Effects of Waterborne Zinc on Reproduction

International Journal for Parasitology 37 (2007) 375–381 www.elsevier.com/locate/ijpara

Eﬀects of waterborne zinc on reproduction, survival and morphometrics of Gyrodactylus turnbulli (Monogenea) on guppies (Poecilia reticulata)

Cristina Gheorghiu a,*, Joanne Cable b, David J. Marcogliese c, Marilyn E. Scott a

a Institute of Parasitology, Macdonald Campus of McGill University, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Que., Canada H9X 3V9 b School of Biosciences, Cardiﬀ University, Cardiﬀ CF10 3TL, UK c Fluvial Ecosystem Research – St. Lawrence Centre Research Group, Aquatic Ecosystem Protection Research Division, Water Science and Technology Directorate, Science and Technology Branch, Environment Canada, 105 McGill, 7th Floor, Montreal, Que., Canada H2Y 2E7

Received 17 July 2006; received in revised form 6 September 2006; accepted 7 September 2006

Abstract

Recent reviews indicate that pollutants in the surrounding macroenvironment directly influence the population dynamics, distribution and dispersal of fish ectoparasites, often leading to increased parasitism. The aim of the current study was to explore the effects of sublethal concentrations of waterborne zinc (up to 240 lg Zn/L) on survival, reproduction and morphometrics of Gyrodactylus turnbulli,a viviparous monogenean infecting the skin and fins of the guppy, Poecilia reticulata. Parasite survival and reproduction on the fish were recorded daily for individual parasites maintained in isolated containers. Both survival and reproduction were reduced in 30 and 120 lg Zn/L, compared with 0, 15, and 60 lg Zn/L indicating direct toxic effects of Zn on the parasite. However, as generation time was unaf- fected by Zn, we attribute the reduced reproduction to the shorter lifespan. Parasite survival off the fish was monitored hourly. Average lifespan of the detached parasites decreased linearly from 19.5 h in 0 lg Zn/L to 17.3 h in 240 lg Zn/L, further supporting the direct toxic effect of Zn to the parasite. In addition, temporal dynamics of parasite morphometrics were monitored from mini-epidemics sampled after 1, 5, 10, and 15 days exposure to various Zn concentrations. All morphological parameters decreased significantly in response both to concentration and duration of exposure to waterborne Zn. Together these data clearly indicate that concentrations as low as 120 lg Zn/L are directly toxic to G. turnbulli. 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Heavy metal toxicity; Waterborne zinc; Guppy; Gyrodactylids; Reproduction; Lifespan; Survival; Morphometrics

1. Introduction ter is probably an asexual clone of her mother, whereas subsequent daughters may be either sexually or partheno- Gyrodactylus species are monogenean ectoparasites liv- genetically-derived (Cable and Harris, 2002). Newborn ing on the skin, fins and gills of many families of marine parasites attach to the fish, adjacent to their mother (Scott, and freshwater teleost fish (Harris et al., 2004). There are 1982). Although dispersal occurs mainly during direct con- over 400 species of Gyrodactylus species ranging between tact between fish, detached gyrodactylids can survive off 0.2–0.8 mm in length, all with similar morphology (Bakke the fish for several hours and are able to re-attach to new et al., in press). The young newborn contains within its hosts (Bakke et al., 1992; Cable et al., 2002a). Gyrodacty- uterus several generations of embryos in sequential stages lids ingest both epidermal cells and mucus (Buchmann and of development (Cable and Harris, 2002). The first daugh- Lindenstrom, 2002), but monogeneans may supplement their diet with organic nutrients of low molecular weight * Corresponding author. Tel.: +1 514 398 8382; fax: +1 514 398 7857. that are absorbed directly from the water across the tegu- E-mail address: [email protected] (C. Gheorghiu). ment (Smyth and Halton, 1983). Embryos within the uterus

