Role of confounding factors in assessing immune competence of bivalves ( Mya arenaria , Mytilus edulis ) exposed to pollutants

C. Brousseau-Fournier,1,2,3 G. Alix,1,2,3 A. Beaudry,1,2,3 S. Gauthier-Clerc,4 M. Duchemin,5 M. Fortier,2 M. Auffret,5 Michel Fournier,1,2,3 P. Brousseau1,2,3 1Parc de la rivière Mitis, Ste-Flavie, Quebec, Canada; 2Institut National de la Recherche Scientifique-Institut-ArmandFrappier (INRS-IAF), Laval, Quebec, Canada; 3Université du Québec à Rimouski, Rimouski, Quebec, Canada; 4Institut des Sciences de la mer de Rimouski (ISMER), Rimouski, Quebec, Canada; 5Université de Bretagne Occidentale, Brest, France


Introduction
The efficacy of bio-monitoring programs could be improved through addition of sensitive biomarkers.Among various aspects that could be included in these programs, the immune system is certainly a judicious selection.Indeed, the immune system is considered as a key physiological component in risk assessment, whatever the nature of the contamination. 1 Moreover, this system is essential towards organism health and interacts intimately with the function of many organs and organ systems.For example, the role of endocrine disruptors, especially Bisphenol-A on the immune system has been previously investigated. 2The main role of the immune system is to provide protection to organism against infectious diseases and neoplasic cells.To do so, the immune system is constituted by numerous lymphoid organs as well as cells and a huge number of soluble factors and receptors which render this system highly sophisticated. 3,4The efficiency of this complex system is dependent on the optimal balance and communication of these components in organisms.It is well documented that the complexity of the immune system decreases in lower vertebrates and in invertebrate species.The immune defenses of invertebrates, such as mollusks, crustaceans, sponges and oligochaetes rely mainly on non-specific immunity. 4Disruption of the immunoregulation in invertebrates could lead to undesirable responses such as immunosuppression. 5lthough complete elucidation of hematopoiesis in bivalves is not yet fully understood, 6,7 the effects of environmental contaminants on the immune system of bivalves have been the subject of a rapidly expanding body of literature. 4 Another key characteristic in bivalves relies in their ability to bioaccumulate chemicals due to their filterfeeding ability and their sedentary life. 8ndeed, bivalves are recognized as sentinel species for aquatic ecosystem. 91][12][13][14][15][16] This aspect has stimulated great interest because these effects generally occur at levels that are lower than those associated with acute toxicity. 3The sensitivity of various immunological responses and the degree to which they agree with host resistance models have been also addressed 1,[17][18][19] and this work has led to the concept of immunomarkers. 20mong immunomarkers, the evaluation of the sensitivity of phagocytosis to a variety of chemicals in bivalves has been under scrutiny since a while because, phagocytic activity is a well conserved function maintained through evolution and therefore is present in all living species.As far as bio-monitoring programs are concerned, phagocytosis of hemocytes from Mya arenaria and Mytilus edulis may represent an ideal immunomarker endpoint.
However, the immune system, because of it close interaction with other main physiological systems may also be the target of source of modulation different from environmental pollutants, that should be taking in account as confounding factors.In previous study our group has demonstrated that different habitat conditions such location on the beach and stress related to tides and temperature, can modulate both the immune competence and sensitivity to chemicals. 21,22In the present paper, we test in a pilot study the impact on immune competence of seasonal variations and the gender difference in sensitivity to mercury.

Animals and protocols
Clams and blue mussels were collected at low tide on South shore of the St. Lawrence River, in Metis Bay (48°40' N, 68°00' W).This is a remote site exempt from any direct sewage outputs and harbor activities.All of the specimens measured between 50-70 mm in shell length.Upon arrival to the laboratory, clams and mussels were transferred and maintained into different aquaria filled with clean sand with flowing sea water (7°C) for an acclimation period of two weeks.After acclimation influence of four parameters on the immune response were verify.Experiments were performed twice.

