metal contamination 
 of freshwater systems occurs worldwide , in 
 some cases reaching concentrations known to cause acute toxicity , 
 yet a few of these rivers and lakes support viable populations of 
 fish .
yellow perch populations inhabiting lakes in north america contaminated 
 through industrial and mining activity with a number of metals ( particularly 
 copper , cadmium and nickel ) are an exceptionally well studied example . 

gradients in contamination have been used to demonstrate correlations 
 between chronic metal exposure and a number of physiological changes 
 associated with metal toxicity and/or tolerance , including alterations 
 in metabolic processes , the antioxidant system and metal transporting / sequestering 
 pathways [ e.g. , refs ( 17 ) ] .
additionally , these metal exposures have resulted in impaired 
 growth , reproduction and genetic diversity , with potentially adverse 
 implications for the health of these populations .
brown trout ( salmo trutta ) inhabiting the river 
 hayle in cornwall ( southwest england ) are another population of fish 
 chronically exposed to elevated metal concentrations .
historically , 
 mining in the surrounding area dates back to neolithic times , peaking 
 during the 1800s and drainage from the disused mines continues to 
 contaminate the river with a mixture of metals .
the 
 middle region of the river hayle has extremely high metal concentrations , 
 where little fish or invertebrate life is found , however this does 
 not prevent brown trout migration and gene flow between the upper 
 and lower sections .
concentrations of 
 metals in the lower region , where brown trout are readily found , have 
 been documented to cause acute toxicity in metal - naive brown trout 
 [ e.g. , refs ( 1013 ) ] , including total zinc , copper 
 and iron which averaged 639 , 42 and 200 g / l respectively ( data 
 kindly provided from the environment agency , supporting 
 information table s1 ) . despite the persistent and significant 
 levels of metal contamination , the river hayle appears to support 
 a sustainable population of brown trout that exhibits no evidence 
 of reduced genetic diversity .
therefore , 
 it is expected that the brown trout population in the river hayle 
 may exhibit mechanisms allowing them to tolerate chronic metal exposure . 

populations of brown trout inhabiting water systems contaminated with 
 metals are not unique to the river hayle and other examples include 
 populations found in norway and the usa . despite this ,
very little is known about the physiological and molecular 
 adaptations that allow this species to survive high concentrations 
 of metals in their environment .
we aimed to address this knowledge 
 gap , using brown trout from 
 the river hayle as a case study .
we adopted an integrative approach 
 combining genomics with analysis of tissue metal accumulation , to 
 understand the molecular mechanisms employed by fish from this population 
 to tolerate the high concentrations of metals in their environment . 

there is relatively limited gene sequence information for brown trout , 
 therefore our first goal was to sequence , assemble and annotate the 
 transcriptome for this species , using the illumina sequencing platform 
 to perform rna - seq .
we then used this resource to investigate molecular 
 pathways differentially regulated in fish from the river hayle , compared 
 to a metal naive brown trout population originating from a relatively 
 un - impacted river within the same geographical region .
our data demonstrated 
 the very significant metal accumulation in tissues of fish originating 
 from the river hayle and proposes a number of molecular mechanisms 
 employed by this species to cope with the high concentrations of metals 
 in their environment , including the regulation of metal and ion homeostasis 
 pathways and activation of antioxidant systems .
to obtain a comprehensive sequence 
 data set for assembly of the brown trout transcriptome , we collected 
 five embryos at ten different stages of development and a range of 
 tissues from adult fish . in a second phase , to investigate the mechanisms 
 of tolerance to metals in the brown trout population from the river 
 hayle , five adult fish from this river and ten from a control river 
 ( river teign ) were sampled for analysis of tissue metal content and 
 for transcriptomic analysis .
a full description of the samples collected 
 is presented in supporting information ( table 
 s2 ) .
metal concentrations in the river hayle 
 and teign were kindly provided by the environment agency and are presented 
 in supporting information ( table s1 ) .
portions 
 of gill , gut ( stomach and intestine ) , kidney and liver were dissected 
 from fish obtained from the river hayle and river teign for determination 
 of the metal content in these tissues .
samples were dried to a constant 
 weight and digested with 1 ml of concentrated hno3 ( for 
 trace metal analysis , fisher chemicals ) for 24 h at 60 c and 
 treated with 60 l h2o2 .
the samples were 
 diluted with 9 ml of milli - q water and analyzed for cu , pb , zn , as , 
 cd , fe and ni by inductively coupled plasma mass spectrometry ( icp - ms ; 
 e : an 6100drc , perkin - elmer , cambridge , u.k . ) .
cluster analysis was 
 performed on the metal concentrations in individual fish tissues from 
 the river hayle ( h1h5 ) and river teign ( t1t10 ) using 
 euclidean distance measure and heatmaps were produced using the pheatmap 
 package in r / bioconductor .
associations 
 between individual fish length / weight and tissue metal concentrations 
 were tested using regression analysis .
total rna was extracted from 
 all individual embryos and adult tissues 
 using tri reagent ( sigma - aldrich ) , according to the manufacturer s 
 instructions .
the concentration and quality of rna in each sample 
 was determined using a nanodrop nd-1000 spectrophotometer ( nanodrop technologies , u.s.a . ) and an agilent 2100 bioanalyzer ( agilent technologies , inc .
equal amounts of rna 
 from individual samples were pooled to obtain the samples described 
 in supporting information table s2 .
cdna 
 libraries were prepared for each pooled sample using the illumina 
 truseq rna sample preparation kit and sequencing was conducted using 
 an illumina gaiix genome analyzer , generating 100 bp paired end reads 
 for the embryonic library and 76 bp paired end reads for 12 
 multiplexed adult libraries .
raw sequences were processed to remove illumina adapter sequences 
 and filter out sequences that did not meet the quality thresholds . 

