Metal Contamination and Biomarkers in Cerastoderma Glaucum: A Multi Levels Approach?
Sahar Karray1,2*, Justine Marchand1, Alain Geffard3, Tarek Rebai5, Francoise Denis1,4,Benoit Chenais1 and Amel Hamza-Chaffai2
1University of Maine - Le Mans, EA 2460 Sea Molecules Health, University Institute Sea and Littoral - FR3473 CNRS, 72085 Le Mans Cedex, France
2University of Sfax, Marine and Environmental Ecotoxicology Laboratory, Tunisia
3University of Reims-Champagne Ardenne, EA 4689 Animal Environment Interactions, BP 1039, 51687 Reims Cedex 2, France
4UMR 7208 CNRS-MNHN-IRD-UPMC Biology of Aquatic Organisms and Ecosystems (BOREA), Concarneau, France
5University of Sfax, Histology Laboratory at the Faculty of Medicine of Sfax, Tunisia
*Address for Correspondence: Sahar Karray, University of Sfax, Marine and Environmental Ecotoxicology Laboratory, Tunisia, 92706657; E-mail: firstname.lastname@example.org
Submitted: 24 October 2017 2017; Approved: 30 October 2017; Published: 31 October 2017
Citation this article: Karray S, Marchand J, Geffard A, Rebai T, Denis F, et al. Metal Contamination and Biomarkers in Cerastoderma Glaucum: A Multi Levels Approach. Adv J Toxicol Curr Res. 2017;1(2): 074-083.
Copyright: © 2017 Karray S, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited
This study investigates the response of the cockle Cerastoderma glaucum to metallic stressors at different levels of biological organisation in three differently impacted sites through the study of 1) the global physiological state of organisms, 2) metal bioaccumulation in natural cockles, 3) the gametogenesis and the reproductive cycle, and 4) the responses at the transcriptional level of several key genes. Results show a global decrease in the global physiological status of cockles (stress-on-stress, condition index and sex ratio) in the polluted sites compared to the "reference" site. Results of metal accumulation show that cockles from the contaminated site present the highest values of different metals (Cd, Cu, Zn and Ni) followed by those from the intermediate site and the "reference" site. Results of gametogenesis and maturity index show few differences in the reproductive cycle of cockles collected from the three studied sites. The expression of seven genes encoding enzymes or proteins involved in response to different types of environmental stress resulted in different patterns that could be grouped into three groups.
1) Genes involved in metals detoxification (ABCB1 and MT) and superoxide dismutase (MnSOD and CuZnSOD) do not show any difference in their levels of expression.
2) HSP70 gene expression decrease when environmental pollution increases.
3) CAT and COI showed higher expression levels in the intermediate and the contaminated site compared to the" reference" site. So, HSP70, Catalase and COI gene expression could be selected as relevant biomarkers for in situ studies of the cockle C. glaucum but they should be coupled to proteomics studies.
The gulf of Gabes located in the southern coast of Tunisia, has particular topographic and biologic characteristics (shallow waters, weak currents, high salinity and temperature). Moreover, it is considered as a refuge for larvae and juveniles of animals . This ecosystem is known to contribute to 42% of the halieutic Tunisian national production . At the same time, this area is exposed to a high anthropogenic pressure linked to a considerable human demographic growth. In fact, many industrial activities, such as crude phosphate treatment, chemical industry, tannery, etc, release in the marine environment their pollutant-containing effluents [3-5]. Therefore this region is convenient for ecotoxicological studies. Since mollusc bivalves are the most successful organisms used to monitor pollution along coasts, we used the cockle Cerastoderma glaucum, which is largely distributed in the gulf of Gabes and Mediterranean coasts. C. glaucum is living in close contact with sediment that is the final sink for many organic and inorganic contaminants introduced into the environment as a consequence of anthropogenic pressure. This species is a sedentary, filter-feeding marine bivalve, thus satisfying criteria for a good bioindicator of pollution [3,4,6-9]. Thus it can be used as a biological model for the determination of biomarkers at different levels of biological organization. The methodology of biomarkers has been extensively developed since the last 20 years, and several biomarkers of exposure/effects of contaminants have been extensively used as early warning signals, with the potential to identify the presence of specific classes of toxicants, and to evaluate deleterious effects induced by pollutants . Ideally these biomarkers should be correlated with adverse effects at higher biological organization levels and so be interpretable as early warnings of the presence of stressors.
