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Environmental Research

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Spatio-temporal variation of trematode parasites community inCerastoderma edule cockles from Ria de Aveiro (Portugal)

Luísa Magalhãesa,b, Simão Correiaa, Xavier de Montaudouinb, Rosa Freitasa,⁎

a Departamento de Biologia & CESAM, Universidade de Aveiro, 3810-193 Aveiro, PortugalbUniv. Bordeaux, EPOC, UMR 5805 CNRS, 2, rue du Pr Jolyet, F-33120 Arcachon, France

A R T I C L E I N F O

Keywords:IntertidalHost-parasite systemSpatio-seasonal homogeneityInterannual heterogeneityIndicators

A B S T R A C T

Cerastoderma edule (edible cockle) is among the most exploited bivalves in Europe playing an important socio-economic role. Cockles live in estuaries and lagoons where their population is controlled by several environ-mental factors including parasitism. Parasites represent an important part of the world known biodiversity butare often neglected. Trematodes are the most prevalent macroparasites of cockles being able to exert an impactboth at the individual and population levels. Therefore, it is of prime relevance to recognize and understand theparasite-host system dynamics in order to better predict potential conservation threats to bivalve populationsand to maximize the success of stock and disease episodes management.

Cockle monitoring was conducted in 2012 and 2016, in six and eight stations, respectively, at the Ria deAveiro coastal lagoon, Portugal. Cockles were sampled in one single occasion in 2012 and seasonally in 2016.The tested hypothesis is that the trematode community in cockles was spatially and seasonally heterogeneousbut stable over time. The main result showed that despite a relative homogeneity of the parasite communitystructure in cockles, the among-years heterogeneity of trematode communities was higher than among-stationsand among-seasons heterogeneity rejecting the postulated hypothesis. Results demonstrated that trematodecommunities from the Ria de Aveiro are characterized by low abundance, which resulted in a spatial and sea-sonal trematode homogeneity (despite an overall channel difference and a slight downstream-upstream gra-dient). The interannual analysis showed a worrisome loss of trematode diversity and prevalence which conse-quently indicates an important loss of overall diversity and/or environmental conditions reflecting the negativeeffects of global change (mean temperature rise and overharvesting, among others). The present study high-lighted the importance of trematodes in characterising their associated environment and respective biodiversitywhich might be helpful to assess ecosystem ecological status and to identify threatened areas.

1. Introduction

The recognition of species presence and distribution is very im-portant in coastal and estuarine science namely to assess ecosystemecological status and to identify priority areas for protection and con-servation (McLusky, 1999). Macroparasitic fauna comprises among themost important species within these ecosystems, representing 40% oftotal metazoan species richness (Dobson et al., 2008) although, it isoften neglected with more taxonomic (Bartoli et al., 2000) and ex-perimental (Studer and Poulin, 2013) studies than studies presentingquantitative field data (de Montaudouin et al., 2000).

In coastal waters, trematode is the most abundant and prevalentclade of macroparasites (Lauckner, 1983). These parasites have acomplex life cycle using vertebrates as final host, where the adultparasitic stage develops, sexually reproduces and spawns its eggs. Eggs

are released in the environment, develop into the miracidium free-living stage that will infect a mollusc as first intermediate host (spor-ocyst or rediae parasitic stage). From mature sporocysts (or rediae),cercariae larvae are released in the environment, another free-livingform, which rapidly penetrates an invertebrate or vertebrate species(second intermediate host) and settles as metacercariae. During thiscycle, the parasite experiences different habitats with different abioticand biotic drivers that will affect parasite success and consequently itsdistribution pattern. Among these drivers, temperature (deMontaudouin et al., 2016), light: dark cycle (de Montaudouin et al.,2016), hydrodynamics (de Montaudouin et al., 1998), diversity of hostspecies (Thieltges and Reise, 2007) and target host density (Magalhãeset al., 2017) are considered important drivers. The annual fluctuation ofthese parameters usually leads to a seasonal pattern with an optimalinfection window occurring in the warmer season (Desclaux et al.,

https://doi.org/10.1016/j.envres.2018.02.018Received 23 November 2017; Received in revised form 29 January 2018; Accepted 13 February 2018

⁎ Corresponding author.E-mail address: rosafreitas@ua.pt (R. Freitas).

Environmental Research 164 (2018) 114–123

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2004; Thieltges and Rick, 2006; de Montaudouin et al., 2016).At the individual level and by definition, the parasite exerts a ne-

gative impact on the host and can alter its biological functions(Carballal et al., 2001; Babirat et al., 2004). The impact of a parasite ona particular organism, or its pathogenicity, is specific and also dependson host and parasite abundance. However, impact on host population isusually reported when there is obvious disease symptoms and massmortality playing a significant role in host population regulation(Marcogliese, 2004). On the other hand, the complexity of the trema-tode life cycle described above, namely its multi-host nature, makestrematodes indicators of ecosystem diversity and health indicators(Hechinger et al., 2006; Hudson et al., 2006). Trematodes were alsoused to assess habitat stability over time, at the scale of several years(de Montaudouin et al., 2012) or to detect global changes effectsthrough fish long-term monitoring (Dzikowski et al., 2003; Zander,

2005).Among the different host-parasite systems taking place in marine

environment, the present study investigated a bivalve-trematodemodel: firstly because bivalves (along with several other molluscs) aresuitable and favourite first and/or second intermediate hosts for tre-matode parasites (Lauckner, 1983); secondly because bivalves re-present the major proportion of the benthic fauna biomass in manycoastal systems, occurring also at high densities (Sousa et al., 2009);thirdly because some species represent the basis of important com-mercial fisheries (Beukema and Dekker, 2006); and finally due to theirimportant role on the ecosystem functioning (Morgan et al., 2013).Thus, bivalves are keystone species, they act as ecosystem engineerscreating, modifying and maintaining habitat for other species(Philippart et al., 2007). They provide structural conditions for otherinvertebrates to settle and occupy a crucial position within food webs

Fig. 1. Study area. The Ria de Aveiro coastal lagoon (Northwest Portugal) indicating the positions of the seasonal sampling stations (São[HYPHEN]Jacinto[HYPHEN]Ovar channel: O1,O2, O3, O4 and Espinheiro channel: E1, E2, E3, E4) with indication of the lagoon division into five transitional water bodies (WB). Compatible stations used for 2012-2016 comparisonsare identified by a black square.

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(Rakotomalala et al., 2015). Bivalves are connected to primary produ-cers by their suspension-feeding activity and related to higher trophiclevels as prey for many bird, fish, crustacean and echinoderm species(Rakotomalala et al., 2015). In fact, bivalves contribute to biodiversityand ecosystem resilience, and therefore the identification of factors thatmodulate their population dynamics is of upmost importance. Amongbivalves, Cerastoderma edule (Linnaeus, 1758), the edible cockle, is adominant species in coastal waters and is infected by several trematodespecies both as first and second intermediate host (de Montaudouinet al., 2009). This species can be found along the north-eastern coast ofthe Atlantic Ocean, from the Barents Sea to Mauritania (Tebble, 1966;Honkoop et al., 2008). It is an exploited living resource with particularimportant socio-economic relevance in Portugal (Pereira et al., 2014).

The present study focused on spatio-temporal variability of thestructure of trematode community infecting C. edule at the scale of theRia de Aveiro coastal lagoon (Portugal), with two main objectives.Firstly, we proposed to map trematode parasites in the Ria de Aveiroand to hierarchize some environmental drivers of infection. Based onformer studies highlighting the importance of abiotic factors (tem-perature, salinity) (Koprivnikar and Poulin, 2009; Lei and Poulin, 2011;Studer and Poulin, 2012; Born-Torrijos et al., 2014; Koprivnikar et al.,2014), we postulate that the structure of parasite communities incockles should follow an oceanic-continental gradient and seasonalcycles. Secondly, we compared trematode communities in cockles froma previous field study (Freitas et al., 2014) with similar sampled sta-tions and season in the Ria de Aveiro. The hypothesis is that the parasitecommunity structure is relatively stable over time as long as environ-mental parameters remain stable too (de Montaudouin et al., 2012).Thus, a significant difference in trematode parasite community struc-ture could alert for environmental changes and alterations in ecosystemfunctioning.

2. Material and methods

2.1. Study area description

The Ria de Aveiro is a coastal lagoon (Northwest of Portugal) withfour main channels which radiate from the ocean mouth with severalbranches, islands and mudflats (Fig. 1). Tides are semi-diurnal andconstitute the main forcing water circulation agent. The minimum andmaximum tidal height ranges are about 0.6 m and 3.2m at neap andspring tides, respectively (Dias et al., 2000). The most importantfreshwater input (the Vouga River) of the Ria de Aveiro flows throughthe Espinheiro channel, one of the present study areas (Fig. 1) that isabout 17 km long and is characterized by a strong horizontal gradient ofsalinity and water temperature which migrates back and forth with thespring/neap cycle (Vaz et al., 2005; Lillebø et al., 2015). The otherfreshwater sources are smaller, namely the Boco, Mira and Cásterrivers, the latter flowing through the 29 km long São Jacinto-Ovarchannel, the other study area (Fig. 1). According to the Water Frame-work Directive, the Ria de Aveiro is divided in five water bodies (WB),identified in Fig. 1, with WB2 gathering both study areas (the twochannels) and is classified with a “moderate” water ecological status(MAMAOT/ARHCentro, 2012).

