Ecotoxicological Evaluation of the Immunocompetence of Two Bivalves Species (Mya arenaria and Mytilus edulis) in the Saguenay Fjord Including a Salinity Gradient ^†

Introduction

Bivalves are filter feeders widely used in ecotoxicological monitoring because of their sensitivity to contaminants present in the environment. [1,2] Their immunity depends of their freely open blood cells, which are directly in contact with xenobiotics make them good species to monitor environmental contamination. Decreased immunotoxicity can affect host resistance and result in an increase in disease incidence with potential effects at the population level, reinforcing the ecological relevance of studying this endpoint in bivalves. [3] However, this endpoint can be subjected to natural variability related to environmental abiotic factors. The presence of two species of bivalves in the Saguenay Fjord, the soft-shell clam (Mya arenaria) and the blue mussel (Mytilus edulis), allows us to compare the immunological status of these two species.

The Saguenay Fjord is the main tributary of the St. Lawrence Estuary and has an important variation of salinity. This gradient allows the establishment of a rich biodiversity. However, it also limits the distribution of some species, such as M. edulis, which is absent upstream of Anse St-Étienne (ASE), given that the salinity is under 18 psu. This tributary is also subject to various sources of contamination. [4,5] A complex mixture of urban, industrial and agricultural xenobiotics arising from upstream Saguenay watershed and atmospheric deposition contaminate the Saguenay-St. Lawrence. [6,7,8,9] Because of its industrial past, the sedimentary matrix of Saguenay is contaminated with toxic and potentially bioaccumulative substances like polycyclic aromatic hydrocarbons, heavy metals and tributyltin. [4,5,10] In addition, municipal wastewater from local villages may not be properly treated and flows directly into the watershed. [6,11] It is therefore crucial to assess the toxicity of all these xenobiotics on sentinel species. In this context, we have assessed the immunological response of two bivalves: the soft-shell-clams and the blue mussels and compared their status according to the sampling site.

We first evaluated the impact of the salinity on the immunocompetence of M. edulis and M. arenaria, between two reference stations, [12] ASE (18 psu) and Baie du Moulin à Baude (BMB) at salinity corresponding to the estuary (28 psu). Then, we compared the impact of corresponding harbors in two bivalve species with different habitats in the attempt to examine the cumulative effects of pollution and salinity in these bivalves.

Materials and Methods

Site location of bivalve collection

Bivalves were collected in 2013 at 4 different stations [ASE, BMB, Baie de Tadoussac (BT), Baie Sainte-Catherine (BSC)] in the Saguenay River, Québec, Canada (48° 15’ N, 70° 09’ W) (Figure 1). ASE and BMB were not exposed to any direct contamination (reference sites) but differed in salinity at 18 and 28 part per thousand respectively. The other two stations, BT and BSC were the two polluted stations because of the intensive commercial and recreational boating activities. [4,12] At each site, 15 clams and 15 mussels were sampled and maintained at 4°C in icebox.

Viability and phagocytosis assessments

Hemolymph was extracted from the adductor muscle using 3 mL syringe with 23 G needle. The viability was determined by flow cytometry using a BD Accuri™ C6 (Becton Dickinson, San Jose, CA, USA) and by propidium iodide (PI) staining. Phagocytosis was evaluated according to the method developed by our laboratory. [13] Briefly, hemocytes were mixed with yellow-green latex FluoSpheres (Molecular Probes Inc., Eugene, OR, USA) at a ratio of 1:100 (hemocytes:beads) in flat-bottom 96 wells plate. The mixtures were incubated at 20°C in the dark. After 18 h, the supernatant was delicately removed. The cells were fixed with 200 µL of 0.5% formalin in sterile water. Phagocytosis was measured by flow cytometry, BD Accuri™ C6 (Becton Dickinson) following the analysis of hemocytes according to their scattering properties of forward and right angle. A total of 3000 events were acquired to analyze fluorescence frequency distribution on FL1 and determined phagocytic activity (one bead and more) and phagocytic efficacy (three beads and more). Data collection and analysis were performed with BD Accuri™ C6 (Becton Dickinson) software.

Statistical analysis

The difference between each station in the Saguenay River was evaluated by analysis if variance (ANOVA) followed by Tukey’s test for pairwise comparisons. Statistical analyses were performed using SigmaStat (version 3.5; Systat Software, Inc., San Jose, CA, USA). Significance was set at P<0.05.

