Ecotoxicology and Environmental Safety 105 (2014) 43–50

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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Development of a non-lethal method for evaluating transcriptomic endpoints in Arctic grayling (Thymallus arcticus) Nik Veldhoen a, Jean E. Beckerton b, Jody Mackenzie-Grieve c, Mitchel R. Stevenson a, Robert L. Truelson b, Caren C. Helbing a,n a

Department of Biochemistry and Microbiology, University of Victoria, P.O. Box 3055, STN CSC, Victoria, BC, Canada V8W 3P6 Water Resources Branch, Environment Yukon, Government of Yukon, Box 2703 (V-310), Whitehorse, Yukon, Canada Y1A 2C6 c Fisheries and Oceans Canada, 100-419 Range Road, Whitehorse, Yukon, Canada Y1A 3V1 b

art ic l e i nf o

a b s t r a c t

Article history: Received 12 October 2013 Received in revised form 15 January 2014 Accepted 25 March 2014

With increases in active mining and continued discharge associated with former mine operations, evaluating the health of watersheds in the Canadian Yukon Territory is warranted. Current environmental assessment approaches often employ guidelines established using sentinel species not relevant to Arctic monitoring programs. The present study focused on the successful development of a quantitative real-time polymerase chain reaction (qPCR) assay directed towards the indigenous Arctic grayling (Thymallus arcticus) and examines the feasibility of using non-lethal sampling from the caudal fin as a means for evaluation of mRNA abundance profiles reflective of environmental conditions. In a proof of concept study performed blind, qPCR results from animals in an area with elevated water concentrations of cadmium (Cd) and zinc (Zn) and higher body burdens of Cd, Zn, and lead (Pb) were compared to a reference location in the Yukon Territory. Lower condition factor and a higher abundance of hepatic and caudal fin gene transcripts encoding the metallothionein isoforms (mta/mtb), in addition to elevated heat shock protein 70 (hsp70) and catalase (cat) mRNAs in liver, were observed in fish from the test site. The strong positive correlation between metal body burden and caudal fin mta/mtb mRNA abundance demonstrates a high potential for use of the Arctic grayling assay in non-lethal environmental monitoring programs. & 2014 Elsevier Inc. All rights reserved.

Keywords: Non-lethal assay Fish Sentinel species Wildlife conservation Quantitative real time polymerase chain reaction Mining

1. Introduction The Yukon Territory of northern Canada supports a diversity of currently active and legacy mining operations (http://miningyu kon.com). With such industry arises the potential for release of anthropogenic environmental pollutants into aquatic environs resulting in deleterious impact on various wildlife species (Brinkman and Johnston, 2012; Dubé et al., 2005). Such contaminants may include, but are not restricted to, heavy metals that leach from mining sites into the surrounding hydrologic system. Thus, continued development of effective and highly sensitive monitoring tools that are applicable to the northern mining industry, in particular, and human activities in general, is warranted.

n

Corresponding author. Fax: þ 1 250 721 8855. E-mail addresses: [email protected] (N. Veldhoen), [email protected] (J.E. Beckerton), [email protected] (J. Mackenzie-Grieve), [email protected] (M.R. Stevenson), [email protected] (R.L. Truelson), [email protected] (C.C. Helbing). http://dx.doi.org/10.1016/j.ecoenv.2014.03.030 0147-6513/& 2014 Elsevier Inc. All rights reserved.

For many living systems exposure to significant levels of heavy metals such as zinc, cadmium, cobalt, and copper can prove toxic, and biological defense pathways have evolved to combat metal uptake (Chowdhury et al., 2005; Jezierska et al., 2009; Valko et al., 2005). One of the most studied of these systems is the metal-induced increase in expression of a family of proteins called metallothioneins (MTs) which function to bind metals taken up by an organism and reduce their harmful chemical reactivity (Andrews, 2000; Chowdhury et al., 2005; Hollis et al., 2001; Shariati and Shariati, 2011). Two forms of metallothionein have been described in salmon species, referred to as mta and mtb, and up-regulation of mRNA expression concomitant with increased protein levels can be detected in a number of animal tissues following exposure to sub-lethal concentrations of water-borne heavy metal (Heikkila et al., 1982; McClain et al., 2003; Price-Haughey et al., 1986; Roch and McCarter, 1984; Shariati and Shariati, 2011). Exposure to biologically disruptive heavy metals can also increase more general stress-associated pathways related to oxidative and thermal stress (Ait-Aïssa et al., 2003; Hansen et al., 2006; Heikkila et al., 1982; Misra et al., 1989). Such additional molecular markers that contribute information related to heavy metal and anthropogenic chemical contaminant exposure

