Trematode communities in cockles (Cerastoderma edule) of the Ria de Aveiro (Portugal): Influence of inorganic contamination

Marine Pollution Bulletin 82 (2014) 117–126

Contents lists available at ScienceDirect

Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Trematode communities in cockles (Cerastoderma edule) of the Ria de Aveiro (Portugal): Influence of inorganic contamination R. Freitas a,⇑, R. Martins a, B. Campino b, E. Figueira a, A.M.V.M. Soares a, X. Montaudouin c a

Departamento de Biologia & CESAM, Universidade de Aveiro, 3810-193 Aveiro, Portugal Departamento de Biologia, Universidade de Aveiro, 3810-193 Aveiro, Portugal c Université de Bordeaux, EPOC, UMR 5805 CNRS, 2, rue du Pr Jolyet, F-33120 Arcachon, France b

a r t i c l e

i n f o

Keywords: Western Iberia Parasitism Trematodes Cerastoderma edule Ecological assessment Contaminants

a b s t r a c t This work aims to assess the trematode parasites infecting the edible cockle Cerastoderma edule, collected in the Ria de Aveiro lagoon, one of the most relevant biodiversity hotspots of the Western Iberia, and evaluate the relationship between the observed patterns and environmental descriptors. A total of 11 of the 16 trematode species known to infect C. edule were identified, including Himasthla continua and Psilostomum brevicolle as new occurrences in this lagoon. Parvatrema minutum was the most abundant and dominant species. Species richness and prevalence were high. The relationship between trematode species abundance, intensity and prevalence, and also environmental variables, showed that most parasites preferred muddy sand areas with euhaline conditions in opposition to areas with contamination and/or distant from the lagoon entrance. This study highlighted the good ecological status of the ecosystem and the transitional biogeographic characteristics of the western Portuguese coast where northern and subtropical faunas can coexist. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction In coastal ecosystems, trematodes are frequent macroparasites of many faunal species (Mouritsen and Poulin, 2002; de Montaudouin et al., 2009). They are characterized by a complex life cycle that usually involves three free-living host species. The parasite sexually reproduces inside a vertebrate host, like fishes or birds. Eggs are emitted in the ecosystem and develop into miracidium larvae that will infect a mollusk as first intermediate host. In the host’s gonad and/or digestive gland, miracidium transforms into sporocysts/rediae where cercariae larvae are produced (asexual reproduction) before being emitted in the water mass. These cercariae have few hours to penetrate and infect the second intermediate host, where they become metacercariae. The cycle is closed when the final host predates the second intermediate host (e.g. Niewiadomska and Pojman´ska, 2011). Thus, there are three important trematode-related processes that are useful for the knowledge of the ecosystem: (1) each trematode parasite species requires three host species to accomplish its lifecycle. Consequently, the presence of many parasite species (i.e. high trematode parasite ⇑ Corresponding author. Address: Departamento de Biologia & CESAM, Universidade de Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal. Tel.: +351 234370782; fax: +351 234372587. E-mail address: [email protected] (R. Freitas). http://dx.doi.org/10.1016/j.marpolbul.2014.03.012 0025-326X/Ó 2014 Elsevier Ltd. All rights reserved.

species richness) is often considered as a proxy of a high general diversity (Hudson et al., 2006; Hechinger et al., 2007,2008) and can even actively boost free-living diversity (Mouritsen et al., 2005); (2) trematodes infect some target species and may impact their host’s population dynamics. First intermediate host´s infection is considered severe and may impact host populations, when the prevalence is high (Jensen and Mouritsen, 1992; Jonsson and André, 1992; Thieltges and Reise, 2006; de Montaudouin et al., 2012). As for second intermediate host, the population is affected when individuals undergo a high metacercariae abundance (Desclaux et al., 2004; Gam et al., 2009); (3) trematode life cycle includes two free-swimming stages (i.e. miracidium and cercariae stages). These larvae may be sensitive to pollution (Morley et al., 2003; Pietrock and Marcogliese, 2003) and a lack of infection can be interpreted as the presence of contaminated conditions (MacKenzie et al., 1995; Lafferty, 1997; MacKenzie, 1999). Finally the interpretation of the presence of trematode as an indicator of environmental fitness can be tricky, with contradictory results between effects at the population level and at the community level (Do et al., 2011). In coastal environments, bivalves are often ecologically and economically important. The edible cockle Cerastoderma edule, is a common inhabitant of bays and estuaries that can be found along the Atlantic shores between Barents Sea and Mauritania. In these environments, cockles are known to undergo high trematode infec-

118

R. Freitas et al. / Marine Pollution Bulletin 82 (2014) 117–126

tion (de Montaudouin et al., 2000, 2009; Fermer et al., 2011; Thieltges and Reise, 2006) and/or metal contamination (Cheggour et al., 2001; Lobo et al., 2010; Freitas et al., 2012). Experimentally, PaulPont et al. (2010) demonstrated that metal contamination (cadmium) did not exert any modification of infection efficiency by trematodes (Himasthla elongata). Conversely, infection by H. elongata can increase the accumulation of cadmium (Baudrimont and de Montaudouin, 2007). Finally, these studies concerning host-parasite interactions within a given environment highlight many knock-on consequences on the ecosystem, with many complex feed-backs. Thus, it becomes difficult to predict the dynamics of the co-occurrence of trematode parasites and pollutants in the environment. Former studies in other coastal systems monitored metal contamination and parasite load separately. Here, in order to test the hypothesis that a polluted area is also an area deprived of parasitic trematodes, we performed a large survey along the Ria de Aveiro lagoon (Portugal), comparing contamination and trematode infection of the cockle metapopulation. This coastal ecosystem forms an ideal study area to conduct such an investigation since cockles represent the most commercially valuable resource comprising more than 90% of the shellfish harvested (250 licensed professionals) in the lagoon (DGPA, 2011), and also because cockles spread over most of the euhaline area of the Ria de Aveiro, in unpolluted areas as well as in sites near industrial activity where metal contamination is higher (Figueira et al., 2011). Thus the present study aims to: (1) provide the first extensive survey of trematodes in the Ria de Aveiro; (2) correlate trematode intensity with environmental parameters, with a special focus on metal contamination. 2. Material and methods 2.1. Study area Ria de Aveiro is a shallow coastal lagoon, with 45 km long and 10 km wide, located on the Northwestern coast of Portugal and connected to the sea by a single channel (Fig. 1). It comprises four main channels (Mira, S. Jacinto/Ovar, Ílhavo and Espinheiro) and extensive intertidal zones, covering a minimum area of approximately 66 km2 at low spring tide and a maximum of 83 km2 at a high spring tide (Dias, 2001). In general, water depth is less than 3 m (Dias et al., 1999). The hydrology is essentially dominated by tidal forcing, responsible for a strong mixture of the water masses. The major sources of freshwater inflow are the Vouga River (50 m3/ s) and the Antuã River (5 m3/s) (Dias et al., 1999). The vertical salinity and temperature gradients are minimal compared to the longitudinal gradients (Dias et al., 1999). Mira and Ílhavo channels present salinities ranging from 30 to 38 near the mouth and 1–10 at the head while in S. Jacinto and Espinheiro channels salinity can range from 0 to 35 depending on the freshwater input (Dias et al., 1999; Dias, 2001; Vaz et al., 2005). 2.2. Biological model, the edible cockle (C. edule) The cockle C. edule is an infaunal suspension-feeder of coastal ecosystems. This species preferably lives in fine to medium sediments and can support low salinities (>11). After a planktonic larval stage, recruitment generally occurs in spring and, in a lesser extent in autumn. Lifespan is about 5–6 years with a 50 mm maximal shell length. Adult density can reach up to 2–3000 ind m 2. 2.3. Sampling strategy Cockles were sampled in October 2012, during low tide, in twenty eight sheltered intertidal stations, along the Ria de Aveiro