all paired reads 
 of the adult tissue and embryo libraries were pooled and assembled de novo using velvet ( version 1.2.08 ; ref ( 17 ) ) and oases ( version 0.2.08 ; 
 ref ( 18 ) ) using a range 
 of k - mers ( see supporting information for full details ) .
the resulting transcripts were annotated using 
 blastn and blastx and a selection of fish and mammalian nucleotide 
 and protein databases and using an e - value cut off 
 < 1  10 .
gene expression was determined 
 in the gill , gut , kidney and liver of fish inhabiting the metal - contaminated 
 river hayle and the reference river teign using rsem .
reads were mapped against the brown trout reference transcriptome 
 using the  --no_polya  parameter and using default settings . 

subsequent analyses in rsem were conducted using a selection of scripts 
 provided as part of the trinity assembly package ( version r20121005;. statistical differences in gene expression levels 
 between tissues of the two rivers were calculated using edger .
genes were considered differentially expressed 
 when fdr < 0.1 ( benjamini  hochberg correction ) .
a 4-way 
 venn diagram showing overlapping differentially expressed genes was 
 produced using venndiagram in r / bioconductor . 

all analyses were carried out on a local server running under the 
 nebc bio - linux 7 environment ( 23 ) unless 
 stated otherwise . a flow diagram describing the transcriptome assembly 
 and gene expression analysis
is presented in figure 1 and a full description of the methodology is presented in 
 the supporting information .
flow diagram illustrating 
 the workflow employed for sequencing , 
 assembling and annotating the brown trout transcriptome and for determining 
 changes in gene expression profiling between brown trout populations . 

the sequence data have been deposited 
 in ncbi s gene expression 
 omnibus and are accessible through geo series accession number gse45637 .
functional analysis was then 
 performed for differentially expressed genes from each tissue using 
 the database for annotation , visualization and integrated discovery 
 ( david version 6.7 ; ( 24 ) ) , using the brown 
 trout transcriptome as background list .
gene ontology ( go ) fat terms 
 for biological process , cellular component and molecular function 
 were considered significantly over - represented when p < 0.05 . in order to validate the quantitative analysis 
 of differential gene expression between hayle and teign trout ,
a selection 
 of four transcripts ( mtb , gpx1b , cat , slc40a1 ) were 
 analyzed via real time quantitative pcr ( rt - qpcr ) on gill , gut , kidney 
 and liver samples from all individual fish , according to previously 
 described methods .
these transcripts encode proteins 
 involved in metal homeostasis and oxidative stress response and are 
 therefore potentially differentially regulated by metal exposure . 