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N. Veldhoen et al. / Ecotoxicology and Environmental Safety 105 (2014) 43–50

136oW

63o30’N

63o30’N

64oN

64oN

137oW

0

137oW

10 km

20

136oW

Fig. 1. Arctic grayling sampling locations investigated in the central Yukon Territory. The positions of Moose Creek site near Stewart Crossing and Christal Creek site near Keno City are indicated by stars.

include catalase, which functions in reduction of reactive hydrogen peroxide levels in living systems (Eyckmans et al., 2011; Hansen et al., 2006), and the heat shock protein family, that serve as chaperones to maintain proteins in a correctly folded state (Ait-Aïssa et al., 2003). Typical tissue collection methods for gene expression studies have most often relied on lethal sampling. Yet a highly desirable refinement is to utilize a minimally-invasive, non-lethal sampling methodology, particularly pertaining to wildlife species where conservation is of concern (Arukwe and Røe, 2008; Jin et al., 2008; Rees et al., 2005; Veldhoen et al., 2013). To this end, we recently developed a caudal fin biopsy approach in rainbow trout that is capable of detecting exposure to metal (cadmium) and estrogen (17α-ethinyl estradiol) (Veldhoen et al., 2013). The detection of activated biological defense pathways within indigenous sentinel species exposed to metals forms the basis of an environmental monitoring assay amenable to supervision of active mine operations as well as evaluation of progress made in mine reclamation. In this context, the current initiative demonstrates the potential of a newly developed quantitative real-time polymerase chain reaction (qPCR) assay directed towards the Arctic grayling (Thymallus arcticus) to identify metal exposure. Additionally, the possibility of applying this assay in a non-lethal manner is demonstrated with a view towards animal conservation. Arctic grayling represent an excellent candidate freshwater sentinel species due to their presence in streams, rivers, and lakes throughout the Arctic regions of North America and Asia combined with a moderate (o 100 km) annual migratory range and territorial behavior during summer (Craig and Poulin, 1975; West et al., 1992). To our knowledge, this work represents the first qPCRbased molecular assay developed for this northern fish species that is a highly valued in both the recreational fishery and as a traditional food source.

2. Materials and Methods 2.1. Animals and sampling locations Through a combination of angling and electroshock methods mixed juvenile and adult Arctic grayling were harvested from the Christal Creek location (21 animals) and Moose Creek site (15 animals) over five collection days between July 23 and August 29, 2012 (Fig. 1). One animal from each location had a mass greater than the scale limit (400 g) and was not used for the calculation of condition factor. Determination of animal sex using morphological criteria was not definitive in the juvenile animals and attempts at applying genotypic sexing methods for Arctic grayling as was previously done for other salmonids (Veldhoen et al., 2010) were unsuccessful. Therefore the contribution of relationship between sex and other measured biological endpoints could not be investigated in the present study. Christal Creek drains the historic, and presently active, hard rock mining district of Keno Hill (near Mayo, Yukon Territory, Canada). Water temperature ranged from 6.0 to 8.7 1C during the collection period and creek water was collected for additional water quality measures as well as evaluation of total metals content (Tables S1 and Table 1). Water chemistry and total metal concentrations determined using inductively-coupled plasma mass spectrometry (ICP-MS) were performed by Maxxam Analytics Inc (Burnaby, BC, Canada). Upon capture, liver (  0.5 cm3) and caudal fin (1  0.5 cm) samples were immediately collected and stored in RNAlater tissue preservation solution, as described by the manufacturer (Life Technologies Inc., Burlington, ON, Canada), in order to maintain the integrity of the RNA in situ. Samples were shipped to the University of Victoria for further processing. The remaining carcasses were stored at  80 1C. Five fish from each site with similar masses (Christal Creek 2357 30 g; Moose Creek 2377 26 g) were selected and their carcasses (minus the small amounts of tissue samples removed above) were individually tested for metals content using ICP-atomic emission spectroscopy (ICP-AES) by Exova, Surrey, BC, Canada.

