Cadmium in vivo exposure alters stress response and endocrine-related genes in the freshwater snail Physa acuta. New biomarker genes in a new model organism

Cadmium in vivo exposure alters stress response and endocrine-related genes in the freshwater snail Physa acuta. New biomarker genes in a new model organism

Environmental Pollution xxx (2016) 1e10 Contents lists available at ScienceDirect Environmental Pollution journal homepage: www.elsevier.com/locate/...

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Environmental Pollution xxx (2016) 1e10

Contents lists available at ScienceDirect

Environmental Pollution journal homepage: www.elsevier.com/locate/envpol

Cadmium in vivo exposure alters stress response and endocrine-related genes in the freshwater snail Physa acuta. New biomarker genes in a new model organism*  nica Morales a, Paloma Sa nchez-Argüello b, Gloria Morcillo a, Pedro Martínez-Paz a, *, Mo a  Luis Martínez-Guitarte Jose a tica y de Fluidos, Facultad de Ciencias, Universidad Nacional de Educacio n a Grupo de Biología y Toxicología Ambiental, Departamento de Física Matema Distancia (UNED), Senda del Rey 9, 28040 Madrid, Spain b n y Tecnología Agraria y Alimentaria (INIA), Ctra. La Laboratorio de Ecotoxicología, Departamento de Medioambiente, Instituto Nacional de Investigacio ~ a km 7, 28040 Madrid, Spain Corun

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 June 2016 Received in revised form 18 August 2016 Accepted 5 October 2016 Available online xxx

The freshwater snail Physa acuta is a sensitive organism to xenobiotics that is appropriate for toxicity testing. Cadmium (Cd) is a heavy metal with known toxic effects on several organisms, which include endocrine disruption and activation of the cellular stress responses. There is scarce genomic information on P. acuta; hence, in this work, we identify several genes related to the hormonal system, the stress response and the detoxification system to evaluate the effects of Cd. The transcriptional activity of the endocrine-related genes oestrogen receptor (ER), oestrogen-related receptor (ERR), and retinoid X receptor (RXR), the heat shock proteins genes hsp70 and hsp90 and a metallothionein (MT) gene was analysed in P. acuta exposed to Cd. In addition, the hsp70 and hsp90 genes were also evaluated after heat shock treatment. Real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) analysis showed that Cd presence induced a significant increase in the mRNA levels of ER, ERR and RXR, suggesting a putative mode of action that could explain the endocrine disruptor activity of this heavy metal at the molecular level on Gastropoda. Moreover, the hsp70 gene was upregulated after 24-h Cd treatment, but the hsp90 gene expression was not affected. In contrast, the hsp70 and hsp90 genes were strongly upregulated during heat shock response. Finally, the MT gene expression showed a non-significant variability after Cd exposure. In conclusion, this study provides, for the first time, information about the effects of Cd on the endocrine system of Gastropoda at the molecular level and offers new putative biomarker genes that could be useful in ecotoxicological studies, risk assessment and bioremediation. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Physa acuta Cadmium Endocrine-responsive genes hsp genes Metallothionein gene

1. Introduction The freshwater snail Physa acuta is an invasive and globally distributed species (Ait Alla et al., 2006; Albrecht et al., 2009; Guo et al., 2009). Snails are ubiquitous members of freshwater communities and play important roles in aquatic ecosystems. They are sensitive to pollutants and suitable for ecotoxicity testing; therefore, mollusc-based toxicity data can be directly applied in the ecological risk assessment of chemicals (S anchez-Argüello et al.,  et al., 2013; Hossain 2009; Musee et al., 2010; De Castro-Catala

*

This paper has been recommended for acceptance by Prof. von Hippel Frank A. * Corresponding author. E-mail address: [email protected] (P. Martínez-Paz).

and Aditya, 2013; Seeland et al., 2013). Recent critical reviews of the Organisation for Economic Co-operation and Development (OECD) guidelines (OECD, 2010; Bandow and Weltje, 2012) discussed the need to develop standardised test protocols using species of molluscs to complement the existing test guidelines on aquatic organisms, as they are uniquely sensitive to certain endocrine disruptor chemicals (EDCs), though there is still a relatively poor understanding of molluscan endocrinology. Snails were reported to be excellent test organisms for the determination of endocrine-disrupting effects (Oehlmann et al., 2000; SchulteOehlmann et al., 2000; Schmitt et al., 2008). Some gastropod species were reported to be tolerant to various stressors but especially sensitive to EDC pollution (Schmitt et al., 2008). Molluscan hormone systems appear to be unique among invertebrate species and

http://dx.doi.org/10.1016/j.envpol.2016.10.012 0269-7491/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Martínez-Paz, P., et al., Cadmium in vivo exposure alters stress response and endocrine-related genes in the freshwater snail Physa acuta. New biomarker genes in a new model organism, Environmental Pollution (2016), http://dx.doi.org/10.1016/ j.envpol.2016.10.012