0020-7519/$30.00 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.09.004 376 C. Gheorghiu et al. / International Journal for Parasitology 37 (2007) 375–381 probably obtain nutrients across the uterine wall which is The strain was maintained in the laboratory by weekly situated close to the parental gastrodermis (Cable et al., addition of naı¨ve fish into tanks with the infected guppies. 2002b). A Nikon dissecting microscope equipped with a fibre optic As in other aquatic ectoparasites, environmental pollu- light source was used for experimental infection and subse- tants influence Gyrodactylus survival, reproduction and quent monitoring of fish anaesthetized for a maximum of population dynamics in a concentration-dependent manner 5 min in 50 ml of 0.02% tricaine methanesulfonate (Finquel that is specific to the pollutant. For example, the preva- MS222, Argent Chemical Laboratories, Washington) buf- lence and intensity of gyrodactylids increased in response fered to a neutral pH with NaHCO2. All procedures were to polyaromatic hydrocarbons (Khan and Kiceniuk, approved by a McGill University Animal Care Committee, 1988). In contrast, aqueous aluminium (Al; 50–200 lg/L) in accordance with the Canadian Council on Animal Care and zinc (Zn; 50–400 lg/L) impaired Gyrodactylus salaris Guidelines (2005). population growth (Soleng et al., 1999; Poleó et al., Experimental Zn solutions were prepared as described 2004), whereas neither copper (10–80 lg/L), iron (25– by Gheorghiu et al. (2006). In brief, Zn was added to arti- 200 lg/L), nor manganese (100–800 lg/L) affected these ficial freshwater to give concentrations of 0, 15, 30 (the parasites (Poleó et al., 2004). Recently, we reported a con- maximal admissible limit for aquatic life according to centration-dependent increase in mortality of guppies in Canadian Council of Ministers of the Environment, response to the combined effects of aqueous zinc and infec- 2005), 60, 120, or 240 lg/L above the baseline concentration by Gyrodactylus turnbulli (Gheorghiu et al., 2006). We tion of 8 lg/L. Solutions were changed every 2 days to further reported that among those fish that survived these ensure that Zn concentration in the water remained combined stresses, peak parasite burden increased with relatively constant. increasing Zn concentration up to 120 lg/L, then declined. However, it was unclear whether parasites directly benefit- 2.2. Experiment 1: Lifetime survival and reproduction of ed from increased Zn concentration or whether elevated Zn individual parasites impaired the host response, thus indirectly promoting parasite survival. In order to evaluate the concentration-dependent effects The present study was designed to determine whether of Zn on parasite survival and reproduction, we followed waterborne Zn has direct effects on the survival, reproduc- individual F2 parasites from embryonic exposure to zinc, tion or morphometrics of G. turnbulli, a parasite on the through their lifespan to death. Isolated guppies were kept skin and fins of the guppy, Poecilia reticulata (Peters). This in a transparent, covered plastic container in 200 ml of 0, experimental model is useful because guppies are easy to 15, 30, 60, or 120 lg Zn/L (assigned at random). Each fish maintain and breed under laboratory conditions and the was initially infected with a single gravid specimen of G. parasite has a short generation time (days) and can be turnbulli and then monitored every morning. If visual monitored without destructive sampling of the host. In inspection suggested that the parasite was likely to give the first experiment, we monitored lifetime survival and birth within a few hours, the fish was re-examined later reproduction of individual parasites on isolated guppies the same day. Immediately after the initial parasite had giv- (Experiment 1) where host response against the parasite en birth (F1 generation), the mother was removed and was minimized by using fish with only one or two individ- killed with fine forceps. Similarly, immediately after her ual parasites. In Experiment 2, we examined