Real-time Quantitative PCR (RT-qPCR) verification
Amplification products for the 15 aforementioned potential biomarker genes in the digestive gland of the C. fluminea following 30 days exposure to 0.05, 0.5 and 5 μg/L fluoxetine exposure are shown in Figure 6.
10.1371/journal.pone.0079516.g006 Figure 6  Real-time qPCR analyses of expression profiles of the 15 functional genes after 30 days fluoxetine exposure.
Experiments were performed in triplicate and repeated three times with similar results. Bars display mean±S.D. One-way ANOVA (p<0.05) was performed using OriginPro® to test the differences of gene expressions between control and fluoxetine treated clams.   For antioxidase genes, the mRNA expression levels of (Cu/Zn) SOD and GPx-A were significantly up-regulated in the 0.5 and 5 μg/L (p<0.05) fluoxetine groups. GST-mu was significantly increased (p<0.05) at 5 μg/L, while TPX1 was only up-regulated (p<0.05) at the 5 μg/L. No significant change was observed in TPX2 (Figure 6), indicating that TPX2 gene is not sensitive to fluoxetine. Previous studies have reported that low concentration of fluoxetine (0.075 μg/L) exposure can affect antioxidant system in M. galloprovincialis [46]. In the present study, 0.5 and 5 μg/L fluoxetine exposure led to significant upregulation of the antioxidase genes (except TPX2) suggesting that the antioxidase genes will be useful biomarkers for exposure to environmental stress. 
CYP4 expression peaked following 5 μg/L fluoxetine exposure with a 4.81-fold increase in transcript levels. CYP30 was significantly (p<0.05) up-regulated at the 0.5 and 5 μg/L by 4.76-fold and 9.24-fold, respectively (Figure 6). As was previously reported, the CYP3-like-2 gene was up-regulated in the digestive gland of M. edulis by PCB126 [58]. Our study found that the gene expressions of CYP4 and CYP30 were up regulated in digestive after fluoxetine exposure. Therefore, further studies are needed to discover the function of CYPs in the C. fluminea.
In this study, the C. fluminea GABARAPs genes were significantly up-regulated (p<0.05) with 5 μg/L fluoxetine exposure, while GABARAPL2 was significantly decreased (p<0.05) (Figure 6). Although the actual biological functions of GABARAPs remain elusive [62], the results of our study showed the putative function of GABARAPs in C. fluminea.
Our present study found that the mRNA transcript for Hsp22, Hsp40, Hsp60 and Hsp90 were significantly increased by 0.5 and 5 μg/L with increases of 2.47-fold and 3.45-fold, 33.93-fold and 67.01-fold, 2.34-fold and 5.83-fold, 4.61-fold and 5.14-fold, respectively. The gene expression of Hsp90 was significantly increased in the 0.05 μg/L (p<0.05) and 5 μg/L (p<0.05) fluoxetine exposure groups. No significant differences were observed in the most of the genes (except Hsp90) at the 0.05 μg/L fluoxetine exposure group (Figure 6). Many previous studies reported that various stressors (such as temperature, metal toxicity and infection) can affect the expression of Hsp70 and Hsp90 [66-70], However, the small Hsps (Hsp22, Hsp40) and Hsp60 have been poorly studied. The present study showed that small Hsps, and Hsp60 were also useful biomarkers when exposure to fluoxetine in C. fluminea.
Over all, the RT-qPCR results of the 15 selected genes after fluoxetine exposure confirmed that the 15 functional genes are linked to environmental stress. We can use the 15 genes as environmental biomarkers to monitor the environmental pollutants in the future. However, further research is needed to better understand the molecular mechanisms of C. fluminea following the contamination exposure.