2.2. Isolation of total RNA and preparation of cDNA All tissue samples were randomized prior to further processing. Total RNA was isolated from Arctic grayling tissue samples using 700 μL TRIzol reagent as described by the manufacturer (Life Technologies) and homogenization for

N. Veldhoen et al. / Ecotoxicology and Environmental Safety 105 (2014) 43–50

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Table 1 Total dissolved metal concentrations at Christal Creek and Moose Creek locations determined by ICP-MS. Values above water quality guidelines are indicated in bold. Metal

Aluminum Arsenic Boron Cadmium Chromium Copper Iron Lead Molybdenum Nickel Selenium Silver Thallium Uranium Zinc Total Hardness (CaCO3)

Units

μg/L μg/L μg/L μg/L μg/L μg/L μg/L μg/L μg/L μg/L μg/L μg/L μg/L μg/L μg/L mg/L

Guideline

Christal Creek August 16, 2012

Moose Creek August 17, 2012

QC/QA % Recovery

Year

Detected

RDLa

CEQGb

Detected

RDLa

CEQGb

1987 1997 2009 1996 NA 1987 1987 1987 1999 1987 1987 1987 1999 2011 1987

27.7 2.23 o50 0.566 o0.50 0.58 156 1.44 0.372 1.22 0.480 0.0120 o0.0020 1.85 66.3 290

3.0 0.020 50 0.0050 0.50 0.20 5.0 0.050 0.050 0.10 0.040 0.0050 0.0020 0.0050 1.0 0.50

100c 5 1500 0.083c NA 4c 300 7c 73 150.00c 1 0.1 0.8 15 30 NA

25.0 1.79 o50 0.0060 o0.50 1.40 711 0.087 0.274 0.68 0.135d o0.0050 o0.0020 0.541 1.4d 140

3.0 0.020 50 0.0050 0.50 0.20 5.0 0.050 0.050 0.10 0.040 0.0050 0.0020 0.0050 1.0 0.50

100c 5 1500 0.044c NA 3.15c 300 4.88c 73 123.43c 1 0.1 0.8 15 30 NA

101 85 NA 88 98 98 105 100 104 98 82 100 104 104 84 94

NA ¼no data available. a

RDL ¼Reportable Detection Limit. Canadian Environmental Quality Guidelines for freshwater long-term exposure. Corrected for total water hardness and/or pH. d Matrix Spike outside acceptance criteria (10% of analytes failure allowed). b c

6 minutes at 20 Hz in a Retsch MM301 mixer mill (ThermoFisher Scientific Inc., Ottawa, ON, Canada) in microtubes containing a 3 mm stainless steel bead. Mixer mill racks were rotated 180 degrees halfway through the homogenization procedure and 20 μg of glycogen was added to each caudal fin sample during RNA isolation. Isolated RNA concentrations were determined by spectrophotometry using a Nanodrop ND-1000 as per the manufacturer's protocol (ThermoFisher Scientific). Hepatic and caudal fin total RNA were normalized to 100 ng/μL and one microgram of each sample used to produce cDNA employing the High Capacity cDNA Reverse Transcription kit as described by the manufacturer (Life Technologies). Each cDNA sample was diluted 20-fold prior to use for isolation of speciesspecific expressed gene sequences and qPCR analysis.