2.2. Sampling procedure and parasite identification

Field monitoring occurred during one year (from December 2015 toNovember 2016) with seasonal sampling in 8 stations along the twochannels, Espinheiro and São Jacinto-Ovar (Fig. 1), identified withdifferent codes: first two letters represent the season (WI: winter, SP:spring, SU: summer, FA: fall); third letter represents the channel (E:Espinheiro channel, O: São Jacinto-Ovar channel); and the numberrepresents the sampling station (from 1, the most oceanic station, to 4,the most continental station). At each station, temperature and salinitywere measured and two sediment samples (2 replicates each) were

collected in order to estimate median grain size and total organic matter(TOM) content. At each station, cockles were collected by sampling sixquadrats (0.25m2 each) and sieving them through a 1-mm mesh sieveto estimate density (cockles m−2). Cockle shell length was measured atthe least mm with a calliper. Size-histogram analysis allowed dis-criminating different cohorts. In order to compare infection in cockleswith similar age, we decided to concentrate on the most representedcohort, i.e. 2014. Twenty cockles per station and season were dissected.All trematodes were identified to the species level following severalauthors descriptions (Bowers, 1969; Bowers et al., 1996; Bartoli et al.,2000; de Montaudouin et al., 2009). Metacercariae identified in each ofthe observed cockles were counted to assess parasite abundance(number of metacercariae per cockle) and prevalence (percentage ofinfected cockles) (Bush et al., 1997). For parasite using cockles as firstintermediate host, only prevalence was calculated.

A survey of the trematode species infecting cockles in the Ria deAveiro has already been conducted in 2012 (Freitas et al., 2014). Inorder to assess interannual variability of the structure of trematodecommunities, 2012 (October sampling) and 2016 (September sampling)databases were compared. Overlapping areas from both studies wereidentified and selected to perform the temporal comparison, corre-sponding to six comparable stations (Fig. 1).

2.3. Sediment analysis

Sediment samples for grain-size analysis were dry and wet sievedthrough a column of sieves with decreasing mesh sizes following theprocedure described by Quintino et al. (1989). Sediment total organicmatter content (TOM) was measured as the percent weight loss in 1 g ofdried sediment, after combustion at 450 °C, during 5 h (Kristensen andAndersen, 1987).

2.4. Data analysis

Prevalence, i.e. the percentage of hosts infected with 1 or moreindividuals of a particular parasite species (Bush et al., 1997), wascalculated for all trematode species found: Diphterostomum brusinae(Stossich, 1889), Himasthla elongata (Mehlis, 1831), H. interrupta Loos-Frank, 1967, H. quissetensis (Miller and Northup, 1926) Stunkard, 1938,Renicola roscovitus (Stunkard, 1932), Parvatrema minutum (Cobbold,1859), Bucephalus minimus (Stossich, 1887) and Monorchis parvus Looss,1902. Parasite abundance, i.e. the total number of individuals of aparticular parasite species in a sample of a particular host species (Bushet al., 1997), was calculated for 6 out of 8 species found, excludingthose infecting cockles as first intermediate host (B. minimus and M.parvus).

Regarding 2016 data, due to high heterogeneity of metacercariaeabundance in cockles and because data from fall season were missing,we performed two separated ANOVAs with mean metacercariae abun-dance per cockle in each station or season as dependent variable. Priorto analysis, data were log (x+ 1) transformed in order to achievehomogeneity of variance which was verified with Cochran test. The firstwas a two-way ANOVA in order to test the effect of the channel (E vs.O), the proximity to ocean (from station 1 (close) to 4 (remote)) and theinteraction between factors on the total number of metacercariae incockles. The second test was a one-way ANOVA assessing seasonal ef-fect (with all stations as replicates) on the mean number of meta-cercariae per cockle. The same analysis (two-way ANOVA with channeland stations followed by a one-way ANOVA with seasons) was repeatedfor P. minutum, the most abundant species.

Comparison of trematode communities in cockles among stations ineach season was performed by a cluster analysis, i.e. an exploratorytechnique that aims to join together objects into successively largerclusters, using some measure of community similarity or distance: ashort distance between two stations means that the community ofparasites in cockles is similar in terms of species composition and

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species abundance. Dataset consisted of a “season/station (categoricalfactor) × trematode species (dependent variables)” matrix. In orderto include the two trematode species found using cockles as first in-termediate host (B. minimus and M. parvus), each dependent variablecorresponded to the mean infection prevalence (mean percentage ofinfected hosts) of a given trematode species in cockles from a givenstation at a given season. The cluster analysis was performed on thematrix of Euclidean distance between variables (Ward method of ag-gregation, ascendant hierarchical method). Due to heterogeneity oftrematode infection in relation with the scale of analysis, we assigned athreshold at 30% of dissimilarity to separate the different groups.Groups were then characterized in terms of parasite community, en-vironmental and host population features. For each of these parameters(dependent variables), differences among affinity groups (categoricalfactor) given by the cluster 30% dissimilarity threshold were testedusing one-way ANOVAs followed by post-hoc Tukey test for comparisonof means, identifying homogenous groups which are represented intables with superscript lower case letters. Prior to ANOVA, homo-geneity of variance was verified with Cochran test.

To compare trematode communities in cockles between October2012 and September 2016, a cluster analysis followed by one-wayANOVAs were performed as previously described for 2016 data.

All statistics were performed with STATISTICA 7.0 (StatSoft) soft-ware.

3. Results

3.1. Trematode community: spatial and seasonal patterns

A total of 8 stations and 4 seasons were sampled but cockles (fromthe 2014-cohort) were absent at 9 occasions, especially during fallcampaign (in 6 stations). During the present study 387 cockles weredissected, 166 of them were uninfected. Eight trematode species (be-longing to 6 different families, Table 1) were observed in 221 cockles,six species using cockles as second intermediate host: Diphterostomumbrusinae (mean prevalence (P) = 0.5%), Himasthla elongata(P= 23.9%), H. interrupta (P=0.9%), H. quissetensis (P= 0.9%), Par-vatrema minutum (P= 48.9%) and Renicola roscovitus (P=0.2%) andtwo using cockles as first intermediate host: Bucephalus minimus(P= 1.1%) and Monorchis parvus (P=0.2%, registered in the presentstudy only in sporocyst form) (Table 1). Among the sampling stations,species richness varied from 0 to 4 species per cockle. Overall, trema-tode mean abundance ranged between 0 and 132 metacercar-iae.cockle−1, with an average of 43 metacercariae.cockle−1 (Fig. 2).The most abundant species was P. minutum which represented 65–100%of total metacercariae in cockles from each station, occurring in 96% ofthe sampling occasions (station × season). H. elongata was present incockles in 83% of sampling occasions but with a very low averageabundance (< 1 metacercariae.cockle−1). H. quissetensis, H. interrupta,D. brusinae and R. roscovitus were rare, infecting 4, 3, 2 and 1 out of the387 total number of cockles dissected, respectively, without spatialand/or seasonal significant differences.

Spatially, the mean number of metacercariae per cockle in 2016 was

significantly higher in Espinheiro channel (47.9 ± 32.3 (standard de-viation (SD)) metacercariae.cockle−1) than in São-Jacinto-Ovarchannel (36.6 ± 42.1 (SD) metacercariae.cockle−1) (two-way ANOVA:F (1) = 6.6, p=0.02) with no significant differences related to oceanproximity (two-way ANOVA: F (3) = 1.3, p=0.32, Table 2) and nointeraction between both independent factors (two-way ANOVA: F (3)= 2.1, p=0.14, Table 2). Seasonally, the mean number of meta-cercariae per cockle ranged between 26.2 ( ± 30.3(SD)) and 63.8( ± 46.5(SD)), with no significant difference among seasons (one-wayANOVA: F (3) = 1.3, p=0.31, Table 2). The most abundant species, P.minutum, presented the highest mean abundance in WIO1, SPO1 andSPE1 (on average, 87, 107 and 132 metacercariae.cockle−1 respec-tively) following the same pattern as total metacercariae, with sig-nificantly higher abundance in Espinheiro channel than São Jacinto-Ovar channel, with no significant differences related to ocean distancenor interaction between factors (two-way ANOVAs: F (1) = 6.2,p=0.02, F (3) = 1.1, p=0.40 and F (3) = 2.0, p=0.16 respectively,Table 2) as well as no seasonal trend (one-way ANOVA: F (3) = 1.3,p=0.31, Table 2).