they include transcripts that were found to be both differentially 
 expressed and not differentially expressed in the rna - seq data , to 
 corroborate both of these scenarios .
transcript expression levels 
 were normalized using the control gene actin - related protein 2/3 complex 
 3 ( arpc3 ) which was selected from the rna - seq data 
 set based on its consistent expression between hayle and teign fish 
 in all tissues ( supporting information table 
 s3a ) .
in the gills , liver and kidney 
 the concentration of all seven metals measured ( cu , pb , zn , as , cd , 
 fe , ni ) was significantly higher in the hayle trout than the teign 
 trout . across all metals
the fold change was highest in the gill ( mean 
 62.6-fold ) followed by the liver ( mean 33.7-fold ) , then the kidney 
 ( mean 18.5-fold ) .
in contrast , in the gut there was no significant 
 difference in the concentration of any of the metals measured ( figure 2 ) .
the considerable increase in hayle gill metal 
 concentration contrasts sharply with the lack of difference in the 
 gut and suggests that the gills are the principal route of metal uptake 
 in these fish .
this is because of their large surface area in direct 
 contact with water and abundance of metal specific carriers ( e.g. , 
 for essential metals copper , zinc and iron ) , as well as other ion / metal 
 transporters that allow uptake of a number of metals through ionic 
 mimicry ( e.g. , cu via na uptake routes and 
 zn and cd via ca uptake routes ) .
concentration 
 of six metals measured by icp - ms , in the gill , gut , 
 kidney and liver of fish from the rivers hayle and teign .
blue bars represent data from fish originating 
 from the metal contaminated river hayle ( n = 5 ) and 
 green bars represent data from fish originating from the relatively 
 unimpacted river teign ( n = 10 ) .
asterisks indicate 
 significant differences in concentration of each metal between fish 
 from each population , * p < 0.05 , * * p < 0.01 , * * * p < 0.001 .
after uptake , metals are transported in the bloodstream throughout 
 the body . the considerable accumulation and greatest total concentration , 
 of metals in the kidney and liver reflects the essential role of these 
 tissues in metal processing , detoxification , storage and excretion . 
 in both hayle and teign fish , zinc was the most abundant metal in 
 the gill , gut and kidney , while copper was found at the highest concentration 
 in the liver .
copper and zinc were also the metals that increased 
 to the greatest extent , in terms of absolute concentration , in the 
 gills , liver and kidney .
corresponding with this , water concentrations 
 of zinc and copper were elevated to the greatest extent of all metals 
 in the river hayle compared to the teign , by approximately 60- and 
 40-fold , respectively ( supporting information table s1 ) .
the tissue distribution and accumulation patterns of 
 iron , cadmium and arsenic reveal some striking correlations between 
 these three metals and this is supported by cluster analysis on individual 
 fish ( supporting information figure s1 ) . 

this strongly suggests that the uptake , storage and metabolism of 
 these metals , in particular , are linked .
no significant correlation 
 was found between fish length or weight and metal concentration in 
 any tissue , suggesting the difference in size / age of the sampled populations 
 is unlikely to have influenced the metal accumulation patterns .
the levels of metal accumulation in the tissues of brown trout 
 from the river hayle were considerably higher than that measured in 
 other chronically exposed fish from metal - contaminated regions .
for 
 example , hayle trout had accumulated 156 , 1800 and 18 g / g copper 
 and 929 , 229 and 1020 g / g zinc in the kidney , liver and gills 
 respectively , compared with 4.88 , 242 and 3.85 g / g copper and 
 186 , 47.9 and 73.7 g / g zinc in the same tissues of brown trout 
 in copper and zinc rich norwegian rivers .
highest recorded values of 256.6 g / g copper and 157 g / g 
 zinc in liver of yellow perch from metal contaminated canadian lakes ( 30 ) are also far lower than the concentrations of 
 these metals in the liver of the hayle brown trout ( 1800 and 229 g / g 
 for copper and zinc , respectively ) .
this highlights both the extent 
 of metal contamination in the river hayle and the high degree of metal 
 tolerance of its resident brown trout population .
sequencing 
 generated 68.8 m 100 bp reads from the embryonic library and a total 
 of 78.7 m 76 bp reads from the multiplexed libraries ( ranging from 
 5.1 to 7.7 m reads per library ) .
following raw sequence read processing 
 and quality filtering a total of 60.1 m ( 9.7% orphans ) embryonic reads 
 and 66.5 m ( 8.7% orphans ) multiplexed reads were retained and input 
 into the transcriptome assemblies .
the de novo assembly 
 consisted of 202,994 transcripts ( 136,848 loci ) , with an average length 
 of 821 bp and an n50 of 1853 bp , 48% of which were annotated by blast 
 ( e - value < 1  10 ) ( figure 1 ) .
the final transcriptome assembly provides a high 
 quality template for global gene expression profiling in this study 
 and also provides a valuable tool for wider research on brown trout , 
 which is an ecologically and economically important fish species with 
 limited existing genomic resources .
transcript profile analysis 
 revealed that 73  881 transcripts were expressed in at least 
 one of the eight hayle and teign libraries tested .
the total number 
 of genes present and their expression level distribution were generally 
 consistent between libraries , although some tissue - specific differences 
 were evident .
for example , liver and gut tissues expressed a greater 
 proportion of rare genes and fewer genes in total , than gill and kidney 
 ( supporting information table s5 ) .
the 
 gene expression patterns for the four selected tissues for the hayle 
 and teign fish were examined to identify potential mechanisms of toxicity 
 and/or tolerance to the metal exposure in the river hayle . a total 
 of 998 transcripts
the greatest number of differentially 
 expressed genes ( 792 ) occurred in the kidney .
perhaps surprisingly , 
 given its role in metal uptake , substantial metal accumulation and 
 known susceptibility to acute metal toxicity , fewest genes ( 183 ) were 
 differentially expressed in the gill .
in contrast , despite no increase 
 in metal accumulation a considerable number of genes ( 288 ) were differentially 
 expressed in the gut , but a large proportion of these genes are linked 
 to digestion and likely to be related to dietary differences between 
 the sites ( see below ) .
rt - qpcr analysis was in full agreement with 
 the rna - seq transcriptional profiling data , confirming the reliability 
 of the quantitative data obtained from sequencing analysis on pooled 
 samples ( supporting information table s6 ) . 