Biomarkers used in this study are involved in diverse pathways such as metal and xenobiotic detoxification (metallothionein, MT; ATP-binding cassette xenobiotic transporter, ABCB1), protection against oxidative stress (superoxide dismutases, MnSOD and CuZnSOD; catalase, 63 CAT), general stress (heat shock protein 70, HSP70), and mitochondrial alterations (cytochrome c oxidase1, CO1). Pollution can also affect development, sexual maturation and reproduction of organisms living in contaminated environments [11-13]. Therefore, when the reproduction of a species is altered, population, community and the whole ecosystem are under threat.
The aim of the present study was to investigate the impact of metallic stressors on the cockle C. glaucum at different levels of biological organisation in three differently impacted sites through the study of 1) the global physiological state of organisms (stress-on-stress, condition index and sex-ratio), 2) metal bioaccumulation in natural cockles, 3) the gametogenesis and the reproductive cycle 4) the responses at the transcriptional level of several key genes, and (5) the relationships between gene expression and metal contents.
Materials and Methods
Studied sites and sampling
Cockles C. glaucum were collected at three sites differing by their contamination level along the gulf of Gabes Ellouza, Sidi Mansour and Gargour sites (Table 1 and Figure 1) Specimens of C. glaucum were collected by hand at low tide from littoral sediment (upper 20 cm depth), then transported to laboratory in cool boxes with approximately 2 cm of seawater from the collection site. In order to examine the physiological and reproduction parameters, specimens of C. glaucum (n = 20 for each sampling date, shell length ranging from 28 to32 mm) were collected from each site in mid-October, November, mid-November and December. To study the responses at transcriptional levels and metal bioaccumulation, twenty cockles samples (sell length ranging from 28 to 32 mm) were collected from the three selected sites.
Twenty animals for each site were sampled and submitted to anoxia by air exposure at 15°C in the laboratory. For each sample, survival was assessed daily according to the method of . Death symptoms were considered to be open valves and absence of muscular activity. Lethal time corresponding to 50% of dead animals (LT50) was measured and the results expressed in days.
Twenty cockles per site were used for the study of the reproductive cycle. Condition Index (CI) of cockles was calculated individually on twenty cockles from each sampling site. Before dissection, all cockles were weighted (total and soft weight), and the CI was expressed as a percentage of the ratio of fresh weight of soft tissues to total weight. Gonads from each cockle were then fixed for 48h in the aqueous Bouin's fixative for histological determination. Tissues were then dehydrated through increasing alcohol concentrations progressively (70°C, 80°C, 90°C and 95°C) for one hour and thirty minutes for each concentration and embedded in paraffin at 56°C. Sections of 3 µm were cut with a mechanical microtome (type HM315). Hematoxylin, Light green and Eosin were used for staining. The reproductive stages were determined on the basis of criteria described in . Pictures and oocytes measurements were taken with a Nikon Eclipse 80i microscope coupled to a Nikon DXM1200-C camera (Nikon, Champigny-sur-Marne, France). In order to quantify the reproductive status of cockles, numbers from 0 to 5 were given to codify the different stages . Each stage was categorized by a maturity factor (1, resting; 2, development; 3, gametogenesis; 4, maturity; 5, spawning; 2, spent) then, a Sexual Maturity Index (SMI) was calculated according to the equation established by : SMI = ∑ (% of each sexual state*maturity factor).
Metals analysis (Cd, Cu, Ni and Zn) were carried out on sediments collected directly from the three sites, and individually on 20 cockles from the three studied sites on the remaining animal tissues (including digestive gland, muscle, mantle and foot). Determination of trace elements on sediments was performed according to  using Inductively Coupled Plasma Atomic Emission Spectrophotometry (ICP AES, Thermo Scientific iCAP 6000 series) and measurement of metal concentrations in cockles was carried out as previously described [17,18] by flameless (Cd, Cu and Ni) or flame (Zn) atomic absorption spectrophotometry with Zeeman correction, using a graphite furnace (SpectrAA Zeeman 220).
RNA extraction and Real time quantitative PCR
Gene transcription analyses were carried out individually on the same individuals for which metal analyses were measured and at each site. The gills of each sample were dissected, conserved in RNA later (Sigma-Aldrich), and stored at −20°C. Total RNA was isolated from 100 mg of gills using Tri-Reagent (Invitrogen) following the manufacturer‘s instructions. RNA concentration was determined spectrophotometrically at 260 nm, and RNA quality was checked using the Experion system (Bio-Rad). Three micrograms of total RNA were added to dT Race primer and nuclease free water in a final volume of 12.25 μl. A denaturation at 70°C was carried out for 5 min and then the mixture was kept on ice. First strand complementary DNAs (cDNAs) were then synthesized using dTRace primer (5-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTT-3), dNTPs, MMLV reverse transcriptase (Promega), RNAs in (Promega), and nuclease-free water in a final volume of 25 μl for 90 min at 42°C. Real-time PCR reactions were performed using a Step-One-plus apparatus (Applied Biosystems) with Fast SYBR Green Master Mix (Applied Biosystems) and specific primers (0.1µM) designed on the basis of previously characterized sequences as described in details in  Efficiencies calculated for all genes (90 ≤ E ≤ 110) indicated correct PCR reactions without inhibition . Three (β-Actin, α- Tubulin, β-Tubulin) out of the 4 housekeeping genes studied were used to normalize the 7 target genes. A Bestkeeper index compiling these 3 housekeeping genes was calculated and used to normalize the target gene transcriptions , and the relative expression was determined by the comparative Ct method  using RNA sample from cockles from the Ellouza site as a calibrator.