In terms of parasite community in cockles, cluster analysis dis-criminated 3 groups at a threshold at 30% of dissimilarity (Fig. 3). Totalmean prevalence was not significantly different among groups (one-way ANOVA: F (2) = 1.7, p=0.20, Table 3). D. brusinae and H. elongatawere the main species contributing for clusters dissimilarity, the firstwas exclusive of Group 1 (3 sampling stations, 2 of them in the mostoceanic part, Fig. 4) where H. elongata presented the highest meanprevalence. Group 2 (5 sampling stations) included mainly the stationslocated in the middle of the sampled channels (stations E3 and O3,Fig. 4). This group displayed intermediate values of H. elongata meanprevalence compared to groups 1 and 3 (Table 3). Finally, group 3gathered 13 sampling stations, including all the upstream positions,characterized by the lower values in terms of H. elongata prevalence(Fig. 3, Table 3). Comparing mean temperature, mean salinity, meantotal organic matter (TOM) content in the sediment, sediment mediangrain-size, mean cockle shell length and cockle density, only grain-sizeand cockles density showed significant differences among cluster affi-nity groups (one-way ANOVA: F (2) = 3.5, p=0.05 and one-wayANOVA: F (2) = 4.1, p=0.03, respectively), with group 3 displayingthe lowest (statistically significant) median grain-size and the highest(statistically significant) cockle density values (Table 3). In terms ofseasonal patterns, spring, summer and fall were relatively dispersed inall groups but group 3 (lower trematode prevalence) included all wintersamples.

3.2. Trematode community: 2012–2016 comparison

In the 2012 survey (Freitas et al., 2014), in the six sampling stationsin common with the present study, cockles were infected by 8 differenttrematode species: D. brusinae, H. elongata, H. interrupta, H. quissetensis,P. minutum and R. roscovitus (metacercaria) and B. minimus and M.parvus (sporocyst). Comparing to 2012 survey, in the 2016 samplesthree species were lacking (H. interrupta, M. parvus and R. roscovitus).Gathering both surveys data (6 stations per survey), cockles were

Table 1List of trematode species identified using Cerastoderma edule as their first and/or second intermediate host and the other hosts of the life cycle.

Trematoda species Family 1st intermediate host 2nd intermediate host Final host

Diphterostomum brusinae Zoogonidae Tritia reticulata Cerastoderma edule FishHimasthla elongata Himasthlidae Littorina littorea Cerastoderma edule Water birdsHimasthla interrupta Himasthlidae Hydrobia spp. Cerastoderma edule Water birdsHimasthla quissetensis Himasthlidae Tritia reticulata, T. neritea Cerastoderma edule Water birdsRenicola roscovitus Renicolidae Littorina littorea Cerastoderma edule Water birdsParvatrema minutum Gymnophallidae Scrobicularia plana Cerastoderma edule Haematopus ostralegusBucephalus minimus Bucephalidae Cerastoderma edule Pomatoschistus sp. Dicentrarchus labraxMonorchis parvus Monorchiidae Cerastoderma edule Cerastoderma edule Diplodus spp.

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distributed by 3 clusters that displayed 30% of dissimilarity (Fig. 5).Groups A and B gathered three sampling stations each and included all2012 sampling year, group C assembled 2016 sampling stations. H.

elongata (significantly higher prevalence in group A), H. quissetensis(significantly higher prevalence in group A) and D. brusinae (sig-nificantly lower prevalence in group C) were the main species con-tributing for the separation of the three cluster groups (70% similarity).Group C (2016 samples) was characterized by lower mean speciesrichness compared to group A (one-way ANOVA: F(2) = 12.3,p < 0.01, Table 4) and lower trematode mean prevalence (one-wayANOVA: F(2) = 16.1, p < 0.01) compared to groups A and B (2012).Regarding host population characteristics and environmental variables(Table 4), only total organic matter content displayed a significantvariation, showing to be significantly higher (one-way ANOVA: F(2)= 4.8, p=0.03) in group A (2012) than in group C (Table 4).

4. Discussion

In its distribution area, Cerastoderma edule is known to be infectedby sixteen different trematode parasite species (de Montaudouin et al.,2009). Eight species were identified in the present study, performed inthe Ria de Aveiro. Similar values of species richness were registered inthe south coast of Ireland (Fermer et al., 2010, eight species) and in theMerja Zerga, a Moroccan coastal lagoon (Gam et al., 2008, nine spe-cies). Ten trematode species were found infecting cockles from theNorthern Wadden Sea, Germany (Thieltges and Reise, 2006). Elevenspecies were recorded in the Ria de Aveiro, Portugal (Russell-Pintoet al., 2006; Freitas et al., 2014) and a maximum of 13 species in Ar-cachon bay, France (de Montaudouin et al., 2009). Parasite communityfrom the Ria de Aveiro seasonal campaign was dominated by Parva-trema minutum (Gymnophallidae) presenting the highest prevalence andmean metacercariae abundance per cockle. Former studies performedin the Ria de Aveiro revealed a similar trend (Russell-Pinto, 1990;Freitas et al., 2014) as well as in the Exe Estuary, England (Goater,1993), Arcachon bay, France (de Montaudouin et al., 2000, 2010) andsouth coast of Ireland (Fermer et al., 2009, 2010). There are somespecies that display a restricted distributional range: Asymphylodorademeli Markowski, 1935, registered only for the German Baltic coastand Gymnophallus somateriae (Levinsen, 1881) only for the GermanNorth Sea coast showing thus a northern distribution (Kesting et al.,1996; Thieltges and Reise, 2006) and an undescribed species registeredonly in Morocco (de Montaudouin et al., 2009). However overall spe-cies composition of the trematode communities is similar among thecockle populations sampled in different locations from south Ireland toMorocco (including the present study area). This suggests a high dis-tributional range of these different trematode species, possibly relatedto final hosts migration (mainly birds) (Feis et al., 2015).

At lower scale, comparing different stations within the Ria deAveiro, mean parasite species richness per cockle and mean

Fig. 2. Mean metacercariae abundance (± standard deviation (SD)) per cockle and per season (WI: winter, SP: spring, SU: summer, FA: fall) in each sampling station for Parvatremaminutum, Himasthla elongata and others (gathering the other four less represented species). NC: absence of cockles.

Table 2Two two-way ANOVAs results performed to test differences among Channels, Oceanproximity (stations 1–4) and interaction between factors in terms of total metacercariaeabundance (total number of metacercariae in cockles) and Parvatrema minutum abun-dance. Two one-way ANOVAs results performed to test differences among seasons interms of total metacercariae abundance and P. minutum abundance.

Dependent variable Statisticaltest

Factors df MS F p

Total metacercariaeabundance

Two-wayANOVA

Channel 1 1.9 6.6 0.02Ocean proximity 3 0.4 1.3 0.32Channel*Oceanproximity

3 0.6 2.1 0.14

Error 15 0.3One-wayANOVA

Season 3 0.5 1.3 0.31Error 19 0.4

Parvatrema minutumabundance

Two-wayANOVA

Channel 1 1.9 6.2 0.03Ocean proximity 3 0.3 1.1 0.40Channel*Oceanproximity

3 0.6 2.0 0.16

Error 15 0.3One-wayANOVA

Season 3 0.5 1.3 0.31Error 19 0.4

Fig. 3. Cluster analysis on trematode parasites community in adult cockles sampledseasonally in 2016 (WI: winter, SP: spring, SU: summer, FA: fall) in eight different stationsin the Ria de Aveiro (E1, E2, E3, E4, O1, O2, O3, O4) coastal lagoon. The 30 % distance ofdissimilarity threshold is drawn.

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metacercariae abundance per cockle displayed low values, especially insummer. The mean and maximal number of metacercariae per cocklereached 43 and 132, respectively, contrasting with other works wheremetacercariae number per cockle frequently surpasses thousands (e.g.Fermer et al., 2010 5585 metacercariae cockle−1, among others). Lowmean number of metacercariae per cockle can be due to parasite-de-pendent mortality, i.e. mortality of heavily parasitized cockles beforesampling. However, infection levels observed in the present work werealways too low to induce mortality outbreak. Cockle mortality thresh-olds were reported at 10–50 for Himasthlidae (Desclaux et al., 2004,2006; Gam et al., 2009) and 500 metacercariae cockle−1 for Gymno-phallidae (Gam et al., 2009). Low metacercariae abundance could alsobe the result of low diversity of the potential intermediate and finalhosts. However, this hypothesis is doubtful because the Ria de Aveiro isconsidered as a hotspot of biodiversity, is part of the Natura 2000network (EU habitats directive), designated Special Protected Area andprotected by the EU Birds Directive (79/409/CEE). The scarcity oftrematode parasites in the Ria de Aveiro could therefore be related tomuch more multifactorial habitat characteristics, comparing to the fewother coastal systems where spatial distribution of these parasites were

performed. The success of infection processes, i.e. the efficiency to ac-complish trematode parasite life cycle, appears related to the more orless sheltered status of the habitat. In inner areas of coastal ecosystemswith more continental influence, more pronounced seasonal variationof temperature and salinity (more extreme values), less hydrodynamicsand lower water mass turnover, and sometimes seagrass occurrence andsalt marsh proximity, trematode parasite abundance is often low. This isthe case in the inner part of Arcachon Bay (France), Merja Zerga(Morocco) (Gam et al., 2008; de Montaudouin and Lanceleur, 2011)and the present area (all stations and seasons) where parasite abun-dance is< 30 metacercariae.cockle−1 (excluding P.minutum). Con-versely, more oceanic influenced habitats with more buffered tem-perature and salinity fluctuations, and higher hydrodynamics featureslike the outer part of Arcachon Bay, Merja Zerga or Sylt area (Germany)are generally characterized by higher metacercariae abundance(Thieltges and Reise, 2007; Gam et al., 2009). These observations arenot true for the gymnophallid P. minutum which can be very abundantin cockles in highly contrasted environments as long as first inter-mediate host (Scrobicularia plana) is present (Fermer et al., 2010).