significantly over - represented go fat terms ( p < 
 0.05 ) , among the differentially expressed gene lists are shown in supporting information table s7 .
( a ) number of differentially 
 expressed genes between populations 
 obtained using edger with a fdr < 0.1 in each tissue .
numbers within 
 bars represent the percentage of the total that were up / down - regulated 
 in brown trout originating from the river hayle compared to the river 
 teign .
( b ) venn diagram displaying the number of differentially expressed 
 genes in each tissue and the overlay between these gene lists across 
 tissues .
fundamentally , 
 metal homeostasis 
 consists of ensuring an adequate supply of essential metals for metabolic 
 processes and controlling the level of essential and nonessential 
 free metal ions to prevent toxicity .
this involves regulating uptake 
 from the environment , distribution through the bloodstream and delivery 
 to target organs , supply to metabolic pathways , biotransformation , 
 storage and excretion .
exposure to elevated 
 metal concentrations in the river hayle and the resulting increase 
 in metal tissue accumulation , would therefore be expected to be associated 
 with changes in the activity of components involved in this homeostatic 
 system .
a number of cellular metal binding proteins serve to 
 detoxify and store metal ions through binding and removal of their 
 redox potential .
glutathione and metallothioneins ( mts ) act as buffers 
 for metal ions entering cells ; both have very high affinity for most 
 metals and glutathione is generally present at high concentrations . 

mts are cysteine - rich , thiol - containing proteins and are widely acknowledged 
 to account for a major portion of the cellular storage of zinc , copper , 
 cadmium and to a lesser extent iron , lead and nickel in fish .
mt - bound metals contribute to the metabolically 
 detoxified cellular fraction and can also be stored in metal rich 
 granules for even more stable , long - term storage . increased mt synthesis
has been extensively shown to occur 
 in response to many metals , in both short - term laboratory exposures 
 and in chronically exposed wild fish and it is the most consistent 
 and sometimes only , mechanism of metal tolerance in fish [ e.g. , refs ( 14 , 15 and 3234 ) ] . a single mt isoform is predominantly induced by metals in fish ; 
 free metal ions bind mtf1 transcription factors , 
 which then bind metal response elements ( mres ) in its promoter region , 
 stimulating transcription .
corresponding 
 with this , we found that one mt ( metallothionein b ) was among the 
 most strongly up - regulated genes in the hayle trout ( significantly 
 up - regulated by 8.2- , 7.7- and 5.6-fold in the gill , gut and liver , 
 respectively , as well as 2.2 fold in the kidney ) , indicating sequestration 
 of metals by mt represents a very important mechanism of metal tolerance 
 in this population .
another mt isoform was also present in the brown 
 trout transcriptome assembly but only expressed at very low levels .
information available in the existing literature shows that acute 
 metal exposures alter the expression of genes encoding metal - specific 
 transporting proteins in the gill , gut , kidney and liver .
these include 
 the main cellular transporters of copper ( copper transporter 1 ( slc31a1 ) , divalent metal transporter 1 ( slc11a2 ) , copper - transporting atpases ( atp7a , atp7b ) ) ; zinc 
 ( various members of the znt ( or slc30 ) and zip ( or slc39 ) families ) ; and iron ( slc11a2 , ferroportin 
 ( slc40a1 ) ) .
this suggests limiting uptake of metals from the 
 environment , to slow onset of toxicity and increasing delivery to 
 organs involved in metal metabolism , storage and excretion may be 
 a mechanism of metal tolerance in short - term exposures .
less information 
 is available on response to chronic exposure , but xie and klerks found a reduced rate of cadmium uptake in a 
 tolerant laboratory population of killifish and gale et al
. showed a reduction in copper uptake rates in 
 the gill contributed to copper tolerance of rainbow fish .
in contrast , 
 other studies have found no evidence of altered metal uptake and distribution 
 kinetics during acclimation [ e.g. , ref ( 34 ) ] . for hayle trout , we hypothesized that gene 
 pathways related to metabolism of copper and zinc were the most likely 
 to be altered , given that concentrations of these metals were the 
 most elevated in both river water and tissues .
of these genes , only 
 the zinc transporter slc39a2 was differentially expressed 
 ( down - regulated in the kidney ) .
there were no apparent trends in altered 
 regulation of the other copper and zinc specific transporting proteins 
 listed above .
however , potential changes in copper and zinc transporters 
 at the protein level , for example through post - transcriptional modification 
 or changes in tissue / cellular localization , should not be ruled out . 