the impact of daughter had given birth (F2 generation), the F1 daughter waterborne Zn on the survival and reproduction of para- was killed. We defined day 0 as the day of birth of the F2 sites that were detached from their fish host. Finally, in daughter and this worm was monitored until its death. Experiment 3, we followed the temporal dynamics of para- Each time the F2 daughter gave birth, its daughter was site morphometrics in small mini-epidemic tanks where killed. We recorded the total number of daughters, the parasite transmission among the fish ensured the persis- age of the F2 daughter at each birth and the lifespan of tence of the parasite (Scott and Anderson, 1984). the F2 daughter (n = 13, 14, 19, 13, and 16, respectively, for each concentration of Zn). Generation time was esti- 2. Materials and methods mated as the age of the F2 daughter at the time of birth of her average daughter. 2.1. General methods 2.3. Experiment 2: Survival and reproduction of detached Experimental fish (standard body length 0.8–1.5 cm) parasites were laboratory bred from a strain of feeder guppies obtained from a pet store in Montreal’s West Island and As detached parasites are able to re-attach to fish, it was were naı¨ve to gyrodactylids. All fish were kept at 25 C, important to consider the influence of Zn concentration on in a 16 h light–8 h dark cycle, and were fed a Nutrafin parasites isolated from their host. This also enabled us to Max Complete Flake diet once a day. G. turnbulli obtained assess direct effects of Zn on the parasite, independent of from infected guppies purchased from the same local pet host effects. For this experiment, individual parasites were supplier were identified according to Harris et al. (1999). removed from recently killed fish, being careful to avoid C. Gheorghiu et al. / International Journal for Parasitology 37 (2007) 375–381 377 contact between the parasite and any host body fluids. The morphometrics, infected fish were kept individually in parasites were individually transferred in 20 ll of artificial 200 ml of 0, 30, 120, or 240 lg Zn/L for 24 h before freshwater to the bottom of a 96-well microtitre plate worms were measured as described above (n =35 (Costar Clear Polystyrene 96-Well Plates, Fisher Scientific, parasites/concentration). Montreal, Canada) containing 80 ll of artificial freshwater. To each well, 100 ll of Zn solution was added to give a 2.5. Statistical analysis final concentration of 0, 15, 30, 60, 120, or 240 lg Zn/L above baseline Zn concentration. Each parasite was exam- The effect of Zn concentration on individual lifespan, ined 1 h after transfer and any that showed signs of damage number of births, the intervals between births, generation or were dead were discarded and excluded from the data time and parasite morphology was assessed using v2, Krus- set. Parasites were then examined every 2 h, and the timing kal–Wallis non-parametric ANOVA, one- and two-way and number of births as well as the time of death of both ANOVAs with Tukey’s post hoc test, depending on the the initial parasite and any daughters were recorded. parameter. Average lifespans were estimated by Probit Non-motile parasites that did not respond to a gentle water analysis. Linear regression analysis was used to examine current created by moving a needle through the water were the relation between lifespan and number of offspring, considered dead; if they responded to the water current and between zinc concentration and lifespan. The mean they were classified as moribund. The lifespan of each par- and SEM as well as the binomial 95% confidence limits asite was recorded as well the percent of lifespan in a mor- for percentages are reported (Rohlf and Sokal, 1981). ibund state. This experiment was replicated five times to Analyses were performed using SAS Version 9.1 software. give a total of 48–56 parasites/concentration. The level of significance was established at P < 0.05; statistics are reported only for significant effects. 2.4. Experiment 3: Temporal dynamics of morphometrics in parasite populations 3. Results