There are two immediate consequences of trematode scarcity in theRia de Aveiro. Firstly, and conversely to what is generally expected,sampled trematode community showed no evident seasonality.Typically, trematode parasites using cockles as second intermediatehost present a seasonal pattern of increasing infection in the warmerseasons and decreasing or no infection in the cold season (Goater, 1993;Desclaux et al., 2004). For example, in Arcachon bay, a synchrony wasobserved between parasite emergence from the first intermediate hostand infection on the second intermediate host, in May-October periodwhen water temperature was above 15 °C (de Montaudouin et al.,2016). Absence of seasonality in the present study could result fromnarrow abiotic (e.g. temperature, salinity) changes occurringthroughout the year as the example of the Cachoeira estuary, Brazil(Boehs et al., 2010). However, it is known that the Ria de Aveiro un-dergoes sharp seasonal salinity and temperature fluctuations (Vaz et al.,2005). In fact, the absence of seasonal fluctuation in parasite infectionis rather interpreted as the result of a general low infection level incockles preventing any discrimination between high and low infectionperiods.

Secondly, another consequence of general low trematode infectionin cockles is the lack of distinct trematode community between stations.In other words, our results showed a great homogeneity of trematodecommunity in the Ria de Aveiro, characterized by poor cockle infection

Table 3Characterization of the affinity groups identified in the Ria de Aveiro spatial and seasonal analysis, in 2016. Each variable is represented by mean and standard deviation (SD). Bold valuesindicate significant differences in the ANOVA main test while superscript lower case letters indicate the homogenous groups identified by the Tukey post-hoc comparison of means test.

Affinity groups One-way ANOVAs results

Group 1 Group 2 Group 3 F pNr. of sampling stations 3 5 13

Parasites Trematoda abundance (mean±SD) 54.03± 49.11 11.88±11.74 51.99± 35.32 2.8 0.09Species richness (mean± SD) 2.33±0.58 2.80± 0.84 2.15±0.99 0.9 0.43Prevalence (mean± SD, %) 92.67± 12.70 66.00±19.49 64.54± 26.40 1.7 0.20Diphterostomum brusinae 3.70±6.42a 0.00±0.00a,b 0.00±0.00b 3.9 0.04Himasthla elongata 80.09±6.56a 41.69±11.60b 7.69±7.53c 99.1

compared to less sheltered sites along the Atlantic coasts. A generalhomogeneity was observed, especially influenced by the wide andprevalent distribution of P. minutum, which in turn could be related toS. plana (first intermediate host) presence and density, one of the mostabundant taxa in the Ria de Aveiro (Nunes et al., 2008). Nevertheless,we evidenced a significant downstream-upstream gradient of pre-valence, with the highest overall values registered on the ocean sidesampling stations that was mainly shaped by the H. elongata prevalence.Taking into account that the first intermediate host of H. elongata(Littorina littorea) is also widely distributed in the Ria de Aveiro lagoon(Laranjeiro et al., 2015) and beyond arguments concerning the effect ofabiotic factors in more or less oceanic areas (as developed above), thehost population itself can regulate parasite infection by dilution effect(Magalhães et al., 2017), also referred as interference effect (Goedknegtet al., 2016). In the present study, target host (cockle) density showedto be negatively linked with trematode prevalence and could have beenan important driver of the trematode distribution. This results may be

corroborated by those stating that the negative consequences of in-traspecific competition at high density can be mitigated by lowerparasite burden (Magalhães et al., 2017).

Knowing that trematodes are useful ecological indicators and can beused even in poorly studied systems (Hechinger et al., 2006), the pre-sent study compared 2012 and 2016 (4 years gap) trematode commu-nities in cockles from the same sampling stations and season. Cocklessampled in the present study (group C), together with cockles from2012 affinity group B (O1, E1 and E3), presented lower trematodespecies richness compared to group A (O2, O3 and E4 from 2012), i.e. amaximum of five species instead of eight and a 2-fold decrease inspecies mean prevalence. On one hand this result could indicate anaturally strong interannual variability in terms of trematode commu-nities. Dynamics of trematode larvae community was mostly describedwith snails as first intermediate hosts, i.e. in a situation where multi-infection is rather rare (Esch and Fernandez, 1994; Esch et al., 2001;Soldánová et al., 2012). In this case, it is expected that some trematodespecies, especially those of migratory birds, undergo some periodicalextinctions (Esch et al., 2001), therefore temporal factors are likely toaffect the trematode prevalence as well as overall species richness andcomposition until the ecosystem reaches a dynamic equilibrium (Eschand Fernandez, 1994). However, in the second intermediate host, theinteraction is different and multispecies infection is generally the rule.Few studies dealing with metacercariae temporal dynamics reportedthat, in a low impacted/stable environment, trematode community alsotends to display significant stability (Thieltges and Reise, 2006;Campbell et al., 2007; de Montaudouin et al., 2012). Actually, theecological quality status (EcoQS) of the Ria de Aveiro water bodies,particularly the WB2 where the study area is inserted, registered animprovement from “Moderate EcoQS” (MAMAOT/ARHCentro, 2012)to “High EcoQS” (Marín et al., 2015) based only on benthic habitatsdensity and species composition. Parasites presence is mainly depen-dent on the diversity and density of any other organisms that partici-pate in the various parasites life cycles (Fredensborg et al., 2006;Hechinger et al., 2006; Hudson et al., 2006; Morley and Lewis, 2007)which is not the limiting factor in the Ria de Aveiro (Rodrigues et al.,2011). However, the parasite infection success is also negatively im-pacted by several anthropogenic activities (such as roads and con-sequent nitrogen and metals input (Altman and Byers, 2014)), i.e. lowwater quality, for example contamination with metals and acidification(Blanar et al., 2009). Indeed, human population in the watershed area

Fig. 5. Cluster analysis on trematode parasites communities in adult cockles sampled in2012 (Freitas et al. 2014) and in 2016 (present study) in six stations of the Ria de Aveirocoastal lagoon (E1, E3, E4, O1, O2, O3). The 30 % distance of dissimilarity threshold isdrawn.

Table 4Characterization of the affinity groups identified in the Ria de Aveiro interannual analysis. Each variable is represented by mean and standard deviation (SD). Bold values indicatesignificant differences in the ANOVA main test while superscript lower case letters indicate the homogenous groups identified by the Tukey post-hoc comparison of means test.

Affinity groups One-way ANOVAs results

Group A Group B Group C F pNr. of sampling sites 3 3 6

Parasites Trematoda abundance (mean± SD) 549.20± 849.46 17.09±7.41 30.21± 31.16 3.4 0.08Species richness (mean± SD) 6.00±1.00a 4.33±1.15a,b 2.83±0.75b 12.3

of the Ria de Aveiro increased in the last decades, with 250,020 in-habitants registered in 2001 and 353,688 in 2011 (National Censusreports, data available at https://www.ine.pt/) which resulted into anincrease of exposure to several anthropogenic-derived pressures such aspharmaceuticals (Calisto et al., 2011), metals and other elements con-tamination (Velez et al., 2015), endocrine disruptor compounds (Rochaet al., 2016) and non-point nitrogen sources (Lopes et al., 2017), amongothers, not included in the EcoQS evaluation. Besides, official datashowed that fisheries effort has been increasing with 127 licensedshellfish fishermen in 2006 and 208 in 2016 (National Fisheries Sta-tistics, data available at https://www.ine.pt/). Then, on the other hand,and considering trematodes as early warning indicators of deterioratingconditions (MacKenzie, 1999) there is a strong possibility of the Ria deAveiro being a less healthy ecosystem in 2016 than it was 4 years be-fore. This presumable ecosystem ecological quality loss, taking trema-tode community changes as indicator, could be already noticed whencomparing 2012 results (Freitas et al., 2014) to a former work per-formed in the same coastal lagoon (Russell-Pinto et al., 2006). Speciesrichness was the same (11 species) but prevalence decreased from 2006to 2012. However, it is important to refer that while Freitas et al.(2014) analysed cockles from 28 stations, Russell-Pinto et al. (2006)study was performed in a single sampling station which could not re-present the entire system. The 2016 lower trematode species richnessand prevalence comparing to 2012 data can also represent an evidenceof climate change phenomena occurring in the Ria de Aveiro coastallagoon. Trematode infection is dependent on temperature (and closelyrelated physico-chemical parameters) thresholds (de Montaudouinet al., 2016) and the data from the Portuguese Institute of the Sea andAtmosphere (IPMA, 2012, 2016) indicated that 2016 fall was the se-venth hottest since 1931 (0.8 °C higher comparing to average values)with mean temperature 1.3 °C higher than 2012 fall.