in contrast , there were increasing trends in expression levels of 
 iron transporters in hayle trout ; slc11a2 was up - regulated 
 2.2 and 3.5 fold in the liver and kidney respectively , while slc40a1 was up - regulated 4.3 fold in the kidney , although 
 these differences were not statistically significant .
a number of 
 genes encoding proteins involved in wider iron transport and storage 
 were differentially expressed , particularly in the kidney , liver and 
 gut .
these include transferrin , a precursor of serotransferrin , as 
 well as transferrin receptor 1b , which is responsible for cellular 
 uptake of metals from transferrin and a form of ferritin , the main 
 cellular iron - binding protein .
additionally , hemopexin , heme - binding 
 protein 2 and heme transporter , which are involved in wider iron homeostasis 
 though hemoglobin regulation , were differentially expressed . moreover , 

the main regulator of iron homeostasis , the hormone hepcidin , was 
 down - regulated in the liver .
these changes in iron - metabolism related 
 genes are particularly marked in contrast to the lack of change in 
 those specific to copper and zinc .
furthermore , these changes are 
 occurring in the absence of long - term significant elevation in the 
 concentrations of fe in the hayle , compared to the teign , river water 
 ( supporting information table s1 ) , but 
 in the presence of significant accumulation of fe in the liver , kidney 
 and gill of hayle fish ( figure 2 ) .
we hypothesize 
 that these iron - homeostasis genes may be regulated by other metals 
 present in the water , or that iron - homeostasis is a target of metal 
 toxicity
. an alternate hypothesis may be that a peak in fe in the 
 river water occurred close to the sample collection and was not recorded 
 in the water sampling conducted by the environment agency .
it is impossible 
 to ascertain if this was the case and , therefore , we can not conclusively 
 interpret the reasons for the alterations of iron related pathways 
 in the hayle fish . despite this , the striking association between 
 the concentrations of fe and several other metals ( particularly as 
 and cd ) , together with the alteration in the expression of genes involved 
 in iron - homeostasis , suggest that these iron - handling pathways play 
 an important role in the response of the hayle fish to metal exposure . 

this is supported by previous reports suggesting an association of 
 other metals , including cd , cu and pb , with binding and regulation 
 of transcription of various components of the iron homeostatic system .
less is known about arsenic distribution pathways in fish , although 
 arsenic is capable of being transported by transferrin in human plasma .
one major mechanism 
 of toxicity common 
 to a number of metals is disruption of ion homeostasis , particularly 
 in the gills .
zn , cd and pb inhibit ca uptake , through competition for ca uptake pathways and direct inhibition of ca atpase , 
 leading to hypocalcaemia .
cu reduces na uptake , 
 both competitively and by interference with na / k atpase .
copper , zinc , cadmium and lead also inhibit carbonic anhydrase , 
 reducing supply of h and hco3 ions for na and cl uptake exchange . 