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are comparable, to some extent, to those of vertebrates (Thornton et al., 2003), especially with regard to steroid hormones. Hence, the development of mollusc tests will complement the aquatic test guidelines for annelids, chironomids and daphnids, providing new alternatives for the replacement of vertebrate animal testing. Cadmium (Cd) is widely used in batteries, electrical conductors, alloys, pigments and stabilisers for plastics. Cd is a common pollutant in natural waterbodies and ultimately causes severe damage to humans and wildlife. This heavy metal is highly toxic and was ranked sixth on the European Union list of priority hazardous substances (Water Framework Directive, 2008/105/EC). Recently, its endocrine-disruptive activity was described in vertebrates (Lafuente, 2013; Rana, 2014; Luo et al., 2015a; Marettov a et al., 2015) and in several aquatic invertebrates (Ketata et al.,  et al., 2010; Gay et al., 2013; Luo et al., 2015b). 2007; Planello Previous studies have shown that Cd modulates growth and reproduction success of freshwater snails (Wadaan, 2007; Sieratowicz et al., 2011; Kimberly and Salice, 2014). However, to date, available information regarding the molecular-level endocrine-disrupting effects of this chemical in snails is scarce. Oestrogen receptors (ERs) belong to the nuclear receptor superfamily. They bind oestrogen and act as the transcription factors that interact with specific DNA elements of different genes to induce biological effects. Oestrogen-related receptors (ERRs) also belong to this superfamily, but the ligand is still unknown, although they bind to their own DNA elements. ERs and ERRs have a high sequence identity, and comparison analyses show that they belong re, 2002). These reto the same branch of steroid receptors (Gigue ceptors were found in diverse species throughout the animal kingdom; however, the simultaneous presence of both receptors has only been reported in two snails (Bannister et al., 2007; Kaur et al., 2015). Moreover, ER has been described in other snails (Kajiwara et al., 2006; Sternberg et al., 2008a; Castro et al., 2007a; Stange et al., 2012; Hultin et al., 2014). Retinoid X receptors (RXR) are also nuclear receptors that bind to their own DNA elements as either homodimers or with other nuclear receptors as heterodimers. RXRs are described in a wide variety of metazoan phyla, including snails (Bouton et al., 2005; Castro et al., 2007b; Janer et al., 2007; Sternberg et al., 2008b; Carter et al., 2010). Moreover, it has been suggested that RXRs play an important role in the development of imposex in gastropods (Horiguchi, 2006). Heat shock proteins (HSPs) are a family of highly conserved proteins expressed in response to a wide range of biotic and abiotic stressors (e.g., chemical, temperature, radiation) and are also known as stress proteins (Srivastava, 2002). Under normal growth conditions, the hsp genes are expressed at low levels, and their transcription increases in response to different stressors, including heat, hypoxia, chemical exposure, infection, magnetic fields or tumorigenesis (Parsell and Lindquist, 1993). During stress response, these proteins work as chaperones to maintain the functional protein folding and prevent any irreversible aggregation among other physiological processes (Nolen and Morimoto, 2002; Sorensen et al., 2003; Silver and Noble, 2012). As HSPs induction is critical to maintain cellular homeostasis in response to changes in the environment, these proteins were proposed as the general biomarkers for environmental monitoring (Gupta et al., 2010). HSPs are classified into several families according to their molecular mass, including HSP90 (85e90 kDa), HSP70 (68e73 kDa), HSP60, HSP40 and low-molecular-weight HSPs (10e30 kDa). The HSP70 family is one of the most conserved proteins and shows tissue-, time- and dose-dependent expressions from chemical and physical stressors (Srivastava, 2002; Sorensen et al., 2003). The hsp70 gene is characterised in several gastropod species (e.g., Biomphalaria glabrata) (Laursen et al., 1997), Haliotis tuberculata (Farcy et al., 2007), Haliotis discus hannai (Cheng et al., 2007), Cellana toreuma

(Han et al., 2013) and Pomacea canaliculata (Song et al., 2014) as showing overexpression in response to environmental stress. Finally, HSP90 is one of the most abundant cytosolic proteins in eukaryotes but is less studied than HSP70 in gastropods. This environmental perspective further highlights the convenience of studying the relevance of hsp70 and hsp90 genes as the toxicological endpoints. Metallothioneins (MTs) are non-enzymatic proteins characterised by the absence of aromatic amino acids and high cysteine content. The thiol groups allow them to sequester heavy metals , 2014); hence, they are involved in bio(Isani and Carpene accumulation and detoxification of metals, metal ion homeostasis, protection against oxidative stress and neuroprotective mechanisms (Mao et al., 2012). Hence, MTs are being used as the biomarkers to analyse the effects of pollutants in different aquatic organisms (Sarkar et al., 2006). Moreover, some studies have demonstrated that Cd could alter the MT levels in the freshwater snail Lymnaea stagnalis and land snails (Leung et al., 2003; Włostowski et al., 2014; Baurand et al., 2015). Finally, it is important to note that MTs are the major ligands for Cd and are involved in the acquisition of tolerance to this metal (Tsui and Wang, 2007). The aims of this study were to evaluate the molecular effects of Cd in the freshwater snail P. acuta, understanding its mode of action and obtaining information about the suitability of this animal as a sentinel organism for environmental risk assessment of aquatic ecosystems. Initially, endocrine-related genes ER, ERR and RXR, stress genes hsp70 and hsp90 and an MT gene were identified in this species and their transcriptional activity was analysed in adult snails exposed to Cd. This heavy metal altered the cellular homeostasis and disrupted the endocrine system in this organism, suggesting that these genes could be good biomarkers in gastropods. Furthermore, this study provides new data at the molecular level for a mollusc species and could be helpful for covering the gaps in the physiological processes involved after chemical injury in this group of invertebrates. 2. Materials and methods 2.1. Chemicals and reagents Cadmium chloride (CdCl2) was purchased from Fluka (USA). Stock solution of CdCl2 was prepared in test medium (2 mM CaCl2, 0.5 mM MgSO4, 0.77 mM NaHCO3 and 0.08 mM KCl) and then diluted in test medium to obtain the experimental concentrations of Cd. The commercial kit TRIzol and M-MLV enzyme were obtained from Thermo-Fisher (USA), oligonucleotide dT20 primer and genespecific primers were supplied by Sigma (UK), RNase-free DNase was purchased from Roche (Germany) and SsoFast EvaGreen Supermix was provided from Bio-Rad (USA). 2.2. Snails P. acuta (Gastropoda, Pulmonata, Basommatophora) is a hermaphroditic species that mainly practises outcrossing. Snails were maintained in a controlled-climate room (20  C) for several generations prior to the experiment. The culture conditions were nchez-Argüello et al., 2009). Briefly, 100 described previously (Sa adult snails were maintained in 2 L of spring water for breeding under constant aeration at 20  C and a standard lightedark period of 16:8. The snails were fed 200 mg of shrimp food flakes (Sera®). Adult snails lay egg masses containing encapsulated embryos, which directly developed into juveniles. The juveniles were fed progressively increasing doses of fine particulate fish food (Sera Micron®), from 65 to 200 mg. Juveniles developed into adults and