(Fig. 1). Stations were selected in order to cover the widest range of the Ria’s habitats and different environmental characteristics as possible. Given this, the following six areas were assessed (Esinheiro, Ílhavo, Laranjo, Mira, Ovar and S. Jacinto). At each station, 20 adult cockles of similar shell length were randomly handpicked, 15 of which were used for parasites identification and the remaining 5 for elements quantification. Cockles used for the parasitological survey were transported alive to the laboratory, in seawater, while cockles for element quantification were transported on ice and preserved in the laboratory at 20 °C until analysis. At each sampling station, salinity, pH and conductivity were measured and sediment was collected for sediment grain size analysis, organic matter content determination and elements quantification (Pb, Cr, Cu, Zn, Ni, As, Cd and Hg). 2.4. Sediment grain size and total volatile solids Sediment grain size analysis was performed by wet and dry sieving, following the procedure described by Quintino et al. (1989). The fines particles fraction (diameter below 0.063 mm) was obtained by wet sieving through a 0.063 mm mesh screen and expressed as a whole, in terms of percentage of the total sediment (dry weight). Sand (diameter from 0.063 to 2 mm) and gravel (diameter above 2 mm) fractions were obtained by dry sieving through a battery of sieves spaced at 1U size intervals (U = log2 the particle diameter expressed in mm). The amount of sediment in each sieve was expressed for each site as a percentage of the whole sediment, dry weight. P50 is the median value of sediment grain size expressed in phi (U) units. The median and the percent content of fines were used to classify the sediment, according to the Wentworth scale: very fine sand (median between 3 and 4 U); fine sand (2–3 U); medium sand (1–2 U); coarse sand (0–1 U); very coarse sand ( 1 – 0 U) (Doeglas, 1968). Samples with more than 50% fines content were classified as mud. Total volatile solids in sediments were determined by loss on ignition at 450 °C (Byers et al., 1978). 2.5. Metal quantification The concentration of elements (Pb, Cr, Cu, Zn, Ni, As, Cd and Hg) was determined in sediments and cockles (soft tissues) and were expressed in lg g 1 wet weight (ww). Samples were digested overnight (±18 h) at 115 °C in digestion Teflon bombs (sealed chambers) with 10 mL of 65% HNO3 (Suprapur, Merck) in the case of the wet sediment and 2 mL of 65% HNO3 in the case of the biological samples (cockles soft-tissue). To prevent the loss of elements by volatilization, chambers were only opened when completely cooled. The cooled digest was made up to 5 ml using 1 M HNO3, and the concentration of all elements was determined by ICP-MS (Inductively Coupled Plasma-Mass Spectroscopy) in a certified laboratory at the University of Aveiro. Regarding the quality controls, the calibration of the apparatus was made with IV standards, and they were verified with standard reference material (National Institute of Standards and Technology, NIST SRM 1643e). During element analysis, the accuracy observed ranged between 90% and 110% (information given by the laboratory). All samples below this accuracy level were rejected and the analysis repeated. Determinations were performed using 3 replicates. 2.6. Parasite diversity and intensity Cockle shell length was measured with a calliper, dissected and the tissues were squeezed between two glass slides for trematode observation under a stereomicroscope. All macroparasites found in each cockle were identified and counted. Parasite species were

R. Freitas et al. / Marine Pollution Bulletin 82 (2014) 117–126

119

Fig. 1. Study area: the Ria de Aveiro lagoon showing the positioning of the sampling sites (circles).

identified following the method described by de Montaudouin et al. (2009) and using different bibliographic references on parasites in cockles (Bowers, 1969; Bowers et al., 1996; Bartoli et al., 2000; Desclaux et al., 2006; Russell-Pinto et al., 2006). For each area, data on parasite species were given in terms of intensity (abundance of a parasite species per infected cockles), prevalence (percentage of infected cockles) and species richness (number of parasite species per cockle) (Bush et al., 1997). For each of the 6 selected areas, c-diversity (total number of  -diversity (mean species richness number trematode species), a  ) (Gotelli and Colwell, 2001; per station) and b-diversity (c/a Anderson et al., 2011) were calculated.

2.7. Data analysis To evaluate the element accumulation by cockles, the Bioaccumulation Factor (BAF) was calculated dividing the concentration of each metal present in the organism by the concentration of that element found in the sediment of the sampling site (McGeer et al., 2003). According to McGeer et al. (2003), BAF should be derived from measurements in natural environments. The matrix gathering the number of trematode parasites (metacercariae) per station was log-transformed and normalized and the Euclidean distance similarity matrix calculated. This similarity matrix was simplified through the calculation of the distance

120

R. Freitas et al. / Marine Pollution Bulletin 82 (2014) 117–126

among centroids matrix based on the six areas, which was then submitted to ordination analysis, performed by Principal Coordinates (PCO). Pearson correlation vectors of biometric data and environmental descriptors were provided as supplementary variables being superimposed on the top of the PCO graph. The null hypotheses (H0) of no significant environmental differences among areas were tested for the following fixed factors: median grain-size (H01), fines content (H02), total organic matter content (H03), salinity (H04), conductivity (H05), pH (H06), bioaccumulation factor (H07), total elements content in sediments (H08) and total elements content (Pb, Cr, Cu, Zn, Ni, As, Cd, Hg) in cockles soft-tissue (H09). The significance in the main and pair-wise tests was obtained through a one-way model in PERMANOVA+ (Anderson et al., 2008), following unrestricted permutation of the raw data (9999 permutations) and the calculation of type III sums of squares. The null hypotheses were rejected at p < 0.05. The null hypotheses of no significant differences among areas were tested based on the [species X site] Euclidean distance similarity matrix and using biometric data as co-variables. The significance in the main and pair-wise tests were obtained following permutation of residuals under a reduced model (9999 permutations) and the calculation of type I sums of squares. The null hypotheses were rejected at p < 0.05. BIOENV routine was used to assess the relationship between the environmental data and the species abundance, intensity, and prevalence per site (square rooted [species X site] Euclidean distance similarity matrix), through the calculation of the Spearman correlation coefficient. All multivariate analyses were performed using PRIMER v.6 software (Clarke and Gorley, 2006). The study area and the spatial distribution of abundance, prevalence, intensity and species richness were plotted with the software ArcGis 10.1.

