Claudia Gérard
email: claudia.gerard@univ-rennes1.fr

Maxime Hervé

Ryan F Hechinger

John E Havel

Sidinei M Thomaz

Lee B Kats

Katya E Kovalenko

Luciano N Santos

Long-term population fluctuations of the exotic New Zealand mudsnail Potamopyrgus antipodarum and its introduced aporocotylid trematode in northwestern France

Keywords:

published or not. The documents may come

Introduction

Long-term studies of species invasions are relatively rare. This is unfortunate, because although invasive species do undergo periods of rapid population growth and spread [when we deem them "invasive" [START_REF] Kolar | Progress in invasion biology: predicting invaders[END_REF]], they can then strongly fluctuate in abundance over time, and sometimes even spontaneously collapse [START_REF] Simberloff | Now you see them, now you don't! -Population crashes of established introduced species[END_REF][START_REF] Strayer | Understanding the long-term effects of species invasions[END_REF]Simberloff, 2013 for reviews). Hence, the preponderance of short-term studies of introduced species will not provide the full understanding of the impacts of invasive species. The demographic success of invasive species can be affected by many factors such as abiotic stresses due to environmental change (e.g., drought, pollution), impacts of parasitism (by parasites introduced after the host introduction and/or native parasites acquired after introduction), and other interactions [START_REF] Simberloff | Now you see them, now you don't! -Population crashes of established introduced species[END_REF][START_REF] Strayer | Understanding the long-term effects of species invasions[END_REF]Simberloff, 2013 for reviews).

The freshwater snail, Potamopyrgus antipodarum [START_REF] Gray | Fauna of New Zealand: materials towards a fauna of New Zealand, Auckland Island, and Chatham Islands[END_REF] (Caenogastropoda), is a globally invasive species, which is a member of the largely Australasian family Tateidae [previously a subfamily of the Hydrobiidae, but now recognized as being a separate family [START_REF] Zielske | Origin and radiation of rissooidean gastropods (Caenogastropoda) in ancient lakes of Sulawesi[END_REF][START_REF] Wilke | Pushing short DNA fragments to the limit: phylogenetic relationships of 'hydrobioid' gastropods (Caenogastropoda: Rissooidea)[END_REF]]. Native to New Zealand, P. antipodarum has invaded streams and lakes of most of the world's continents including Australia, Europe, America, and Asia [START_REF] Alonso | The exotic aquatic mud snail Potamopyrgus antipodarum (Hydrobiidae, Mollusca): state of the art of a worldwide invasion[END_REF][START_REF] Collado | Out of New Zealand: molecular identification of the highly invasive freshwater mollusk Potamopyrgus antipodarum (Gray, 1843) in South America[END_REF]. Its introduction on these different continents likely has multiple independent origins, but has involved only a small number of parthenogenetic clones (e.g., [START_REF] Hughes | Evolutionary ecology of parthenogenentic strains of the prosobranch snail, Potamopyrgus antipodarum (Gray) (=P. jenkinsi (Smith))[END_REF][START_REF] Jacobsen | Clonal variation in life-history traits and feeding rates in the gastropod, Potamopyrgus antipodarum: performance across a salinity gradient[END_REF][START_REF] Städler | Mitochondrial haplotypes and the New Zealand origin of clonal European Potamopyrgus, an invasive aquatic snail[END_REF][START_REF] Hoy | Salinity adaptation of the invasive New Zealand mud snail (Potamopyrgus antipodarum) in the Columbia River estuary (Pacific Northwest, USA): physiological and molecular studies[END_REF]. In Europe, only two haplotypes have been reported to date: "t" and "z." The most widely distributed is haplotype t, which appears to be a generalist in fresh waters, whereas haplotype z is restricted to brackish waters [START_REF] Städler | Mitochondrial haplotypes and the New Zealand origin of clonal European Potamopyrgus, an invasive aquatic snail[END_REF]. As for many invasive species, the successful spread of P. antipodarum is at least partly explained by inadvertent human introductions, but also by the snail's mode of reproduction (i.e., ovoviviparity and parthenogenesis), wide physiological tolerances (e.g., euryhalinity), efficient dispersal, and enemy release (predators, parasites) [START_REF] Alonso | What explains the invading success of the aquatic mud snail Potamopyrgus antipodarum (Hydrobiidae, Mollusca)?[END_REF], 2012 for reviews). In its native range, P. antipodarum is first intermediate host for as many as 20 species of highly host-specific trematodes [START_REF] Hechinger | Faunal survey and identification key for the trematodes (Platyhelminthes: Digenea) infecting Potamopyrgus antipodarum (Gastropoda: Hydrobiidae) as first intermediate host[END_REF], which, as parasitic castrators, generally can substantially impact host populations (e.g., [START_REF] Lafferty | Effects of parasitic castration on growth, reproduction and population dynamics of the marine snail Cerithidea californica[END_REF][START_REF] Gérard | Importance du parasitisme dans la communauté de Gastéropodes de l'étang de Combourg (Bretagne, France)[END_REF][START_REF] Gérard | Structure and temporal variation of trematode and gastropod communities in a freshwater ecosystem[END_REF][START_REF] Negovetich | Life history cost of trematode infection in Helisoma anceps using mark-recapture in Charlie's pond[END_REF]. In contrast, in Europe, populations are rarely parasitized. Previous studies in France and Poland revealed that P. antipodarum was infected as first intermediate host by only a single trematode species and at very low levels (≤ 0.48% in France, 0.007% in Poland) [START_REF] Gérard | Invader and invaded -colonization by, and of, Potamopyrgus antipodarum Gray (Mollusca, Hydrobiidae)[END_REF][START_REF] Gérard | Establishment of a new host-parasite association between the introduced invasive species Potamopyrgus e[END_REF]Gérard et al., 2003;[START_REF] Zbikowski | Invaders of an invader -Trematodes in Potamopyrgus antipodarum in Poland[END_REF]. This trematode was reported for the first time in 1999 in western France (Gérard et al., 2003), where P. antipodarum was first recorded in 1912 [START_REF] Lucas | Progrès récents en Europe d'une espèce envahissante : Hydrobia jenkinsi[END_REF]. DNA sequence analyses indicate that the trematode from French P. antipodarum corresponds to Aporocotylid sp. I (sensu [START_REF] Hechinger | Faunal survey and identification key for the trematodes (Platyhelminthes: Digenea) infecting Potamopyrgus antipodarum (Gastropoda: Hydrobiidae) as first intermediate host[END_REF], a New Zealand blood fluke with a two-host life cycle that infects fishes as final hosts (Gérard et al., 2017). Both in New Zealand and in Europe, the average prevalence of Aporocotylid sp. I in P. antipodarum is very low (0.07%), suggesting that this trematode probably does not regulate native or introduced snail host populations (Gérard et al., 2017).

Here, we investigate the long-term population dynamics of P. antipodarum and its aporocotylid parasite in a French stream over a 14-year period, with intensive sampling during 2000-2004 [just after the first record in 1999 of the parasite in the same region (Gérard et al., 2003)] and 2009-2013 (10 years after). We had three aims. First, we sought to better understand temporal fluctuations of the introduced population of P. antipodarum in relation with abiotic and biotic conditions. Therefore, we examine the temporal fluctuations of two major abiotic parameters; water temperature, which is crucial for ectotherms such as molluscs and which can correlate with other physicochemical parameters, and depth, which reflects the availability of habitat/space resources. We also examine the long-term dynamics of the whole molluscan assemblage, which may include potential competitors with the invasive snail. Second, we wished to observe whether there was a delayed regulation or even a crash of the invasive mudsnail following its introduction. Third, we sought to examine details of parasitism by the introduced aporocotylid parasite in the P. antipodarum population. Gérard et al. (2017) presented limited information concerning the parasite's prevalence, using pooled samples from this same dataset. Here, we reported higher-resolution details of the prevalence of Aporocotylid sp. I in P. antipodarum throughout the entire 14-year time period, and we examined the occurrence of parasitic trematodes (including Aporocotylid sp. I) in the rest of the molluscan assemblage.