Given the critical role of Zn in cell division, growth and 3.1. Experiment 1: Lifetime survival and reproduction of development (Eisler, 1993), we examined the potential individual parasites effect of Zn on the size of parasites exposed for 5, 10, and 15 days to various concentrations of waterborne Zn. On average, the F2 daughters maintained in 0 lg Zn/L This experiment focused on soft body parts because we survived for 7.9 days and had 3.2 daughters, with a gener- anticipated more rapid effects of Zn on these tissues rather ation time of 3.6 days (Table 1). Poorer survival and repro- than hooks and hamuli. Moreover our primary interest duction occurred in parasites kept in 30 lg Zn/L; they concerned parasite reproduction and survival rather than survived only for 5.8 days and had 2.5 daughters, with an attachment and movement. A set of mini-epidemics was average generation time of 2.7 days. Between these two established by placing one infected guppy together with extremes of life history traits, parasites kept in 15 or three uninfected guppies in 1 L plastic tanks containing 120 lg Zn/L had intermediate values of average lifespan, one of six waterborne Zn solutions (0, 15, 30, 60, 120, number of births and generation time. To explore this fur- or 240 lg/L). When an infected fish died, it was replaced ther, we classified parasite lifespan as short (1–4 days), with an uninfected one to ensure maintenance of the par- medium (5–8 days) or long (9–12 days). Comparison across asite population for 15 days. On days 5, 10, and 15 p.i. at Zn concentrations confirmed that parasites survived longer least one infected fish in each Zn concentration was killed in 0 lg Zn/L than in 30 or 120 lg Zn/L (Table 1), a differ- in 50 ml of 0.03% MS222. Between 20 and 33 parasites/ ence driven by the very low percentage of parasites that concentration were removed from the fish and transferred survived for more than 9 days at the higher concentrations 2 with 2 ml of MS222 into a well of a 6-well tissue culture (v8 = 16.72, P < 0.05). Surprisingly, survival and reproduc- plate. Aqueous neutral red vital stain (4 ml of 0.005% tion of F2 parasites kept in 60 lg Zn/L were similar to solution) was added to each well and the plate was kept parasites kept in 0 lg Zn/L (Table 1). at 4C for 4–5 h to immobilize the parasites and improve As with lifespan, the total number of births was lower at the reliability of measurements. Each parasite was then 30 and 120 lg Zn/L than at 0 lg Zn/L. At 30 and 120 lg placed on a microscope slide under cover slip pressure. Zn/L, low fecundity (0–2 daughters) was more common Total body length, body width at the widest part of the than higher fecundity (3–5 daughters) whereas the two cat- uterus, pharynx diameter and opisthaptor length and egories were equally common in parasites maintained at width were measured using a Nikon compound micro- 0 lg Zn/L (Table 1). The number of offspring increased scope at 125· magnification. Body area was estimated as linearly with lifespan (P < 0.0001), such that longer-lived the product of body length and body width. For each parasites had more offspring and this was observed at each Zn concentration and for each time point, measurements Zn concentration. Although generation time appeared to were made on parasites from three replicate mini-epidem- be lower for parasites kept at 30 lg Zn/L, neither the aver- ics. Moreover, to determine whether the addition of Zn in age generation time, nor the average age of the F2 daughter artificial freshwater induced an osmotic effect on parasite at birth of her first daughter nor the average inter-birth 378 C. Gheorghiu et al. / International Journal for Parasitology 37 (2007) 375–381