3. Results 3.1. Environmental characterization of the selected areas The study area was divided in six main areas, taking into account the geographical distribution of the sampling stations and their environmental characteristics within the Ria de Aveiro: Espinheiro Channel, Ílhavo Channel, Laranjo Bay, Mira Channel, Ovar Channel and S. Jacinto Channel (Fig. 1). Table 1 summarizes the environmental characteristics of each area. Espinheiro is an eastern channel with a NE–SW orientation that connects the Vouga River and the lagoon entrance. It is represented by three stations characterized by fine sands with the lowest fines particles content (5.1%), low TOM content (2.63%), the highest pH (7.56), low elements content in sediments (38.99 lg g 1 ww) and the highest Pb content (2.82 lg g 1 ww) in cockles soft-tissues (Table 1). Ílhavo Channel connects the Boco River to the Espinheiro Channel, close to the Port of Aveiro and the entrance of the lagoon. This area comprises five stations characterized by muddy fine sands with moderate fines content (16.9%), the lowest pH (5.65), low total elements content in sediment (36.61 lg g 1 ww) and the highest As content in cockles soft-tissues (12.33 lg g 1 ww) (Table 1). Laranjo Bay is a highly anthropogenic influenced area fed by the Antuã River, located far from the lagoon entrance. Five stations were included in this area, with sediments ranging from medium and fine sands to muds, with high fines and TOM content (28.3% and 4.1%, respectively). All elements showed here the highest concentration in sediments, specially the metals Hg, Pb, Zn and Cd content. The BAF value was the lowest when comparing to the remaining areas (Table 1).

Mira is a channel parallel to the coastline, connected to the Ria de Aveiro entrance channel. The four stations sampled in this area were characterized by medium and fine sands with moderate fines content (8.3%), the highest TOM content (4.5%), the lowest total elements content in sediments and in cockles soft-tissues (32.08 and 10.48 lg g 1 ww, respectively; Table 1). Ovar Channel is a N–S channel parallel to the coastline, in the northern branch of the Ria de Aveiro, connected to the entrance channel. Due to its environmental and morphodynamic differences it is divided in Ovar and S. Jacinto areas. The Ovar area is the most distant of the entrance channel being represented by four sampling stations. Sediments ranged from medium and fine sands to mud, with high fines content (25.3%) and the lowest salinity (26). The elements total content in cockles was high, particularly Ni and Zn, and the BAF was the highest (Table 1). S. Jacinto is the southern area of the Ovar Channel and is represented by seven stations. Sediments were mainly fine sands with a mean content of 16.8% of fines particles and 2.69% of TOM. Elements contents in sediments were high while in the cockles softtissue were low which corresponded to a low BAF (Table 1). 3.2. Parasite diversity In the Ria de Aveiro, 14 metazoan taxa were identified, 11 of which were trematode species (Table 2). Among trematodes, one species infected cockles as first intermediate hosts, one species as first and second hosts, and eight as second intermediate hosts (Table 2). Six species were present in the six studied areas, althought one showed clear dominance in intensity, Parvatrema minimus (92% of the total abundance of metacercariae found in Ria de Aveiro) (Table 3). Ílhavo and S. Jacinto exhibited the highest c-diversity (10 out of the 11 species that were identified in this study) (Table 3). Both areas were characterized by fine sands, showing the same fine content (17%). These areas were also among the less polluted in terms of cockle metal contamination (Table 1). However S. Jacinto displays a higher b-diversity (1.8 vs. 1.5) and a 10-fold higher parasite intensity (501 vs. 51 metacercariae per cockle for S. Jacinto and Ílhavo, respectively) (Table 3 and Fig. 2). Ovar showed the highest b-diversity (2.2) related to the lowest a-diversity (3.3). Comparing to the Ria’s overall parasite intensity, Ovar values were medium (99 metacercariae per cockle). This area also showed the lowest salinity values. The less contaminated area (Mira) is the less infected in terms of diversity (c-diversity = 6) but displays a rather high a-diversity (5.3) and one of the highest parasite intensity (222 metacercariae per cockle). It is characterized by coarser sands. The most contaminated area (Laranjo) has one of the lowest adiversity (4.2) (and a moderate c-diversity, i.e. 7), the lowest parasite intensity (21 metacercariae per cockle) and one of the highest prevalence of Bucephalus minimus (9%) (Fig. 3). Espinheiro shelters the most contaminated cockles, despite a low parasite intensity (44 metcercariae per cockle) and the highest B. minimus prevalence (11%, with one station (#11) reaching 27%). 3.3. Multivariate analysis The relationship between the mean abundance, prevalence, intensity and species richness determined by areas and biometric and environmental data were assessed through multivariate analysis. Fig. 4 shows the centroids PCO ordination graph, based on the species mean abundance, intensity and prevalence matrix ([species X sites]). The PCO axis 1 explained 48.7% of the total variation of data separating Laranjo Bay and Ovar Channel (in the negative side of the axis), from the other areas (in the positive side of the axis).

121

R. Freitas et al. / Marine Pollution Bulletin 82 (2014) 117–126

Table 1 Environmental characterization of the six main areas identified in the Ria de Aveiro, in terms of median grain-size (/), fines content (%), total organic matter content (TOM;%), salinity (g L 1), conductivity (mS/m), pH, bioaccumulation factor (BAF: ratio between the total element concentration in the organism and the element concentration in the sediment) and elements content in determined in sediments and cockles soft-tissue (lg g 1, wet weight). Environmental variables Sampling stations

Espinheiro 4, 8, 11

Ílhavo 1–3, 12, 44

Laranjo 31, 32, 36–38

Mira 24, 25, 27, 46

Ovar 39–42

S. Jacinto 15, 16, 20–23, 29

Sediment Median grain-size Fines content TOM content Salinity Conductivity pH BAF Cr Ni Cu Zn As Cd Pb Hg Total metal content

2.37 ± 0.28 5.08 ± 5.06 2.63 ± 1.24 33.33 ± 0.58 2.36 ± 0.21 7.56 ± 0.11 0.53 ± 0.28 6.24 ± 3.55 3.69 ± 2.02 2.47 ± 1.52 19.50 ± 11.73 3.03 ± 1.69 0.05 ± 0.04 3.98 ± 2.05 0.03 ± 0.03 38.99 ± 22.53

2.55 ± 0.43 16.92 ± 12.08 2.22 ± 0.60 31.00 ± 5.52 1.64 ± 0.96 5.65 ± 3.18 0.43 ± 0.23 5.78 ± 3.46 3.36 ± 1.89 2.47 ± 1.61 17.99 ± 9.47 2.71 ± 1.20 0.05 ± 0.02 4.24 ± 2.33 0.02 ± 0.01 36.61 ± 19.55

2.51 ± 0.98 28.34 ± 34.33 4.08 ± 4.35 34.60 ± 2.97 1.86 ± 0.34 7.30 ± 0.21 0.20 ± 0.09 7.77 ± 5.24 5.13 ± 3.02 7.47 ± 8.35 58.01 ± 42.80 11.03 ± 11.02 0.19 ± 0.20 8.71 ± 7.06 1.90 ± 3.74 100.22 ± 81.06

1.86 ± 0.15 8.28 ± 7.00 4.46 ± 3.15 31.00 ± 4.76 14.72 ± 4.30 6.74 ± 0.31 0.37 ± 0.14 4.80 ± 2.12 2.89 ± 1.19 2.30 ± 1.28 15.43 ± 6.72 2.92 ± 1.15 0.04 ± 0.02 3.69 ± 1.24 0.01 ± 0.01 32.08 ± 13.51

2.37 ± 1.16 25.32 ± 41.04 2.58 ± 1.29 26.00 ± 10.46 13.91 ± 6.65 7.28 ± 0.09 0.56 ± 0.41 5.25 ± 1.68 2.85 ± 0.82 5.88 ± 9.36 25.33 ± 20.33 1.98 ± 1.20 0.06 ± 0.04 2.44 ± 1.55 0.05 ± 0.03 43.85 ± 34.34