Materials and methods

Study site

We conducted monitoring in a fourth-order oligotrophic stream (Le Petit Hermitage, in Ille-et-Vilaine, eastern Brittany, France, 48°32′N, 1°34′W) draining a 10 ha-catchment area in a riparian wetland at an altitude of ca. 20 m above sea level (see [START_REF] Clément | Seasonal dynamics of denitrification along toposequences in three different riparian wetlands[END_REF][START_REF] Lefebvre | Stream regulation and nitrogen dynamics in sediment interstices: comparison of natural and straightened sectors of a third-order stream[END_REF][START_REF] Gruau | Controls on the distribution of rare earth elements in shallow groundwater[END_REF] for site details). Stream flow is characterized by a base-flow period from June to November (mean monthly discharge ≤ 40 L s ), and a high-flow period from December to May (> 40 L s ). Stream substratum is primarily schist, consisting mainly of fine gravel and coarse sand. We ).

Water had medium calcium levels (10-40 mg/l Ca ), known to provide the most favorable conditions for freshwater molluscs [START_REF] Dussart | The ecology of freshwater mollusks in North West England in relation to water chemistry[END_REF][START_REF] Dussart | The ecology of Potamopyrgus jenkinsi (Smith) in North West England with a note on Marstoniopsis scholtzi (Schmidt)[END_REF].

Sample collection and measurements

To ensure a standard search effort, the same person (CG) sampled molluscs at 62 different dates on a 14-year interval during two broad time periods. On each occasion, we measured water temperature (°C) and maximum water depth (cm). Then, we trawled a pond-net (nylon mesh: 1 mm, square aperture: 0.5 × 0.5 m) from the surface sediment through the water column for three minutes along a 20 m long × 2 m wide area, as in previous studies [START_REF] Blanc | Etude préliminaire de la malacofaune de deux hydrosystèmes interconnectés dans une zone humide[END_REF][START_REF] Gérard | Invader and invaded -colonization by, and of, Potamopyrgus antipodarum Gray (Mollusca, Hydrobiidae)[END_REF][START_REF] Gérard | Ecological impacts of toxic cyanobacteria on community structure and microcystin bioaccumulation of freshwater molluscs[END_REF]. We carefully examined the contents in tap water in the laboratory, and we counted and identified all the molluscs, following [START_REF] Glöer | Süsswassermollusken. Deutscher Jugendbund für Naturbeobachtung[END_REF], to the species level for gastropods and to the family level for bivalves. We measured the shell size of each gastropod to the nearest 0.1 mm, but we only measured bivalve size in March 2003, May 2004, and September 2004. We dissected each mollusc under a stereoscopic microscope to detect infection by larval trematodes (sporocysts or rediae, and cercariae) and to record sex for gonochoristic species (i.e., P. antipodarum). When present, parasites were observed alive under light glass coverslip pressure using bright-field and phase-contrast microscopy.

We dissected females of P. antipodarum with shell height ≥ 2.5 mm to count the number of brooded embryos in March, April, and May 2002 (1008 females), in May 2004 (69), and in March, April, and May 2013 (37). Fecundity was not investigated in females < 2.5 mm because the smallest size of gravid females from the stream was previously shown to be about 3.0 mm [START_REF] Gérard | Variation in the response of the invasive species Potamopyrgus antipodarum (Smith) to natural (cyanobacterial toxin) and anthropogenic (herbicide atrazine) stressors[END_REF].

We

AQ1

Statistical analysis

We report central tendencies as the mean ± 95% confidence interval (CI). We describe parasitism by prevalence (number of hosts infected with a particular parasite species/number of examined hosts) [START_REF] Bush | 22 sur 31 24/10/2017 09:35 Parasitology meets ecology on its own terms: Margolis et al. revisited[END_REF]. For prevalence, we calculated CI using the score method [START_REF] Newcombe | Two-sided confidence intervals for the single proportion: comparison of seven methods[END_REF].

We performed analyses using R software (R Core Team, 2014). We analyzed temporal variation of the P. antipodarum population using a linear model (LM) based on generalized least squares (GLS) to take into account temporal autocorrelation (autoregressive process of order 1) [R package nlme (Pinheiro et al., 2016 [START_REF] Lenth | Least-squares means: the R package lsmeans[END_REF]]. When there was a significant interaction between time and monitoring period, we tested the slope of the P. antipodarum-time relationship against the null value for each monitoring period. When there was a significant effect of monitoring period, we computed the LSMean abundance and 95% CI of P. antipodarum at the middle point of each monitoring period in order to control for possible trends over time within periods. The three CI were Bonferroni-adjusted and examined for overlap.

AQ2

We analyzed the abundance of Sphaeriidae (log -transformed) in the exact same way as for the abundance of P. antipodarum. We tested the general relationship between these two abundances (P. antipodarum and Sphaeriidae) independently of the time period using a Pearson correlation test.

Due to the high proportion of zeroes and the very low non-zero values in parasite prevalence and abundance data, it was not possible to fit a proper model on these data, and so we performed no statistical analysis.

We analyzed the fecundity of P. antipodarum using a likelihood-ratio test based on a generalized linear model (family: negative binomial, link: log ) where the independent variables were the female's size treated as an ordinal factor with five levels: 2.5-2.99, 3.0-3.49, 3.5-3.99, 4.0-4.49, and 4.5-4.99 mm), sampling year (2002, 2004, and 2013), month nested in year (March, April, and May), and the female size × year interaction. We tested the relationship between the mean monthly fecundity and the total number of P. antipodarum sampled the same months using a Pearson correlation test.

We compared mean monthly water temperature between monitoring periods (June 2000-June 2001, March 2002-September 2004, April 2009-March 2012) using an ANOVA based on an LM. We chose mean monthly temperature in order to take into account that temperature was not consistently monitored among the three periods. We applied the same procedure to water depth.

Differences were considered statistically significant at P ≤ 0.05.

Results

Temporal fluctuations of temperature and depth

Temperature, which varied from 0 to 17°C during the whole study, was different between the three monitoring periods according to the LM model, (F = 27.93, P < 0.0001) (Fig. 1a). Specifically, temperature was significantly lower during the last monitoring period (April 2009-March 2012, 6.4 ± 0.4°C) compared to both the first (June 2000-June 2001, 9.0 ± 0.4°C) and second periods (March 2002-September 2004, 11.7 ± 0.7°C), and temperature was also different between these two periods (Fig. 1a).

Temporal fluctuations of P. antipodarum and its life history characteristics

Potamopyrgus antipodarum abundance showed strong periodicity, with positive autocorrelation being strongest at time intervals of 1-2 months and about 1 year (10-13 months). The LM-GLS model revealed no significant effect of depth on P. antipodarum abundance (F = 2.83, P = 0.098), but a significant effect on abundance of time (F = 89.38, P < 0.0001), monitoring period (F = 18.99, P < 0.0001), and their interaction (F = 3.51, P = 0.037). The mean abundance of snails was significantly different between the three monitoring periods. Indeed, it was sixfold higher during the second period [March 2002-September 2004, back-transformed LSMean (95% CI): 1,992 (1021-3886) snails] compared to the first period [June 2000-June 2001, 319 (154-664) snails], and 87-fold higher compared to the third period [April 2009-March 2012, 23 (15-36) snails] (Fig. 2a). Moreover, we observed a significant temporal trend of the snail population only during the first monitoring period, with a tenfold increase in population size from June 2000 to June 2001 (t = 2.42, P = 0.019) (Fig. 2a). No male P. antipodarum was encountered. 2. Different letters (a, b, c) in bar plots indicate statistically significant differences between monitoring periods. When a statistically significant relationship between mollusc abundance and time was found, this is represented by the corresponding regression line According to the GLM, the number of embryos per female significantly increased with shell size (χ = 818.90, df = 4, P < 0.0001), and did so similarly in each year fecundity data were taken (2002, 2004, and 2013) (χ = 10.50, df = 7, P = 0.162) (Table 1). The GLM also revealed a significant increase of fecundity from March to April (χ = 48.24, df = 4, P < 0.0001) (Table 1). Moreover, the mean number of embryos per female was significantly different between 2002, 2004, and 2013 (χ = 10.36, df = 2, P = 0.006). Specifically, in 2002 and 2004, the mean embryo number per female, respectively, 15.25 ± 0.79 and 16.87 ± 2.86, was significantly lower than in 2013, i.e., 27.78 ± 7.59 (Table 1). Despite there being a hint that fecundity was highest when P. antipodarum density was lowest (Table 1), no correlation was found between the mean monthly fecundity and the P. antipodarum abundance (r = -0.21, t = -0.47, P = 0.657). Additionally, P. antipodarum populations consistently had adults (individuals > 3 mm), with new snail recruitment at several points throughout the year, and two or even three generations were observed in most months but without obvious period of recruitment [Supplementary Fig. 2). However, no significant relationship was found between the abundance of P. antipodarum and the abundance of Sphaeriidae (Pearson correlation: r = 0.14, t = 1.11, P = 0.272).