For the statistical analysis, we used the statistical package SPSS program (version 13). To check the equality of the variances, a Levene test was performed. The normal distribution was then checked by the P-Plot function. A one-way ANOVA test was performed to examine differences between the means. After normalization of the data by log(x+ 1) transformation and stabilization of their variances by square-root transformation, influences of inter- sites metallic pollution on gene transcription, metals bioaccumulation, sex ratio and CI were assessed by independent ANOVA significance test. Principal Component Analyses (PCAs) were performed and correlations were tested statistically using Pearson’s method to highlight correlations between gene transcription and metals bioaccumulation. The data were finally submitted to a Tukey’s HDS test to determine the critical differences between the groups. Significance was set at p < 0.05 in all cases.
Polymetallic contamination of the sites
Metals contamination in sediments: Sediments were collected from the Ellouza, Sidi Mansour and Gargour sites (Table 2) and analyzed by ICP (Cd, Cu, Zn, Ni, Cr, Mn and Pb). Gargour sediment presented the highest values for different metals studied, followed by Sidi Mansour and Ellouza sites. Only Cd presented values above the threshold established by  (Table 2). For all other metals values from the three sites were below those thresholds.
Metals accumulation in cockles’ tissues: Bioaccumulation of metals (Cd, Cu Zn and Ni) in cockles tissues from the three studied sites were presented in figure 2. Results show that Zn level was the most important followed by Ni and Cu. The lowest values are for Cd. Inter-site variations for the different metals show that the highest values for all metals (p < 0.05) are registered in Gargour site while the lowest ones (p < 0.05) are for the Ellouza site.
Global physiological state of cockles
Stress-on-stress: Survival under anoxia was assessed for the three studied sites (Fig. 3). Cockles from Gargour site presents the lowest values of lethal time (LT50) (3 days < LT50 < 4 days) followed by cockles from Sidi Mansour site (LT50 = 5 days). Cockles from Ellouza site presents the highest values of LT50 (LT50 = 6 days).
Condition index: Condition Index (CI) was assessed bi-monthly between October and December 2009 in cockles collected from the three studied sites (Figure 4). Results show that variations in CI were synchronous in the three studied sites: CI decrease significantly between October and November and no variation was observed for the remaining sampling dates. Inter-sites CI variations show that cockles from the Gargour site presented the lowest values of CI followed by cockles from Sidi Mansour and then those from Ellouza site.
Sex-ratio: Figure 5 outlines the percentage of males and females from the three studied sites. Results show that in Gargour site the sex ratio was highly unbalanced in favour of females. However, in the Ellouza site, percentages of males and females are equivalent.
Gametogenesis and reproductive cycle
Previous data dealing with the reproductive stages of C. glaucum demonstrated five stages in males and females: development, gametogenesis, maturity, spawning, and spent stages . Figure 6 illustrate the distribution between each of the categories of gonad development stages and the seasonal Sexual Maturity Index (SMI) cycle of C. glaucum from the three studied sites. No difference was detected in the reproductive stages between males and females at the three studied sites. The evolution of the gametogenesis was quite the same at all three sites. The majority of cockles were at the development stage in the first sampling date, at maturity for the second sampling, spawning in the third and finally spent in the fourth. However, two differences were detected: the first in the beginning of gametogenesis cycle in the contaminated site (Gargour) with a majority of individuals in gametogenesis stage (stage 3), while on the reference (Ellouza) and the intermediate (Sidi Mansour) sites, the majority of specimens are in development stage (stage 2). The second difference was observed in the last sampling date (December) in Sidi Mansour and Gargour sites with specimens in spent stages while specimens in Ellouza are still in spawning stage. The SMI provided a quantitative evaluation of the successful reproductive cycle. Evolution of SMI index (Figure 6) was similar in the three studied sites at the three first sampling periods. An increase in this index was observed between October and November, corresponding to an evolution of gametogenesis that took place synchronously in the three studied sites. In the last sampling date, a decrease of SMI was observed in cockles from the intermediate site Sidi Mansour and from the contaminated site Gargour, however it continues to increase in cockles from the reference site.