Please cite this article in press as: Martínez-Paz, P., et al., Cadmium in vivo exposure alters stress response and endocrine-related genes in the freshwater snail Physa acuta. New biomarker genes in a new model organism, Environmental Pollution (2016), http://dx.doi.org/10.1016/ j.envpol.2016.10.012

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first oviposition occurred approximately two months after hatching. 2.3. Treatments For experimental treatments, the snails were heat shocked at 39  C for 5, 15, 90 and 120 min in the preheated and aerated test medium. Control snails were maintained at culture temperature (20  C) for corresponding exposure times. For Cd treatments, adult snails were exposed for 5, 24, and 48 h at 20  C to nominal concentrations of 0.1 and 1 mM (18.33 and 183.32 mg/L). Concentrations were selected on the basis of previous experiments in other freshwater snails (Gomot, 1998; Coeurdassier et al., 2003; Schirling et al., 2006; Vincent-Hubert et al., 2012; Ducrot et al., 2014; Kimberly and Salice, 2014; Kokkinopoulou et al., 2014). Three independent experiments were performed in each analysis. Control snails were maintained in test medium. After shell separation, the snails were recovered and stored at 80  C until RNA isolation was carried out. 2.4. RNA isolation Total RNA was extracted from control and exposed adult snails (five animals per experiment) using a guanidine isothiocyanatebased method performed with TRIzol according to the manufacturer's protocol. Briefly, snails were homogenised in 1 mL of TRIzol, frozen at 80  C and thawed at room temperature for 5 min; this procedure was repeated three times. Subsequently, 200 mL of chloroform was added to each sample, which was then mixed and incubated at room temperature for 5 min. The samples were then centrifuged for 15 min at 4  C and 15,000 g. Following the transfer of the aqueous phase, the RNA was finally recovered by isopropyl alcohol precipitation (500 mL), washed with 1 mL of 70% ethanol and resuspended in DEPC water. The RNA was then treated with RNase-free DNase, followed by phenolisation and resuspension in DEPC water. The quality and quantity of the total RNA were assessed by agarose electrophoresis and absorbance spectrophotometry (Biophotomer, Eppendorf). Finally, the purified RNA was stored at 80  C. 2.5. Retrotranscription Reverse transcription was performed using 0.5 mg of the isolated RNA, 0.5 mg oligonucleotide dT20 primer (Sigma) and 100 units of M-MLV enzyme (Invitrogen) in a final volume of 20 mL according to the manufacturer's instructions. 2.6. Characterisation and isolation of cDNA To amplify a fragment of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13 (rpL13), tubulin, ER, ERR, RXR, hsp70, hsp90 and MT genes from P. acuta, primers were obtained using three strategies. All primers used in this work are presented in Table 1. GAPDH, ER, and ERR primers were designed based on the P. acuta sequences from Romiguier et al. (2014). Briefly, a transcriptome project employed in this article was obtained from the public web of the authors (http://kimura.univ-montp2.fr/PopPhyl/ index.php?section¼data#dataset_0) and used to compare all the contigs using Blast2GO software (Conesa et al., 2005). From the results, the sequences for GAPDH, ERR and ER (Contigs 506, 1634 and 3836, respectively) were identified and used to design the primers. An enriched P. acuta whole body cDNA library present in GenBank EST database was used to perform a Blastp search to identify the different sequences included the library. From this search rpL13, hsp90 and MT were identified. The accession numbers