Molluscan assemblage and overall parasite prevalence

Potamopyrgus antipodarum was the only molluscan species found parasitized (Table 2) and, as reported by Gérard et al. (2017), it was only infected by the trematode species Aporocotylid sp. I, with an overall prevalence of 0.09% (CI 0.06-0.14%). Below, we report details on the temporal fluctuations of prevalence. model demonstrated a significant and negative effect of depth (F = 10.23, P = 0.002), and a significant effect of time (F = 10.49, P = 0.002), monitoring period (F = 8.36, P = 0.0007), and time x period interaction ((F = 3.82, P = 0.028). The abundance of Sphaeriidae was also higher during the two last monitoring periods [March 2002-September 2004, back-transformed LSMean (95% CI): 31 (13-74) bivalves; April 2009-March 2012, 32 (16-64) bivalves] compared to the first [June 2000-June 2001, two (0-6) bivalves] (Fig. 2b). Moreover, we observed a significant temporal trend only during the second monitoring period with a 27-fold increase of Sphaeriidae from March 2002 to September 2004 (t = 2.84, P = 0.006) (Fig. 2b).

Temporal fluctuations of Sphaeriidae

Temporal fluctuations of Aporocotylid sp. I

The monthly prevalence of Aporocotylid sp. I infecting P. antipodarum varied from 0.00% (CI 0.00-0.13%, n = 2871) in April 2003 to a maximum of 1.52% (CI 0.65-3.50%, n = 330) in September 2000 (Fig. 3). The abundance of infected P. antipodarum per monitoring period decreased over time, whatever the total abundance of P. antipodarum (Table 2). However, the overall prevalence of infection did not significantly vary between monitoring periods, given the generally overlapping 95% CI calculated on the prevalence data [June 2000-June 2001 prevalence = 0.32% (0.19-0.52%), March 2002-September 2004 prevalence = 0.04% (0.02-0.08%), April 2009-May 2012 prevalence = 0.07% (0.01-0.41%), March-May 2013 prevalence 0% (0.00-5.07%)].

Discussion

Molluscan assemblage in Le Petit Hermitage

We recorded seven species of gastropods (the caenogastropod P. antipodarum and six pulmonates) and two taxa of bivalves belonging to Sphaeriidae in Le Petit Hermitage during the whole study. However, the diversity was very low over time (1.2 ± 0.1 gastropod species per sampling date) as was the mean abundance of most species (≤ 0.2 ± 0.2 individuals of each pulmonate species per sampling date). The molluscan assemblage in the study site was characterized by a high dominance of the exotic mudsnail P. antipodarum, especially during the period of 2000-2004 (97%). By the period of 2009-2013, the relative abundance of P. antipodarum decreased to 44% of the molluscan assemblage, which was then dominated by Sphaeriidae (56%). These cosmopolitan bivalves are very common and abundant in Europe, occurring in all types of running and still waters [START_REF] Boycott | The habitats of freshwater mollusca in Britain[END_REF][START_REF] Dussart | Sphaerium corneum (L.) and Pisidium spp. Pfeiffer -the ecology of freshwater bivalve molluscs in relation with water chemistry[END_REF][START_REF] Holopainen | Bathymetric distribution and abundance of Pisidium (Bivalvia: Sphaeriidae) in Lake Esrom, Denmark from 1954 to 1988[END_REF]. Interestingly, while Sphaeriidae were rare (3%) in Le Petit Hermitage during the period of 2000-2004, they were abundant (~ 50% of the molluscan assemblage) at the same time in a nearby pond (about 50 m away), which was connected to the stream during the flood season and where P. antipodarum has never been recorded [START_REF] Blanc | Etude préliminaire de la malacofaune de deux hydrosystèmes interconnectés dans une zone humide[END_REF][START_REF] Gérard | Ecological impacts of toxic cyanobacteria on community structure and microcystin bioaccumulation of freshwater molluscs[END_REF]Gérard, pers. obs.). We rarely found pulmonates, all native species, in the stream during the study (0.07%) and, when encountered, e. 14 sur 31 24/10/2017 09:35 they were always juveniles. Most of these individuals probably originated from the above mentioned nearby pond, where most of the pulmonate species were commonly recorded and abundant [START_REF] Blanc | Etude préliminaire de la malacofaune de deux hydrosystèmes interconnectés dans une zone humide[END_REF][START_REF] Gérard | Ecological impacts of toxic cyanobacteria on community structure and microcystin bioaccumulation of freshwater molluscs[END_REF]Gérard, pers. obs.). Environmental conditions in Le Petit Hermitage appeared unfavorable for these pulmonates compared to the pond [START_REF] Blanc | Etude préliminaire de la malacofaune de deux hydrosystèmes interconnectés dans une zone humide[END_REF][START_REF] Gérard | Ecological impacts of toxic cyanobacteria on community structure and microcystin bioaccumulation of freshwater molluscs[END_REF], whereas they seemed to become favorable for Sphaeriidae based on their dominance during the period of 2009-2013.

Temporal fluctuations of abundance and fecundity of P. antipodarum

Change in the dynamics of the P. antipodarum population was evident during our 14-year field investigation. After an exponential growth, the introduced population strongly declined before the last monitoring period and the snail abundance was about 14-fold lower compared to its initial abundance. Given the long history of the P. antipodarum invasion in France [over 100 years, first recorded in 1912 [START_REF] Lucas | Progrès récents en Europe d'une espèce envahissante : Hydrobia jenkinsi[END_REF]], we cannot know whether the dynamics we documented in Le Petit Hermitage reflect periodic cycling of a long-established population [START_REF] Ricklefs | Ecologie[END_REF], or a post-invasion "boom and bust," similar to that described for P. antipodarum in a North American California stream, 7 years after its first report there [START_REF] Moore | Stream community and ecosystem responses to the boom and bust of an invading snail[END_REF]. According to relatively short-term (< 5 years) demographic studies, population dynamics of P. antipodarum in invaded ranges can be unstable, with unexplained rapid increases and sudden extinctions (e.g., [START_REF] Dorgelo | Density fluctuations in populations (1982-1986) and biological observations of Potamopyrgus jenkinsi in two trophically differing lakes[END_REF][START_REF] Mcmillan | The history of alien freshwater mollusca in North-West England[END_REF][START_REF] Dahl | Life-history and growth of the prosobranch snail Potamopyrgus jenkinsi in Lake Esrom, Denmark[END_REF][START_REF] Levri | The distribution of the invasive New Zealand mud snail (Potamopyrgus antipodarum) in Lake Ontario[END_REF][START_REF] Cross | Ecosystem ecology meets adaptive management: food web response to a controlled flood on the Colorado River, Glen Canyon[END_REF][START_REF] Dorgelo | Dynamics of natural populations of the detritivorous mudsnail Potamopyrgus antipodarum (Gray) (Hydrobiidae) in two interconnected Lakes differing in trophic state[END_REF]. These extinctions, for which the causes remain unknown, occur despite the generally large physiological tolerance characterizing the small number of clones that have invaded most of the world's continents, particularly the generalist clone (haplotype t) from European fresh waters (e.g., [START_REF] Hughes | Evolutionary ecology of parthenogenentic strains of the prosobranch snail, Potamopyrgus antipodarum (Gray) (=P. jenkinsi (Smith))[END_REF][START_REF] Jacobsen | Clonal variation in life-history traits and feeding rates in the gastropod, Potamopyrgus antipodarum: performance across a salinity gradient[END_REF][START_REF] Städler | Mitochondrial haplotypes and the New Zealand origin of clonal European Potamopyrgus, an invasive aquatic snail[END_REF][START_REF] Hoy | Salinity adaptation of the invasive New Zealand mud snail (Potamopyrgus antipodarum) in the Columbia River estuary (Pacific Northwest, USA): physiological and molecular studies[END_REF], which probably occurs in our study site.