In conclusion, the present study showed a spatial and seasonalhomogeneity in terms of trematode parasites prevalence in cocklesliving in the Ria de Aveiro. However, authors described an influence ofabiotic factors and target host population density on the trematodespatial distribution. This work most important outcome was the effec-tive use of trematode communities as possible early warning indicatorsof global changes and deteriorating conditions occurring in an eco-system. This finding is comparable to what was demonstrated by Turner(1985) using trematodes infecting oysters as indicators of water qualityand Schmidt et al. (2003) using several parasite species infectingflounders as a valuable tool for the assessment of chemical con-tamination in a habitat, as also reviewed by MacKenzie et al. (1995)and MacKenzie (1999).

Acknowledgments

Luísa Magalhães benefited from PhD grant (reference: PD/BD/52570/2014) given by the National Funds through the PortugueseScience Foundation (FCT), supported by FSE and Programa OperacionalCapital Humano (POCH) and European Union. Rosa Freitas benefitedfrom a research position funded by the Integrated Programme of SR&TD“Smart Valorization of Endogenous Marine Biological Resources Undera Changing Climate” (reference: Centro-01–0145-FEDER-000018), co-funded by Centro 2020 program, Portugal 2020, European Union,through the European Regional Development Fund. This work wassupported by the research project COCKLES (EAPA_458/2016 COCKLESCo-Operation for Restoring CocKle SheLlfisheries & its Ecosystem-Services in the Atlantic Area). Thanks are also due, for the financialsupport to CESAM (UID/AMB/50017), to FCT/MEC through nationalfunds, and the co-funding by the FEDER, within the PT2020 PartnershipAgreement and Compete 2020. Authors are grateful to Anthony Moreirafor correcting the English and to anonymous reviewers for pertinentsuggestions and improvement of the manuscript.

References

Altman, I., Byers, J.E., 2014. Large-scale spatial variation in parasite communities in-fluenced by anthropogenic factors. Ecology 95, 1876–1887. http://dx.doi.org/10.1890/13-0509.1.

Babirat, C., Mouritsen, K.N., Poulin, R., 2004. Equal partnership: two trematode species,not one, manipulate the burrowing behaviour of the New Zealand cockle Austrovenusstutchburyi. J. Helminthol. 78, 195–199. http://dx.doi.org/10.1079/joh2003231.

Bartoli, P., Jousson, O., Russell-Pinto, F., 2000. The life cycle of Monorchis parvus(Digenea: monorchiidae) demonstrated by developmental and molecular data. J.Parasitol. 86, 479–489. http://dx.doi.org/10.1645/0022-3395(2000)086[0479:TLCOMP]2.0.CO;2.

Beukema, J.J., Dekker, R., 2006. Annual cockle Cerastoderma edule production in theWadden Sea usually fails to sustain both wintering birds and a commercial fishery.Mar. Ecol. Prog. Ser. 309, 189–204. http://dx.doi.org/10.3354/meps309189.

Blanar, C.A., Munkittrick, K.R., Houlahan, J., MacLatchy, D.L., Marcogliese, D.J., 2009.Pollution and parasitism in aquatic animals: a meta-analysis of effect size. Aquat.Toxicol. 93, 18–28. http://dx.doi.org/10.1016/j.aquatox.2009.03.002.

Boehs, G., Villalba, A., Ceuta, L.O., Luz, J.R., 2010. Parasites of three commerciallyexploited bivalve mollusc species of the estuarine region of the Cachoeira river(Ilhéus, Bahia, Brazil). J. Invertebr. Pathol. 103, 43–47. http://dx.doi.org/10.1016/j.jip.2009.10.008.

Born-Torrijos, A., Holzer, A.S., Raga, J.A., Kostadinova, A., 2014. Same host, same la-goon, different transmission pathways: effects of exogenous factors on larval emer-gence in two marine digenean parasites. Parasitol. Res. 113, 545–554. http://dx.doi.org/10.1007/s00436-013-3686-7.

Bowers, E.A., 1969. Cercaria Bucephalopsis haimeana (Lacaze-Duthiers, 1854) (Digenea:bucephalidae) in cockle, Cardium edule L. in south Wales. J. Nat. Hist. 3, 409–422.http://dx.doi.org/10.1080/00222936900770351.

Bowers, E.A., Bartoli, P., Russell-Pinto, F., James, B.L., 1996. The metacercariae of siblingspecies of Meiogymnophallus, including M. rebecqui comb. nov. (Digenea: gymno-phallidae), and their effects on closely related Cerastoderma host species (Mollusca:bivalvia). Parasitol. Res. 82, 505–510. http://dx.doi.org/10.1007/s004360050153.

Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostak, A.W., 1997. Parasitology meets ecology onits own terms: margolis et al revisited. J. Parasitol. 83, 575–583. http://dx.doi.org/10.2307/3284227.

Calisto, V., Bahlmann, A., Schneider, R.F., Esteves, V.I., 2011. Application of an ELISA tothe quantification of carbamazepine in ground, surface and wastewaters and vali-dation with LC–MS/MS. Chemosphere 84, 1708–1715. http://dx.doi.org/10.1016/j.chemosphere.2011.04.072.

Campbell, N., Cross, M.A., Chubb, J.C., Cunningham, C.W., Hatfield, E.C., MacKenzie, K.,2007. Spatial and temporal variations in parasite prevalence and infracommunitystructure in herring (Clupea harengus L.) caught to the west of the British Isles and inthe North and baltic Seas: implications for fisheries science. J. Helminthol. 81,137–146. http://dx.doi.org/10.1017/s0022149x07747454.

Carballal, M.J., Iglesias, D., Santamarina, J., Ferro-Soto, B., Villalba, A., 2001. Parasitesand pathologic conditions of the cockle Cerastoderma edule populations of the coast ofGalicia (NW Spain). J. Invertebr. Pathol. 78, 87–97. http://dx.doi.org/10.1006/jipa.2001.5049.

de Montaudouin, X., Binias, C., Lassalle, G., 2012. Assessing parasite community structurein cockles Cerastoderma edule at various spatio-temporal scales. Estuar. Coast. ShelfSci. 110, 54–60. http://dx.doi.org/10.1016/j.ecss.2012.02.005.

de Montaudouin, X., Blanchet, H., Desclaux-Marchand, C., Lavesque, N., Bachelet, G.,2016. Cockle infection by Himasthla quissetensis - I. From cercariae emergence tometacercariae infection. J. Sea Res. 113, 99–107. http://dx.doi.org/10.1016/j.seares.2015.02.008.

de Montaudouin, X., Kisielewski, I., Bachelet, G., Desclaux, C., 2000. A census of mac-roparasites in an intertidal bivalve community, Arcachon Bay, France. Oceanol. Acta23, 453–468. http://dx.doi.org/10.1016/s0399-1784(00)00138-9.

de Montaudouin, X., Lanceleur, L., 2011. Distribution of parasites in their second inter-mediate host, the cockle Cerastoderma edule: community heterogeneity and spatialscale. Mar. Ecol. Prog. Ser. 428, 187–199. http://dx.doi.org/10.3354/meps09072.

de Montaudouin, X., Paul-Pont, I., Lambert, C., Gonzalez, P., Raymond, N., Jude, F.,Legeay, A., Baudrimont, M., Dang, C., Le Grand, F., Le Goic, N., Bourasseau, L.,Paillard, C., 2010. Bivalve population health: multistress to identify hot spots. Mar.Pollut. Bull. 60, 1307–1318. http://dx.doi.org/10.1016/j.marpolbul.2010.03.011.

de Montaudouin, X., Thieltges, D.W., Gam, M., Krakau, M., Pina, S., Bazairi, H.,Dabouineau, L., Russell-Pinto, F., Jensen, K.T., 2009. Digenean trematode species inthe cockle Cerastoderma edule: identification key and distribution along the north-eastern Atlantic shoreline. J. Mar. Biol. Assoc. U. Kingd. 89, 543–556. http://dx.doi.org/10.1017/s0025315409003130.

de Montaudouin, X., Wegeberg, A.M., Jensen, K.T., Sauriau, P.G., 1998. Infection char-acteristics of Himasthla elongata cercariae in cockles as a function of water current.Dis. Aquat. Org. 34, 63–70. http://dx.doi.org/10.3354/dao034063.

Desclaux, C., de Montaudouin, X., Bachelet, G., 2004. Cockle Cerastoderma edule popu-lation mortality: role of the digenean parasite Himasthla quissetensis. Mar. Ecol. Prog.Ser. 279, 141–150. http://dx.doi.org/10.3354/meps279141.

Desclaux, C., Russell-Pinto, F., de Montaudouin, X., Bachelet, G., 2006. First record anddescription of Metacercariae of Curtuteria arguinae n. sp. (Digenea: echinostoma-tidae), parasite of cockles Cerastoderma edule (Mollusca: bivalvia) in Arcachon Bay,France. J. Parasitol. 92, 578–587. http://dx.doi.org/10.1645/ge-3512.1.

