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Environmental Pollution 243 (2018) 1469e1478

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Environmental Pollution

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Interactive effects of contamination and trematode infection in cocklesbiochemical performance*

Luísa Magalh~aes a, b, Xavier de Montaudouin b, Etelvina Figueira a, Rosa Freitas a, *

a Departamento de Biologia & CESAM, Universidade de Aveiro, 3810-193, Aveiro, Portugalb Universit�e de 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

Article history:Received 15 May 2018Received in revised form19 September 2018Accepted 19 September 2018Available online 22 September 2018

Keywords:Cercariae doseArsenicExperimental infectionInfection successHost susceptibility

* This paper has been recommended for acceptanc* Corresponding author. Departamento de Biolog

Campus Universit�ario de Santiago, 3810-193, Aveiro,E-mail address: rosafreitas@ua.pt (R. Freitas).

https://doi.org/10.1016/j.envpol.2018.09.1020269-7491/© 2018 Elsevier Ltd. All rights reserved.

a b s t r a c t

Anthropogenic activities, especially those involving substances that pollute the environment can inter-fere with bivalve populations, as well as parasitism, a fundamental ecological interaction often neglected.In marine environments, organisms are concomitantly exposed to pollutants and parasites, a combina-tion with synergistic, antagonistic or additive effects representing a potential threat to aquatic com-munities sustainability. In the present study, Cerastoderma edule (the edible cockle)eHimasthla elongata(trematode) was used as hosteparasite model. Cockles are worldwide recognized as good sentinel andbioindicator species and can be infected by several trematodes, the most abundant macroparasites incoastal waters. Tested hypotheses were: 1) cockles exposed to increasing parasite pressure will presentgreater stress response; 2) cockles exposure to arsenic (single concentration test: 5.2 mg L�1) will changeparasite infection success and cockles stress response to infection. Arsenic was used for being one of themost common pollutants in the world and stress response assessed using biochemical markers ofglycogen content, metabolism, antioxidant activity and cellular damage. Results showed that intensity ofparasite pressure was positively correlated to biochemical response, mainly represented by highermetabolic requirements. Contamination did not affect parasite infection success. Compared to arsenic,trematode infection alone exerted a stronger impact: higher glycogen storage, metabolism and cellulardamage and antioxidant activity inhibition. In interaction, parasitism and arsenic reduced hosts meta-bolism and cellular damage. Therefore, to a certain extent and in a contamination scenario, cockles maybenefit from trematode infection, working as a protection for the pollutant accumulation in the or-ganisms, reducing overall ROS production, which can consequently led to less toxic effects. These find-ings highlighted the deleterious effects of trematode infection in their hosts and showed the importanceof including parasitology in ecotoxicological studies.

© 2018 Elsevier Ltd. All rights reserved.

1. Introduction

Pollution is present in aquatic ecosystems worldwide, particu-larly in areas of industrial and agricultural intensification resultingfrom high population density (de Sherbinin et al., 2007). Arsenic(As), is one of the most widely distributed pollutants in the world(Mandal and Suzuki, 2002), with known negative effects in aquaticenvironments namely on the oxidative stress balance of inhabitingorganisms (Ventura-Lima et al., 2011), including bivalves (e.g.mussels Mytilus galloprovincialis exposed to 1 mgL�1 As in

e by Maria Cristina Fossi.ia, Universidade de Aveiro,Portugal.

combination to thermal stress (Coppola et al. (2018)) and Crassos-trea spp. oysters exposed to 4 mgL�1 As (Moreira et al. (2016)))..Several anthropogenic activities have been contributing to theincreasing As concentration in the environment, including woodpreservation, insecticides and herbicides use as well as coal burningand mining (Corzo and Gamboa, 2018; Mandal and Suzuki, 2002;Ranft et al., 2003). In the marine environment, As occurs in bothseawater and sediment fractions (Mandal and Suzuki, 2002).Average As concentration in seawater is typically around 1.5 mg L�1

reaching up to 8.8 mg L�1 (Smedley and Kinniburgh, 2002) inestuarine and more industrial influenced areas. In sediments, Asconcentrations range is wider, varying between 0.82mg kg�1

(Velez et al., 2015) and 33.4mg kg�1 (Wang et al., 2010). In theenvironment, As is commonly present in its inorganic forms(arsenite and arsenate) where it may be transformed into less toxic

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L. Magalh~aes et al. / Environmental Pollution 243 (2018) 1469e14781470

organic forms, such as arsenobetaine and arsenocholine, andaccumulated by marine organisms (Fattorini et al., 2006).