In Le Petit Hermitage, the absence of male P. antipodarum indicates that the population was exclusively composed of parthenogenetic females. The lack of sexual reproduction is consistent with what is seen for introduced P. antipodarum from Europe, Australia, and America, where males are rare or completely absent (e.g., [START_REF] Wallace | Notes on the distribution of sex and shell characters in some australian populations of Potamopyrgus (Gastropoda : Hydrobiidae)[END_REF][START_REF] Wallace | On the distribution of the sexes of Potamopyrgus jenkinsi (Smith)[END_REF][START_REF] Hughes | Evolutionary ecology of parthenogenentic strains of the prosobranch snail, Potamopyrgus antipodarum (Gray) (=P. jenkinsi (Smith))[END_REF][START_REF] Schreiber | Life-history and population dynamics of the exotic snail Potamopyrgus antipodarum (Prosobranchia: Hydrobiidae) in lake Purrumbete, Victoria, Australia[END_REF]Gérard et al., 2003;[START_REF] Gaino | The invader mudsnail Potamopyrgus antipodarum in the Tiiber River basin (Central Italy)[END_REF][START_REF] Mckenzie | New Zealand mud snail (Potamopyrgus antipodarum) in Boulder Creeck, Colorado: environmental factors associated with fecundity of a parthenogenic invader[END_REF][START_REF] Collado | Out of New Zealand: molecular identification of the highly invasive freshwater mollusk Potamopyrgus antipodarum (Gray, 1843) in South America[END_REF]. increased from March to May in each year, and also increased with snail size, as previously demonstrated (e.g., [START_REF] Winterbourn | Population studies on the New Zealand freshwater gastropod, Potamopyrgus antipodarum (Gray)[END_REF][START_REF] Gérard | Variation in the response of the invasive species Potamopyrgus antipodarum (Smith) to natural (cyanobacterial toxin) and anthropogenic (herbicide atrazine) stressors[END_REF][START_REF] Gaino | The invader mudsnail Potamopyrgus antipodarum in the Tiiber River basin (Central Italy)[END_REF][START_REF] Mckenzie | New Zealand mud snail (Potamopyrgus antipodarum) in Boulder Creeck, Colorado: environmental factors associated with fecundity of a parthenogenic invader[END_REF]. Females were reproductively mature at shell height from 2.5 to 3.0 mm, with up to 74 embryos in the largest females. These results are also consistent with observations for other introduced populations, which mature at size between 2.5 and 3.5 mm, and have maximum brood size ranging from 60 to 89 embryos (e.g., [START_REF] Strzelec | Population ecology of Potamopyrgus antipodarum (Gray, 1843) in a recently colonized area: Upper Silesia (Southern Poland) (Gastropoda: Prosobranchia: Hydrobiidae)[END_REF][START_REF] Gaino | The invader mudsnail Potamopyrgus antipodarum in the Tiiber River basin (Central Italy)[END_REF][START_REF] Mckenzie | New Zealand mud snail (Potamopyrgus antipodarum) in Boulder Creeck, Colorado: environmental factors associated with fecundity of a parthenogenic invader[END_REF].

Based on the size-frequency distributions (Supplementary Fig. ), both adult and newly recruited, growing P. antipodarum were present throughout the year, but without obvious, constrained periods where most recruitment occurred. In general, parthenogenetic forms of P. antipodarum produce offspring throughout the year, with seasonal fluctuations in reproductive output depending on the clonal population considered and environmental conditions such as temperature, hardness (dissolved calcium), phosphorus level, food availability, and population density (e.g., [START_REF] Winterbourn | Population studies on the New Zealand freshwater gastropod, Potamopyrgus antipodarum (Gray)[END_REF][START_REF] Dussart | The ecology of Potamopyrgus jenkinsi (Smith) in North West England with a note on Marstoniopsis scholtzi (Schmidt)[END_REF][START_REF] Dahl | Life-history and growth of the prosobranch snail Potamopyrgus jenkinsi in Lake Esrom, Denmark[END_REF][START_REF] Schreiber | Life-history and population dynamics of the exotic snail Potamopyrgus antipodarum (Prosobranchia: Hydrobiidae) in lake Purrumbete, Victoria, Australia[END_REF][START_REF] Neiman | Embryo production in a parthenogenetic snail (Potamopyrgus antipodarum) is negatively affected by the presence of other parthenogens[END_REF][START_REF] Neiman | Complex consequences of increased density for reproductive output in an invasive freshwater snail[END_REF][START_REF] Tibbets | Phosphorusmediated changes in life history traits of the invasive New Zealand mudsnail (Potamopyrgus antipodarum)[END_REF][START_REF] Mckenzie | New Zealand mud snail (Potamopyrgus antipodarum) in Boulder Creeck, Colorado: environmental factors associated with fecundity of a parthenogenic invader[END_REF]. Females in Le Petit Hermitage had a higher fecundity in 2013 when population densities were low (from 12 to 48 snails sampled per month) than in 2002 and 2004 when densities were very high (from 1,623 to 2,993 snails per month), although no significant correlation was found between monthly embryo number per adult female and snail abundance. If the high fecundity in 2013 represents fecundity during the last monitoring period (2009-2012), it would indicate that higher mortality rates, not lower birth rates, are what drove population density down. However, it is possible that 2013 represented recently increased individual fecundity preceding the beginning of a new population increase. According to [START_REF] Neiman | Embryo production in a parthenogenetic snail (Potamopyrgus antipodarum) is negatively affected by the presence of other parthenogens[END_REF], a nearly twofold increase in the embryo number per female was observed in low versus high density populations maintained in the laboratory with similar food availability, suggesting that reproductive output may be influenced by intraspecific competition. In contrast, the reproductive output of P. antipodarum was much higher in high versus low density conditions in other experiments with no food limitation [START_REF] Neiman | Complex consequences of increased density for reproductive output in an invasive freshwater snail[END_REF], as also observed in the Italian Topino River [START_REF] Gaino | The invader mudsnail Potamopyrgus antipodarum in the Tiiber River basin (Central Italy)[END_REF]. Thus, further investigations are needed for a more comprehensive view of the complex connections between environmental quality, resource availability, density, and fecundity of parthenogenetic females, and how they may influence the e.

16 sur 31 24/10/2017 09:35 population dynamics of P. antipodarum.

What possible environmental changes may influence the dynamics of P. antipodarum?

Despite the difficulty of identifying the causes of P. antipodarum population decline in Le Petit Hermitage, we explore two potential explanations.

First, the snail abundance may have been negatively influenced by changing abiotic conditions, such as the substantial decrease in water temperature and depth. Indeed, mean water temperature decreased about 3°C between the first and the last monthly monitoring periods (2000-2001 and 2009-2012), and very low winter temperatures (0-5°C) were frequently observed during 2009-2012 (vs. 2000-2001). Such temperatures (0-5°C) have been associated with lower performance and population densities of P. antipodarum in a Danish estuary (Europe) and the Silver Creek watershed (Idaho, USA) [START_REF] Siegismund | Dispersal-mediated coexistence of mud snails (Hydrobiidae) in an estuary[END_REF]Moffitt & James, 2012a, b). In fact, temperature is generally considered to drive P. antipodarum dynamics even if temperature tolerance may vary between invasive clones [START_REF] Gangloff | The New Zealand mud snail in Western North America[END_REF][START_REF] Kerans | Potamopyrgus antipodarum: distribution, density, and effects on native macroinvertebrate assemblages in the Greater Yellowstone Ecosystem[END_REF]. Temperature is one of the most significant physicochemical variables positively related to P. antipodarum density, as demonstrated in 15 European sites from North West England and the French Bay of Mont St. Michel (including Le Petit Hermitage) [START_REF] Dussart | The ecology of Potamopyrgus jenkinsi (Smith) in North West England with a note on Marstoniopsis scholtzi (Schmidt)[END_REF][START_REF] Gérard | Invader and invaded -colonization by, and of, Potamopyrgus antipodarum Gray (Mollusca, Hydrobiidae)[END_REF] and in two California (USA) lakes [START_REF] Bennett | Ecology of the invasive New Zealand mud snail, Potamopyrgus antipodarum (Hydrobiidae), in a mediterranean-climate stream system[END_REF]. Hence, it is possible that the temperature decrease explains the substantial decrease in P. antipodarum population abundance observed over the 14-year time period.