Results

The hemocytes viability of each species was not significantly different for both species between ASE and BMB. However, the phagocytic activities significantly differed for Mya arenaria. No variations were observed for M. edulis in salinity gradient (Figure 2).

The hemocytes viability and phagocytosis were then evaluated for the polluted stations (BT and BSC) compared with reference stations but only with BMB for M. arenaria because of salinity impact observed in Figure 3. For both species, no variations were observed in the hemocytes viability despite the anthropic activities and known contamination present at BT and BSC. For M. arenaria, no differences were observed in hemocytes phagocytic activity and efficacy, but significant variation occurs for M. edulis. This variation was observed between ASE and only one polluted station (BSC), where the phagocytosis significantly increased. However, no modulation of the phagocytic competence has been observed when BMB was compared with BT and BSC for both species. On the other side, the phagocytosis of mussel hemocytes of the two polluted stations differs significantly and was increased at BSC (Figure 3). The hemocytes viability and phagocytosis of each species were compared to determine the immunological modulation at each station. In the reference stations, ASE and BMB, no significant differences were observed between clams and mussels. In contrast, the hemocytes viability showed no differences in contaminated stations, at BT and BSC, but the immune response was significantly modulated. In BT, the phagocytic competence of soft-shell clams was higher than for mussels. In BSC, mussels’ hemocytes had higher phagocytic capacity (1 bead and more) but no differences other were observed for the phagocytic efficacy (3 beads and more).

Discussion

The large distribution of bivalves in the intertidal zone and in the Saguenay makes them good species to follow environmental conditions and contamination. However, the impact of xenobiotics must be evaluated for each species because they are not subject to the same types of exposure. [12,14] The analysis of the immune-modulation in field studies is complex due to the multitude of confounding factors in the habitat of these species may influence this reaction. [12,14] In this study, we want to assess the immunomodulation of M. edulis and Mya arenaria in four different stations in Saguenay Fjord including a salinity gradient between two reference stations. By this investigation, we initially evaluated the impact of this confounding factor, then the anthropic pollution impact in BT and BSC and finally, if both species showed the same immunological response.

The reduction of salinity is already known to induce many physiological changes like growth rate, [15] heart rate, [16,17] respiration [18] and energy acquisition. [19,20] Moreover, a reduction of salinity from 32 psu to 16 psu induced a decrease in the immune response of mussels in controlled conditions. [20] However, our results demonstrate no significant difference between ASE (18 psu) and BMB (28 psu) for this species (Figure 2). This may be explained by the natural adaptation of the mussels from ASE. In contrast, the soft-shell clams hemocytes phagocytic efficacy is significantly lower at ASE (Figure 2). Despite this observed modulation in hemocytes, there are no differences between the immune response of these two bivalves at ASE and BMB.

In contaminated stations, it is noteworthy that M. arenaria and M. edulis seem to be affected differently by the xenobiotics present in the environment, principally at BT. This variation may be caused by a difference in sensitivity for each species facing contamination, [21] but also by the difference in the way of exposure to xenobiotics. [1] Indeed, M. arenaria is exposed directly to sediments and water column contaminants, while M. edulis is only exposed to the xenobiotics when filtering water. [1,4,5,22] Furthermore, the properties of some xenobiotics can change with salinity and affect their biodisponibility enable them to be absorbed by the sediments particles and accumulate in the organisms. [23] Because of this chemical variation, all comparisons with ASE, need to be done with a lot of cautiousness despite the absence of immunological variation in the salinity gradient for mussels (Figure 2). Indeed, both species absorb contaminants in their tissues by filtering water, but the clams also accumulate them through direct contact with sediment and porewater [23] resulting in increased transfer. [24] The sediment matrix in the Saguenay Fjord is more heavily polluted than the water column for a given station, thus exposing the clams to a broader range of contaminants with higher concentrations. [10]

Conclusions

In this study, we demonstrated that mussels living in natural environment and exposed to different salinity (18 psu and 28 psu) adapt their immunocompetence and show no difference in basal immunocompetence. However, the clam is slightly affected by this salinity reduction by a decreased in their phagocytic efficacy. Moreover, stations heavily affected by human activities (BT and BSC), reveal a clear different modulation of the phagocytosis between clams and mussels, highlighting their own sensitivity to pollution. This shows the relevance of using multiple sentinel species in field studies to have a better overview and comprehension of the impact of human activities.