Dias, J.M., Lopes, J.F., Dekeyser, I., 2000. Tidal propagation in Ria de Aveiro lagoon,Portugal. Phys. Chem. Earth Part B-Hydrol. Oceans Atmos. 25, 369–374. http://dx.doi.org/10.1016/s1464-1909(00)00028-9.

Dobson, A., Lafferty, K.D., Kuris, A.M., Hechinger, R.F., Jetz, W., 2008. Homage to

L. Magalhães et al. Environmental Research 164 (2018) 114–123

121

https://www.ine.pt/https://www.ine.pt/http://dx.doi.org/10.1890/13-0509.1http://dx.doi.org/10.1890/13-0509.1http://dx.doi.org/10.1079/joh2003231http://dx.doi.org/10.1645/0022-3395(2000)086[0479:TLCOMP]2.0.CO;2http://dx.doi.org/10.1645/0022-3395(2000)086[0479:TLCOMP]2.0.CO;2http://dx.doi.org/10.3354/meps309189http://dx.doi.org/10.1016/j.aquatox.2009.03.002http://dx.doi.org/10.1016/j.jip.2009.10.008http://dx.doi.org/10.1016/j.jip.2009.10.008http://dx.doi.org/10.1007/s00436-013-3686-7http://dx.doi.org/10.1007/s00436-013-3686-7http://dx.doi.org/10.1080/00222936900770351http://dx.doi.org/10.1007/s004360050153http://dx.doi.org/10.2307/3284227http://dx.doi.org/10.2307/3284227http://dx.doi.org/10.1016/j.chemosphere.2011.04.072http://dx.doi.org/10.1016/j.chemosphere.2011.04.072http://dx.doi.org/10.1017/s0022149x07747454http://dx.doi.org/10.1006/jipa.2001.5049http://dx.doi.org/10.1006/jipa.2001.5049http://dx.doi.org/10.1016/j.ecss.2012.02.005http://dx.doi.org/10.1016/j.seares.2015.02.008http://dx.doi.org/10.1016/j.seares.2015.02.008http://dx.doi.org/10.1016/s0399-1784(00)00138-9http://dx.doi.org/10.3354/meps09072http://dx.doi.org/10.1016/j.marpolbul.2010.03.011http://dx.doi.org/10.1017/s0025315409003130http://dx.doi.org/10.1017/s0025315409003130http://dx.doi.org/10.3354/dao034063http://dx.doi.org/10.3354/meps279141http://dx.doi.org/10.1645/ge-3512.1http://dx.doi.org/10.1016/s1464-1909(00)00028-9http://dx.doi.org/10.1016/s1464-1909(00)00028-9

Linnaeus: how many parasites? How many hosts? Proc. Natl. Acad. Sci. USA 105,11482–11489. http://dx.doi.org/10.1073/pnas.0803232105.

Dzikowski, R., Diamant, A., Paperna, I., 2003. Trematode metacercariae of fishes assentinels for a changing limnological environment. Dis. Aquat. Org. 55, 145–150.http://dx.doi.org/10.3354/dao055145.

Esch, G.W., Curtis, L.A., Barger, M.A., 2001. A perspective on the ecology of trematodecommunities in snails. Parasitology 123, S57–S75. http://dx.doi.org/10.1017/S0031182001007697.

Esch, G.W., Fernandez, J.C., 1994. Snail-trematode interactions and parasite communitydynamics in aquatic systems - a review. Am. Midl. Nat. 131, 209–237. http://dx.doi.org/10.2307/2426248.

Feis, M.E., Thieltges, D.W., Olsen, J.L., de Montaudouin, X., Jensen, K.T., Bazairi, H.,Culloty, S.C., Luttikhuizen, P.C., 2015. The most vagile host as the main determinantof population connectivity in marine macroparasites. Mar. Ecol. Prog. Ser. 520,85–99. http://dx.doi.org/10.3354/meps11096.

Fermer, J., Culloty, S.C., Kelly, T.C., O'Riordan, R.M., 2009. Intrapopulational distribu-tion of Meiogymnophallus minutus (Digenea, Gymnophallidae) infections in its firstand second intermediate host. Parasitol. Res. 105, 1231–1238. http://dx.doi.org/10.1007/s00436-009-1545-3.

Fermer, J., Culloty, S.C., Kelly, T.C., O'Riordan, R.M., 2010. Temporal variation ofMeiogymnophallus minutus infections in the first and second intermediate host. J.Helminthol. 84, 362–368. http://dx.doi.org/10.1017/s0022149x09990708.

Fredensborg, B.L., Mouritsen, K.N., Poulin, R., 2006. Relating bird host distribution andspatial heterogeneity in trematode infections in an intertidal snail-from small to largescale. Mar. Biol. 149, 275–283. http://dx.doi.org/10.1007/s00227-005-0184-1.

Freitas, R., Martins, R., Campino, B., Figueira, E., Soares, A.M.V.M., Montaudouin, X.,2014. Trematode communities in cockles (Cerastoderma edule) of the Ria de Aveiro(Portugal): Influence of inorganic contamination. Mar. Pollut. Bull. 82, 117–126.http://dx.doi.org/10.1016/j.marpolbul.2014.03.012.

Gam, M., Bazairi, H., Jensen, K.T., De Montaudouin, X., 2008. Metazoan parasites in anintermediate host population near its southern border: the common cockle(Cerastoderma edule) and its trematodes in a Moroccan coastal lagoon (Merja Zerga).J. Mar. Biol. Assoc. U. Kingd. 88, 357–364. http://dx.doi.org/10.1017/s0025315408000611.

Gam, M., De Montaudouin, X., Bazairi, H., 2009. Do trematode parasites affect cockle(Cerastoderma edule) secondary production and elimination? J. Mar. Biol. Assoc. U.Kingd. 89, 1395–1402. http://dx.doi.org/10.1017/s0025315409000599.

Goater, C.P., 1993. Population biology of Meiogymnophallus minutus (Trematoda,Gymnophallidae) in cockles from the Exe Estuary. J. Mar. Biol. Assoc. U. Kingd. 73,163–177.

Goedknegt, M.A., Feis, M.E., Wegner, K.M., Luttikhuizen, P.C., Buschbaum, C.,Camphuysen, K.C.J., Van Der Meer, J., Thieltges, D.W., 2016. Parasites and marineinvasions: ecological and evolutionary perspectives. J. Sea Res. 113, 11–27. http://dx.doi.org/10.1016/j.seares.2015.12.003.

Hechinger, R.F., Lafferty, K.D., Huspeni, T.C., Brooks, A.J., Kuris, A.M., 2006. Canparasites be indicators of free-living diversity? Relationships between species rich-ness and the abundance of larval trematodes and of local benthos and fishes.Oecologia 151, 82–92. http://dx.doi.org/10.1007/s00442-006-0568-z.

Honkoop, P.J.C., Berghuis, E.M., Holthuijsen, S., Lavaleye, M.S.S., Piersma, T., 2008.Molluscan assemblages of seagrass-covered and bare intertidal flats on the Bancd'Arguin, Mauritania, in relation to characteristics of sediment and organic matter. J.Sea Res. 60, 235–243. http://dx.doi.org/10.1016/j.seares.2008.07.005.

Hudson, P.J., Dobson, A.P., Lafferty, K.D., 2006. Is a healthy ecosystem one that is rich inparasites? Trends Ecol. Evol. 21, 381–385. http://dx.doi.org/10.1016/j.tree.2006.04.007.

IPMA, 2012. Boletim Climatológico Sazonal - outono 2012. Technical report available at〈https://www.ipma.pt/resources.www/docs/im.publicacoes/edicoes.online/20140428/RCIUABQhNWvVRVtRRjRa/cli_20120901_20121130_pcl_sz_co_pt.pdf〉(Accessed 13 October 2017).

IPMA, 2016. Resumo Climatológico – outono 2016, Portugal Continental. Technical re-port available at 〈https://www.ipma.pt/resources.www/docs/im.publicacoes/edicoes.online/20161209/ukUbZKPgHKPZfKnYwCxK/cli_20160901_20161130_pcl_sz_co_pt.pdf〉 (Accessed 13 October 2017).

Kesting, V., Gollasch, S., Zander, C.D., 1996. Parasite communities of the Schlei Fjord(Baltic coast of northern Germany). Helgol. Meeresunters. 50, 477–496. http://dx.doi.org/10.1007/bf02367162.

Koprivnikar, J., Ellis, D., Shim, K.C., Forbes, M.R., 2014. Effects of temperature andsalinity on emergence of Gynaecotyla adunca cercariae from the intertidal gastropodIlyanassa obsoleta. J. Parasitol. 100, 242–245. http://dx.doi.org/10.1645/13-331.1.

Koprivnikar, J., Poulin, R., 2009. Effects of temperature, salinity, and water level on theemergence of marine cercariae. Parasitol. Res. 10, 957–965. http://dx.doi.org/10.1007/s00436-009-1477-y.