In addition to the temperature decrease, the water depth decreased by almost half from 2000-2001 to 2009-2012, resulting in a reduced water volume and a more restricted habitat area. In general, the increase in predator pressure and interspecific competition related to the restricted habitat size during drought leads to the elimination of most freshwater gastropods, and new generations typically appear after the water level rises again (Dudgeon, 1982;[START_REF] Gérard | Structure and temporal variation of trematode and gastropod communities in a freshwater ecosystem[END_REF]. In small Mediterranean streams characterized by seasonal droughts and floods, P. antipodarum populations are generally incapable of achieving very high densities, and this has been attributed mainly to harsh hydrologic conditions [START_REF] Múrria | Effects of the invasive species Potamopyrgus antipodarum (Hydrobiidae, Mollusca) on community structure in a small Mediterranean stream[END_REF]. In conditions of much deeper depths (≥ 10 m), as in the North American Great Lakes, the density of P. antipodarum is influenced by water depth and highest densities occurred between 15 and 25 m, no individuals being detected at less than 13 m and at more than 40 m [START_REF] Levri | The distribution of the invasive New Zealand mud snail (Potamopyrgus antipodarum) in Lake Ontario[END_REF]. Hence, it is possible that in the shallow studied stream, the water depth (habitat area) decrease may have combined with lower temperatures to negatively influence P. antipodarum abundance, explaining its longer-term decline.

Secondly, it is possible that P. antipodarum may have been negatively affected by sphaeriid bivalves, or vice versa. Some studies (e.g., [START_REF] Kerans | Potamopyrgus antipodarum: distribution, density, and effects on native macroinvertebrate assemblages in the Greater Yellowstone Ecosystem[END_REF][START_REF] Hall | Extremely high secondary production of introduced snails in rivers[END_REF][START_REF] Moore | Stream community and ecosystem responses to the boom and bust of an invading snail[END_REF] have shown negative interactions between P. antipodarum density (and/or biomass) and those of native macroinvertebrates. In a North American California stream for instance, as P. antipodarum populations rose and fell, the abundance of native grazing herbivores crashed and then recovered [START_REF] Moore | Stream community and ecosystem responses to the boom and bust of an invading snail[END_REF]. In our study, sphaeriid bivalves were rarely recorded during 2000-2001, corresponding to the period of increasing abundance of P. antipodarum. Then, sphaeriid abundance rapidly increased about 27-fold from 2002 to 2004, and high abundance was maintained in 2009-2012 and until the end of the study in 2013, coinciding with the decline of the P. antipodarum population. Even if no direct relationship was found between the abundance of Sphaeriidae and P. antipodarum, the increase of Sphaeriidae may have amplified the decline of P. antipodarum or may have resulted from the decline. Indeed, competitive interactions both for space and nutrients probably occur between them, given their great ecological and physiological similarities. Occurring in a great variety of lotic and lentic waters, Sphaeriidae inhabit the bottom (mud, sand) or bury in the substratum, and often climb up plants [START_REF] Boycott | The habitats of freshwater mollusca in Britain[END_REF][START_REF] Dussart | Sphaerium corneum (L.) and Pisidium spp. Pfeiffer -the ecology of freshwater bivalve molluscs in relation with water chemistry[END_REF][START_REF] Holopainen | Bathymetric distribution and abundance of Pisidium (Bivalvia: Sphaeriidae) in Lake Esrom, Denmark from 1954 to 1988[END_REF][START_REF] Dillon | The Ecology of Freshwater Molluscs[END_REF][START_REF] Korniushin | Non-unionid freshwater bivalves (Sphaeriidae, Corbiculidae, Dreissenidae) of North American fauna[END_REF], as is also observed for P. antipodarum [START_REF] Alonso | What explains the invading success of the aquatic mud snail Potamopyrgus antipodarum (Hydrobiidae, Mollusca)?[END_REF][START_REF] Hechinger | Faunal survey and identification key for the trematodes (Platyhelminthes: Digenea) infecting Potamopyrgus antipodarum (Gastropoda: Hydrobiidae) as first intermediate host[END_REF] for reviews). To feed, Sphaeriidae filter phytoplankton, bacteria, and particulate organic matter from the water column (Vaughn & Hakenkamps, 2001;[START_REF] Dillon | The Ecology of Freshwater Molluscs[END_REF]Korniushin, 2007 for reviews). Interestingly and unlike most other bivalves, they also remove bacteria and organic detritus from the sediment by filtering interstitial water and pedal deposit feeding (i.e., a form of deposit feeding using cilia on the foot to collect buried organic matter) [START_REF] Vaughn | The functional role of burrowing bivalves in freshwater ecosystems[END_REF] for review). P. antipodarum is a generalist feeder, simultaneously a deposit-feeder, grazer and detritivore, which uses periphytic algae (e.g., green algae, diatoms), bacteria, and detritus, and thus, the same resources as many other macroinvertebrates found in the French stream, including Sphaeriidae [START_REF] Fenchel | Factors determining the distribution patterns of mud snails (Hydrobiidae)[END_REF][START_REF] Haynes | Food finding and food preference in Potamopyrgus jenkinsi[END_REF][START_REF] Schreiber | Life-history and population dynamics of the exotic snail Potamopyrgus antipodarum (Prosobranchia: Hydrobiidae) in lake Purrumbete, Victoria, Australia[END_REF][START_REF] Kerans | Potamopyrgus antipodarum: distribution, density, and effects on native macroinvertebrate assemblages in the Greater Yellowstone Ecosystem[END_REF]. Because food limitation is shown to be a e.

18 sur 31 24/10/2017 09:35 primary source of negative density-dependence for P. antipodarum [START_REF] Fenchel | Factors determining the distribution patterns of mud snails (Hydrobiidae)[END_REF][START_REF] Neiman | Complex consequences of increased density for reproductive output in an invasive freshwater snail[END_REF], potential decrease of resource availability (not only food but also space) possibly induced by the increased abundance of Sphaeriidae may have contributed to the decline of P. antipodarum in Le Petit Hermitage. Hence, further studies are warranted to carefully examine the extent to which competition occurs between P. antipodarum and sphaeriids. Such studies might use biomass as a currency, instead of abundance, which could be important given that the sizes of these taxa can substantially differ (e.g., P. antipodarum is usually ~ 2-5 mm length, whereas S. corneum can be ~ 10-12 mm) [START_REF] Glöer | Süsswassermollusken. Deutscher Jugendbund für Naturbeobachtung[END_REF].

We may explain the increased abundance of Sphaeriidae by them responding positively to changing environmental conditions, as is documented elsewhere. For instance, the great temporal fluctuations of Sphaeriidae (both Pisidium and Sphaerium) observed in a Danish Lake through a 34-year investigation were possibly due to eutrophication and food availability [START_REF] Holopainen | Bathymetric distribution and abundance of Pisidium (Bivalvia: Sphaeriidae) in Lake Esrom, Denmark from 1954 to 1988[END_REF]. In Le Petit Hermitage, the increase of Sphaeriidae may be related to the substantial decrease of water depth because sphaeriid density was negatively influenced by water depth. More precisely, the rapid increase of sphaeriid abundance from 2002 to 2004 (about 27-fold), followed by consistently high abundance from 2009 to the end of the study, coincided with the lower water levels in Le Petit Hermitage (29 cm in 2002-2004 and 25 cm in 2009-2012 vs 41 cm in 2000-2001). Our results are consistent with the gradual reduction of Pisidium spp. abundance in the North American Ottawa River as depth increased beyond 50 cm [START_REF] Kilgour | Factors affecting the distribution of sphaeriid bivalves in Britannia Bay of the Ottawa River[END_REF]. Moreover, Sphaeriidae can survive both temporary air exposure and freezing of the sediment they inhabit (Jurkiewicz-Karnkowska [START_REF] Jurkiewicz-Karnkowska | Long-term changes and spatial variability of mollusc communities in selected habitats within the e[END_REF][START_REF] Kubíková | The occurrence of Pisidium species (Bivalvia: Sphaeriidae) in oligotrophic springs of the Blanice River catchment (Czech Republic) in relation to ecological conditions[END_REF]), yet may perform poorly at increased temperatures [e.g., the declines of Pisidium amnicum in the French Saône and Iberian Minho Rivers during heat waves [START_REF] Mouthon | Population dynamics and life cycle of Pisidium amnicum (Müller) (Bivalvia: Sphaeriidae) and Valvata piscinalis (Müller) (Gastropoda: Prosobranchia) in the Saône river, a nine-year study[END_REF][START_REF] Sousa | Rapid decline of the greater European peaclam at the periphery of its distribution[END_REF]]. This all suggests that the substantially lower mean and minimum water temperatures and lower depths may have favored the population performance of Sphaeriidae in Le Petit Hermitage, and disfavored the population performance of P. antipodarum.