2.51 ± 0.41 16.77 ± 14.25 2.69 ± 0.93 34.57 ± 8.70 26.25 ± 38.96 7.30 ± 0.22 0.25 ± 0.07 7.46 ± 3.96 4.31 ± 2.03 2.53 ± 0.87 24.15 ± 6.07 3.47 ± 1.40 0.07 ± 0.03 4.15 ± 0.86 0.03 ± 0.02 46.17 ± 12.93

Cockles soft-tissue Cr Ni Cu Zn As Cd Pb Hg Total metal content

0.80 ± 0.23 2.43 ± 0.36 0.72 ± 0.09 7.55 ± 0.58 2.11 ± 0.61 0.06 ± 0.01 2.82 ± 4.45 0.01 ± 0.00 16.51 ± 4.22

0.76 ± 0.32 2.15 ± 0.49 0.51 ± 0.18 6.21 ± 1.04 2.42 ± 1.20 0.05 ± 0.02 0.23 ± 0.09 0.01 ± 0.00 12.33 ± 2.25

1.37 ± 1.15 3.66 ± 0.30 0.71 ± 0.20 7.49 ± 1.55 1.81 ± 0.59 0.06 ± 0.03 0.34 ± 0.17 0.07 ± 0.06 15.50 ± 2.85

0.53 ± 0.22 1.98 ± 0.34 0.57 ± 0.17 5.73 ± 0.81 1.33 ± 0.09 0.04 ± 0.01 0.29 ± 0.12 0.00 ± 0.00 10.48 ± 0.70

0.66 ± 0.08 4.09 ± 0.75 0.62 ± 0.17 8.99 ± 4.27 1.13 ± 0.14 0.03 ± 0.01 0.25 ± 0.11 0.00 ± 0.00 15.78 ± 4.71

0.57 ± 0.18 1.90 ± 0.55 0.62 ± 0.20 6.03 ± 0.69 1.34 ± 0.17 0.05 ± 0.02 0.21 ± 0.06 0.00 ± 0.01 10.73 ± 1.42

Table 2 List of trematode species using Cerastoderma edule as first and/or second intermediate hosts and their final host. Mean species intensity (I) and prevalence (P, %) at Ria de Aveiro scale, with standard deviation.

Bucephalus minimus Diphterostomum brusinae Himasthla continua Himasthla elongata Himasthla interrupta Himasthla quissetensis Monorchis parvus Parvatrema fossarum Parvatrema minutum Psilostomum brevicolle Renicola roscovita

First intermediate host

Second intermediate host

Final host

Prevalence

Intensity

Cerastoderma edule Nassarius reticulatus Hydrobia ulvae Littorina littorea Hydrobia ulvae Nassarius reticulatus Cerastoderma edule Scrobicularia plana Scrobicularia plana Hydrobia ulvae Littorina littorea

Pomatoschistus spp. Cerastoderma edule Cerastoderma edule Cerastoderma edule Cerastoderma edule Cerastoderma edule Cerastoderma edule Cerastoderma edule Cerastoderma edule Cerastoderma edule Cerastoderma edule

Dicentrarchus labrax Fishes Water birds Water birds Water birds Water birds Diplodus spp. Haematopus ostralegus Haematopus ostralegus Water birds Water birds

5±8 54 ± 40 32 ± 26 48 ± 43 30 ± 34 32 ± 26 0.2 ± 1 4±9 52 ± 34 2±5 4±7

– 4±3 1±2 6 ± 10 3±6 2±2 – 1±1 172 ± 398 0.2 ± 1 1±1

82 ± 30

189 ± 407

ALL

Biometric data presented high positive correlation with axis 1 (r > 0.7). Most of the superimposed environmental variables showed high negative correlation with axis 1 (r > 0.7), namely the content of Cu and Zn in sediments, Ni and Zn in cockles and fine particles, highlighting that higher abundance of parasites was recorded in sites with the lowest elements and/or fines content. Axis 2 described 27.6% of the total variation isolating Mira and S. Jacinto (in the positive axis), from Ílhavo and Espinheiro (in the negative axis). Conductivity has a high positive correlation with axis 2 (r > 0.7) while the total content of elements in cockles showed high negative correlation (r > 0.7). According to the PERMANOVA main-test, the null hypotheses of no significant differences among areas based on the species mean abundance, prevalence and intensity, and using biometric data as co-variables, was rejected at p < 0.05. From the three biometric co-variables only weight was statistically significant (p = 0.006), explaining part of the abundance, intensity and prevalence variability.

The combination of environmental variables best related to the species abundance, prevalence and intensity data were the BAF values and the content of Cd and Cu in sediments and Ni and Hg in soft-tissues (Spearman rho = 0.559, in the BIOENV routine).

4. Discussion The present study revealed a highly diverse and abundant parasite fauna in cockles collected in the Ria de Aveiro. The parasite community hosted by C. edule dominated by Parvatrema minutum, is similar to communities found in other Atlantic lagoons, particularly in France, despite the moderate abundance, intensity and prevalence values recorded in the present study area (e.g. Russell-Pinto et al., 2006; Thieltges and Reise, 2006; Gam et al., 2008; de Montaudouin et al., 2009; Fermer et al., 2011). From the 16 trematodes species known to infect C. edule cockles

122

R. Freitas et al. / Marine Pollution Bulletin 82 (2014) 117–126

Table 3 Characterization of the study area as whole (‘‘overall’’) and each area individually, in terms of mean species intensity (I) and prevalence (P, %) and mean values of intensity, prevalence, species richness (SR), Alpha-, Beta- and Gamma-diversity and biometric data. Bucephalus minimus was only analyzed in terms of prevalence). The highest values for each species are highlighted in italics. Standard deviation is provided. Species