Bivalves constitute an essential component of estuarine andcoastal ecosystems, including many keystone species (Taylor et al.,2018). Representing a major proportion of the benthic faunabiomass (Sousa et al., 2009), bivalves play an important role on theecosystem functioning (Rakotomalala et al., 2015) and are the basisof important commercial fisheries (Oliveira et al., 2013; Rowleyet al., 2014). Therefore, bivalves are often used as indicators ofenvironmental conditions, being frequently labelled as goodsentinel organisms in monitoring programs (Beyer et al., 2017). Tothis end, it largely contributes their low capacity of locomotion(Tebble, 1966), i.e. living near in the same place all their life, theirability to accumulate both metal(oid)s and organic contaminants(Luna-Acosta et al., 2015; Marques et al., 2016), their wide andabundant distribution among habitats (McKeon et al., 2015),including those relatively heavily polluted (Fishelson et al., 1999),and their easy collection. Cerastoderma spp. cockles, are among themost used sentinel (Cheung et al., 2006; Freitas et al., 2012; Karrayet al., 2015) considered also as good bioindicator bivalve species(Cheggour et al., 2001; Velez et al., 2016).

In the environment, besides contamination, bivalve populationscan also be affected by several biotic factors such as parasitism(Gam et al., 2009) which can seriously interfere with host popu-lation dynamics (Friesen et al., 2017). Usually, cockles are infectedby several trematode species both as first and second intermediatehost and multispecies infection is frequent (de Montaudouin et al.,2009). Impacts of parasites on bivalves include disrupting basicfunctions such as growth (de Montaudouin et al., 2012a) and bur-rowing capacity (Babirat et al., 2004), but may also lead tomortalityoutbreaks (Desclaux et al., 2004; Marcogliese, 2004). It is describedthat Himasthla elongata infection can impair byssus production inblue mussels (M. edulis) and alter the burrowing capacity or evencause mortality in juvenile cockles (Lauckner, 1984, 1987). More-over, by inducing host defence mechanism against oxidative stressand increasing its metabolism and energy demand (Magalh~aeset al., 2017), parasites can increase host vulnerability to otherstressors (Magalh~aes et al., 2018b).

In the aquatic environment, abiotic and biotic drivers rarely actindividually, with inhabiting organisms being exposed to a com-bination of several factors, with a wide range of impacts (Sures,2008). Regarding contamination and infection levels, the relation-ship between the bivalve contaminant body burden and the para-site infection is a complex question to address. Pollution can eitherfavour or impair parasite infection success depending on a highnumber of interacting variables (Sures, 2008). Certain contami-nants may increase parasite infection by excluding their naturalpredators (Long et al., 2012), by reducing the resistance of theirhosts, or by providing improved conditions for their intermediatehosts to live. For example, Lymnaea truncatula (gastropod) sus-ceptibility to Fasciola hepatica miracidia (trematode) increaseswhen exposed to detergent contamination at low concentration(according to authors description: 4 drops in 3 L of spring water)(Abrous et al., 2001) and prevalence of Levinseniella byrdi (trema-tode) in the amphipod Orchestia grillus increases when this speciesis chronically exposed to nutrient enrichment (Johnson and Heard,2017). On the other hand, contaminants can interfere with parasitetransmission within their hosts or exert deleterious effects on in-termediate hosts of the parasites with complex life cycles and thuscan reduce the parasite abundance. As an example, F. hepaticainfectivity reduced when the host L. truncatula was exposed tocopper (Rondelaud, 1995).

Nevertheless, the available information concerning the effect ofcontaminants on host susceptibility to infection is still scarce,especially regarding the combined impact of pathogen and

contamination on host basic functions. To the best of our knowl-edge, this is the first study that comprehensively assessed hostbiochemical response, through energy reserves, metabolism andoxidative stress biomarkers, 1) when challenged by differentparasite burdens, and 2) when performing a combined exposure toparasite infection and As contamination. C.erastoderma edule (theedible cockle) and H. elongata (a trematode parasite) were used as ahost e parasite model to test the hypotheses “The greater theinfection burden, the greater the stress response” and “As willchange parasite infection levels and, consequently, cockles stressresponse to infection”.