To conclude, both water temperature and depth decrease, and competitive interactions between P. antipodarum and Sphaeriidae may contribute to explain the longer-term decline of P. antipodarum in Le Petit Hermitage. Other environmental factors, not studied here, may have also contributed to this decline. For instance, microcystins (hepatotoxic cyanotoxins) known to induce the decline of molluscan assemblages in stagnant waters subject to recurrent cyanobacterial blooms (e.g., [START_REF] Gérard | Long-term dynamics and community structure of freshwater gastropods exposed to parasitism and other environmental stressors[END_REF] were previously detected in tissues of P. antipodarum from Le Petit Hermitage despite the absence of cyanobacteria [START_REF] Gérard | Ecological impacts of toxic cyanobacteria on community structure and microcystin bioaccumulation of freshwater molluscs[END_REF]. The large, long-term population drop of P. antipodarum in Le Petit Hermitage is similar in magnitude to the decrease documented by [START_REF] Moore | Stream community and ecosystem responses to the boom and bust of an invading snail[END_REF] for invasive P. antipodarum in a North American California stream. Although the drivers of that population crash are also not known, the collapse strongly influenced the impacts of the invasive snail on the surrounding ecosystem [START_REF] Moore | Stream community and ecosystem responses to the boom and bust of an invading snail[END_REF]. The extent to which such strong fluctuations in population size and impacts occur for P. antipodarum more broadly in Europe is unknown given the lack of other long-term data sets on the abundance of this invasive species.

Temporal fluctuations of Aporocotylid sp. I prevalence in P. antipodarum and number of infected snails

Despite the decreasing abundance of P. antipodarum in Le Petit Hermitage, the aporocotylid prevalence did not significantly vary between the monitoring periods and appeared to remain stable over the 14-year study with a mean prevalence of 0.09% and a maximal monthly prevalence of 1.52%. Both the occurrence of a single parasite species in the introduced mudsnail and the low prevalence of this parasite are in accordance with the Enemy Release Hypothesis, namely that the success of introduced species is at least partly driven by them having relatively low levels of overall parasitism [START_REF] Torchin | Introduced species and their missing parasites[END_REF]. The low prevalence of Aporocotylid sp. I in Le Petit Hermitage is also consistent with that observed in Poland [START_REF] Zbikowski | Invaders of an invader -Trematodes in Potamopyrgus antipodarum in Poland[END_REF]. Interestingly, this specific parasite (Aporocotylid sp. I) also has similarly low prevalence in populations of P. antipodarum in the native range (New Zealand) (Gérard et al., 2017). As Gérard et al. (2017) noted, this pattern is also consistent with the general pattern characterizing invasive parasites, where, for those few parasites that make it to an invaded range with their host, there is no general tendency for prevalence to change compared to that in the native range [START_REF] Torchin | Introduced species and their missing parasites[END_REF]. However, the persistence of this host-parasite association in Le Petit Hermitage may be threatened, at least in the long term, if there is a real decline of the host population. Indeed, as the snail host population strongly declined during the study period, so did the number of snails infected by Aporocotylid sp. I in Le Petit Hermitage (15 in 2000-2001, nine in 2002-2004, one in 2009-2012, and (Gérard et al., 2003)], we cannot infer that the parasite is no longer present. It is possible that the parasite is still present because the low abundance of the snail during that time period (2009)(2010)(2011)(2012)(2013) translated into lower sample sizes compared with the early part of the temporal study (2000)(2001)(2002)(2003)(2004). Hence, although we dissected 1,321 snails after June 2009 (from July 2009 to May 2013), the low overall prevalence of the parasite (0.09%) means that we had a 30% chance of missing it [(1 -0.0009) = 0.30]. In any case, the population size of Aporocotylid sp. I decreased (assuming the river site reflected the broader population) and it remains an open question how much of this drop was driven by the decline in P. antipodarum abundance, or by other factors, including those involving the parasite's unknown final host fish in New Zealand, as well as in Europe (Gérard et al., 2017).

Conclusion

In summary, we document a substantial population decline for the invasive P. antipodarum in a French stream, indicating that the ecological impacts of this invader have changed over time. This population decline was potentially driven directly or indirectly by documented environmental changes in water temperature and depth and competitive interactions with sphaeriid clams. Further studies are needed to (i) confirm what mechanisms underlie negative density-dependence in P. antipodarum, (ii) determine whether the population decline of P. antipodarum is more widespread, and (iii) understand how the population collapse has influenced the impacts of this invader on species in the surrounding communities, including the dynamics of its "co-invasive" aporocotylid parasite.

  Fig. 1 Temporal fluctuations of (a) water temperature and (b) maximal water depth in the French stream (Le Petit Hermitage) during the three monitoring periods included in statistical analyses, i.e., June 2000-June 2001, March 2002-September 2004, and April 2009-March 2012. Full circles are observed values, empty circles are predicted values

Fig. 2

 2 Fig. 2 Temporal fluctuations of (a) P. antipodarum abundance (log -values) and (b) Sphaeriidae abundance (log -values) Please, the legend of Y axis has to be corrected on Fig. 2b : it is the number of Sphaeriidae sampled (not P. antipodarum). in the French stream (Le Petit Hermitage) during the three monitoring periods included in statistical analyses, i.e., June 2000-June 2001, March 2002-September 2004, and April 2009-March 2012. Sample sizes are indicated in Table2. Different letters (a, b, c) in bar plots indicate statistically significant differences between monitoring periods. When a statistically significant relationship between mollusc abundance and time was found, this is represented by the corresponding regression line

  Relative size-frequency distributions from June 2000 to September 2004 (a), and from April 2009 to May 2013 (b)].

A

  total of 29,865 molluscs were collected during the study, among which 90.80% (27,118) were P. antipodarum. The other mollusc species corresponded to Sphaeriidae (9.13%), a family of Bivalvia [namely, Pisidium spp. Pfeiffer, 1821 and Sphaerium corneum (Linnaeus, 1758)], and to Pulmonata (0.07%) belonging to five families (Acroloxidae, Ancylidae, Lymnaeidae, Physidae, and Planorbidae) (Table2). All the pulmonate snails were juveniles according to their shell size, and included 14 individuals of Planorbis planorbis (Linnaeus, 1758), two individuals of Ancylus fluviatilis Müller, 1774 and Aplexa hypnorum (Linnaeus, 1758) and one individual of Acroloxus lacustris (Linnaeus, 1758), Physa heterostropha (Say, 1817), and Radix ovata (Draparnaud, 1805). Mean species richness of gastropods (seven species in total) over the 62 sampling dates of the study was very low: 1.23 ± 0.13 species.

Fig. 3

 3 Fig. 3 Temporal fluctuations of Aporocotylid sp. I prevalence (%) in the French stream (Le Petit Hermitage) during the three monitoring periods included in statistical analyses, i.e., June 2000-June 2001, March 2002-September 2004, and April 2009-March 2012. Sample sizes are indicated in Table2. Note that, despite temporal fluctuations, prevalence was always low (≤ 1.52%) throughout the monitoring periods

Table 1

 1 Mean

	Size classes	2002

number (± CI) per size class and maximal number (Max) of embryos per female of

(March, April, May), 2004 (May), and 2013 (March, April, May)

Table 2

 2 Molluscan

	Total	NMS

assemblage (total abundance, A, and relative abundance, A %) in the French stream Le Petit Hermitage from June 2000 to May 2013 according to the monitoring period (NMS = number of monthly samples per period) Total NMS e. 11 sur 31 24/10/2017 09:35 Despite its high overall relative abundance (90.80%), P. antipodarum was numerically dominant only during the first broad time period (June 2000-June 2001, March 2002-September 2004) when it comprised 96.81% of mollusc individuals. In contrast, during the second time period (April 2009-March 2012, March 2013-May 2013), P. antipodarum represented only 43.51% of all encountered molluscs, and the Sphaeriidae become the numerically dominant taxon, with 56.34% relative abundance (Table

Table 2

 2

	. Note that, despite

  no infected snails in Although no infected P. antipodarum were detected after June 2009 [i.e., 10 years after the first record in the same region

	.
	2013).
	20 sur 31	24/10/2017 09:35

24/10/2017 09:35

Acknowledgements

We thank Valérie Briand for bibliographical assistance.

Compliance with ethical standards

Conflict of interest All of the authors read and approved the paper that has not been published previously nor is it being considered by any other peer-reviewed journal. The authors declare that there are no conflicts of interest. Below is the link to the electronic supplementary material.