Espinheiro

Ílhavo

Laranjo

Mira

Ovar

S. Jacinto

Bucephalus minimus I P

n.a. 11±14

n.a. 4±9

n.a. 9 ± 10

n.a. 2±3

n.a. 2±3

n.a. 4±5

Diphtherostomum brusinae I P

4.3 ± 3.6 84 ± 21

5.3 ± 1.5 84±12

0.2 ± 0.4 1±3

2.6 ± 1.2 55 ± 35

0.6 ± 0.8 10 ± 13

6.3±4.1 83 ± 21

Himasthla continua I P

0.0 ± 0.0 0±0

2.7±5.1 35±48

0.0 ± 0.0 0±0

0.0 ± 0.0 0±0

0.0 ± 0.0 0±0

0.0 ± 0.0 0±0

Himasthla elongata I P

2.3 ± 2.5 44 ± 51

8.5 ± 8.7 79±33

0.6 ± 0.9 4±6

9.3 ± 12.3 58 ± 43

2.1 ± 2.7 12 ± 19

10.8±14.5 74 ± 36

Himasthla interrupta I P

2.4 ± 0.4 47 ± 13

1.5 ± 1.3 19 ± 20

0.4 ± 0.5 3±4

12.1±12.3 60±26

1.4 ± 2.8 22 ± 43

2.9 ± 6.0 39 ± 44

Himasthla quissetensis I P

0.7 ± 1.2 2±4

1.9 ± 2.4 36 ± 36

1.2 ± 0.4 16 ± 11

4.2±3.6 42 ± 21

1.3 ± 0.4 27 ± 15

2.4 ± 1.0 50±24

Monorchis parvus I P

0.0 ± 0.0 0±0

0.0 ± 0.0 0±0

0.0 ± 0.0 0±0

0.0 ± 0.0 0±0

0.0 ± 0.0 0±0

0.1±0.4 1±3

Parvatrema minutum I P

33.1 ± 20.5 38 ± 17

29.5 ± 27.8 57 ± 21

18.0 ± 9.1 29 ± 28

193.4 ± 309.7 55 ± 36

93.3 ± 165.9 13 ± 22

477.5±700.0 90±8

Parvatrema fossarum I P

0.0 ± 0.0 0±0

1.1±1.5 7±8

0.5 ± 0.8 5±9

0.0 ± 0.0 0±0

0.4 ± 0.8 5 ± 10

0.5 ± 1.3 5 ± 13

Psilostomum brevicolle I P

1.2±1.1 9±10

0.4 ± 0.5 4±6

0.0 ± 0.0 0±0

0.0 ± 0.0 0±0

0.0 ± 0.0 0±0

0.1 ± 0.4 1±3

Renicola roscovita I P

0.8 ± 0.8 9±8

1.2±0.8 7±5

0.0 ± 0.0 0±0

0.0 ± 0.0 0±0

0.0 ± 0.0 0±0

0.6 ± 0.6 9 ± 11

I P

44.7 ± 20.2 98 ± 0

52.1 ± 43.1 95 ± 10

21.0 ± 10.7 45 ± 30

221.7 ± 329.7 100 ± 0

99.1 ± 167.1 50 ± 40

501.2 ± 711.9 100 ± 0

Alpha-diversity Gamma-diversity Beta-diversity

5.3 8 1.5

6.6 10 1.5

4.2 7 1.7

5.3 6 1.1

3.3 7 2.2

5.7 10 1.8

Weight (g)

10.5 ± 1.3

8.0 ± 1.5

6.4 ± 1.7

8.0 ± 3.8

6.3 ± 1.1

10.0 ± 1.7

Length (mm)

29.5 ± 1.3

26.8 ± 2.2

25.5 ± 2.5

26.3 ± 2.8

23.4 ± 1.6

28.8 ± 1.9

Width (mm)

26.3 ± 1.3

24.6 ± 1.9

22.7 ± 2.2

23.5 ± 1.9

20.9 ± 1.5

25.8 ± 1.8

Fig. 2. Intensity (log (x + 1) of metacercariae: D.b. Diphterostomum brusinae; H.c. Himasthla continua; H.e. Himasthla elongata; H.i. Himasthla interrupta; H.q. Himasthla quissetensis; P.b. Psilostomum brevicolle; P.f. Parvatrema fossarum; P.m. Parvatrema minutum; R.r. Renicola roscovita.

R. Freitas et al. / Marine Pollution Bulletin 82 (2014) 117–126

123

Fig. 3. Bucephalus minimus prevalence (%) and standard deviation values, in each area.

Fig. 4. Centroids ordination diagram (PCO) based on the species abundance, intensity, and prevalence dataset determined per area. Pearson correlation vectors are superimposed as supplementary variables, namely biometric data, as gray dashed vectors, and environmental data (r > 0.7) data, as black dashed vectors.

(de Montaudouin et al., 2009), 11 were identified in the present study. This value is higher comparing to similar studies in the northwestern Spain (Carballal et al., 2001), southern coast of Ireland (8 spp.; Fermer et al., 2011), northern Morocco (9 spp.; Gam et al., 2008) and northern Wadden Sea (10 spp.; Thieltges and Reise, 2006). On the other hand, the number of trematod species found was similar to a study carried out in cockles from one sampling site in S. Jacinto Channel (Ria de Aveiro) (11 spp; Russell-Pinto et al., 2006), and lower when compared to Arcachon Bay cockles (13 spp; de Montaudouin et al., 2009). Most of these species are commonly found in northwestern European coast, apart from Parvatrema fossarum, Himasthla quissetensis and Diphterostomum brusinae, which are species distributed in the Lusitanean and/or Mediterranean and/or Northern African biogeographic provinces, closely related to the meridional distribution of the first intermediate host, such as Nassarius reticulatus for H. quissetensis and D. brusinae (Russell-Pinto and Bartoli, 1992; Russell-Pinto et al., 2006; Thieltges and Reise, 2006; Gam et al., 2008; de Montaudouin et al., 2009; Fermer et al., 2011). A closer comparison between the present study and the previous study conducted in this area (1 site at the S. Jacinto Channel, Russell-Pinto et al., 2006) demonstrated that: (a) the parasite species prevalence decreased, excepting for B. minimus, D. brusinae, H. elongata and

H. interrupta; (b) the number of trematode species known in the Ria de Aveiro increased from 11 to 13, confirming H. continua and P. brevicolle as new occurrences in this lagoon and equaling the parasites diversity of Arcachon Bay cockles (de Montaudouin et al., 2009). Both parasite species have seabirds as final host and Hydrobia ulvae. and C. edule as first and second intermediate host, respectively. A deeper screening as consequence of an increase of expertise in this field and a geographically wide survey may explain those new occurrences. This high diversity was expected since the Ria de Aveiro lagoon, a large estuarine area with saltmarshes, freshwater marshes and alluvial forest associated to several rivers, represents an important hotspot of biodiversity, particularly for fishes,, shorebirds and marine birds. It is located in the northern Atlantic coast, between the colder northern European waters and warmer African and Mediterranean waters, allowing the breading and feeding of large-scale migratory or resident birds (e.g. Purple Heron or Western Marsh-harrier). Due to its ecological importance, it was classified as an Important Bird Area (PT007) and a Special Protection Area under the Birds Directive (PTZPE0004), among other protection status (BirdLife International, 2013). The combination of abiotic characteristics and high biotic productivity together with the biogeographic transitional characteristics of this area, which are shared with those prevalent in the western Iberian coast (e.g. Martins et al., 2013), ensures a high diversity of microfauna (e.g. Russell-Pinto et al., 2006; this study), macrofauna (e.g. Rodrigues et al., 2011) and megafauna (e.g. Pombo and Rebelo, 2002). Among the macroinvertebrates species present in the Ria de Aveiro, C. edule is one of the most abundant and with highest biomass and economic interest (Rodrigues et al., 2011). Nearly 80% of the studied cockles were infected and most of them held a high diversity of parasites, which may be explained by the parasites spatial niche segregation within the host (e.g. Thieltges and Reise, 2006). Therefore, the high diversity of trematode species is associated to the complexity and dynamics of the ecosystem that ensures the success of the entire life cycle of the parasite species since intermediate and final host species can be found here. For most parasite species, the level of infection observed in Ria de Aveiro (i.e. parasite intensity) was too low to induce severe cockle mortality. Himasthla spp. impacted adult cockle populations (growth, mortality) when a threshold of a minimum of 20–30 metacercariae per cockle was reached in the case of H. quissetensis (Desclaux et al., 2004; Gam et al., 2009), and ca. 230 metacercariae per cockle for H. interrupta (de Montaudouin et al., 2012). However, the threshold beyond which a host impact is observed varies among ecosystems (Desclaux et al., 2004; Gam et al., 2009), due to interactions among stressors (Paul-Pont et al., 2010).