Temperature
Animals were collected on June 11 th 2012 and the temperature at collection time was 7.0°C.Temperature was gradually raised from 7 to 13°C over a period of 6 weeks (9, 11, 13°C).Animals were left at each new temperature for two weeks before assays.

Seasonal variation
Blue mussels were collected from Metis Bay from June until November, brought back to the laboratory and assays were done immediately upon arrival.

Gender effect
Hemocytes collected from 40 males and 40 females blue mussels were used in an in vitro exposure to mercuric chloride (HgCl 2 ) (Sigma Chemical Company, St. Louis, MO, USA) to verify their sensitivity to toxic.Briefly, individual cell suspensions were distributed in plate (5¥10 5 /well) and cells were incubated at final concentrations of HgCl 2 ranging from 10 -9 up to 10 -3 for 18 h before assays.

N o n -c o m m e r c i a l u s e o n l y
cells was determined microscopically.The viability was determined by flow cytometry using propidium iodide.Briefly, 1 µL of a 1 mg mL -1 stock solution (Sigma Chemical Company) was added to each cell suspension and flow cytometric acquisition of these suspensions was performed.The results were expressed in percent of viable cells.

Phagocytosis
Hemocytes were mixed with yellow-green latex FluoSpheres (Molecular Probes Inc., Eugene, OR, USA), at a ratio of 1:30 (hemocytes:beads) in microplates.After an incubation of 18 h in the dark, the microplates were emptied by inversion.Cells were resuspended and fixed in phosphate buffered saline containing 0.5% formalin.A FACScan (Becton Dickinson, San Jose, CA, USA) with an air cooled argon laser providing an excitation at 488 nm was used.Fluorescence emission was collected at 520 nm.Results were expressed in percentage of phagocytic activity (1 bead and more), phagocytic efficacy (3 beads and more) or in percentage of normal response.

Statistical analysis
Results were expressed as mean±SD.Each parameter was assessed using two-way analysis of variance (ANOVA), considering the treatment (water temperature … and metal exposure), the sampling time and their interaction as main factors and the measured parameters as dependent variables.Tukey's multiple comparison tests (P≤0.05) were used where significant differences were detected in the ANOVA (SAS software version 9.1, SAS Institute Inc., Cary, NC, USA).

Results and Discussion
To our knowledge, only a few studies have investigated so far the effects of changing environmental parameters on the immune responses of bivalves.In this pilot experiment, the range of temperatures tested (7-13°C) did not significantly influence the viability of hemocytes or modify the immune response.However, significant changes for seasonal variations (Figure 1) and gender effects (Figure 2) for phagocytosis activity and efficacy were observed.
For seasonal variations, the results obtained showed that phagocytic activity as well as efficacy was significantly reduced for four time points out of six with a quite important reduction in July as well in November.Results from Crassostrea gigas 23 showed that phagocytosis index was significantly reduced during spawning in June-July and November-December.In the present study, we have observed early summer a decrease of the phagocytosis activity which coincide with the spawning. 24However, a more dramatic reduction of phagocytosis activity was observed in November, This could be the result of a combined action of spawning and cold water temperature.Though, confirmation of spawning in the fall shall be done.
For gender difference, we have observed that the phagocytic activity in females was significantly reduced when the cells were exposed to 10 -5 M of mercuric chloride, while significant reduction for males was observed 10 fold higher (10 -4 M).It has already been published similar results in vertebrates, following in vitro incubations of immune cells with heavy metals. 25However, to our knowledge, this is the first demonstration of a gender difference to mercury, in bivalves.
In conclusion, this pilot study has reinforced the fact that when environmental studies are designed, special care must be applied in the identification of confounding factors such as temperature, seasonality and gender, which could increase the variability of phagocytosis.

Figure 1 .
Figure 1.The phagocytic activity of blue mussel is not significantly different between June and August but significantly lower for the other four months.

Figure 2 .
Figure 2. Phagocytic activity of blue mussel, expressed as percent of normal response, shows a higher sensitivity for female than male to mercury with a significant suppression of this function at 10 -5 M for females and 10 -4 M for males.