3

for each sequence are BW985970 (rpL13), BW986187 (hsp90) and BW986274 (MT). These sequences were used to design primers for each gene and amplify by PCR a sequenced DNA fragment. Then, a new pair of primers were designed for each gene. Finally, tubulin, RXR and hsp70 were obtained through the comparison of sequences from other molluscs. Different sequences were obtained from GenBank and used for multiple sequence alignment with CLUSTALW software (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat. pl?page¼/NPSA/npsa_clustalwan.html) for each gene, and a pair of primers were designed based on consensus sequences observed. Subsequently, PCRs were performed and a DNA fragment amplified and sequenced. From these sequences, gene-specific primers were designed for expression analysis. The sequences used for tubulin were Lymnaea stagnalis (X15542), Chlamys farreri (AY395571) and Crassostrea ariakensis (DQ437571); for RXR were Lymnaea stagnalis (AY846875) and Haliotis diversicolor (HM484209) and for hsp70 were Haliotis discus hannai (DQ324856), Biomphalaria glabrata (L44127) and Cellana toreuma (JX169849). In each case, PCRs were performed in 20 mL with 2 mM of MgCl2, 0.2 mM dNTPs (Biotools, Spain), 0.4 mM of each primer and 0.2 mL of Taq polymerase (Biotools, Spain) under the following conditions: 1 cycle of 94  C for 5 min, followed by 35 cycles at 94  C for 30 s, 57  C for 30 s, 72  C for 30 s and, finally, 72  C for 10 min. 2.7. Real-time polymerase chain reaction (RT-PCR) A total of 25 ng of cDNA obtained was used as a template for the quantitative real-time polymerase chain reaction (qRT-PCR). qRTPCR was used to evaluate the mRNA expression profile of the ER, ERR, RXR, hsp70, hsp90 and MT in response to Cd exposure and hsp70 and hps90 in response to heat shock. qRT-PCR was performed on a CFX96 thermocycler (Bio-Rad) using the SsoFast EvaGreen Supermix, and the following cycling conditions: initial denaturation at 95  C for 3 min and 35 cycles of denaturation at 95  C for 5 s; annealing at 58  C for 15 s and then elongation at 65  C for 10 s. To verify the accuracy of each amplicon, a melting curve analysis was performed after amplification. In this study, rpL13 and MT were employed as the reference genes in heat shock treatments, whereas GAPDH, rpL13, and tubulin were used in Cd exposure. These reference genes presented a coefficient of variation <0.25 and an Mvalue < 0.5 (Hellemans et al., 2007). In each case, the gene expression patterns were compared with those observed in control animals after normalisation with the reference genes. The PCR amplification efficiency was established using calibration curves. For each gene, a standard curve based on five dilutions from an equimolar mix of cDNA samples was produced in triplicate to verify the amplification efficiency (Table 1). To confirm the amplification of only one fragment of DNA, a melting curve analysis was performed after amplification. Each sample was run in duplicate wells and three independent PCR replicates were used for each experiment. The cycle threshold (Ct) values were converted to relative gene expression levels using the 2-DDCt method and the BioRad CFX Manager 3.1 software to analyse gene expression. 2.8. Statistical analysis The mRNA levels of ER, ERR, RXR, hsp70, hsp90 and MT genes in response to Cd exposure were normalised against the reference genes in the same samples by using standard curves. Normal distribution and variance homogeneity of data were assessed using the Kolmogorov-Smirnov and Levene tests, respectively. The normalised levels of the specific gene transcripts in treated groups were compared to those of the non-exposed controls using ANOVA. All statistical tests were performed using SPSS 22® (IBM). A p  0.05 (*) was considered to indicate significant differences. All results are

Please cite this article in press as: Martínez-Paz, P., et al., Cadmium in vivo exposure alters stress response and endocrine-related genes in the freshwater snail Physa acuta. New biomarker genes in a new model organism, Environmental Pollution (2016), http://dx.doi.org/10.1016/ j.envpol.2016.10.012

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Table 1 Primers used for RT-PCR of genes from P. acuta. Gene

Oligo name

Primer DNA sequence

rpL13

L13 F1 L13 R1 Tub F1 Tub R1 RXR F RXR R hsp70 F1 hsp70 R1 hsp90 F1 hsp90 R1 Met F1 Met R1 GAPDH rtF GAPDH rtR L13 F2 L13 R2 Tub rtF Tub rtR ER rtF ER rtR ERR rtF ERR rtR RXR rtF RXR rtR hsp70 F3 hsp70 R3 hsp90 F2 hsp90 R2 Met F2 Met R2

50 -CATAAGGATTGGCAGCGTATGG-30 50 -CATCACCCTTTCGTGGTTTGCC-30 50 -TAGCACCGCCATCCAGGAGC-30 50 -AATTCACCCTCATCCTCGGC-30 50 -CGAGCAGGTTGGAATG-30 50 -ATCTTCATCTCTCTCATCTTGG-30 50 -GACGAAGCTGTAGCTTATGGTGCTGC-30 50 -ACCTCAATCTGTGGGACGCCTCTTGG-30 50 -AGCAGCTGAAAGAGTATGAGGG-30 50 -TGAGATGTGACAATACAGCATGG-30 50 -TGCACTGGACCAAGCACATGC-30 50 -TCTGGAGTTGCCATTCAAGACG-30 50 -ATACATCAGGAACAGGGACTC-30 50 -GACTTATGACAACCGTGCA-30 50 -ATGAGCTTAAGGCTGCAGG-30 50 -CCAACTTGGTCTTGTACTCTTTAAG-30 50 -TTCAAACGTGTCTCTGA-30 50 -GTGGCATCCTGGTACTGTT-30 50 -AAGGCTATGGCAGTTGG-30 50 -TGATGGGTGATGTTGATGT-30 50 -GATGCTCTCTCAGACTGTGTC-30 50 -AGCATTTCCAGGAAGAGC-30 50 -CTCATTGCTGGCTTCTCC-30 50 -CAACAAGCTCTGTCAGGACTC-30 50 -CTGGAGGCGTTATGACTG-30 50 -AGGTGAAATCGACCCAAG-30 50 -GTTTGTGTCACTAAAGAAGGCC-30 50 -TGTCACTAGCCTATTTGATACAACC-30 50 -AAGCTCTGATTGTGCATGC-30 50 -AGATCATCTGAAGCCTTCAATC-30

Tubulin RXR hsp70 hsp90 MT GAPDH rpL13 Tubulin ER ERR RXR hsp70 hsp90 MT

expressed as the mean ± standard error of the mean (SEM) of three experiments.