Kristensen, E., Andersen, F.O., 1987. Determination of organic-carbon in marine sedi-ments - a comparison of 2 CHN-analyzer methods. J. Exp. Mar. Biol. Ecol. 109, 15–23.http://dx.doi.org/10.1016/0022-0981(87)90182-1.

Laranjeiro, F., Sanchez-Marin, P., Galante-Oliveira, S., Barroso, C., 2015. Tributyltinpollution biomonitoring under the Water Framework Directive: proposal of a multi-species tool to assess the ecological quality status of EU water bodies. Ecol. Indic. 57,525–535. http://dx.doi.org/10.1016/j.ecolind.2015.04.039.

Lauckner, G., 1983. Diseases of mollusca: bivalvia. In: Kinne, O. (Ed.), Diseases of MarineAnimals. Hamburg, Biologische Helgoland, Germany, pp. 477–879.

Lei, F., Poulin, R., 2011. Effects of salinity on multiplication and transmission of an in-tertidal trematode parasite. Mar. Biol. 158, 995–1003. http://dx.doi.org/10.1007/s00227-011-1625-7.

Lillebø, A.I., Ameixa, O.M.C.C., Sousa, L.P., Sousa, A.I., Soares, J.A., Dolbeth, M., Alves,F.L., 2015. The physio-geographical background and ecology of Ria de Aveiro. In:

Lillebø, A.I., Stålnacke, P., Gooch, G.D. (Eds.), Coastal Lagoons in Europe: IntegratedWater Resource Strategies. International Water Association (IWA), London, UK, pp.21–28.

Lopes, M.L., Marques, B., Dias, J.M., Soares, A., Lillebø, A.I., 2017. Challenges for theWFD second management cycle after the implementation of a regional multi-muni-cipality sanitation system in a coastal lagoon (Ria de Aveiro, Portugal). Sci. TotalEnviron. 586, 215–225. http://dx.doi.org/10.1016/j.scitotenv2017.01.205.

MacKenzie, K., 1999. Parasites as pollution indicators in marine ecosystems: a proposedearly warning system. Mar. Pollut. Bull. 38, 955–959. http://dx.doi.org/10.1016/s0025-326x(99)00100-9.

MacKenzie, K., Williams, H.H., Williams, B., McVicar, A.H., Siddall, R., 1995. Parasites asindicators of water quality and the potential use of Helminth Transmission in MarinePollution Studies. Adv. Parasitol. 35, 85–144. http://dx.doi.org/10.1016/s0065-308x(08)60070-6.

Magalhães, L., Freitas, R., Dairain, A., Montaudouin, X., 2017. Can host density attenuateparasitism? J. Mar. Biol. Assoc. U. Kingd. 97, 497–505. http://dx.doi.org/10.1017/S0025315416001107.

MAMAOT/ARHCentro, 2012. Plano de Gestão das Bacias Hidrográficas dos rios Vouga,Mondego e Lis Integrados na Região Hidrográfica 4, Parte 2 – Caracterização Geral eEspecífica, 1.4.1 – Caracterização das Massas de Águas Superficiais. Ministério daAgricultura, Mar, Ambiente e Ordenamento do Território/Administração da RegiãoHidrográfica do Centro I.P. (official report regarding the implementation of the WFD,in Portuguese).

Marcogliese, D.J., 2004. Parasites: small players with crucial roles in the ecologicaltheater. EcoHealth 1, 151–164. http://dx.doi.org/10.1007/s10393-004-0028-3.

Marín, A., Lloret, J., Velasco, J., Bello, C., Lillebø, A.I., Sousa, A.I., Soares, A.M.V.M.,Tuchkovenko, Y., Tuchkovenko, O., Warzocha, J., Kornijów, R., Gromisz, S., Drgas,A., Szymanek, L., Margoński, P., 2015. Lagoons response using key bio-indicators andimplications on ecological status (WFD). In: Lillebø, A.I., Stålnacke, P., Gooch, G.D.(Eds.), Coastal Lagoons in Europe: Integrated Water Resource Strategies.International Water Association (IWA), London, UK, pp. 167–178.

McLusky, D.S., 1999. Estuarine benthic ecology: a European perspective. Aust. J. Ecol. 24,302–311. http://dx.doi.org/10.1046/j.1442-9993.1999.00983.x.

Morgan, E., O'Riordan, R.M., Culloty, S.C., 2013. Climate change impacts on potentialrecruitment in an ecosystem engineer. Ecol. Evol. 3, 581–594. http://dx.doi.org/10.1002/ece3.419.

Morley, N.J., Lewis, J.W., 2007. Anthropogenic pressure on a molluscan–trematodecommunity over a long-term period in the Basingstoke Canal, UK, and its implicationsfor ecosystem health. EcoHealth 3, 269–280 (doi 0.1007/s10393-006-0058-0).

Nunes, M., Coelho, J.P., Cardoso, P.G., Pereira, M.E., Duarte, A.C., Pardal, M.A., 2008.The macrobenthic community along a mercury contamination in a temperate es-tuarine system (Ria de Aveiro, Portugal). Sci. Total Environ. 405, 186–194. http://dx.doi.org/10.1016/j.scitotenv.2008.07.009.

Pereira F., Maia F., Gaspar M., 2014. Gepeto project, Case study: Bivalve harvesting in theRia de Aveiro. Distribution, abundance and biomass of bivalves with higher com-mercial interest in the Ria de Aveiro. Technical report available at 〈http://www.repositorio.ieo.es/e-ieo/bitstream/handle/10508/9665/GEPETO_Activity-2_final-report_WP5.pdf?Sequence=1&isAllowed=y〉 (Accessed 4 September 2017).

Philippart, C.J.M., Beukema, J.J., Cadee, G.C., Dekker, R., Goedhart, P.W., van Iperen,J.M., Leopold, M.F., Herman, P.M.J., 2007. Impacts of nutrient reduction on coastalcommunities. Ecosystems 10, 95–118. http://dx.doi.org/10.1007/s10021-006-9006-7.

Quintino, V., Rodrigues, A.M., Gentil, F., 1989. Assessment of macrozoobenthic com-munities in the lagoon of Óbidos, western coast of Portugal. Sci. Mar. 53, 645–654.

Rakotomalala, C., Grangere, K., Ubertini, M., Foret, M., Orvain, F., 2015. Modelling theeffect of Cerastoderma edule bioturbation on microphytobenthos resuspension to-wards the planktonic food web of estuarine ecosystem. Ecol. Model. 316, 155–167.http://dx.doi.org/10.1016/j.ecolmodel.2015.08.010.

Rocha, M.J., Cruzeiro, C., Reis, M., Pardal, M.A., Rocha, E., 2016. Pollution by endocrinedisruptors in a southwest European temperate coastal lagoon (Ria de Aveiro,Portugal). Environ. Monit. Assess. 188. http://dx.doi.org/10.1007/s10661-016-5114-9.

Rodrigues, A.M., Quintino, V., Sampaio, L., Freitas, R., Neves, R., 2011. Benthic biodi-versity patterns in Ria de Aveiro, Western Portugal: environmental-biological re-lationships. Estuar. Coast. Shelf Sci. 95, 338–348. http://dx.doi.org/10.1016/j.ecss.2011.05.019.

Russell-Pinto, F., 1990. Differences in infestation, intensity and prevalence of hinge andmantle margin Meiogymnophallus minutus metacercariae (Gymnophallidae) inCerastoderma edule (Bivalvia): possible species coexistence in Ria de Aveiro. J.Parasitol. 76, 653–659. http://dx.doi.org/10.2307/3282978.

Russell-Pinto, F., Goncalves, J.F., Bowers, E., 2006. Digenean larvae parasitizingCerastoderma edule (Bivalvia) and Nassarius reticulatus (Gastropoda) from Ria deAveiro, Portugal. J. Parasitol. 92, 319–332. http://dx.doi.org/10.1645/ge-3510.1.

Schmidt, V., Zander, S., Korting, W., Broeg, K., von Westernhagen, H., Dizer, H., Hansen,P.D., Skouras, A., Steinhagen, D., 2003. Parasites of flounder (Platichthys flesus L.)from the German Bight, North Sea, and their potential use in biological effectsmonitoring - C. Pollution effects on the parasite community and a comparison tobiomarker responses. Helgol. Mar. Res. 57, 262–271. http://dx.doi.org/10.1007/s10152-003-0159-x.

Soldánová, M., Kuris, A.M., Scholz, T., Lafferty, K.D., 2012. The role of spatial andtemporal heterogeneity and competition in structuring trematode communities in thegreat pond snail, Lymnaea stagnalis (L.). J. Parasitol. 98, 460–471. http://dx.doi.org/10.1645/ge-2964.1.

Sousa, R., Gutierrez, J.L., Aldridge, D.C., 2009. Non-indigenous invasive bivalves asecosystem engineers. Biol. Invasions 11, 2367–2385. http://dx.doi.org/10.1007/s10530-009-9422-7.