124

R. Freitas et al. / Marine Pollution Bulletin 82 (2014) 117–126

Conversely, the parasite intensity reached by P. minutum in S. Jacinto (477 metacercariae per cockle) was close to what was observed at Merja Zerga (Morocco) and Arcachon (France) where parasite-dependent mortality was suggested (Gam et al., 2009). Finally, a more alarming situation concerns cockles that are infected as first intermediate hosts (B. minimus), when the prevalence is high as observed in Espinheiro and Laranjo. Indeed, this type of infection is reputed as deleterious for host populations (Calvo-Ugarteburu and McQuaid, 1998; de Montaudouin et al., 2012; Jonsson and André, 1992; Thieltges, 2006) and could negatively control cockle population in these areas. Besides, these parasites are reputed to interact with metals like cadmium in enhancing host vulnerability (Baudrimont et al., 2006). This study also intended to understand the relationship between the abundance, intensity, prevalence and species richness patterns with environmental descriptors. A recent study showed a clear relationship between warmer waters (20–25 °C) and the infection of the Crassostrea ariakensis by the parasite Bonamia sp., overwhelming oyster host defenses (Carnegie et al., 2008). The northwestern Portuguese coast is integrated in the Lusitanean biogeographic province with cool water, ranging between 14 and 16 °C during autumn, in the Ria de Aveiro (http://neptuno.fis.ua.pt/). This range of temperature is noticeably below the temperature that can affect the bivalve immunological defenses (Matozzo and Marin, 2011) and may not fully explain simultaneously the high diversity and the lower abundance, intensity and prevalence comparing with other areas, which points to other causes. Pech et al. (2010) defended that rainfall deeply influences the abundance and prevalence patterns of metazoan parasites in aquatic gastropods and fishes. The authors demonstrated that rapid changes in the proportion of infected hosts and host density were positively related with changes in the rainfall patterns. During 2012, Portugal was submitted to a severe drought event (MAMAOT, 2013) that provoked a decrease of the input of freshwater in the coastal lagoon and consequent increase of salinity, in agreement with the euhaline conditions found in all areas, except Ovar. Cockles are known to be organisms quite resistant to salinities ranging from 5% to 35°%, with the optimum at 25% (Kater et al., 2006), and therefore it can be assumed that the higher salinity affected marginally the survival of infective parasitic stages (Rigby and Moret, 2000; Lafferty and Holt, 2003). However, admitting that the low salinity could explain the low infection rates in Ovar, this abiotic factor cannot explain by itself the results of the semi-enclosed Laranjo Bay, which recorded the highest salinity and the lowest infection rates. This study showed that higher values of species abundance, intensity and prevalence were recorded in areas without element contamination, being these patterns mostly related with the bioaccumulation factor and the content of some elements in sediments and cockles. The most determinant elements explaining these patterns were Cd and Cu in sediments and Ni and Hg in soft-tissues. Also, the mean variables determined by area, showed negative correlation with all metals, metalloids (As) and fines content, and positive correlation with conductivity and salinity. This points to a combination of abiotic variables ruling the spatial distribution of the host cockles and affecting the parasites distribution, which was never previously shown in past studies dealing with parasites infection in cockles. Leite et al. (2004) showed an increase of the susceptibility of the clam Ruditapes decussatus to be infected by Perkinsus sp. in areas contaminated mostly by TBT and hydrocarbons along the Iberian coast. High content of contaminants in sediments, particularly metals, are known to be deleterious for bivalves causing biochemical and physiological stress and inhibition of the mechanisms of defense (Chu, 1999; Chu et al., 2002; Freitas et al., 2012) and consequent propensity to be infected by parasites. The United States National Oceanic and Atmospheric Administration and United States Environmental

Protection Agency defined a large database on sediment chemistry and toxicity (Long et al., 1995; Buchman, 1999). A detailed comparison between those guidelines and the sedimentary geochemistry shows that Laranjo Bay exceeds the Arsenic ERL threshold (8.2 lg g 1; concentration above which toxicity may begin to affect the most sensitive species) and largely exceeds the Mercury ERM threshold (0.71 lg g 1; content above which adverse biological effects will be more frequently observed) (Long et al., 1995; Buchman, 1999). This area is historically contaminated due to untreated mercury and arsenic rich effluents from chlor-alkali, pyrite roasting and smelters located in the vicinity of this area (Pereira et al., 1997, 2009; Coelho et al., 2014). Despite the settlement of a sewage treatment plant, the contaminants concentration in the Laranjo Bay sediments, remain above pre-industrial levels which is mostly caused by the spring tides, channels morphology and currents and at less extent the extreme weather events and channels dredging operations (Coelho et al., 2014). The contaminants seem to play a key role in the growth and abundance of the C. edule cockles (this study) and in the spatial distribution of the benthic communities dominated by other potential intermediate hosts (Rodrigues et al., 2011), which in turn seems to affect the survival and performance of the parasites, unlike the other uncontaminated areas where cockles grow and harbor a very diverse parasite community. In conclusion, the diversity of habitats within Ria de Aveiro explains the high species richness of trematode species, while the cold waters, occasionally associated with contaminants and other types of anthropogenic disturbances, explain the low parasite intensity and relatively low a-diversity. The high diversity and a community dominated by P. minutum, combined with higher abundance, intensity and prevalence of parasites in cockles from areas without contamination, in contrast with heavily contaminated areas (e.g. Laranjo Bay), highlights the potential use of cockles as an indicator of good ecological status. The high diversity is a sign of ecosystem health and highlights the transitional characteristics of the western Portuguese coast where northern and subtropical faunas can coexist. Acknowledgements This work was supported by CESAM and Department of Biology own funding and through the programme ‘‘Ações Integradas LusoFrancesas’’, funded by CRUP, under the project (Ref. TC-07_12) ‘‘Inventory, dynamics and impact of the parasites trematodes and bacteria in bivalves of high economic importance in Portugal’’. The authors would like to thank to Anthony Peter Moreira for the English editing. References Anderson, M.J., Gorley, R.N., Clarke, K.R., 2008. PERMANOVA+ for PRIMER: Guide to Software and Statistical Methods. University of Auckland and PRIMER-E, Plymouth. Anderson, M.J., Crist, T.O., Chase, J.M., Vellend, M., Inouye, B.D., Freestone, A.L., Sanders, N.J., Cornell, H.V., Comita, L.S., Davies, K.F., Harrison, S.P., Kraft, N.J.B., Stegen, J.C., Swenson, N.G., 2011. Navigating the multiple meanings of b diversity: a roadmap for the practicing ecologist. Ecol. Lett. 14, 19–28. 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. Baudrimont, M., de Montaudouin, X., 2007. Evidence of an altered protective effect of metallothioneins after cadmium exposure in the digenean parasite-infected cockle (Cerastoderma edule). Parasitology 134, 237–245. Baudrimont, M., de Montaudouin, X., Palvadeau, A., 2006. Impact of digenean parasites infection on metallothionein synthesis by the cockle (Cerastoderma edule): a multivariate field monitoring. Mar. Poll. Bull. 52, 494–502. BirdLife International, 2013. Important Bird Areas factsheet: Ria de Aveiro. Downloaded from on 10.12.2013. Bowers, E.A., 1969. Cercaria bucephalopsis haimeana (Lacaze-Duthiers, 1854) (Digenea: Bucephalidae) in the cockle, Cardium edule L. in South Wales. J. Nat. Hist. 3, 409–422.