Inter- sites variation of relative gene expression
Relative gene expression of seven-regulated genes was investigated in cockles from the three sites (Figure 7-9). No difference was found in the expression of these genes between males and females and the results obtained for the two sexes have been therefore grouped. Results do not showed inter-sites variations in the expressions of MT, ABCB1, MnSOD and CuZnSOD genes (Figure 7). HSP70 expression decreased when pollution increased. Indeed, the highest mRNA level of HSP70 was recorded in the Ellouza site while the lowest one was recorded in the Gargour site (Fig. 8). Catalase expression level was similar in the intermediate and the the contaminated sites, but it was significantly over expressed (3 fold) in the "reference" site (Figure 9). The same expression pattern was observed for the COI gene (Figure 9). Expression of this gene was significantly over expressed (1000 fold) in the intermediate and contaminated sites with respect to the "reference" site.
Principal components analysis
To highlighted relationships between mRNA levels (Ct = Ct sample–CHKG) and bioaccumulation of metals (Cd, Cu, Zn and Ni) two PCAs were carried out. The first PCA (Figure 10-A) showed relationships between metal bioaccumulation level and gene expression. The second PCA (Figure 10-B) corresponds to a projection of individuals from the three sites on the two axes of the PCA. Results of the first PCA showed the presence of two correlated groups: the first group contained Cd, Cu, Zn and Ni accumulations and expression of catalase and COI genes. The second group is composed by the expression of CuZnSOD, MnSOD, HSP70 and MT genes. Projections of the samples on the two main axes of the PCA (Figure 10- B) indicate the separation of cockles from the three studied sites. Samples from Ellouza site were quite clustered on the left of the figure with the lowest metal level but the highest mRNA expression of MT, HSP70, MnSOD and CuZnSOD genes. Cockles from Gargour site were grouped on the right at the top of the figure with an important expression of COI and CAT genes, while samples from Sidi Mansour site are found on the right lower part of the figure with the highest metals bioaccumulation.
This study was conducted on three sites differing by their levels of contaminations, Gargour site was considered as the most polluted site that is affected by industrial activity and effluents carried out by North-south streams according to the studies of  and . The Ellouza site was considered as a" reference" site according to the studies of [24,25] conducted both in sea water and in sediments. Sidi Mansour was considered as an intermediate site. In this study, composition of the three sediment shows that Gargour sediment presented the highest values for almost the different metals studied, followed by the Sidi Mansour and Gargour sites. Among studied metals, only Cd presented values above the threshold established by  (Table 2). In general, contaminated sediment reflects a polluted site and a real risk to organisms living in contact with the sediment since sediment serves as nursery and breeding place for many benthic species including cockles [16,26]. The global physiological state of cockles was assessed in the three studied sites by performing stress-on-stress test and measuring both condition index and sex ratio. The stress-on-stress test reveals the physiological capacity of cockles to survive under anoxia. Our data demonstrate that exposure to natural metallic pollution affects the LT50 of cockles, Indeed, the highest values of LT50 were recorded in cockles from the Ellouza site reflecting an important resistance to anoxia in cockles from this site compared to cockles from Gargour site in which cockles presents the lowest values of LT50 reflecting a metabolic perturbations and a general lower health status of cockles from this site. Our results are in concordance with previous data performed on the same species demonstrating that cockles from a reference site are more resistant that cockles from a contaminated site . Other studies carried out in controlled conditions on C. glaucum [4,9,27] and on others bivalves [14, 28] showed that, following an exposure to metallic contamination, a decrease in LT50 was observed with exposure time and increasing metals concentrations. The CI reflects the physiological and nutritional state of animals. For organisms collected at the same period of reproduction, this index can highlights differences between control and contaminated sites . In this study, considering animals sampled at the same period of reproduction on the three studied sites, the highest CI was recorded for cockles from the reference site Ellouza reflecting the good physiological state of cockles. However, the lowest CI was recorded in cockles from the contaminated Gargour site, reflecting the physiological perturbation state of cockles. Our results are in accordance with previous data in marine organisms demonstrating a decrease in CI when increasing metallic pollution [30,31] However, variations in CI could be also related to hydro- climatic conditions in studied sites (temperature, salinity, oxygen availability) and food availability . Moreover CI could be also strongly related to seasonal variations and age of animals. Therefore, this parameter must be considered carefully in ecotoxicological studies .