Amplification efficiency

Amplicon length

PCR objective

e

374 bp

Gen amplification

e

197 bp

Gen amplification

e

182 bp

Gen amplification

e

332 bp

Gen amplification

e

217 bp

Gen amplification

e

199 bp

Gen amplification

101.8%

206 bp

103.1%

138 bp

102.9%

154 bp

90.0%

221 bp

92.5%

173 bp

101.8%

139 bp

103.2%

166 bp

111.0%

159 bp

110.6%

148 bp

Real-time PCR Real-time PCR Real-time PCR Real-time PCR Real-time PCR Real-time PCR Real-time PCR Real-time PCR Real-time PCR

Table 2 Results of the normalisation procedures using rpL13 and MT as the reference genes. Treatment

rpL13 CV

M-value

CV

M-value

CV

M-value

Heat shock

0.1225

0.3465

0.1177

0.3465

0.1201

0.3465

3. Results

MT

Mean value

3.1. In vivo Cd exposure and snail survival Concentrations of 0.1 and 1 mM of Cd did not induce mortality for all exposure times. These doses were selected for further treatments to analyse possible alterations in gene expression profiles of selected genes, with the aim of identifying the early response of sensitive genes related to endocrine systems, cell stress and detoxification systems as the general biomarkers for environmental monitoring. 3.2. Effects of heat shock treatments on hsp70 and hsp90 mRNA levels The identification of the hsp70 and hsp90 genes in this animal offered an opportunity to obtain information related to heat shock response of the P. acuta snails. To examine the transcriptional response of these hsp genes to thermal stress, snails were exposed to 39  C for 5, 15, 90 and 120 min, and the mRNA levels were analysed. Coefficients of variation and M-values of reference genes are shown in Table 2. As shown in Fig. 1, the temperature shifts clearly upregulated the expression levels of hsp70 and hsp90. The Hsp70 mRNA levels significantly increased after as few as 5 min and continued to increase, reaching the maximum at 90 min. It is important to note that the maximum increase was more than 1000 folds of that in the control. Similarly, hsp90 was also significantly activated by temperature, but in this case, the increase was moderate, reaching a maximum at 120 min. As expected, the mRNA levels of both genes were increased upon heat shock, but with

different kinetics and response intensity. 3.3. Effects of cadmium exposure on endocrine-related genes Information regarding the molecular-level effects of Cd on the endocrine system in gastropods is scarce. Thus, to obtain information about the mode of action of this heavy metal on the endocrine system, three endocrine-related genes coding for ER, ERR and RXR were identified, and their expression profiles in the exposed animals were assessed. The snails were exposed to 0.1 and 1 mM of Cd for 5, 24 and 48 h. As shown in Fig. 2, while ER was downregulated at 48 h for both Cd concentrations, the transcriptional activity of RXR and ERR was increased when the animals were exposed to 1 mM for 5 and 24 h. The obtained data showed different kinetics for these genes, suggesting a late inhibition for ER, while RXR and ERR presented a similar response with an early increase of transcription at 1 mM and recovering to the normal condition at 48 h. 3.4. Effects of cadmium exposure on hsp70 and hsp90 transcriptional activity HSP70 and HSP90 are related to the endocrine system as they participate in the maturation of steroid hormone receptors (Echeverria and Picard, 2010). Furthermore, it is known that Cd can affect the transcription of these heat shock genes in invertebrates

Please cite this article in press as: Martínez-Paz, P., et al., Cadmium in vivo exposure alters stress response and endocrine-related genes in the freshwater snail Physa acuta. New biomarker genes in a new model organism, Environmental Pollution (2016), http://dx.doi.org/10.1016/ j.envpol.2016.10.012

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Fig. 1. Effects of 5, 15, 90 and 120 min exposure to 39  C on mRNAs levels of the hsp70 (A) and hsp90 (B) genes in control and treated snails, as measured by RT-PCR with primers and reference genes, as indicated in Section 2.7. The means ± SEMs of three independent experiments are shown, and each sample consisted of three replicates. Values were normalised to the reference genes and are presented in relation to the values of the control snails, which were set to 1. Note the logarithmic y-axis in Fig. 1A. The asterisks indicate significant differences between control snails and heat shock-exposed snails: *p  0.05.

Fig. 2. Effects of Cd exposure on the mRNA levels of ER (A), ERR (B) and RXR (C) in control and treated snails as measured by qRT-PCR with primers and reference genes, as indicated in Section 2.7. The means ± SEMs of three independent experiments are shown, and each sample consisted of three replicates. Values were normalised to the reference genes GAPDH, rpL13 and tubulin and are presented in relation to the values of the control snails, which were set to 1 (dashed line). The asterisks indicate significant differences between control snails and Cd-exposed snails: *p  0.05.

(Choi et al., 2008; Morales et al., 2011; Liu et al., 2015). To analyse the transcriptional response of hsp70 and hsp90, the mRNA levels were analysed by qRT-PCR under both normal snail growth conditions and Cd exposure. Although the results did not show statistical significance, we observe that two different kinetics exist,

similar to those observed for endocrine-related genes. For both Cd concentrations, Hsp70 showed a transient upregulation with an increase at 24 h and recovering to the normal levels, or even below the mRNA levels observed in control animals, at 48 h (Fig. 3A). In contrast, Cd did not affect hsp90 transcription (Fig. 3B). These

Please cite this article in press as: Martínez-Paz, P., et al., Cadmium in vivo exposure alters stress response and endocrine-related genes in the freshwater snail Physa acuta. New biomarker genes in a new model organism, Environmental Pollution (2016), http://dx.doi.org/10.1016/ j.envpol.2016.10.012

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Fig. 3. Relative mRNA levels of hsp70 (A) and hsp90 (B) in P. acuta after 0.1 and 1 mM Cd exposure for 5, 24 and 48 h. Values were normalised to the reference genes GAPDH, rpL13 and tubulin and are presented in relation to the values of the control snails, which were set to 1 (dashed line). The means ± SEMs from three independent experiments are shown, and each sample consisted of three replicates.

results imply the different responses of HSPs.