2. Material and methods

2.1. Live material

Snails (Littorina littorea) infected only by Himasthla elongata asfirst intermediate host were collected from the NIOZ harbour, Texel,the Netherlands (53�00032.100N, 4�47036.500E). Potential second in-termediate hosts (Cerastoderma edule) were collected from theMirachannel, Ria de Aveiro coastal lagoon, Portugal (40�38031.700N,8�44010.900W). H. elongata was identified in its metacercariae stagefollowing de Montaudouin et al. (2009) description. Hosts andparasites were obtained and kept as described in Magalh~aes et al.(2018b). Shell length of the collected cockles ranged between 14and 17mm, i.e. young individuals, in order to limit natural trema-tode former infection (de Montaudouin et al., 2012b; Mouritsenet al., 2003; Wegeberg et al., 1999).

2.2. Infestation levels experiment

After two weeks of acclimation to laboratory conditions,twenty-four cockles were individually placed in glass containersfilled with 50mL seawater, with constant aeration (Fig. 1A) andunder controlled abiotic conditions: salinity¼ 30± 1, tempera-ture¼ 17 �C, pH¼ 8.2 and photoperiod¼ 12:12 h (light/dark).

In order to obtain cercariae, infected snails (kept at ~14 �C) wereindividually transferred to a 6-well plate with ~16mL artificialseawater (salinity¼ 35± 1) per well and exposed to constant illu-mination and consequent temperature boost (~25 �C) during 4e6 h.Cercariae were collected with a pipette, pooled, counted andseparated into groups of twelve, twenty-five and fifty cercariae andthen used for cockle immediate infestation.

Experimental design included one factor (trematode infection)with four levels, i.e. control (CTL¼ 0 cercariae), twelve, twenty-fiveand fifty cercariae (inf 12, inf 25 and inf 50, respectively), with sixreplicates per level/condition (Fig. 1A). At the end of the infestationexperiment (48 h after cercariae addition, to allow encystment (deMontaudouin et al., 2016)) cockles were conserved at - 80 �C forfurther H. elongata infection success calculation (percentage ofadministered cercariae that infected cockles), metacercariaeabundance determination (cockles susceptibility to parasites) andfor biochemical responses evaluation.

2.3. Contamination experiment

After two weeks of acclimation period, twenty-four cockleswere individually placed in glass containers filled with 50mLseawater, with constant aeration and under the same controlledabiotic conditions described previously. These cockles wereexposed to two different As treatments, with 12 replicates pertreatment (Fig. 1B), during 96 h. This exposure period was selectedbased on standard guides for conducting toxicity tests with mac-roinvertebrates (ASTM E729-96, 2002). The tested treatments were0.0 mg L�1 (control) and 5.2 mg L�1, corresponding to maximum As

Fig. 1. Schematic representation of the experimental designs. A: Infestation levelsexperiment and B: Contamination experiment. As: Arsenic.

L. Magalh~aes et al. / Environmental Pollution 243 (2018) 1469e1478 1471

levels found dissolved in thewater of the Ria de Aveiro (Ereira et al.,2015). Arsenic stock solution (10.4mg L�1) was prepared usingSodium Arsenate (Sigma-Aldrich).

After 96 h of contamination, infected snails were exposed to anew temperature boost, cercariae were obtained as mentionedbefore and separated into groups of twenty-five. Then, cercariaewere used to infest six cockles per As treatment (Fig. 1B).

At the end of the experiment (96 h of As exposure þ 48 h ofcombined As and cercariae exposure) cockles were conserved at -80 �C for further H. elongata infection success calculation, meta-cercariae abundance determination and biochemical responsesassessment.

2.4. Biochemical descriptors

After the experimental period, cockles from the two experi-ments (infestation levels and contamination), making a total ofeight treatments, were dissected and observed using a stereomi-croscope to assess H. elongata infection success and metacercariaenumber. Then, in order to obtain enough flesh for further analysis,cockles were pooled in groups of two entire organisms per repli-cate, three replicates (corresponding to six cockles) per treatment.After homogenization with liquid nitrogen, each replicate wasseparated into at least 3 aliquots containing 0.3 g of soft tissue.Eight different biochemical markers were determined after aliquotsextraction using specific buffers (described in Magalh~aes et al.,2018b): protein (PROT) and glycogen (GLY) contents, for energystorage measure; electron transport system (ETS) activity, repre-senting a proxy of cellular respiratory potential; superoxide

dismutase (SOD) activity, the enzyme responsible for the removalof superoxide anion (O2�) with hydrogen peroxide (H2O2) forma-tion; catalase (CAT) activity, the enzyme that reduces the SODproduct to water; glutathione peroxidase (GPx) activity, theenzyme that catalyses the reduction of several hydroperoxides towater; glutathione S-transferases (GSTs) activity, a group ofbiotransformation enzymes that act as cell detoxifiers and lipidperoxidation (LPO) level, an indicator of cellular damage. Details onbiomarkers methods are described in Table 1.