Table 1 Lifetime survival and reproduction of individual Gyrodactylus turnbulli on fish in response to waterborne zinc (Zn) lg Zn/L 0 15 30 60 120 Average lifespan (days) ± SEM 7.9 ± 0.9 6.9 ± 0.8 5.8 ± 0.6 7.6 ± 0.8 6.9 ± 0.6 % with lifespan exceeding 8 days 62 43 10a 46 12a Average number of births ± SEM 3.2 ± 0.4 3.1 ± 0.4 2.5 ± 0.3 3.2 ± 0.4 2.9 ± 0.3 % with three or more births 62 50 21a 54 19a Average generation time (days) ± SEM 3.6 ± 0.4 3.2 ± 0.4 2.7 ± 0.3 3.2 ± 0.4 3.0 ± 0.3 Average age at birth of first daughter (days) ± SEM 1.1 ± 0.1 1.1 ± 0.1 1.1 ± 0.04 1.1 ± 0.1 1.0 ± 0.04 Average inter-birth interval (days) ± SEM 3.4 ± 0.2 3.3 ± 0.2 3.8 ± 0.2 3.2 ± 0.3 3.8 ± 0.2 a Significantly different from 0 lg Zn/L (P < 0.025). interval for subsequent births differed significantly among 3.3. Experiment 3: Temporal dynamics of morphometrics in Zn concentrations (Table 1). parasite populations Together, these data demonstrate that parasite survival responded in a non-linear manner to increasing concentra- For parasites kept at 0 lg Zn/L, none of the morpho- tions of Zn, with lowest values at 30 and 120 lg Zn/L and logical measurements differed over time, whereas all highest values at 0 lg Zn/L. Although parasite reproduc- morphological parameters were significantly affected by tion showed a similar pattern, the shorter lifespan of these waterborne Zn, by duration of Zn exposure and by the parasites may explain the reduced reproduction. interaction between the two factors (Table 2). In order to confirm that the effect of Zn was not due to acute osmotic 3.2. Experiment 2: Survival and reproduction of detached stress on the parasite, we compared the size of parasites parasites after 24 h exposure to 0, 30, 120, and 240 lg Zn/L (Fig. 2) and found no significant effect of Zn concentration Parasites that had been removed from the host had an on any of the morphometric parameters. After 5 days expo- estimated average lifespan (LT50)of19.4hin0lg Zn/L. sure, the body area was markedly lower at all concentra- Lifespan decreased linearly (F1,4 = 20.88, P = 0.0103) with tions compared with 0 lg Zn/L and effects were largely increasing concentrations of Zn to 17.3 h for parasites kept dose-dependent (Fig. 2). With prolonged exposure to Zn, in 240 lg Zn/L (Fig. 1). Parasites not exposed to Zn were the effect of Zn was more pronounced, with a concentra- moribund for only 14.7% of their life off the fish whereas tion-dependent decrease detected both at 10 and 15 days among parasites exposed to Zn, the percent of lifespan in exposure. Similar patterns were seen for all morphometric a moribund state was similar among all concentrations indicators (Table 2). (19.7%) and significantly higher for 120 lg Zn/L than for 0 lg Zn/L (F5,305 = 2.51, P = 0.0301). Approximately 4. Discussion 12% of detached parasites gave birth or aborted their embryos, but given the low sample size, we were unable For gyrodactylids, as for all living organisms, Zn is pre- to draw conclusions about the impact of Zn on reproduc- sumably an essential structural, catalytic and regulatory tion of detached parasites. micronutrient for many enzymes and critical for protein synthesis, cell proliferation, growth, development and reproduction (Vallee and Falchuk, 1993; Hogstrand and Wood, 1996). On the other hand, high concentrations of Zn are likely to be toxic, as Zn is known to impair ion regulation (McGeer et al., 2000) and cellular energy production (Dineley et al., 2003), cause apoptosis and be mutagenic and teratogenic (Eisler, 1993). The potential interactions between gyrodactylids and Zn are further com- plicated by the effects of Zn on the host. Fish regulate uptake of Zn across epithelial tissues by release of mucus that traps Zn, preventing its entry into tissues (Handy et al., 1989; Shephard, 1994). Fish also release mucus as a defence mechanism against pathogens, including gyrodactylids (Lester and Adams, 1974; Buchmann and Lindenstrom, 2002). Thus, the mucus release induced by Zn may indirectly affect the parasite. Fig. 1. Average lifespan of detached Gyrodactylus turnbulli exposed to different waterborne zinc (Zn) concentrations. Solid line indicates best fit We previously reported that parasite population linear regression model (y = À0.0091x + 19.375). growth improved when G. turnbulli was maintained at Zn C. Gheorghiu et al. / International Journal for Parasitology 37 (2007) 375–381 379 ±4 ± 0.8 ± 0.5 ± 0.4 ± 0.6 ± 0.8 E C D D D D ± 4± 0.9± 0.5 312 ± 62.3 0.4± 19.8 0.6± 29.6 0.8 60.1 79.0 B C C C D CD ± 5± 0.8± 0.5± 63.5 0.4 340 ± 0.7 21.8 ± 0.7 31.8 61.9 81.5 B C D centrations. In all cases, the main effects of BC BC CD