3.5. Effects of Cd exposure on MT mRNA levels Metallothioneins (MTs) form a family of cysteine-rich proteins that bind to physiological (such as zinc, copper and selenium) and xenobiotic (such as Cd, mercury, silver and arsenic) heavy metals. An MT gene was identified in this species, and its expression profile was analysed on Cd-exposed snails. A slight tendency for upregulation, with no statistical significance, was observed at 24 h with 0.1 and 1 mM Cd, recovering to similar levels to those observed in control animals at 48 h (Fig. 4). These results suggest that this MT could be Cd responsive, but that the concentrations and exposure times used were not sufficiently strong to induce a sustained response.

Fig. 4. Relative expression of the MT gene in P. acuta exposed to 5-, 24- and 48-h Cd treatments (0.1 and 1 mM). qRT-PCR experiments were carried out with the primers listed in Table 1. Transcript levels were normalised using the reference genes GAPDH, rpL13 and tubulin and presented in relation to the values for the control snails, which were set to 1 (dashed line). The means ± SEMs of three independent experiments are shown, and each sample consisted of three replicates.

4. Discussion We identified several sequences of genes in the gastropod P acuta. These sequences were used to design primers and to analyse the transcription profiles of genes related to the endocrine system and stress response to improve knowledge regarding the mode of action of Cd in molluscs (mainly in gastropods). Using information present in the GenBank ETS database, ER, ERR, RXR, hsp70, hsp90 and MT genes were identified, and their mRNA levels were analysed in animals exposed to Cd concentrations that are close to environmental concentrations. Furthermore, the response to thermal stress was characterised for the two HSPs identified. Nuclear receptors bind to hormones and play an important role in endocrine and metabolic functions, such as reproduction, behaviour, immunity, stress responses and development (Kaur et al., 2015). The identification of nuclear receptors in Biomphalaria glabrata and Lottia gigantea with high homology to human nuclear receptors indicates the potential of molluscs as the model systems to assess putative EDCs, thus avoiding the use of vertebrates (Kaur et al., 2015). Cd is known to disrupt the endocrine system in different organisms but molecular mechanisms remain poorly understood. ERs are nuclear hormone receptors that regulate gene expression in response to oestrogens and it has been reported that they are modulated by different EDCs in invertebrates (Ciocan et al., 2010; Stange et al., 2012; Zhang et al., 2012). In P. acuta, Cd downregulated the transcriptional activity of ER, showing a similar result to that observed in some vertebrates (Ishitobi et al., 2007; Mehinto et al., 2014; Chen and Chan, 2016). In contrast, other studies report that the mRNA levels of ER in vertebrates were upregulated in the presence of this chemical (Luo et al., 2015a; Driessnack et al., 2016). No previous data exist regarding the effect of Cd in ER transcription in molluscs but it has been hypothesised that hexavalent chromium upregulates ER in Mytilus galloprovincialis (Barmo et al., 2011); thus, it could be possible that behaviours differ depending on the species and the heavy metal. Vertebrate-like sex steroid hormones are widely detected in molluscs, but their role is still unknown, although a role in reproductive processes was proposed (Zheng et al., 2014). Moreover, the signalling pathways of oestrogen-like responses in invertebrates are still unknown. As observed in vertebrates, different results are obtained when the compounds with estrogenic activity are tested, showing induction (Hultin et al., 2014) and no response to these

Please cite this article in press as: Martínez-Paz, P., et al., Cadmium in vivo exposure alters stress response and endocrine-related genes in the freshwater snail Physa acuta. New biomarker genes in a new model organism, Environmental Pollution (2016), http://dx.doi.org/10.1016/ j.envpol.2016.10.012