2.5. Data analysis

For the infestation levels experiment, one-way ANOVA wasperformed in order to compare initial cockle shell length betweentreatments and to test the effect of treatments (CTL, 12, 25 and 50cercariae) on the infection success and number of H. elongatametacercariae infecting cockles after exposure. Prior to analysis,homogeneity of variance was verified with Cochran test. One-wayANOVA was followed by post-hoc Tukey test for comparison ofmeans.

For the contamination experiment, two-way ANOVA was per-formed in order to compare initial cockle shell length betweentreatments and to test the effect of treatment (0.0 and 5.2 mg L�1),infection condition (parasitized and non-parasitized) and theinteraction between factors on the number of H. elongata meta-cercariae infecting cockles after exposure. Prior to analysis, homo-geneity of variance was verified with Cochran test. Two-wayANOVA was followed by post-hoc Tukey test for comparison ofmeans.

Due to a lack of homogeneity of variance, PROT, GLY, ETS, SOD,CAT, GPx, GSTs and LPO, were separately submitted to a non-parametric permutational analysis of variance (PERMANOVA Add-on in PRIMER-E software) with one fixed factor (infection level)and four levels (CTL, 12, 25 and 50 cercariae). Main test with pvalues lower than 0.05 were considered as significant and followedby pair-wise tests. Pair-wise tests were used to identify statisticaldifferences and represented in figures with lower case letters. Theeffect of contamination (0.0 and 5.2 mg L�1), infection condition(parasitized and non-parasitized) and the interaction betweenfactors were tested on each biomarker using the same analysisdescribed above but taking into account two fixed factors (Astreatment and infection condition) with two levels each.

The Euclidean distance of the matrix of each experiment con-taining biomarkers results per treatment was calculated aftersamples normalisation. Distances among centroids were thenplotted in a Principal Coordinates Ordination analysis (PCO).Superimposed vectors were used to represent the variables (bio-markers) that better (r> j0.8j) explained samples spatialdistribution.

3. Results

Cockles used on each treatment level of each experimentshowed similar mean shell length (p� 0.05), mean¼ 15.8± 0.8(standard deviation) mm (N¼ 2 experiments x 4 treatments x 6replicates¼ 48).

3.1. Infestation levels experiment

Different infestation levels resulted in similar H. elongatainfection success (One-way ANOVA, F (2)¼ 0.51, p< 0.61, Fig. 2A)and significantly different number of H. elongata metacercariae(One-way ANOVA, F (3, 16)¼ 14.7, p< 0.001). H. elongata abun-dance was the lowest in naturally infected cockles (0.8± 1.0 met-acercariae cockle-1), from hereafter named as “non-parasitized

Table 1Method principle, function and respective reference of the eight biomarkers used in the present study.

Biomarker Method Principle Function Reference

Protein (PROT) Under alkaline conditions, the peptide bonds form a purple complex (measured at 540 nm)with coppersalts contained in the biuret reagent

Energy reserve Robinson andHogden 1940

Glycogen (GLY) Under acidic conditions, carbohydrates are dehydrated forming a colored product with phenolspectrophotometrically measured at 492 nm

Energy reserve Dubois et al., 1956

Electron TransportSystem (ETS)

ETS reduces tetrazolium with formation of formazan, a chromogenic product spectrophotometricallymeasured at 490 nm

Mitochondrialmetabolism

De Coen andJanssen 1997

SuperoxideDismutase (SOD)

Nitroblue tetrazolium (NBT) is converted to NBTdiformazan (formazan dye) via superoxide radical,which is the SOD substrate. SOD activity is measured in decreased absorbance at 560 nm

Antioxidant enzyme Beauchamp andFridovich 1971

Catalase (CAT) CAT reacts with methanol in the presence of H2O2 producing formaldehyde that isspectrophotometrically measured at 540 nm using Purpald as a chromogen

Antioxidant enzyme Johansson andBorg 1988

GlutathionePeroxidase (GPx)