Electronic supplementary material

Supplemental Table

Physicochemical


P Bustamante

J-L Teyssié

S W Fowler

M Warnau

AU TRAVERS DE LA PAROI DES OEUFS DE SEICHES CONTRASTING BIOACCUMULATION AND TRANSPORT BEHAVIOUR OF TWO ARTIFICIAL RADIONUCLIDES ( 241 AM AND 134 CS) IN CUTTLEFISH EGGSHELL

Keywords: RADIONUCLIDES, BIOKINETICS, BIOACCUMULATION, CEPHALOPODS RADIONUCLEIDES, BIOCINETIQUES, BIOACCUMULATION, CEPHALOPODES

Radionuclide experiments were performed to study the accumulation, retention and transport of 241 Am and 134 Cs in eggs of the common cuttlefish Sepia officinalis. Experiments were designed to determine the processes controlling uptake of both radionuclides following exposure via seawater. The bioaccumulation of both radionuclides displayed saturation kinetics with steady state in uptake being reached in 10 d for 241 Am and 6 d for 134 Cs. 241 Am was readily taken up by the eggs which reached a concentration factor (CF) of 20 after 10 d of exposure. During embryonic development, the eggshell acted as an effective shield against 241 Am penetration with 98% of the radionuclide associated with this membrane. In contrast, 134 Cs was transported across the eggshell but was not concentrated to any large extent in the eggs. The mean 134 Cs CF for the whole egg did not exceed 1.3 ± 0.2, indicating a poor affinity of 134 Cs for the embryonic tissues of cuttlefish.

INTRODUCTION

Various contaminants occur in relatively high concentrations in coastal areas which are subjected to release from agricultural, domestic and industrial activities. Increased ambient contaminant concentrations may provoke toxic effects in marine organisms which ultimately could lead to a modification of the entire ecosystem structure [START_REF] Watzin | The effects of zinc contamination on the recruitment and early survival of benthic invertebrates in an estuary[END_REF]. In invertebrates, the early life stages, viz. embryos and juveniles, are very sensitive to contaminants [START_REF] Calabrese | The toxicity of heavy metals to embryos of the American oyster Crassostrea virginica[END_REF][START_REF] Martin | Toxicities of ten metals to Crassostrea gigas and Mytilus edulis embryos and Cancer magister larvae[END_REF], Warnau et al. 1996a). This is particularly obvious when the spawning and the subsequent embryonic development occur directly in seawater, thus allowing a direct contact of waterborne contaminants with the embryos and larvae. This sensitivity is believed to be reduced when the embryo develops within an egg, with the eggshell acting as a potential protective barrier to reduce or prevent the intake of contaminants from the dissolved phase.

The common cuttlefish Sepia officinalis spawns eggs surrounded by a proteinaceous eggshell which is known to prevent the incorporation of some non-essential metals such as Cd, Pb, or V (Bustamante et al. 2002, Miramand et al. in press). However, other toxic elements such as Ag can pass through the eggshell and become incorporated in embryonic tissues [START_REF] Bustamante | Uptake, transfer and distribution of silver and cobalt in tissues of the common cuttlefish Sepia officinalis at different stages of its life cycle[END_REF]. Element transport selectivity in the eggshell is apparently not determined by the metabolic needs of the embryo for essential elements, since the nonbiologically essential element Ag is well known for its enhanced embryotoxicity [START_REF] Calabrese | The toxicity of heavy metals to embryos of the American oyster Crassostrea virginica[END_REF][START_REF] Martin | Toxicities of ten metals to Crassostrea gigas and Mytilus edulis embryos and Cancer magister larvae[END_REF], Warnau et al. 1996a).

In general, knowledge about such selectivity remains very limited and, in particular, is lacking for certain long-lived radionuclides. Therefore, the present work aims at investigating the accumulation of two key radioelements, 241 Am and 134 Cs, in cuttlefish eggs. These radionuclides were selected to investigate any differences in behaviour of a particle-reactive element ( 241 Am) and a biological-reactive element like 134 Cs which can act as an analogue for potassium. This study focuses specifically on the accumulation rates of these radionuclides in the whole eggs and their subsequent distribution among the different egg compartments.

MATERIAL AND METHODS

Biological material and radiotracer exposure

Eggs of the common cuttlefish Sepia officinalis L. were obtained from cultured adults and were maintained in an aquarium with flowing seawater (salinity: 36 p.s.u.; temperature: 16.5 ± 0.5°C; 12/12 h dark/light cycle) until used in the experiments. Approximately 35 days after spawning, the eggs were placed in a 70 L glass aquarium containing natural seawater spiked with 241 Am and 134 Cs (nominal activity: 6 kBq L -1 each) until hatching, i.e. for 11 d. Both radionuclides and seawater were renewed daily in order to maintain seawater quality and radionuclide concentration as constant as possible. The radionuclide activity in seawater was checked daily, before and after each seawater renewal. During the experiment, 241 Am and 134 Cs levels were measured in the eggs on days 1, 2, 3, 6, 9, and 11. At each counting time, 3 eggs were dissected to determine the partitioning of the radiotracers among the three egg compartments: eggshell, peri-embryonic fluid and embryo.

Radionuclides and radioanalyses.

241 Am [t½ = 433 yrs] and 134 Cs [t½ = 2 yrs] were purchased from Amersham, UK, as nitrate and chloride salts, respectively. Stock solutions were prepared in their respective solutions (0.1 N) to obtain radioactivities which would allow using spikes of 20 µL.

Radioactivity of the tracers was measured using a high-resolution γ-spectrometry system consisting of coaxial Ge (N-or P-type) detectors (EGNC 33-195-R, Intertechnique) connected to a multichannel analyser and a computer with spectra analysis software (Interwinner, Intertechnique). The detectors were calibrated with appropriate standards for each of the counting geometries used.

Measurements were corrected for background and physical decay of the radionuclides. Counting times were adapted to obtain relative propagated errors less than 5%. However, in a few cases, this counting precision could not be obtained even after 48 h of counting, due to the very low activity in the smallest fractions.

Data and statistical analyses.

Uptake of 241 Am and 134 Cs from seawater was expressed as change in whole-egg concentration factors (CF) over time (CF = Bq g -1 wet weight (wwt) egg divided by the time-integrated Bq g -1 in seawater). Uptake kinetics in the eggs were described using a singlecomponent first-order kinetic model:

CFt = CFequil (1 -e -k t ),
where CFt and CFequil are concentration factors at time t (d) and steady-state, respectively, and k is the depuration rate constant (d -1 ) [START_REF] Whicker | Radioecology: nuclear energy and the environment[END_REF].

Constants of the model and their statistics were estimated by iterative adjustment and Hessian matrix computation, respectively, using the non-linear curve-fitting routines in the Systat 5.2.1 Software [START_REF] Wilkinson | Systat: the system for statistics[END_REF]. Changes in radiotracer distribution among egg compartments were tested for significance by the G procedure (adapted from the log-likelihood ratio test) for 2xk contingency tables [START_REF] Zar | Biostatistical analysis[END_REF]. Changes in % of radioactivity in a single compartment during the accumulation period were tested by one-way ANOVA followed by the Tukey's multiple comparison test after arcsin transformation of the data [START_REF] Zar | Biostatistical analysis[END_REF]. The significance level for statistical analyses was routinely set at α = 0.05.

RESULTS

Accumulation of the radionuclides

Regular monitoring of radiotracer activities in seawater allowed calculation of time-integrated radioactivities, viz. 6.4 ± 0.3 kBq L -1 for 241 Am and 8.6 ± 0.7 kBq L -1 for 134 Cs. Despite the relatively short experimental exposure (11 d), the uptake of 241 Am and 134 Cs displayed saturation kinetics (Figs 1A &1B).

Estimated steady-state concentration factors (CF equil ) in the eggs were 19.7 ± 3.2 and 1.33 ± 0.21 for 241 Am and 134 Cs, respectively.

Distribution among egg compartments

Table 1 shows the distribution of the nuclides among the different compartments of the eggs. The major proportion of 241 Am was always associated with the egg capsule membrane during the course of the experiment.

In contrast, the distribution of 134 Cs varied greatly among the egg compartments over time. A much lower percentage of 134 Cs was found in the eggshell (always < 38%), whereas the percentages in the peri-embryonic liquid and the embryo differed significantly (P G-test <0.05) from one sampling time to another.