2.3. Isolation of species-specific expressed gene sequences All cloning and qPCR DNA primers were obtained from Integrated DNA Technologies Inc (Coralville, IA, USA). Gene-specific primers were designed using Primer Premier 5 (Premier Biosoft Inc., Palo Alto, CA, USA) based on ClustalW2 alignment (http://www.ebi.ac.uk/Tools/msa/clustalw2/) of comparable Salmonidae sequences available from the NCBI GenBank database (http://www.ncbi.nlm.nih. gov/genbank/) including Pacific and Atlantic salmon species as well as rainbow trout (Table S2; (Veldhoen et al., 2010)). Additional qPCR primers specific for mta and mtb were sourced from the literature (Vergani et al., 2007). Arctic grayling cDNA amplicons were produced in a 15 mL PCR reaction containing 20 pmol of each primer pair, 1.5 mM MgCl2, 2 mL diluted cDNA and 2 units of Immolase DNA polymerase along with additional reagents as recommended by the manufacturer (Bioline USA Inc., Randolph, MA, USA). Reactions were performed on a MyCycler thermal cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) and, in all cases, the thermocycle program consisted of initial activation for 7 min (95 1C), 45 cycles of 15 s (95 1C), 30 s (52 1C), and 45 s (72 1C), and a final extension for 10 min (72 1C). PCR amplicons were evaluated on a 1.5% agarose gel and DNA of the correct predicted size (see Table S3) excised and extracted from the gel using an Ultrafree-DA DNA Extraction kit as described by the manufacturer (Millipore Corp., Billerica, MA, USA). Each amplicon DNA was further purification using a GeneJET PCR Purification kit as described by the manufacturer (Thermo Fisher) and eluted in 30 mL water. Isolated Arctic grayling cDNA fragments were subjected to direct DNA sequencing or subsequently cloned into a plasmid vector prior to sequencing using the CloneJET PCR Cloning kit as described by the manufacturer (Thermo Fisher). The selection of positive clones and preparation of recombinant plasmids for DNA sequencing were as previously described (Boone et al., 2013). Species-specific sequences were obtained using the services of either Sequetech (Mountain View, CA, USA) or Eurofins MWG Operon (Huntsville, AL, USA) and assembled with the CAP3 software (http://pbil.univ-lyon1.fr/cap3.php). Arctic grayling gene information and associated PCR primers used in sequence isolation were either deposited in NCBI GenBank (see Table S3) or presented in Table S4 and subsequently used to confirm the appropriate targeting of qPCR primers directed towards specific expressed gene transcripts (Table S3).

2.4. Development of a species-specific qPCR assay The Arctic grayling qPCR assay was subjected to a three-tier quality assessment which ensured that targeted gene-specific qPCR signal is achieved for each combination of primer pair and tissue sample type and that the collected data is appropriate for use in quantification of mRNA abundance (Bustin et al., 2009; Schmittgen and Livak, 2008; Veldhoen et al., 2009). Details regarding the extensive quality assessment employed to satisfy MIQE standards can be found elsewhere (Veldhoen et al., 2009, 2010). Upon successful completion of the quality assurance evaluation (Table S3), the qPCR assay was used to assess liver and caudal fin cDNA samples where applicable. The qPCR assay was performed as previously described (Veldhoen et al., 2010) on a MX3005P Real-Time PCR System (Stratagene, LaJolla, CA). All samples were randomized and assayed blind to eliminate subjective bias and run in quadruplicate reactions for maximum quantitative accuracy. Each 15 μL qPCR amplification reaction consisted of 10 mM Tris–HCl (pH 8.3 at 20 1C), 50 mM KCl, 3 mM MgCl2, 0.01% Tween 20, 0.8% glycerol, 40,000-fold dilution of SYBR Green I (Thermo Fisher), 69.4 nM ROX Thermo Fisher, 5 pmol of each primer, 200 μM dNTPs (Bioline USA Inc, Taunton, MA, USA), 2 μL of 20-fold diluted cDNA, and one unit of Immolase Hot Start Taq DNA polymerase (Bioline). Amplification reactions were subject to the following thermocycle conditions: an initial activation step of 9 min at 95 1C followed by 40 cycles of 15 s denaturation at 95 1C, 30 s annealing at 60 1C, and 45 s polymerization at 72 1C. Specificity of target amplification was measured by the inclusion of reactions lacking cDNA (no DNA template control) and by subjecting completed runs to thermodenaturation analysis. An additional inter-plate variation control comprising a universal cDNA sample present in all qPCR runs was included for each gene target. Variation between plate runs was observed at less than one cycle threshold (Ct) value for all gene targets. To confirm the absence of genomic DNA contamination in the evaluation of the gene transcripts, we performed qPCR directly on a subset of appropriately diluted total RNA samples using rps10 primers that produce an amplicon of 1.1 kb from genomic DNA template. No evidence of genomic DNA contamination was observed in any sample (data not shown). For each gene, an identical threshold value was set for collection of Ct values across multiple plate runs. All reaction thermodenaturation curves were assessed to confirm correct targeting with removal of Ct values associated with non-target signal capture. Additionally, technical replicate data were evaluated for standard deviation of less than 0.5 with removal of poor replicate values. Data attrition due to application of these quality assurance steps displayed a median of 1.4% with a maximum of 4% across the liver and caudal fin samples. Replicate data for each cDNA sample were subsequently averaged and test gene targets normalized to the geometric mean of eukaryotic translation elongation factor 1 alpha (eef1a), ribosomal protein S10 (rps10), and cytoplasmic β actin (actb) during application of the comparative Ct method (ΔΔCt) (Schmittgen and Livak, 2008). Suitability of these three normalizer transcripts was confirmed using RefFinder (http://www.leonxie.com/referencegene.php). This webbased tool used a combination of currently available normalizer evaluation software including geNorm (Vandesompele et al., 2002), Normfinder (Andersen et al., 2004), BestKeeper (Pfaffl et al., 2004), and comparative ΔCt (Silver et al., 2006).