GPx catalyzes the reduction of cumene hydroperoxide oxidizing reduced glutathione (GSH) to formdisulfide glutathione. The oxidized glutathione is then reduced by glutathione reductase and NADPHforming NADPþ, resulting in decreased absorbance at 340 nm and recycling the GSH

Antioxidant enzyme Paglia andValentine 1967

Glutathione S-transferases(GSTs)

GSTs catalyse the conjugation reaction of CDNB (used as substrate) to GSH with formation of aspectrophotometrically measured (at 340 nm) thioether

Biotransformationenzyme/cell detoxifier

Habig et al., 1974

Lipid peroxidation(LPO)

Malonaldehyde, a product from membrane deterioration, is determined by thiobarbituric acid (TBA)-measurements forming TBA reactive substances, spectrophotometrically measured at 532 nm

Cellular damage Buege and Aust1978

Fig. 2. Mean (±standard deviation) of Himasthla elongata infection success (A) andmetacercariae infection (B) found in control and three levels of experimentally para-sitized cockles (infection levels: 12, 25 and 50). Post-hoc Tukey test homogenousgroups (p< 0.05) are represented by lower case letters.

L. Magalh~aes et al. / Environmental Pollution 243 (2018) 1469e14781472

cockles (NP)” and the highest in experimentally infected cockles,from hereafter named as “parasitized cockles (P)”, infested withfifty cercariae (11.7± 1.2 metacercariae cockle�1) (Fig. 2B). Cockles

infested with twelve and twenty-five cercariae showed interme-diate and similar H. elongata abundance (4.3± 3.6 and 6.5± 2.0metacercariae cockle�1, respectively) (Fig. 2B).

3.2. Contamination experiment

Different As treatments resulted in similar H. elongata infectionsuccess (One-way ANOVA, F (1, 10)¼ 1.1, p¼ 0.33, Fig. 3A). Cocklespresented similar infection when exposed to As compared to non-contaminated cockles (7.0± 4.1 metacercariae cockle�1) (Fig. 3B,Table 2). P cockles presented significantly higher number ofH. elongata metacercariae compared to control, i.e. NP cockles(Fig. 3B, Table 2), regardless As treatment.

3.3. Biochemical descriptors

3.3.1. Infestation levels experimentInfection levels did not affect PROT content (Table 3).GLY content was significantly higher in P cockles compared to

CTL cockles, with significantly higher values at the highest infectionlevel, while infection levels 12 and 25 displayed intermediate andnot significantly different GLY contents (Fig. 4A, Table 3).

Cockles exposed to the highest infection level (50 cercariae)presented significantly higher ETS values compared to CTL andcockles exposed to the lowest infection level (Fig. 4B, Table 3).

SOD activity showed no significant differences between cocklesexposed to 50 cercariae and CTL that presented significantly highervalues compared to cockles from the infection levels 12 and 25(Fig. 4C, Table 3).

CAT activity was significantly higher in P cockles compared toCTL. The highest CAT activity was observed at the highest infectionlevel, with significant differences to the remaining conditions(Fig. 4D, Table 3).

GPx activity showed no significant differences between cocklesexposed to 50 cercariae and CTL that presented higher valuescompared to cockles from the infection levels 12 and 25 (Fig. 4E,Table 3).

GSTs activity showed no significant differences between cocklesexposed to 50 cercariae and CTL. The lowest GSTs levels wereregistered for cockles exposed to 25 cercariae, with significantdifferences to the remaining conditions (Fig. 4F, Table 3).

Regarding LPO levels, no significant differences were observedbetween cockles exposed to the highest level of infection and CTL.LPO was significantly higher in cockles exposed to 25 cercariae and

Fig. 3. Mean (±standard deviation) of Himasthla elongata infection success (A) andmetacercariae infection (B) found in not experimentally parasitized (NP) and experi-mentally parasitized (P) cockles of each Arsenic treatment (0.0 and 5.2 mg L�1). Post-hoc Tukey test homogenous groups (p< 0.05) are represented by lower case letters.

Table 3PERMANOVA results performed to test the effects of infection conditions (0, 12, 25and 50) on the biochemical descriptors. PROT: protein; GLY: glycogen; ETS: electrontransport system; SOD: superoxide dismutase; CAT: catalase; GPx: glutathioneperoxidase; GSTs: glutathione S-transferases; LPO: lipid peroxidation; df: degrees offreedom of the factor, samples; MS: mean square. Bold letters indicate significantdifferences (p< 0.05). N¼ 12.

