history strategy, such as a tion of size classes within this month indicates that the infection decrease in the age at first reproduction. This could be accom- effect in this case is spurious. Specifically, 59% of the unin- plished via increased growth rates so that reproductive size is fected snails were Ͻ8 mm, whereas only 36% of the infected reached earlier in life, or it could arise from decreases in size individuals were represented by the fastest growing size classes. at maturity (Roff, 1992). In Charlie’s Pond, snails became re- Furthermore, no difference in SGR was detected within the size productively mature at 7.5–8.0 mm in mid-1980s (Goater, classes examined. This is surprising given the number of host– 1989), but the latest fecundity experiment demonstrated ovi- parasite associations where the growth rate of the host is af- position by 6-mm snails in August and September. The decrease fected by the trematode (Sorensen and Minchella, 2001). Com- in size at maturity could be an evolutionary response in a sys- peting hypotheses explain alterations in growth schedules. The tem where castration by H. occidualis has been occurring in first states that the available energy for the host is contingent the snail population for nearly 25 yr. The life history estimates on the needs of the parasite (Sousa, 1983). In castrated individ- derived from the current study are being used to construct a 324 THE JOURNAL OF PARASITOLOGY, VOL. 94, NO. 2, APRIL 2008 model of the H. anceps population in Charlie’s Pond. A matrix ———, AND ———. 1991b. Guild structure of larval trematodes in the model will quantify the cost of castration at the population level snail Helisoma anceps: Patterns and processes at the individual host level. Journal of Parasitology 77: 528–539. by determining the population growth rate across a range of ———, AND ———. 1991c. The component community structure of infection probabilities. Furthermore, the effect of adjustments larval trematodes in the pulmonate snail Helisoma anceps. 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