400

Condition can be described as the well-being or robustness of fish. Condition indices are widely used to describe fish health because data requirements are minimal. The condition factor (K) for Moose Creek and Christal Creek fish were calculated according to Fulton's condition factor K (Fulton, 1904):

300

where W ¼ mass (g), L ¼fork length (mm), and 100,000 is a scaling factor used to obtain a value close to unity. The approach assumes that growth is isometric (b ¼ 3) and was selected based on recommendations for small sample sizes (Froese, 2006). Statistical comparisons of mRNA abundance in liver and caudal fin between fish sampling sites were performed using SYSTAT 13 version 13.00.05 (SYSTAT Software Inc., Chicago, IL, USA) with application of the non-parametric Mann–Whitney U test. Condition factor comparisons were made using a two-way ANOVA using SYSTAT. Spearman's rho correlations were calculated using SYSTAT and a program in R (Wessa, 2013). Statistical significance was set at p r 0.05.

3. Results Water chemistry demonstrated clear differences between the conditions present at the two sampling locations (Christal Creek and Moose Creek) with respect to both standard water characteristics (Table S1) and presence of metals (Table S2). In particular, Christal Creek showed higher total dissolved solids and sulfate as well as the presence of higher cadmium (Cd) and zinc (Zn) concentrations. Moose Creek showed higher concentrations of phosphorus and iron (elements that are substantially less toxic to aquatic life relative to Cd and Zn). In preparation for qPCR assay development, eight expressed gene sequences were obtained for Arctic grayling with two representing mRNA transcript variants of rps10 (Table S3). Where cross-species comparisons could be made, the Arctic grayling gene sequences displayed a high degree of conservation in DNA base identity (4 90%) to other members of the Salmonidae family (Table S2). This supported evaluation of the efficacy of qPCR primers designed across different salmon species on their ability to detect mRNA abundance in Arctic grayling tissue samples. Table S3 shows the qPCR primer pairs selected for analysis. Animals assessed at the two field sites did not differ in weight, fork length, or total length (Fig. 2). However, the condition (K) of Moose Creek animals (1.08 7 0.02) was higher than that of the Christal Creek animals (1.01 70.02, p ¼0.028, ANOVA; Fig. 2).Both metal-related transcripts as well as stress-associated mRNA showed different abundance profiles in the liver of animals from Christal Creek compared to those in Moose Creek (Fig. 3). Transcripts encoding mta and mtb were significantly higher in Christal Creek animals (p¼ 0.002 and p o0.001, respectively). Higher levels of hepatic cat and hsp70 transcripts (p o0.001 and p ¼0.045, respectively) were also detected in Christal Creek animals indicating the presence of physiological stress in this group (Fig. 3). We then performed Spearman's rho correlation analyses on the data set. Since the correlation profiles were very similar between sampling locations, we combined the data from the two sites to increase the power of the analysis. Animal mass, total length, and fork length, while strongly correlated with each other, did not correlate with any of the transcripts (data not shown). Comparison of transcript abundance with condition factor revealed similar results with the exception of cat mRNA abundance that had a tendency to increase when condition factor decreased (Table S5). A strong positive correlation between mta and mtb transcript abundance was evident in both tissues (Table S5). The levels of mta/mtb mRNAs also were positively correlated with cat mRNAs in the liver (Table S5). While hsp70 mRNA levels correlated positively with mta/mtb transcripts in the caudal fin, no such relationship was detected in the liver (Table S5). Each transcript type, mta, mtb,

200

100

Total Length (mm)

K ¼ W=L3  100; 000

Mass (g)

2.5. Statistical analyses

ForkL ength (mm)