Infection condition p Error MS

df MS Pseudo-F df

PROT 3, 10 3.2 18.5 0.06 8 0.2GLY 3, 10 3.5 74.0

Fig. 4. Mean values (±standard deviation) and significant differences represented with different lower case letters of A: GLY, glycogen content; B: ETS, electron transport systemactivity; C: SOD, superoxide dismutase activity; D: CAT, catalase activity; E: GPx, glutathione peroxidase activity; F: GSTs, glutathione S-transferases activity and G: LPO, lipidperoxidation levels in four levels of cercariae infection: 0 (CTL), 12, 25 and 50. H: Principal coordinates ordination analysis (PCO) showing the variables that better explained samplesdistribution.

Table 4Biomarkers correlation scores to each axis of the principal coordinates ordinationanalysis (PCO) and each experiment. PCO1: PCO horizontal axis; PCO2: PCO verticalaxis; GLY: glycogen; ETS: electron transport system; SOD: superoxide dismutase;CAT: catalase; GPx: glutathione peroxidase; GSTs: glutathione S-transferases; LPO:lipid peroxidation. Bold letters indicate high correlations (r> j0.8j, p< 0.05).

Infestation experiment Contamination experiment

PCO1 (49%) PCO2 (37%) PCO1 (74%) PCO2 (21%)

GLY ¡0.97 0.18 ¡0.96 �0.04ETS ¡1.00 0.01 ¡0.96 0.24SOD �0.37 �0.64 0.83 0.55CAT ¡0.89 �0.12 0.90 �0.37GPx �0.48 ¡0.86 0.80 0.58GSTs 0.47 ¡0.87 0.22 0.96LPO �0.25 0.83 ¡0.83 0.45

L. Magalh~aes et al. / Environmental Pollution 243 (2018) 1469e14781474

0.8, Table 4) while SOD, CAT and GPx presented high positive cor-relation (r> 0.8, Table 4) to PCO 1. These variables presented themost influence on samples space distribution. The PCO2 explained21% of the total variation, separating cockles from control treat-ment (NP and P), in the positive side of the axis, from cocklesexposed to As contamination (NP and P) in the negative side, with astrong positive correlation to GSTs activity (r> 0.8, Fig. 5H, Table 4).

4. Discussion

Experimental studies on parasite transmission events have

proved to be extremely useful for the knowledge on parasiteecology (Poulin, 2010). In the present study, the experimentalapproach contributed to understand the effects of a classicalcontaminant on the infectious success of a trematode parasite and,for the first time, the repercussions on host biochemical perfor-mance and consequent susceptibility to infection.

In what regards to infection success, different authors agreedthat, in a natural context, few hosts carry the majority of the par-asites (Ebert et al., 2000), i.e. parasites distribute aggregativelywithin their population host, which is possibly explained by a snowball effect, i.e. the more parasites infect a host, the more susceptiblethis particular host is for new infections. However, and experi-mentally speaking, results are controversial with some studiesshowing that infection success is negatively dose-dependent(Poulin, 2010), but others demonstrated that infection intensitytends to proportionally increase with the number of cercariaeexposure (Liddell et al., 2017). Our findings showed that the highestthe cercariae exposure (at least within the tested range of 50cercariae), the highest the trematode infection and consequentmetacercariae encystment in the second intermediate host (Cera-stoderma edule cockles). Moreover, the present study showed thatthe more parasites infect a single host, the stronger are theparasite-induced effects on the host biochemical response.

The bivalve immune system answers to trematode invasion byrecruiting granulocytes. This type of haemocytes are the bivalveimmunoreactive cells that present the highest phagocytic capacity,encapsulating and destroying the pathogen with lysosomal

Table 5PERMANOVA results performed to test the effects of arsenic experimental treatments (0.0 and 5.2 mg L�1), infection condition and interaction of factors on the biochemicaldescriptors. PROT: protein; GLY: glycogen; ETS: electron transport system; SOD: superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; GSTs: glutathione S-transferases; LPO: lipid peroxidation; df: degrees of freedom of the factor, samples; MS: mean square. Bold letters indicate significant differences (p< 0.05). N¼ 12.