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chemicals (Bannister et al., 2013). Finally, it is not clear that mollusc ER binds oestradiol; thus, it could be possible that this receptor would bind to another steroid present in molluscs with a similar function to oestrogens on vertebrates. In contrast, ERR and RXR showed simultaneous increase in mRNA levels in the presence of Cd. These data suggest a temporal coordination in the expression of these two nuclear receptors. ERR is an orphan receptor with homology to ER that is described in several species, both vertebrate and invertebrate. It was evaluated  et al., 2014) and inin vertebrates (Schlecht et al., 2004; Tohme vertebrates as a biomarker for EDC exposure (Park and Kwak, 2010; Bannister et al., 2013; Morales et al., 2013, 2014) and exhibited different behaviour depending on the chemical and the species. RXR is a nuclear receptor that forms heterodimers with the nuclear receptors of subfamily 1 that are involved in several physiological processes. Recently, it was proposed that RXR could act as a mediator in the molecular mechanisms of organotin-induced imposex in gastropods (Nishikawa et al., 2004; Sternberg et al., 2008b; Horiguchi et al., 2010; Abidli et al., 2013). In this sense, Cd increased the transcriptional activity of RXR; thus, it might be possible that this heavy metal could induce imposex in P. acuta. Further research is needed to assess this hypothesis. These combined data support the concept that Cd alters the endocrine system in gastropods by affecting the transcription of endocrine-related genes; however, the mode of action is complex since it can inhibit the ER and upregulate ERR and RXR. Moreover, the coordinated response of ERR and RXR suggests that both receptors could be involved in similar pathways or regulation events. HSPs are abundantly induced by various stressful conditions and environmental changes, which play an important role in protecting cells against a broad spectrum of potentially lethal pollutants (Gupta et al., 2010). Hsp70 and hsp90 were identified and their response to heat shock and Cd exposure was analysed to assess the utility of these genes as biomarkers. As expected, both genes were significantly upregulated by a heat shock at 39  C, showing similar behaviour to other HSPs studied in other mollusc species, including gastropods. Previous studies in Ostrea edulis (Piano et al., 2002), Mytilus galloprovincialis (Cellura et al., 2006), Haliotis discus hannai (Cheng et al., 2007), Haliotis tuberculata (Farcy et al., 2007), Haliotis discus (Wang et al., 2011), Sphincterochila zonata (Mizrahi et al., 2012), Sphincterochila cariosa (Mizrahi et al., 2012) and Pomacea canaliculata (Song et al., 2014) reported thermal induction of HSP70 and HSP90, at both gene and protein levels. In P. acuta, the response of hsp70 was fast and could be detected only 5 min from the start of the heat shock. The high level of transcription (a several thousandfold increase) is almost a qualitative response, and it is sustained for 90 min before it begins to decline. However, hsp90 needed 15 min for an increase in the transcription level, but reached the maximum in a similar time to that observed for hsp70. From these results, it is clear that hsp70 is an early-response gene, while hsp90 shows a delayed activation. Finally, the increase of hsp90 is lower, suggesting that this HSP plays a less significant role in the acute response to heat shock. Further, animals exposed to Cd showed only an increase in hsp70 transcriptional activity. Cd can activate hsp70 in other molluscs (Liu et al., 2015), but it is not altered by this metal (Taylor et al., 2013). It is interesting that in this last report, Cd could induce hsp90 activation, as observed in Crassostrea gigas (Choi et al., 2008) but this is in contrast to our data. Regardless, the induction by Cd was less potent than that observed during heat shock and the recovery of normal levels at 48 h of exposure could be considered faster, taking in account that each stimulus-induced expression occurs at different times. This dynamic may reflect hsp70 gene turnover. Previous studies showed that the accumulation of HSP70 protein promotes the degradation of hsp70 mRNA (Balakrishnan and De

7

Maio, 2006) and Shi et al. (1998) reported that HSP70 acts as a negative regulator of hsp70 transcription. Therefore, the expression of hsp70 needs to be tightly regulated. This gene was also upregulated after in vivo Cd exposure in other invertebrates (Morales et al., 2011; Qian et al., 2012; Kim et al., 2014; Haap et al., 2016). It is important to note that the activation of hsp70 could be related to the role that this protein plays in hormonal receptor functioning. The dynamics observed are very similar to those found with ERR and RXR; thus, the hypothesis that the lower activation was reflecting a lower requirement of the protein in comparison to that required for heat shock response cannot be rejected. Moreover, the fact that hsp90 did not show induction could be because of a higher constitutive level of this protein in relation to HSP70; hence, there was no need to increase the transcription. It is also possible that the stimulus was not sufficient to activate the stress response in a similar way to thermal stress. In contrast to the obtained hsp90 data, previous studies show that Cd upregulates mRNA levels of this gene in other invertebrates such as arthropods (Sonoda et al., 2007; Qian et al., 2012) and platyhelminthes (Plusquin et al., 2012), a difference that could be related to the concentrations used in the study or the metabolism of each species. Nevertheless, HSPs are important tools in environmental studies, and therefore, the hsp70 and hsp90 transcriptional activity observed in P. acuta will help to elucidate the mode of action of Cd in a similar way to the studies carried out in other invertebrates with different xenobiotics tard et al., 2013; Chen (Morales et al., 2011; Qian et al., 2012; Boue et al., 2014). MTs are commonly used as the biomarkers to assess metal exposure in environmental monitoring programmes in aquatic organisms. Recent reports found that the transcriptional activity of MT could be a potential biomarker of Cd exposure in different aquatic invertebrates (Bigot et al., 2011; Pedersen et al., 2014; Geng  et al., 2016). Moreover, part of the snail's stress et al., 2015; Tousova defence strategy is its ability to express MTs, the concentrations of which increased in snails from Cd-polluted sites (Abdel-Halim et al., 2013; Pedrini-Martha et al., 2016). Because this family of proteins is formed of different members, we analysed the MT gene that we identified as responsive to Cd. The results showed a slight induction of the MT gene expression, which could be because the short-term exposure and low concentrations used might be unable to induce expression of this gene or because MT may not necessarily be the main player in this heavy metal detoxification in P. acuta. In this sense, it has been reported that the phytochelatin protein can play a role on Cd detoxification in the aquatic snail Lymnaea stagnalis (Gonçalves et al., 2016). Furthermore, recent reports have shown the physiological and diurnal variability and the age-dependent MT gene expression in land snails exposed to Cd (Baurand et al., 2016; Pedrini-Martha et al., 2016). Hence, the relationship of this MT gene and Cd should be further investigated to analyse if this gene is an appropriate biomarker to short-term heavy metal exposures in the freshwater snail P. acuta. 5. Conclusions Previous studies report on the toxicity and the endocrinedisrupting effects of Cd in various organisms, including fishes (Garcia-Santos et al., 2013), amphibians (Gay et al., 2013), birds,  et al., 2015), molluscs mammals (Siu et al., 2009; Marettova (Lagadic et al., 2007), crustaceans (Rodríguez et al., 2007) and in et al., 2010; Morales et al., 2011; Martínez-Paz et al., sects (Planello 2014). However, there is poor knowledge about its mode of action at the molecular level in invertebrates. This is especially important as invertebrates show high diversity; hence, their response to pollutants can vary greatly. The study presented here facilitated defining the response of endocrine- and stress-related genes on