N. Veldhoen et al. / Ecotoxicology and Environmental Safety 105 (2014) 43–50

300

200

0

100

400

1.5

300

200

100

Condition Factor

46

1.0 a 0.5

0.0

Fig. 2. Animals from Moose Creek (white bars; n¼ 14) and Christal Creek (gray bars; n ¼20) were evaluated for size metrics (mass, fork length, total length) and condition factor. One animal from each location had a mass greater than the scale limit (400 g) and these two animals are not included in the size metrics or condition factor calculations. Statistically significant differences (p o 0.05) are indicated by ‘a’. The medians are shown as solid black lines within the box, and the box indicates the first and third quartiles. The whiskers indicate minimum and maximum values. Outliers (cases between 1.5 and 3.0 box lengths from the upper or lower edge of the box) are indicated by an asterisk.

and hsp70, correlated strongly in abundance between tissues (Table S5). To further investigate the relationship between chronic metal exposure and transcript abundance, a small subset of five fish from either site were selected based upon similar masses (see Section 2 for details) and their metal body burdens were determined. Aluminum, molybdenum, and selenium body burdens were not significantly different between sites, but were higher than the tissue screening concentration (TSC) suggesting possible adverse ecological effects (Table 2; Dyer et al., 2000). Arsenic, copper, nickel, silver (below detection limit), and thallium body burdens were not different between sites and below the TSC (Table 2). The Moose Creek animals showed a significantly higher chromium body burden that was around the TSC (Table 2). In contrast, Cd, Zn, and lead (Pb) body burdens were significantly higher in the Christal Creek fish and above the TSC (Table 2) consistent with the higher levels measured in the water samples at this location (Table 1). Although there was a trend towards higher iron body burdens in Moose Creek fish, there was a high degree of variation resulting in non-significance between the two sites (Table 2). Condition factor did not correlate with any one metal found to be significantly different between sites (data not shown). Comparison of metal body burden and transcript levels in these same fish revealed a strong positive correlation between Cd, Zn, and Pb and hepatic hsp70 and cat mRNA abundance (Table 3). A tendency towards positive correlation of these metals and mtb mRNA was also observed that might be strengthened by higher animal numbers (Table 3). A strong negative correlation was observed between chromium body burden and hepatic hsp70,

N. Veldhoen et al. / Ecotoxicology and Environmental Safety 105 (2014) 43–50

47

Fig. 3. Animals from Moose Creek (white bars; n ¼15) and Christal Creek (gray bars; n¼ 21) were evaluated for relative mRNA abundance in liver and caudal fin tissue. Refer to the Fig. 2 legend for graph details. Extreme values (cases 43.0 box lengths from the upper or lower edge of the box) are indicated by an open circle. ND ¼not determined in this tissue.

Table 2 Measured metal body burden of select study fish (n¼ 5 per site). Metal

Units (mg/g wet weight) Christal Creek

Aluminum Arsenic Cadmium Chromium Copper Iron Lead Molybdenum Nickel Selenium Silver Thallium Zinc

c

17.547 2.00 0.20 70.04 0.197 0.01d 0.137 0.04d 0.687 0.04 52.42 78.07 0.587 0.06d 0.077 0.01 0.127 0.02 1.12 70.06 o 0.10 0.43 70.02 27.50 7 0.90d

RDLa

TSCb

1.0 0.09 0.02 0.04 0.05 1.00 0.14 0.04 0.07 0.30 0.10 0.3 0.10

4.40 1.60 0.04 0.18 3.00 NA 0.06 0.06 0.39 0.56 0.37 NA 20.00

Moose Creek 65.147 30.43 0.31 70.09 0.047 0.01 1.92 7 0.80 0.75 70.17 274.22 797.97 o 0.14e 0.157 0.05 0.25 70.08 0.97 70.10 o 0.10 0.55 70.04 18.50 7 1.48

NA ¼no data available. a

RDL ¼Reportable Detection Limit. TSC ¼ Tissue screening concentration; tissue residue in aquatic biota above which adverse ecological effects may occur according to Dyer et al. (2000). c Values above the TSC are indicated in bold. d Statistically significant between sites with po 0.05. e Limit of detection. b

hepatic cat, and caudal fin mtb transcripts (Table 3). Additional significant negative correlations were also observed between hepatic hsp70 mRNAs and each of iron, molybdenum, and thallium body burdens (Table 3). A positive correlation between hepatic hsp70 mRNA abundance and selenium and between aluminum and hepatic mta mRNA abundance was also observed (Table 3). In contrast, hsp70 mRNA levels in the caudal fin did not correlate with the body burdens of any measured metal (Table 3). Rather, strong correlations were observed between

caudal fin mta/mtb mRNA abundance and Cd or Pb (Table 3) and negative correlations were found between caudal fin mtb mRNAs and chromium or molybdenum (Table 3). A tendency towards positive correlation of Zn and mta mRNA was also observed that might be strengthened by higher animal numbers (Table 3). No correlation was observed between the body burdens of arsenic, copper, or nickel and liver or caudal fin transcripts (Table 3).