L. Magalhães et al. Environmental Research 164 (2018) 114–123

122

http://dx.doi.org/10.1073/pnas.0803232105http://dx.doi.org/10.3354/dao055145http://dx.doi.org/10.1017/S0031182001007697http://dx.doi.org/10.1017/S0031182001007697http://dx.doi.org/10.2307/2426248http://dx.doi.org/10.2307/2426248http://dx.doi.org/10.3354/meps11096http://dx.doi.org/10.1007/s00436-009-1545-3http://dx.doi.org/10.1007/s00436-009-1545-3http://dx.doi.org/10.1017/s0022149x09990708http://dx.doi.org/10.1007/s00227-005-0184-1http://dx.doi.org/10.1016/j.marpolbul.2014.03.012http://dx.doi.org/10.1017/s0025315408000611http://dx.doi.org/10.1017/s0025315408000611http://dx.doi.org/10.1017/s0025315409000599http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref35http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref35http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref35http://dx.doi.org/10.1016/j.seares.2015.12.003http://dx.doi.org/10.1016/j.seares.2015.12.003http://dx.doi.org/10.1007/s00442-006-0568-zhttp://dx.doi.org/10.1016/j.seares.2008.07.005http://dx.doi.org/10.1016/j.tree.2006.04.007http://dx.doi.org/10.1016/j.tree.2006.04.007https://www.ipma.pt/resources.www/docs/im.publicacoes/edicoes.online/20140428/RCIUABQhNWvVRVtRRjRa/cli_20120901_20121130_pcl_sz_co_pt.pdfhttps://www.ipma.pt/resources.www/docs/im.publicacoes/edicoes.online/20140428/RCIUABQhNWvVRVtRRjRa/cli_20120901_20121130_pcl_sz_co_pt.pdfhttps://www.ipma.pt/resources.www/docs/im.publicacoes/edicoes.online/20161209/ukUbZKPgHKPZfKnYwCxK/cli_20160901_20161130_pcl_sz_co_pt.pdfhttps://www.ipma.pt/resources.www/docs/im.publicacoes/edicoes.online/20161209/ukUbZKPgHKPZfKnYwCxK/cli_20160901_20161130_pcl_sz_co_pt.pdfhttps://www.ipma.pt/resources.www/docs/im.publicacoes/edicoes.online/20161209/ukUbZKPgHKPZfKnYwCxK/cli_20160901_20161130_pcl_sz_co_pt.pdfhttp://dx.doi.org/10.1007/bf02367162http://dx.doi.org/10.1007/bf02367162http://dx.doi.org/10.1645/13-331.1http://dx.doi.org/10.1007/s00436-009-1477-yhttp://dx.doi.org/10.1007/s00436-009-1477-yhttp://dx.doi.org/10.1016/0022-0981(87)90182-1http://dx.doi.org/10.1016/j.ecolind.2015.04.039http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref45http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref45http://dx.doi.org/10.1007/s00227-011-1625-7http://dx.doi.org/10.1007/s00227-011-1625-7http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref47http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref47http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref47http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref47http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref47http://dx.doi.org/10.1016/j.scitotenv2017.01.205http://dx.doi.org/10.1016/s0025-326x(99)00100-9http://dx.doi.org/10.1016/s0025-326x(99)00100-9http://dx.doi.org/10.1016/s0065-308x(08)60070-6http://dx.doi.org/10.1016/s0065-308x(08)60070-6http://dx.doi.org/10.1017/S0025315416001107http://dx.doi.org/10.1017/S0025315416001107http://dx.doi.org/10.1007/s10393-004-0028-3http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref53http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref53http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref53http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref53http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref53http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref53http://dx.doi.org/10.1046/j.1442-9993.1999.00983.xhttp://dx.doi.org/10.1002/ece3.419http://dx.doi.org/10.1002/ece3.419http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref56http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref56http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref56http://dx.doi.org/10.1016/j.scitotenv.2008.07.009http://dx.doi.org/10.1016/j.scitotenv.2008.07.009http://www.repositorio.ieo.es/e-ieo/bitstream/handle/10508/9665/GEPETO_Activity-2_final-report_WP5.pdf?Sequence=1�&�isAllowed=yhttp://www.repositorio.ieo.es/e-ieo/bitstream/handle/10508/9665/GEPETO_Activity-2_final-report_WP5.pdf?Sequence=1�&�isAllowed=yhttp://www.repositorio.ieo.es/e-ieo/bitstream/handle/10508/9665/GEPETO_Activity-2_final-report_WP5.pdf?Sequence=1�&�isAllowed=yhttp://dx.doi.org/10.1007/s10021-006-9006-7http://dx.doi.org/10.1007/s10021-006-9006-7http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref59http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref59http://dx.doi.org/10.1016/j.ecolmodel.2015.08.010http://dx.doi.org/10.1007/s10661-016-5114-9http://dx.doi.org/10.1007/s10661-016-5114-9http://dx.doi.org/10.1016/j.ecss.2011.05.019http://dx.doi.org/10.1016/j.ecss.2011.05.019http://dx.doi.org/10.2307/3282978http://dx.doi.org/10.1645/ge-3510.1http://dx.doi.org/10.1007/s10152-003-0159-xhttp://dx.doi.org/10.1007/s10152-003-0159-xhttp://dx.doi.org/10.1645/ge-2964.1http://dx.doi.org/10.1645/ge-2964.1http://dx.doi.org/10.1007/s10530-009-9422-7http://dx.doi.org/10.1007/s10530-009-9422-7

Studer, A., Poulin, R., 2012. Effects of salinity on an intertidal host-parasite system: is theparasite more sensitive than its host? J. Exp. Mar. Biol. Ecol. 412, 110–116. http://dx.doi.org/10.1016/j.jembe.2011.11.008.

Studer, A., Poulin, R., 2013. Cercarial survival in an intertidal trematode: a multifactorialexperiment with temperature, salinity and ultraviolet radiation. Parasitol. Res. 112,243–249. http://dx.doi.org/10.1007/s00436-012-3131-3.

Tebble N., 1966. British Bivalve Seashells. A Handbook for Identification. Trustees of theBritish Museum (Natural History), London.

Thieltges, D.W., Reise, K., 2006. Metazoan parasites in intertidal cockles Cerastodermaedule from the northern Wadden Sea. J. Sea Res. 56, 284–293. http://dx.doi.org/10.1016/j.seares.2006.06.002.

Thieltges, D.W., Reise, K., 2007. Spatial heterogeneity in parasite infections at differentspatial scales in an intertidal bivalve. Oecologia 150, 569–581. http://dx.doi.org/10.1007/s00442-006-0557-2.

Thieltges, D.W., Rick, J., 2006. Effect of temperature on emergence, survival and

infectivity of cercariae of the marine trematode Renicola roscovita (Digenea:Renicolidae). Dis. Aquat. Org. 73, 63–68.

Turner, H.M., 1985. Parasites of eastern oysters from subtidal reefs in a louisiana estuarywith a note on their use as indicators of water quality. Estuaries 8, 323–325. http://dx.doi.org/10.2307/1351493.

Vaz, N., Dias, J.M., Leitao, P., Martins, W., 2005. Horizontal patterns of water tempera-ture and salinity in an estuarine tidal channel: ria de Aveiro. Ocean Dyn. 55,416–429. http://dx.doi.org/10.1007/s10236-005-0015-4.

Velez, C., Figueira, E., Soares, A., Freitas, R., 2015. Spatial distribution and bioaccumu-lation patterns in three clam populations from a low contaminated ecosystem. Estuar.Coast. Shelf Sci. 155, 114–125. http://dx.doi.org/10.1016/j.ecss.2015.01.004.

Zander, C.D., 2005. Four-year monitoring of parasite communities in gobiid fishes of thesouthwest Baltic - III. Parasite species diversity and applicability of monitoring.Parasitol. Res. 95, 136–144. http://dx.doi.org/10.1007/s00436-004-1252-z.

L. Magalhães et al. Environmental Research 164 (2018) 114–123

123

http://dx.doi.org/10.1016/j.jembe.2011.11.008http://dx.doi.org/10.1016/j.jembe.2011.11.008http://dx.doi.org/10.1007/s00436-012-3131-3http://dx.doi.org/10.1016/j.seares.2006.06.002http://dx.doi.org/10.1016/j.seares.2006.06.002http://dx.doi.org/10.1007/s00442-006-0557-2http://dx.doi.org/10.1007/s00442-006-0557-2http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref72http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref72http://refhub.elsevier.com/S0013-9351(18)30086-0/sbref72http://dx.doi.org/10.2307/1351493http://dx.doi.org/10.2307/1351493http://dx.doi.org/10.1007/s10236-005-0015-4http://dx.doi.org/10.1016/j.ecss.2015.01.004http://dx.doi.org/10.1007/s00436-004-1252-z

Spatio-temporal variation of trematode parasites community in Cerastoderma edule cockles from Ria de Aveiro (Portugal)IntroductionMaterial and methodsStudy area descriptionSampling procedure and parasite identificationSediment analysisData analysis

ResultsTrematode community: spatial and seasonal patternsTrematode community: 2012–2016 comparison

DiscussionAcknowledgmentsReferences