R. Freitas et al. / Marine Pollution Bulletin 82 (2014) 117–126 Bowers, E.A., Bartoli, P., Russell-Pinto, F., James, B.L., 1996. The metacercariae of sibling species of Meiogymnophallus, including M. rebecqui comb. nov. (Digenea: Gymnophallidae), and their effects on closely related Cerastoderma host species (Mollusca: Bivalvia). Parasitol. Res. 82, 505–510. Buchman, M.F., 1999. NOAA Screening Quick Reference Tables, NOAA HAZMAT Report 99-1. Coastal Protection and Restoration Division, Seattle WA. Bush, A.O., Lafferty, K.D., Lotz, J.M., Shostak, A.W., 1997. Parasitology meets ecology on its own terms: Margolis, Revisited. J. Parasitol. 83, 575–583. Byers, S.C., Mills, E.L., Stewart, L., 1978. A comparison of methods of determining organic carbon in marine sediments, with suggestions for a standard method. Hydrobiologia 58, 43–47. Calvo-Ugarteburu, G., McQuaid, C.D., 1998. Parasitism and invasive species: effects of digenetic trematodes on mussels. Mar. Ecol. Prog. Ser. 169, 149–163. Carballal, M.J., Iglesias, D., Santamarina, J., Ferro-Soto, B., Villalba, A., 2001. Parasites and pathologic conditions of the cockle Cerastoderma edule populations of the coast of Galicia (NW Spain). J. Invertebr. Pathol. 78, 87–97. Carnegie, R., Stokes, N., Audemard, C., Bishop, M., Wilbur, A., Alphin, T., Posey, M., Peterson, C., Burreson, E., 2008. Strong seasonality of Bonamia sp. infection and induced Crassostrea ariakensis mortality in Bogue and Masonboro Sounds, North Carolina, USA. J. Invertebr. Pathol. 98, 335–343. Cheggour, M., Chafik, A., Langston, W.J., Burt, G.R., Benbrahim, S., Texier, H., 2001. Metals in sediments and the edible cockle Cerastoderma edule from two Moroccan Atlantic lagoons: Moulay Bou Selham and Sidi Moussa. Environ. Pollut. 115, 149–160. Chu, F.L.E., 1999. Effects of field-contaminated sediments and related water soluble components on haemocyte function and Perkinsus marinus susceptibility and expression in oysters. Biomarkers 4, 537–548. Chu, F.L.E., Volety, A.K., Hale, R.C., Huang, Y., 2002. Cellular responses and disease expression in oysters (Crassostrea virginica) exposed to suspended field contaminated sediments. Mar. Environ. Res. 53, 17–35. Clarke, K.R., Gorley, R.N., 2006. PRIMER v6: User Manual/Tutorial. PRIMER-E, Plymouth, UK. Coelho, J.P., Pato, P., Henriques, B., Picado, A., Lillebø, A.I., Dias, J.M., Duarte, A.C., Pereira, M.E., Pardal, M.A., 2014. Long-term monitoring of a mercury contaminated estuary (Ria de Aveiro, Portugal): the effect of weather events and management in mercury transport. Hydrol. Process. 28, 352–360. de Montaudouin, X., Kisielewski, I., Bachelet, G., Desclaux, C., 2000. A census of macroparasites in an intertidal bivalve community, Arcachon Bay, France. Oceanol. Acta 23, 453–468. de Montaudouin, X., Thieltges, D.W., Gam, M., Krakau, M., Pina, S., Bazaïri, H., Dabouineau, L., Russell-Pinto, F., Jensen, K.T., 2009. Review e Digenean trematode species in the cockle Cerastoderma edule: identification key and distribution along the North-East Atlantic shoreline. J. Mar. Biol. Assoc. U.K. 80, 543–556. de Montaudouin, X., Bazairi, H., Culloty, S., 2012. Effect of trematode parasites on cockle Cerastoderma edule growth and condition index: a transplant experiment. Mar. Ecol. Prog. Ser. 471, 111–121. Desclaux, C., de Montaudouin, X., Bachelet, G., 2004. Cockle (Cerastoderma edule) population mortality: the role of the digenean parasite Himasthla quissetensis. Mar. Ecol. Prog. Ser. 279, 141–150. Desclaux, C., Russell-Pinto, F., de Montaudouin, X., Bachelet, G., 2006. First record and description of metacercariae of Curcuteria arguinae n. sp. (Digenea: Echinostomatidae), parasite of cockles Cerastoderma edule (Mollusca: Bivalvia) in Arcachon Bay, France. J. Parasitol. 92, 578–587. DGPA – Direcção-Geral das Pescas e Aquicultura, 2011. Recursos da Pesca, Série Estatística, Ano 2010. vol. 24 A–B, DGPA, Lisboa, Portugal. Dias, J.M., 2001. Contribution to the Study of the Ria de Aveiro Hydrodynamics. PhD Thesis, University of Aveiro, Portugal. Dias, J.M., Lopes, J.F., Dekeyser, I., 1999. Hydrological characterization of Ria de Aveiro, Portugal, in early summer. Oceanol. Acta 22 (5), 473–485. Do, V.T., de Montaudouin, X., Lavesque, N., Blanchet, H., Guyard, H., 2011. Seagrass colonization: knock-on effects on zoobenthic community, populations and individual health. Estuar. Coast. Shelf Sci. 95, 458–469. Doeglas, D.J., 1968. Grain-size indices, classification and environment. Sedimentology 10, 83–100. Fermer, J., Culloty, S., Kelly, T., O’Riordan, R., 2011. Parasitological survey of the edible cockle Cerastoderma edule (Bivalvia) on the south coast of Ireland. J. Mar. Biol. Assoc. U.K. 91 (4), 923–928. Figueira, E., Lima, A., Branco, D., Quintino, V., Rodrigues, A.M., Freitas, R., 2011. Health concerns of consuming cockles (Cerastoderma edule L.) from a low contaminated coastal system. Environ. Int. 37 (5), 965–972. Freitas, R., Costa, E., Velez, C., Santos, J., Lima, A., Oliveira, C., Rodrigues, A.M., Quintino, V., Figueira, E., 2012. Looking for suitable biomarkers in benthic macroinvertebrates inhabiting coastal areas with low metal contamination. Ecotoxicol. Environ. Saf. 75, 109–118. Gam, M., Bazaïri, H., Jensen, K.T., de Montaudouin, X., 2008. Metazoan parasites in an intermediate 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.K. 88, 357–364. Gam, M., de Montaudouin, X., Bazaïri, H., 2009. Do trematode parasites affect cockle (Cerastoderma edule) secondary production and elimination? J. Mar. Biol. Assoc. U.K. 89, 1395–1402. Gotelli, N.J., Colwell, R.K., 2001. Quantifying biodiversity: procedures and pitfalls in the measurement and comparison of species richness. Ecol. Lett. 4, 379–391. Hechinger, R.F., Lafferty, K.D., Huspeni, T.C., Brooks, A.J., Kuris, A.M., 2007. Can parasites be indicators of free-living diversity? Relationships between species