DISCUSSION

Only very limited information concerning trace element metabolism in the eggs of cephalopods is available in the literature [START_REF] Miramand | Variation of heavy metal concentrations (Ag, Cd, Co, Cu, Fe, Pb, V, Zn) during the life cycle of the common cuttlefish Sepia officinalis. Sci Total Environ[END_REF], Guary & Fowler 1982[START_REF] Ueda | Accumulation of 65 Zn by Octopus Octopus vulgaris[END_REF][START_REF] Bustamante | Biokinetics of zinc and cadmium accumulation and depuration at different stages in the life cycle of the cuttlefish Sepia officinalis[END_REF][START_REF] Bustamante | Uptake, transfer and distribution of silver and cobalt in tissues of the common cuttlefish Sepia officinalis at different stages of its life cycle[END_REF]). Among the cephalopods, cuttlefish undertake coastal to open sea migrations as they evolve from juveniles to adults, and then return to coastal waters for reproduction. Contaminant-enrichment of coastal waters due to anthropogenic activities may have adverse effects on the embryos, potentially influencing the recruitment and eventual dynamics of cuttlefish populations. The strong toxicity of various trace elements with respect to the larvae of marine invertebrates is well known (e.g. [START_REF] Calabrese | The toxicity of heavy metals to embryos of the American oyster Crassostrea virginica[END_REF][START_REF] Martin | Toxicities of ten metals to Crassostrea gigas and Mytilus edulis embryos and Cancer magister larvae[END_REF], Warnau et al. 1996a). However, similar information for cephalopods is limited to only a few metals (viz. Ag, Cd, Co and Zn; [START_REF] Bustamante | Biokinetics of zinc and cadmium accumulation and depuration at different stages in the life cycle of the cuttlefish Sepia officinalis[END_REF][START_REF] Bustamante | Uptake, transfer and distribution of silver and cobalt in tissues of the common cuttlefish Sepia officinalis at different stages of its life cycle[END_REF], and to the best of our knowledge nothing concerning radionuclides has been reported to date.

When spawned, cuttlefish eggs are surrounded by an eggshell the thickness of which is approximately 1.5 mm [START_REF] Lemaire | Etude du développement embryonnaire de Sepia officinalis L[END_REF]. This shell, composed of albumin and other proteins, hardens when coming into contact with seawater. It also becomes thinner during embryonic development, and is almost transparent at the moment of hatching [START_REF] Wolf | Respiration of Sepia officinalis during embryonic and early juvenile life[END_REF]. In general, the concentrations of toxic metals within the eggs remain very low until the end of the embryogenesis, and they are in the same concentration range as those in the yolk of early spawned eggs (Miramand et al. in press). Moreover, previous experiments with radiotracers have shown that Cd and Co were accumulated in the eggshell which constituted an effective protective barrier preventing the incorporation of waterborne metals in the embryo during its development [START_REF] Bustamante | Biokinetics of zinc and cadmium accumulation and depuration at different stages in the life cycle of the cuttlefish Sepia officinalis[END_REF][START_REF] Bustamante | Uptake, transfer and distribution of silver and cobalt in tissues of the common cuttlefish Sepia officinalis at different stages of its life cycle[END_REF]. In contrast, Ag and to a lesser extent Zn were incorporated after a few days of exposure, suggesting a limited capacity of retention for Ag in eggshell and a facilitated transport across the eggshell of the biologically-essential element Zn. It is therefore likely that the physical-chemical properties of the different elements determine their capability to pass through the eggshell. It is evident that more detailed tracer studies are needed in order to provide a clearer picture of the specific processes involved in the selectivity of the eggshell towards certain trace elements.

A major objective of our experiments with radionuclides was to compare the behaviour of a particlereactive element which has no known biological function, viz. 241 Am, with a biologically-reactive element such as 134 Cs which acts as an analogue element of potassium (K). During embryonic development, 241 Am was readily taken up from seawater reaching a CF of 20 ± 3 after 11 d of exposure (Fig. 1). Approximately 98% of the incorporated 241 Am remained associated with the capsule membrane of the eggs during the entire experiment (Table 1). Similar to other transuranium nuclides, 241 Am is well known for its specific affinity for the calcareous structures of echinoderms [START_REF] Grillo | Comparative studies on transuranium nuclide biokinetics in sedimentdwelling invertebrates[END_REF], Guary et al. 1982[START_REF] Fowler | Americium biokinetics in benthic organisms as a function of feeding mode[END_REF], Warnau et al. 1996b) and bivalves [START_REF] Guary | Recherches sur les transferts et la fixation du plutonium, de l'américium et du neptunium dans le milieu marin[END_REF][START_REF] Bjerregaard | Biokinetics of americum and plutonium in the mussel Mytilus edulis[END_REF][START_REF] Fisher | Accumulation and retention of metals in mussels from food and water: a comparison under field and laboratory conditions[END_REF], and therefore it might be expected to accumulate in the cuttlebone of the embryo. In fact, our results clearly demonstrate that the eggshell acts as a very effective shield against the incorporation of transuranium nuclides.

In contrast to 241 Am, the 134 Cs CF remained very low with bioaccumulation reaching a steady state after only 6 d of exposure (Fig. 1). In fact, 134 Cs was readily transferred across the eggshell and was not specifically trapped on the membrane, which at any time only contained between 13 and 38% of the total radionuclide activity (Table 1). However, the percentage of 134 Cs in the peri-embryonic fluid and the embryo varied significantly throughout the experiment (p G-test < 0.05), suggesting that this element is not specifically sequestered within the egg. It is therefore likely that rapid exchange between the internal compartments (periembryonic fluid and embryo) and the external medium (seawater) occurred. In general, 134 Cs displayed a quite different behaviour compared to other elements, especially 241 Am, 109 Cd, and 57 Co, which are mainly bound to the eggshell [START_REF] Bustamante | Biokinetics of zinc and cadmium accumulation and depuration at different stages in the life cycle of the cuttlefish Sepia officinalis[END_REF](Bustamante et al. , 2004, this study), this study). In contrast, 134 Cs is readily transferred across the membrane much like K which is a major element occurring in high concentrations in muscular tissues. However, the low concentrations of 134 Cs in the embryo indicate a poor incorporation of this element in muscle during ontogenesis. Any reduction of Cs incorporation in the muscular tissues of cephalopods is likely a reflection of the very low stable Cs concentration relative to K in these organisms, as for example the Cs (7.5 ng g -1 wwt) and K (3,100 µg g -1 wwt) levels measured in the mantle of the purpleback flying squid Sthenoteuthis oualaniensis [START_REF] Ichihashi | Multielemental analysis of purpleback flying squid using high resolution inductively coupled plasma-mass spectrometry (HR ICP-MS)[END_REF]. Therefore, a comparative examination of the results of the few investigations examining metal incorporation during cuttlefish embryogenesis [START_REF] Bustamante | Biokinetics of zinc and cadmium accumulation and depuration at different stages in the life cycle of the cuttlefish Sepia officinalis[END_REF], 2004, Miramand et al. in press, this study) allows defining 3 distinct categories of elements: (1) those efficiently trapped on the eggshell which results in protecting embryos against metal toxicity (i.e. 241 Am, 109 Cd, and 57 Co), (2) those which are firstly trapped on/in the membrane and which subsequently cross the eggshell, probably when both specific and nonspecific sites for binding are occupied (i.e. 110m Ag and 65 Zn), and (3) those which are readily transferred across the eggshell in both directions (i.e. 134 Cs). However the specific mechanisms involved remain poorly understood, and further detailed investigations which specifically address the physiological mechanisms controlling eggshell selectivity in metal accumulation and transfer should be considered in the future. Table I. Sepia officinalis. Distribution (%; mean values ± SD, n = 3) of 241 Am and 134 Cs among the cuttlefish egg compartments at different times during their exposure via seawater.

Fig. 1 .

 1 Fig.1. Sepia officinalis. Whole-body uptake kinetics of 241 Am (A) and 134 Cs (B) in cuttlefish eggs exposed for 11 d to radionuclides in seawater (concentration factor, CF; mean ± SD, n = 3).

ACKNOWLEDGEMENTS -We thank Dr. S. von Boletzky from the Marine Station of Banyuls, France for providing the cuttlefish eggs.

MW is a Honorary Research Associate of the National Fund for Scientific Research (NFSR, Belgium). The Agency is grateful for the support provided to its Marine Environment Laboratory by the Government of the Principality of Monaco.