4. Discussion Despite divergence of the Thymallus and Oncorhynchus genera approximately 60 million years ago (Crête-Lafrenière et al., 2012), Arctic grayling display a high degree of conservation in the protein encoding regions of gene sequences currently targeted for qPCR assay development (Table S2). Such evolutionary constraint across the family Salmonidae allowed for the application of previously established salmon qPCR tools (mta, mtb, actb, and cat) towards this Arctic species (Veldhoen et al., 2010; Vergani et al., 2007) along with inclusion of de novo cross-species designed genespecific primer pairs (eef1a, rps10, and hsp70). The resulting transcriptomics-based assay comprised three candidate normalizer genes (eef1a, rps10, and actb), two metal-associated genes (mta and mtb), and gene markers related to physiological stress (cat and hsp70). This assay focused on the detection of biological response of Arctic grayling to heavy metal exposure and was vetted on two tissue types including liver, a popular target for environmental assessment of molecular endpoints, and caudal fin, a tissue source amenable to conservation-based non-lethal collection methods. Response to heavy metal exposure with increased expression of metallothionein mRNA and/or protein is a biological defense pathway that is conserved across a diverse range of freshwater and marine aquatic species (Andrews, 2000; Hansen et al., 2006,

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N. Veldhoen et al. / Ecotoxicology and Environmental Safety 105 (2014) 43–50

Table 3 Spearman's rho correlation coefficients comparing metal body burden and liver/caudal fin transcript levels (n¼ 10). Caudal fin

Liver

Aluminum Arsenic Cadmiumb Chromiumb Copper Iron Leadb Molybdenum Nickel Selenium Thallium Zincb a b

mta

mtb

hsp70

cat

mta

mtb

hsp70

0.588a  0.013 0.343 0.122 0.564 0.261 0.313 0.196 0.322  0.006 0.164 0.462

0.370  0.100 0.550  0.134 0.333  0.042 0.550  0.092  0.043  0.006  0.030 0.523

 0.503  0.163 0.685a  0.888a 0.067  0.782a 0.738a  0.752a  0.419 0.612a  0.733a 0.699a

 0.115 0.069 0.832a  0.644a 0.442  0.527 0.682a  0.495  0.274 0.115  0.321 0.644a

 0.406 0.533 0.838a  0.505  0.139  0.297 0.794a  0.520 0.067 0.018  0.418 0.559

 0.552 0.395 0.789a  0.644a  0.176  0.455 0.769a  0.679a  0.122  0.030  0.394 0.462

 0.370 0.464 0.440  0.292  0.055  0.067 0.394  0.355 0.207  0.261 0.030 0.134

Statistical significance set at p o 0.05. Silver was not analyzed because the levels were below the detection limit. Statistically significant metal body burden between sites with p o0.05. Refer to Table 2 for details.