Please cite this article in press as: Martínez-Paz, P., et al., Cadmium in vivo exposure alters stress response and endocrine-related genes in the freshwater snail Physa acuta. New biomarker genes in a new model organism, Environmental Pollution (2016), http://dx.doi.org/10.1016/ j.envpol.2016.10.012

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P. acuta, an ecologically relevant aquatic snail, thus providing new information about the mode of action of Cd in gastropods. Furthermore, we characterised the response of two HSP genes to thermal stress. The obtained data showed that Cd alters the endocrine system, but that it can have opposite effects depending on the pathway affected, as demonstrated by the different responses observed for ER and for ERR and RXR. Moreover, the HSP genes show different responses to Cd, which may reflect not only a stress response but also a response according to their roles in other physiological functions related to the hormonal receptors. Finally, specific biomarkers of exposure to EDCs in aquatic snails are still scarce; hence, this work illustrates the potential of the analysed genes to be employed as the sensitive molecular biomarkers in P. acuta, which would be a suitable sentinel organism for evaluating the effect of EDCs on aquatic ecosystems. Acknowledgements We would like to thank Pilar García from Departamento de Medioambiente (INIA) for technical support. This work was supn Científica, Desarrollo e ported by the Plan Nacional de Investigacio  n Tecnolo  gica (Spain), grants CTM2012-37547 and CTMInnovacio 2015-64913-R from the Ciencias y Tecnologías Medioambientales programme. The authors declare that they have no conflicts of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.envpol.2016.10.012. References Abdel-Halim, K.Y., Abo El-Saad, A.M., Talha, M.M., Hussein, A.A., Bakry, N.M., 2013. Oxidative stress on land snail Helix aspersa as a sentinel organism for ecotoxicological effects of urban pollution with heavy metals. Chemosphere 93 (6), 1131e1138. http://dx.doi.org/10.1016/j.chemosphere2013.06.042. Abidli, S., Castro, L.F., Lahbib, Y., Reis-Henriques, M.A., Trigui El Menif, N., Santos, M.M., 2013. Imposex development in Hexaplex trunculus (Gastropoda: Caenogastropoda) involves changes in the transcription levels of the retinoid X receptor (RXR). Chemosphere 93 (6), 1161e1167. http://dx.doi.org/10.1016/ j.chemosphere.2013.06.054. Ait Alla, A., Mourneyrac, C., Durou, C., Moukrim, A., Pellerin, J., 2006. Tolerance and biomarkers as useful tools for assessing environmental quality in Oued Souss estuary (Bay of Agadir, Morocco). Comp. Biochem. Physiol. C Toxicol. Pharmacol. 143 (1), 23e29. http://dx.doi.org/10.1016/j.cbpc.2005.11.015. Albrecht, C., Oliver, K., Terrazas, E.M., Wilke, T., 2009. Invasion of ancient lake Titicaca by the globally invasive Physa acuta (Gastropoda: Pulmonata: Hygrophila). Biol. Invasions 11, 1821e1826. http://dx.doi.org/10.1007/s10530-0089360-9. Balakrishnan, K., De Maio, A., 2006. Heat shock protein 70 binds its own messenger ribonucleic acid as part of a gene expression self-limiting mechanism. Cell Stress Chaperones 11 (1), 44e50. http://dx.doi.org/10.1379/CSC-136R1.1. Bandow, C., Weltje, L., 2012. Development of an embryo toxicity test with the pond snail Lymnaea stagnalis using the model substance tributyltin and common solvents. Sci. Total Environ. 435e436, 90e95. http://dx.doi.org/10.1016/ j.scitotenv.2012.07.005. Bannister, R., Beresford, N., May, D., Routledge, E.J., Jobling, S., Rand-Weaver, M., 2007. Novel estrogen receptor-related transcripts in Marisa cornuarietis; a freshwater snail with reported sensitivity to estrogenic chemicals. Environ. Sci. Technol. 41 (7), 2643e2650. http://dx.doi.org/10.1021/es062565m. Bannister, R., Beresford, N., Granger, D.W., Pounds, N.A., Rand-Weaver, M., White, R., Jobling, S., Routledge, E.J., 2013. No substantial changes in estrogen receptor and estrogen-related receptor orthologue gene transcription in Marisa cornuarietis exposed to estrogenic chemicals. Aquat. Toxicol. 140e141, 19e26. http:// dx.doi.org/10.1016/j.aquatox.2013.05.002. Barmo, C., Ciacci, C., Fabbri, R., Olivieri, S., Bianchi, N., Gallo, G., Canesi, L., 2011. Pleiotropic effects of hexavalent chromium (CrVI) in Mytilus galloprovincialis digestive gland. Chemosphere 83 (8), 1087e1095. http://dx.doi.org/10.1016/ j.chemosphere.2011.01.037. Baurand, P.E., Pedrini-Martha, V., de Vaufleury, A., Niederwanger, M., Capelli, N., Scheifler, R., Dallinger, R., 2015. Differential expression of metallothionein isoforms in terrestrial snail embryos reflects early life stage adaptation to metal stress. PLoS One 10 (2), e0116004. http://dx.doi.org/10.1371/ journal.pone.0116004.

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Please cite this article in press as: Martínez-Paz, P., et al., Cadmium in vivo exposure alters stress response and endocrine-related genes in the freshwater snail Physa acuta. New biomarker genes in a new model organism, Environmental Pollution (2016), http://dx.doi.org/10.1016/ j.envpol.2016.10.012