125

richness and the abundance of larval trematodes and of local benthos and fishes. Oecologia 151, 82–92. Hechinger, R.F., Lafferty, K.D., Kuris, A.M., 2008. Trematodes indicate animal biodiversity in the Chilean intertidal and lake Tanganika. J. Parasitol. 94, 966– 968. Hudson, P.J., Dobson, A.P., Lafferty, K.D., 2006. Is a healthy ecosystem one that is rich in parasites? Trends Ecol. Evol. 21, 381–385. Jensen, K.T., Mouritsen, K.N., 1992. Mass mortality in two common soft-bottom invertebrates, Hydrobia ulvae and Corophium volutator – the possible role of trematodes. Helgol. Meeresunters. 46, 329–339. Jonsson, P.R., André, C., 1992. Mass mortality of the bivalve Cerastoderma edule on the Swedish west coast caused by infestation with the digenean trematode Cercaria cerastodermae I. Ophelia 36, 151–157. Kater, B.J., van Kessel, A.J.M.G., Baars, J.J.M.D., 2006. Distribution of cockles Cerastoderma edule in the Eastern Scheldt: habitat mapping with abiotic variables. Mar. Ecol. Prog. Ser. 318, 221–227. Lafferty, K.D., 1997. Environmental parasitology: what can parasites tell us about human impacts on the environment? Parasitol. Today 13, 251–255. Lafferty, K.D., Holt, R.D., 2003. How should environmental stress affect the population dynamic of disease? Ecol. Lett. 6, 654–664. Leite, R.B., Afonso, R., Cancela, M.L., 2004. Perkinsus sp. infestation in carpet-shell clams, Ruditapes decussatus (L), along the Portuguese coast. Results from a 2year survey. Aquaculture 240, 39–53. Lobo, J., Costa, P.M., Caeiro, S., Martins, M., Ferreira, A.M., Caetano, M., Cesário, R., Vale, C., Costa, M.H., 2010. Evaluation of the potential of the common cockle (Cerastoderma edule L.) for the ecological risk assessment of estuarine sediments: bioaccumulation and biomarkers. Ecotoxicology 19, 1496–1512. Long, E.R., MacDonald, D.D., Smith, S.L., Calder, F.D., 1995. Incidence of adverse biological effects within ranges of chemical concentrations in marine and estuarine sediments. Environ. Manage. 19, 81–97. MacKenzie, K., 1999. Parasites as pollution indicators in marine ecosystems: a proposed early warning system. Mar. Poll. Bull. 38, 955–959. MacKenzie, K., Williams, H.H., Williams, B., McVicar, A.H., Siddall, R., 1995. Parasites as indicators of water quality and the potential use of helminth transmission in marine pollution studies. Adv. Parasitol. 35, 85–144. MAMAOT, 2013. Relatório de Balanco da Seca 2012. Available from: . Martins, R., Quintino, V., Rodrigues, A.M., 2013. Diversity and spatial distribution patterns of the soft-bottom macrofauna communities on the Portuguese continental shelf. J. Sea Res. 83, 173–186. Matozzo, V., Marin, M.G., 2011. Bivalve immune responses and climate changes: is there a relationship? Invertebrate Surviv. J. 8, 70–77. McGeer, J.C., Brix, K.V., Skeaff, J.M., DeForest, D., Brigham, S.I., Adams, W.J., Green, A., 2003. Inverse relationship between bioconcentration factor and exposure concentration for metals: implications for hazard assessment of metals in the aquatic environment. Environ. Toxicol. Chem. 22, 1017–1037. Morley, N.J., Irwin, S.W.B., Lewis, J.W., 2003. Pollution toxicity to the transmission of larval digeneans through their molluscan hosts. Parasitology 126, 5–26. Mouritsen, K.N., Poulin, R., 2002. Parasitism, community structure and biodiversity in intertidal ecosystem. Parasitology 124, 101–117. Mouritsen, K.N., Tompkins, D.M., Poulin, R., 2005. Climate warming may cause a parasite-induced collapse in coastal amphipod populations. Oecologia 146, 476–483. Niewiadomska, K., Pojman´ska, T., 2011. Multiple strategies of digenean trematodes to complete their life cycles. Wiad. Parazytol. 57 (4), 233–241. Paul-Pont, I., Baudrimont, M., Gonzalez, P., de Montaudouin, X., 2010. Interactive effects of metal contamination and pathogenic organisms on the marine bivalve Cerastoderma edule. Mar. Poll. Bull. 60, 515–525. Pech, D., Aguirre-Macedo, M.L., Lewis, J., Vidal-Martínez, V.M., 2010. Rainfall induces time-lagged changes in the proportion of tropical aquatic hosts infected with metazoan parasites. Int. J. Parasitol. 40, 937–944. Pereira, M.E., Duarte, A.C., Millward, G.E., Abreu, S.N., Reis, M.C., 1997. Distribution of mercury and other heavy metals in the Ria de Aveiro. Quim. Anal. 16, 31–35. Pereira, M.E., Lillebø, A.I., Pato, P., Válega, M., Coelho, J.P., Lopes, C., Rodrigues, S., Cachada, A., Otero, M., Pardal, M.A., Duarte, A.C., 2009. Mercury pollution in Ria de Aveiro (Portugal): a review of the system assessment. Environ. Monit. Assess. 155, 39–49. Pietrock, M., Marcogliese, D.J., 2003. Free-living endohelminth stages: at the mercy of environmental conditions. Trends Parasitol. 19, 293–299. Pombo, L., Rebelo, J.E., 2002. Spatial and temporal organization of a coastal lagoon fish community – Ria de Aveiro, Portugal. Cybium 26 (3), 185–196. Quintino, V., Rodrigues, A.M., Gentil, F., 1989. Assessment of macrozoobenthic communities in the lagoon of Óbidos, western coast of Portugal. Sci. Mar. 53, 645–654. Rigby, M.C., Moret, Y., 2000. Life-history trade-offs and immune defenses. In: Poulin, R., Morand, S., Skorping, A. (Eds.), Evolutionary Biology of Host-Parasite Relationships: Theory Meets Reality. Elsevier, Amsterdam, pp. 129–142. Rodrigues, A.M., Quintino, V., Sampaio, L., Freitas, R., Neves, R., 2011. Benthic biodiversity patterns in Ria de Aveiro, Western Portugal: environmental– biological relationships. Estuar. Coast. Shelf Sci. 95, 338–348. Russell-Pinto, F., Bartoli, P., 1992. Sympatric distribution of Meiogymnophallus minutus and M. fossarum (Digenea: Gymnophallidae) in Cerastoderma edule in the Ria de Aveiro estuary in Portugal. Parasitol. Res. 78, 617–618. Russell-Pinto, F., Gonçalves, J.F., Bowers, E., 2006. Digenean larvae parasitizing Cerastoderma edule (Bivalvia) and Nassarius reticulatus (Gastropoda) from Ria de Aveiro, Portugal. J. Parasitol. 92, 319–332.

126

R. Freitas et al. / Marine Pollution Bulletin 82 (2014) 117–126

Thieltges, D.W., 2006. Parasite induced summer mortality in the cockle Cerastoderma edule by the trematode Gymnophallus choledochus. Hydrobiologia 559, 455–461. Thieltges, D.W., Reise, K., 2006. Metazoan parasites in intertidal cockles Cerastoderma edule from the northern Wadden Sea. J. Sea Res. 56, 284–293.

Vaz, N., Dias, J.M., Leitão, P., Martins, I., 2005. Horizontal patterns of water temperature and salinity in an estuarine tidal channel: Ria de Aveiro. Ocean Dynam. 55, 416–429.