Treatment Infection condition Interaction Error

df MS Pseudo-F p df MS Pseudo-F p df MS Pseudo-F p df MS

PROT 1, 10 2.6 11.6 0.06 1, 10 6.5 29.1 0.06 1, 10 6.0.10�2 0.3 0.62 8 0.2GLY 1, 10 0.3 1.0 0.36 1, 10 7.8 22.9 0.002 1, 10 0.2 0.6 0.47 8 0.3ETS 1, 10 1.8 9.3 0.02 1, 10 5.1 26.1 0.001 1, 10 2.6 13.2 0.008 8 0.2SOD 1, 10 1.0 3.3 0.12 1, 10 6.8 22.1 0.001 1, 10 0.7 2.3 0.17 8 0.3CAT 1, 10 3.6 53.4

L. Magalh~aes et al. / Environmental Pollution 243 (2018) 1469e14781476

antioxidant and biotransformation enzymes, cockles that weremoderately infected (infection levels 12 and 25) showed an overall(except for CAT) reduction of their activities compared to CTL andinfection level 50. It is known that the Ria de Aveiro (the coastallagoon where cockles were collected) is characterized by lowtrematode metacercariae abundance and prevalence (Magalh~aeset al., 2018a), which lead us to hypothesise that these cockles im-mune system may be poorly adapted to rapidly react to trematodeinfection. Although bivalves lack the mechanisms conferringadaptive immunity in vertebrates, bivalves immune system isstrongly influenced by endogenous and environmental relatedfactors (Zannella et al., 2017) and there are already some evidencesof a certain level of immune specificity and immune memory(Zhang et al., 2014), or at least some “memory mechannisms”related to the life history of these bivalves (Paul-Pont et al., 2010a).Thus, a moderate infection (represented by 12 and 25 cercariaeaddition) may not be enough for cockles to efficiently activate theirantioxidant system and resulting into higher cellular damage(higher LPO). On the other hand, when these young and smallcockles were exposed to 50 cercariae and, consequently, meta-cercariae infection reached the threshold of more than 10 meta-cercariae per cockle, cockles increased the activity of all enzymesstudied leading to an efficient reduction of the cellular damage.

Regarding contamination experiment results, the present studyrevealed that when cockles were exposed to As contamination andwere then challengedwith a moderate level of H. elongata cercariae(25), they presented similar number of metacercariae compared tonot contaminated cockles (8.8 vs. 6.0 metacercariae cockle�1,respectively). Conversely, several examples from the literatureshowed higher infection in contaminated conditions: snails (Physafontinalis and Lymnaea stagnalis) previously exposed to toxicants(cadmium, zinc and a cadmium/zinc mixture used at concentra-tions of 100, 1000 and 10,000 mg L�1) have shown an increase inparasite Echinoparyphium recurvatum prevalence and intensity(Morley et al., 2002) and M. edulis mussels exposed to neurotoxinsthrough ingestion of harmful algal species showed a significantincrease in the occurrence of Gymnophallidae metacercariae(Galimany et al., 2008). These environmental pollutants may haveweakened host defences and contributed for the higher infectionsuccess of the parasites. However, the cercariae pressure (25) andthe contamination level (5.2 mg L�1 As) used in the present work, aswell as the duration of the exposure (96 h) may not have beensufficient to exert similar effect.

During several cellular pathways, mainly related to aerobicmetabolism, ROS are naturally produced (Murphy, 2009). Undernormal conditions, bivalves can regulate ROS quantity and preventoxidative stress using the antioxidant system. However, chemicaltoxicity can interfere with the cellular balance between proox-idants and antioxidants resulting into antioxidants capacitydepression and increase of intracellular ROS formation (Regoli andGiuliani, 2014). Nevertheless, in the present study, results showedlower impact of As contamination compared to trematode infectioneffects. As mentioned before, this can be explained by the Ascontamination level used (5.2 mg L�1, which is under reportedC. edule bioaccumulated As (Figueira et al., 2011)) and by the shortexposure period (96 h) that was not enough to induce cellular in-juries. Low impact of As (1mg L�1), acting under regular temper-ature conditions (17 �C), was also demonstrated in mussels,M. galloprovincialis,with similar SOD, CAT and ETS activities as wellas similar LPO levels compared to control (no As contamination)(Coppola et al., 2018). In its turn, trematode infection alone caused asignificant increase in cockles GLY content and on ETS activity that,as previously discussed, is related to higher energy storage andmetabolic requirements induced by the parasite presence. More-over, comparable to what was discussed above, regarding the

infestation levels experiment and particularly concerning theinfestation level 25, the antioxidant response was not activated bythe presence of trematode infection leading to higher LPO levels.