2007; Shariati and Shariati, 2011). While fish from both sampling sites had similar body burdens of aluminum, molybdenum, and selenium at levels above which adverse ecological effects may occur, Christal Creek animals had significantly lower condition factor (Fig. 2) and Cd, Zn, and Pb body burdens above the TSC compared to Moose Creek fish (Table 2). These observations combined with the known inductive response of mt genes in Salmonidae following exposure to heavy metals strongly suggest that a higher response to heavy metal stress is being detected in Arctic grayling from the Christal Creek location compared to Moose Creek. That increased environmental Cd and Zn are potentially deleterious to freshwater fish populations is supported by recent risk assessments of brown trout (Salmo trutta) and evaluation of health metrics in bull trout (Salvelinus confluentus) in more southerly regions of North America (Kiser et al., 2010; Toll et al., 2013). A popular animal model for environmental toxicology, rainbow trout (Oncorhynchus mykiss) chronically exposed to waterborne Cd show increased metal burden with significant accumulation in the gills and kidneys and moderate amounts detected in liver and body carcass (Hollis et al., 2001; Szebedinszky et al., 2001). Subsequent increase in MT protein levels following Cd exposure was confirmed along with deleterious impacts on reproductive success (Brown et al., 1994; Chowdhury et al., 2005; Hollis et al., 2001). Environmental exposure to Zn can affect a number of aquatic species with vertebrates generally displaying greater sensitivity compared with invertebrates (Brinkman and Johnston, 2012). Long-term exposure of rainbow trout to Zn results in accumulation of the metal primarily in gill tissue and carcass (skin, muscle, and bone) (Sappal et al., 2009). The reduced water hardness (higher CaCO3 concentration) at Christal Creek compared to Moose Creek is predicted to enhance Zn toxicity (Brinkman and Johnston, 2012). However, the environmental concentration of Zn at Christal Creek was approximately 4-fold lower than levels found to contribute to mortality in cutthroat trout (Oncorhynchus clarkii) under lower water hardness (as CaCO3) conditions (Brinkman and Johnston, 2012). Rainbow trout exposed to Zn display accumulated body burdens that appear to remain bioavailable with minimal sequestration (Sappal et al., 2009) suggesting that the higher levels of mt mRNA detected in Arctic grayling at Christal Creek may primarily serve to accommodate the increased exposure to Cd. This is supported by the relative body burdens measured in the current study (Table 2) where Cd levels were 5-fold higher in Christal Creek compared to Moose Creek fish and 5-fold higher than the TSC (Table 2). Zn body burdens were more closely matched and hovered around the TSC (Table 2). Although below the Canadian

Environmental Quality Guidelines (CEQG) at both sites, the body burdens of Pb suggest an accumulation of this heavy metal in Christal Creek fish above those in which adverse ecological effects may occur (Table 2). Compared to Cd and Zn, Pb is not as effective at eliciting an inductive response of mt in rainbow trout hepatocytes (Risso-de Faverney et al., 2000). Such differential metal effects remain to be tested in Arctic grayling. Protection against metal-associated oxidative damage through increased cat expression is likely a response to the increased presence of the three metals at Christal Creek (Hansen et al., 2006, 2007). The measured levels of total Zn and Cd in Christal Creek in 2012 are twice and seven times, respectively, the CEQG for freshwater long-term exposure of aquatic life set by the Canadian Council of Ministers of the Environment (CCME; http://st-ts.ccme.ca/). Interestingly, the Moose Creek reference location demonstrated 4.5fold higher total iron content compared with Christal Creek which is more than twice the recommended CEQG long-term exposure value. However, the body burden and hepatic mRNA profiles observed for Arctic grayling suggest that the animals at Moose Creek display lower induction of metal defense pathways and stress response compared to their counterparts in Christal Creek despite the higher iron content (see Fig. 3). We were interested to determine if the significantly altered mRNA abundance profiles detected in the liver of animals from Christal Creek could also be observed in an external tissue amenable to nonlethal sampling methods. Fish epidermis represents an adaptive tissue responsive to changes in environmental conditions and rainbow trout epidermal cells have demonstrated altered cellular functions following exposure to Zn (Ní Shúilleabháin et al., 2006). Further support includes changes in mRNA status in Atlantic salmon (Salmo salar) skin observed using qPCR methods following exposure to the estrogenic environmental contaminant nonylphenol (Arukwe and Røe, 2008) and we have recently demonstrated that the caudal fin can act as a viable indicator tissue upon exposure to metal or estrogen in laboratory-exposed rainbow trout (Veldhoen et al., 2013). The present data supports the use of caudal fin in qPCRbased assessment of exposure to an aquatic environment containing increased heavy metal content. This non-lethal collection of molecular biology endpoints from the fin can potentially be combined with similar conservation-based methods for monitoring chemical contaminant uptake in fish muscle (Ouyang et al., 2011; Togunde et al., 2012) providing a wider scope of metrics with minimal environmental impact. Further application of the Arctic grayling qPCR assay at multiple sites across the Canadian Arctic associated with active and legacy mining will help to provide a better understanding of the

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landscape of biological impact that human resource development has on the fragile northern ecosystem (Bowman et al., 2010; Braune et al., 1999). In addition, investigation of environmental variables that may modulate toxicity of heavy metals in freshwater fish species, such as water chemistry, presence of organic matter, and climate change, will benefit from the developed qPCR assay (Hansen et al., 2002; Nimick et al., 2007; Schwartz et al., 2004).

Acknowledgments This work was supported by Yukon Environment, Water Resources Branch, with a grant from the Mining & Petroleum Environment Research Group Fund.

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