Fig. 2. Temporal dynamics of Gyrodactylus turnbulli morphometrics in response to diﬀerent waterborne zinc (Zn) concentrations. ¤,0lg Zn/ L; n,15lg Zn/L; D,30lg Zn/L; h,60lg Zn/L; m, 120 lg Zn/L; e, 240

± 4± 0.8± 0.5± 348 0.4 62.6 ± 0.6 22.2 ± 33.1 0.7 61.9 79.8 lg Zn/L. C C C BC BC CD

concentrations as high as 120 lg Zn/L and had speculated that parasite survival and/or reproduction might be directly enhanced as Zn concentration increased (Gheorghiu ± 5± 0.8± 0.5± 65.5 0.4 338 ± 0.8 22.5 ± 32.4 0.9 61.4 81.6

B B B B B et al., 2006). If true, our present study should have revealed BC prolonged parasite lifespan both on and off the host and/or a increased rates of reproduction as Zn concentration increased up to 120 lg/L. However, this was not the case. In fact, both parasite survival and total number of off- ± 4± 0.6± 0.4± 0.3 354 67.2 ± 0.6 24.3 ± 0.6 33.4 61.8 85.6 spring were significantly lower at 120 lg Zn/L than at A A A A A A 0 lg Zn/L. In addition, survival of detached parasites declined with increasing Zn concentrations, as did parasite size. Thus, it is clear that 120 lg/L Zn is toxic to this parasite. This is consistent with the reduced population growth ± 3± 0.6± 0.4 378 ± 72.2 0.3± 27.6 0.5± 35.5 0.5 67.7 88.9 c c c c b b of G. salaris on Atlantic salmon exposed to 50 to 200 lg Al/L or 50 to 400 lg Zn/L (Poleó et al., 2004). Our most direct measure of the specific effect of Zn on the parasite was the reduced survival of detached parasites that were exposed to Zn but not to host responses. This ± 3± 0.6± 0.4 332 ± 65.8 0.3± 22.2 0.5± 31.5 0.6 61.4 80.6 c c c c b < 0.0001). bc observation is consistent with reduced lifespan of digenean P populations in response to waterborne zinc (Zn) miracidia and cercariae exposed to Zn, in concentrations in the order of 100 lg/L (Asch and Dresden, 1977; Evans, 1982a,b: Morley et al., 2001a,b, 2002). Although the

± 4± 0.6± 0.4± 0.3 341 63.8 ± 0.6 22.2 ± 0.6 31.7 62.2 82.4 decrease in average lifespan between 0 and 240 lg Zn/L a b b b b b was only about 2 h, we suspect that the ability to re-attach to a ﬁsh may have been more dramatically aﬀected than

Gyrodactylus turnbulli survival as suggested by Olstad et al. (2006). Toxicity of Zn to detached parasites may be associated not only with

± 5± 0.9± 0.5 359 ± 66.1 0.4± 24.1 0.8± 35.1 0.9 63.2 84.4 increased apoptosis and impaired ion regulation, but also a a a a a a with energy balance. Detached gyrodactylids continue to Time eﬀects (pooled across Zn concentrations)Day 1 Day 5 Zn eﬀects (pooled across Day time) 10 Day 15 Zn 0givebirth Zn 15 (personal Zn 30 observations) Zn 60 and Zn 120 need Zn 240 to maintain energy stores not only for reproduction but also for re-at- ) 29.1 m) 68.2 2 m) 90.9

m) 35.1 tachment to ﬁsh that may swim by. Furthermore, gyro- l l l dactylids, like digenean larvae, may sequester excessive m) 387 m) 75.0

l metals in secretory structures associated with the tegumen- l tal surface as a means to detoxify Zn (Morley et al., 2003) and this is likely an energy-demanding process. However,