Acting in interaction, As contamination and trematode infectiondid not affect the GLY accumulation pattern described above, i.e.parasitized cockles presented higher GLY content than non-parasitized cockles regardless As exposure. A similar case wasfound in a freshwater clam (Pisidium amnicum) infected by trem-atodes and exposed to petachlorophenol (Heinonen et al., 2001).Conversely, in the present study, the stressors interaction (Ascontamination and parasite infection) induced an effect in the hostmetabolism reducing the ETS activity. This change in the meta-bolism of infected cockles under contaminated conditions canindicate a change in some physiological parameters such as feedingor respiration when cockles were facing more than one stressfulcondition at the same time. Similarly, it was registered a reductionin the cardiac rate of both bivalve and pulmonate molluscs,compared to controls, when under the combined effect of pollut-ants (0.2, 1.0, 1.8mg L�1 of copper sulfate) and trematodes (Morleyet al., 2006). Surprisingly, and taking also into account no signifi-cant activation of the antioxidant system, LPO levels of cocklessimultaneously exposed to As and trematode infection were lowcompared to parasitized cockles not exposed to As, displayingvalues that were even closer to control (NP, 0.0 mg L�1 As). Theselow LPO levels are certainly related to lower metabolic activity andconsequent lower ROS production (Murphy, 2009) induced also byAs and trematode infection interaction. Lower oxidative burst couldbe simultaneously explained by a phagocytosis suppressioninduced by the stressors interaction (Sauv�e et al., 2002). Therefore,to a certain extent and in a contamination scenario, trematodeinfection may be beneficial to cockles, working as a protection forthe pollutant accumulation in the organisms, reducing overall ROSproduction, which consequently led to less toxic effects. Otherauthors with convergent findings showed a decrease in Littorinalittorea iron, copper and nickel accumulation (field monitoring)related to digenean trematode infection (Evans et al., 2001) and asignificant decrease in cadmium (used at 15 mg L�1) bio-accumulation due to the presence of pathogens (trematodeH. elongata and bacteria Vibrio tapetis) on C. edule (Paul-Pont et al.,2010b).

5. Conclusion

Our results allowed acceptance of the first postulated hypoth-esis, i.e. when the infection burden was higher, the cockles stressresponse was also higher. However, concerning the second hy-pothesis, As contamination (at least at the single As concentrationused¼ 5.2 mg L�1) did not change parasite infection success butmodified cockles stress response to infection mostly at the meta-bolism reduction level. The present findings highlighted the dele-terious effects of trematode infection in their hosts biochemicalperformance. Biochemical markers proved to be useful tools inreflecting the invasion effects of pathogens however, may givefalse-positive or false-negative results modulated by several envi-ronmental variables such as contamination. In fact, caution must betaken when extrapolating simplified laboratory experiments intocomplex ecosystems which encounter a variety of stresses.

Acknowledgments

Luísa Magalh~aes benefited from PhD grant (reference: PD/BD/52570/2014) given by the National Funds through the PortugueseScience and Technology Foundation (FCT), supported by FSE andPrograma Operacional Capital Humano (POCH) and EuropeanUnion. Rosa Freitas benefited from a research position funded by

L. Magalh~aes et al. / Environmental Pollution 243 (2018) 1469e1478 1477

the Integrated Programme of SR&TD “Smart Valorisation ofEndogenous Marine Biological Resources Under a ChangingClimate” (reference: Centro-01-0145-FEDER-000018), co-fundedby Centro 2020 program, Portugal 2020, European Union, throughthe European Regional Development Fund. This work was sup-ported 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 finan-cial support to CESAM (UID/AMB/50017), to FCT/MEC through na-tional funds, and the co-funding by the FEDER, within the PT2020Partnership Agreement and Compete 2020. Authors are grateful toDavid Thieltges and his team for helping to collect Littorina littoreaspecimens.

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Interactive effects of contamination and trematode infection in cockles biochemical performance1. Introduction2. Material and methods2.1. Live material2.2. Infestation levels experiment2.3. Contamination experiment2.4. Biochemical descriptors2.5. Data analysis

3. Results3.1. Infestation levels experiment3.2. Contamination experiment3.3. Biochemical descriptors3.3.1. Infestation levels experiment3.3.2. Contamination experiment

4. Discussion5. ConclusionAcknowledgmentsReferences