Our results on sex-ratio demonstrate that in the contaminated site Gargour, the sex ratio is highly unbalanced in favour of females. However, in the reference site Ellouza, percentages of males and females are equal. Previous studies [33-35] reported that an unbalanced sex-ratio is often related to particular environmental conditions (temperature, trophic conditions, "etc") as well as to reproductive stages but rarely to environmental gradient pollution. However the results obtained in our study may suggest that the unbalanced sex-ratio could be due to gradient pollution factor, which can act in synergy with the other factors (temperature, trophic conditions, endocrine distruptors, "etc"). Moreover, our study indicates that the predominance was for females in the contaminated site. This finding is in accordance with the study of  demonstrating a predominance of females in cockles having a size above 24 mm in some sites in the gulf of Gabes region. However, these authors do not relate the predominance of females to pollution factor but to the size of populations. Results of gametogenesis and maturity index show few differences in the reproductive cycle of cockles collected from the three studied sites. However, two differences are detected: the first in the beginning of the gametogenesis cycle in cockles from the contaminated site (Gargour) with a majority of individuals in gametogenesis stage, while in cockles from the "reference" (Ellouza) and intermediate (Sidi Mansour) sites, the majority of specimens are in development stage (stage 2). The second difference is observed for the last sampling date (December) in Sidi Mansour and Gargour sites where animals were in spent stages while cockles from the reference site Ellouza were still in spawning stage. These two differences are recorded between "reference" and contaminated sites and are probably linked to pollution factor. Previous studies reported many effects on reproduction (delayed oogenesis or spermatogenesis, histological alterations, etc) in several bivalves exposed to metallic or chemical contaminants such as Myaarenaria , Itapesdeccussatus [11,34], Mytilusgalloprovincialis  and Donaxtrunculus . Studying metal accumulation in cockles tissues is very important because metals can be accumulated at higher levels than those found in natural environment and can even reach toxic thresholds levels [39,40]. Results in metal accumulation show that cockles from the most contaminated site, Gargour, present the highest values of Cd, Cu, Zn and Ni metals followed by those from the medium contamination site, Sidi Mansour, and finally those from the reference site, Ellouza. This result confirms that bioaccumulation in cockles follows levels of contamination in studied sites. When exposed to a contamination, an organism can modify the expression of its genes to acclimate to this new situation. This change is often considered as an early response from an organism under stress [41-45]. The expression of 7 genes encoding enzymes or proteins involved in response to different types of environmental stress was conducted on cockles from the three studied sites. Considering the complexity of the typologies of the studied sites, it seemed important to study markers involved in multiple stress such as MT, ABCB1, MnSOD and CuZnSOD, CAT, HSP70 and COI. Results show that expression profiles obtained can be grouped into three groups.
1) Genes involved in metals detoxification (ABCB1 and MT) and superoxide dismutase (MnSOD and CuZnSOD) do not display any difference in their levels of expression. This result may be due to the interference of confounding factors such as biotic factors (age, size and sex) and abiotic factors (temperature, salinity turbidity and availability of food).
2) HSP70 gene expression decrease when environmental pollution increases. This gene presents the highest level in cockles from the most contaminated site, Gargour, and the lowest one in samples from the reference site, Ellouza. This fact could be explained by an inhibition of HSP70 synthesis in the contaminated sites following high metallic contamination. Similar results were reported for oysters demonstrating an inhibition of HSP70 synthesis in sites presenting high levels of metals.
3) CAT and COI showed higher expression level in both the intermediate and the most contaminated site compared to the reference site. Moreover, expression of COI gene is 1000 fold higher in cockles from the Gargour and Sidi Mansour sites compared to the reference site Ellouza. This result is related to the energy demand, which is particularly high when exposed to any environmental stress [46-48]. So, the important COI gene expression in the most contaminated sites confirms the importance of energetic metabolism in response to stress.
To our knowledge, this study is the first to use biomarkers at different levels of biological organization (global physiological state of organisms, reproductive cycle and molecular responses) for the assessment of the health status of the cockle C. glaucum exposed to multiple metallic pollution in situ. Results obtained show i) a global decrease in the Physiological status of cockles in the most polluted site. ii) HSP70, Catalase and COI could be selected as relevant biomarkers of natural metallic pollution for the cockle C. glaucum. However, despite a large literature on biomarkers and their use in biomonitoring programs, their incorporation in regulatory legislation is still limited and should be coupled to proteomics studies.
This research was supported by the European Program FP7 PEOPLE-IRSES GENERA (use of genomic and proteomic tools for the development of contaminant-specific biomarkers for the environmental risk assessment of aquatic ecosystems). The author thank Dr Emmanuel Tastard for its statistical help.
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