Means ± SEM; lower case superscripts indicate significant differences over time; upper case superscripts indicate significant differences among Zn con detached gyrodactylids no longer feed and even if they a Time, Zinc and the Time*Zn interaction were highly significant ( Table 2 Temporal dynamics of morphometrics of Body length ( Body width ( Total body area (mm Pharynx diameter ( Opisthaptor length ( Opisthaptor width ( re-absorb nutrients from their in utero embryos (Cable 380 C. Gheorghiu et al. / International Journal for Parasitology 37 (2007) 375–381 and Tinsley, 1991; Cable and Harris, 2002), energy stores When we examined the impact of Zn on parasite size, we will decline more rapidly at higher Zn concentrations. In found that the longer the period of exposure to Zn and the addition, Zn impairs cellular energy production in other higher the concentration, the smaller the parasites were. organisms (Dineley et al., 2005). Together, the reduced This was observed in the mini-epidemics maintained at energy stores and reduced energy production may contrib- concentrations as low as 15 lg Zn/L for at least 5 days. ute to the toxic effect on parasite survival. More prolonged exposure to Zn resulted in smaller para- Not only was Zn toxic to detached parasites, but it was sites that, in turn, gave birth to even smaller offspring indi- also toxic to parasites living on the fish. Parasite survival cating a cumulative toxic effect. In interpreting data from was reduced at Zn concentrations as low as 30 lg Zn/L this experiment, it is important to note the mixed age struc- but interestingly parasites were more tolerant to 60 lg ture of the parasite populations in the mini-epidemics. Zn/L than to 30 lg Zn/L. At 120 lg Zn/L, parasite survival Although we cannot exclude the possibility that the was impaired to a similar extent as in parasites maintained observed effects of Zn on morphometrics reflect a changing at 30 lg Zn/L. While the reduced lifespan at 30 and 120 lg age-structure of the parasite population, Gyrodactylus spp. Zn/L could be explained by direct toxicity to the parasite, are not generally considered to ‘‘grow’’ in a traditional the better survival at 60 lg Zn/L was unexpected according sense; rather, they are reported as full-size at birth but this to classic models of dose-dependence of toxicants (Calabre- has never been tested experimentally. Therefore, the Zn se and Baldwin, 2003). Rather, it is suggestive of a non-lin- dependent effect on parasite size presumably reflects ear hormetic response (Calabrese and Baldwin, 2003) embryonic development of the parasites; hence age-struc- where the net effect of interacting factors results in a more ture of the parasite population should not affect the aver- complex pattern. In this system, the non-linear dose-re- age size of the parasite. sponse may result from the efforts of the fish to exclude Taken together, the present results do not explain the entry of Zn into its tissues, combined with the direct toxic parasite population dynamics observed in our first study effects of Zn on the parasite. When exposed to waterborne (Gheorghiu et al., 2006). We had reported improved toxicants, including Zn, fish immediately release mucus parasite population growth at Zn concentrations up to over epithelial surfaces to trap Zn and prevent its entry into 120 lg/L (Gheorghiu et al., 2006) whereas our present the body (Handy et al., 1989; Shephard, 1994; Hogstrand results indicate reduced parasite survival and reduced and Wood, 1996; Khunyakari et al., 2001), but are unable reproduction over this range of Zn concentrations. There- to sustain mucus release over prolonged periods of time fore, we reject the suggestion that waterborne Zn (Eddy and Fraser, 1982), especially at higher concentra- improves parasite growth and reproduction, and hypothe- tions. Thus, as Zn concentrations increases from 0 to 15 size that the host response against the parasite may be to 30 lg Zn/L, the release of higher amounts of mucus pro- impaired as Zn concentration increases, especially after 1 tects the host, but also removes parasites trapped in week pre-exposure, thus providing a microenvironment sloughed off sheets of mucus (Lester and Adams, 1974). permissive for parasite population growth. This hypothe- When Zn concentrations exceed 30 lg Zn/L, the initial sis is currently under investigation. In conclusion, our high rate of mucus production may not be sustainable data show that elevated concentrations of Zn are toxic (Eddy and Fraser, 1982), in which case the probability of to G. turnbulli. parasites being trapped in mucus is reduced. This would account for the increased survival between 30 and 60 lg Acknowledgements Zn/L. Concurrently, however, Zn exerts direct toxic effects on the parasite, which would be expected to increase as Zn Funding for this research was provided by the Natural concentration increases, thus explaining the reduced life- Sciences and Engineering Research Council, Canada span of the parasite between 60 and 120 lg Zn/L. (NSERC 3585) and a Natural Environment Research The concentration response of parasite reproduction to Council, UK. Advanced Fellowship to JC (NER/J/S/ Zn was very similar to that of parasite lifespan, thus we 2002/00706). The first author also acknowledges an attribute this result to the shortened lifespan of the parasite NSERCR Postgraduate Scholarship. Research at the rather than to any direct toxic effect of Zn on parasite Institute of Parasitology is supported by a Regroupement reproduction. It is not surprising that short-lived parasites Strate´gique from FQRNT, a provincial funding agency. have fewer offspring, but it is intriguing that Zn appears not to affect reproduction of gyrodactylids, given that the References reproductive system is very sensitive to Zn in other organisms (Eisler, 1993). Perhaps the unique reproductive Asch, H.L., Dresden, M.H., 1977. Schistosoma mansoni: effects of zinc on biology of gyrodactylids, involving a combination of asex- cercarial and schistosomule viability. J. Parasitol. 63, 80–86. ual, parthenogenetic and sexual production of multiple Bakke, T.A., Cable, J., Harris, P.D., in press. The biology of gyrodactylid generations within the same individuals and hypervivipari- monogeneans: the ‘‘Russian-Doll Killers’’. Adv. Parasitol. Bakke, T.A., Harris, P.D., Hansen, L.P., Jansen, P.A., 1992. Host ty (Cable and Harris, 2002), protects embryos from Zn specificity and dispersal strategy in gyrodactylid monogeneans with toxicity. 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