lower plasma nacl levels lead to increased blood viscosity and can 
 cause circulatory collapse .
several laboratory studies have demonstrated acclimation 
 of fish to metals , including copper , cadmium and lead , following chronic 
 exposure , characterized by a restoration of plasma ionic balance and 
 physiological condition . increased synthesis of na / k atpase to restore total cellular na / k atpase activity , as well as morphological changes in the gill
although not significant , 
 in the kidney our results show a trend of up - regulation for both of 
 these atpases known to be inhibited by metals , particularly for the 
 most highly expressed ( and therefore probably functionally most important ) 
 isoforms in this tissue .
na / k atpases atp1a and atp1b1a were both up - regulated 
 by 2 fold , while ca atpase atp2b1a was 
 up - regulated by 3.3 fold .
additionally there was a significant up - regulation 
 of carbonic anhydrase in the liver .
this suggests that the up - regulation 
 of these enzymes in the hayle brown trout may be employed to counter 
 their inhibition by metals .
a number of other genes encoding proteins 
 important in maintaining ion balance were differentially expressed . 
 in the kidney na - cl cotransporter ( slc12a3 ) , which reabsorbs nacl from urine ,
there was also a general trend of up - regulation 
 in the kidney of a number of other transporters including those in 
 the slc12 family , although a low - expressed isoform slc12a9 had significantly reduced expression , as well as 
 those in the slc4 ( sodium - bicarbonate transporter ) 
 and slc9 ( nah exchanger ) 
 families .
however , slc24a6 , a nacak exchanger was significantly down - regulated 
 in both the kidney and liver .
several other genes responsible for 
 ion transport were down - regulated including chloride intracellular 
 channel related proteins in the kidney and serum / glucocorticoid regulated 
 kinase , which has a role in activating ion channels , in the liver . 

three aquaporins , which contribute to maintenance of osmotic balance , 
 were also differentially expressed in the kidney and gut .
additionally , 
 a number of genes with a role in maintaining calcium homeostasis were 
 differentially expressed , predominantly being down - regulated in the 
 kidney , gut and liver .
these include two calcium binding proteins 
 and two s100-calcium binding proteins , calmodulin and calmodulin binding 
 transcription activator , which are involved in calcium signaling and 
 three isoforms of stanniocalcin , which regulates calcium flux .
additionally , 
 two chemokine receptors which are related to calcium flux and signaling , 
 together with calcium binding proteins with specific roles in muscle 
 contraction ( calponin , calsequestrin and caldesmon ) were also down - regulated . 

overall , these results indicate an integrated response of the ion - homeostatic 
 system , which may contribute to the metal tolerance of this population 
 through compensation of metal - induced ion balance disturbance . the 
 most pronounced response appears to be in the kidney , reflecting the 
 important role of this organ in regulating plasma ion and water balance . 

however , it is surprising that so few genes related to ion homeostasis 
 were differentially expressed in the gill given that it is the main 
 target of metal - disrupted ion balance .
redox - active metal ions , including copper and iron , generate reactive 
 oxygen species ( ros ) via fenton chemistry .
several metals , including 
 copper , cadmium , arsenic , nickel and lead , can deplete and inhibit 
 the cellular antioxidants . copper and cadmium can also generate ros 
 via disruption of the electron transport chain .
oxidative stress can lead to lipid peroxidation , dna and protein 
 damage with associated adverse health effects at the cellular level . 
 to counteract this , increased cellular ros stimulate an up - regulation 
 of the cellular antioxidant defense system , comprised of reduced glutathione 
 ( gsh ) and a suite of enzymes , to limit oxidative damage .
there is 
 evidence from gene expression studies that various metals stimulate 
 a response of the antioxidant system during short - term exposures [ e.g. , 
 refs ( 11 , 36 and 43 ) ] . in chronically exposed wild fish populations there 
 is some evidence of damage caused by oxidative stress , including lipid 
 peroxidation in brown trout , but gene 
 expression profiling has found inconsistent changes of the antioxidant 
 system .
although gene expression profiling 
 revealed that one isoform of the antioxidant superoxide dismutase 
 was significantly down - regulated in all tissues , other , more highly 
 expressed isoforms showed no change in expression level .
no significant 
 difference in expression was found for any other antioxidants , although 
 in the gill , there was > 2-fold apparent up - regulation of gsh - related 
 antioxidants glutathione peroxidase ( gpx1b ) and glutathione - s - transferases 
 ( gsta5 , gsto1 , gstz1 ) . there were no differences 
 in expression level of glutathione synthetase or reductase , which 
 are essential for gsh synthesis and restoration from its oxidized 
 form , although a number of oxidoreductase enzymes , involved in supplying 
 nadph for restoring gsh , were significantly up - regulated .
we found 
 significant up - regulation of cytochrome c oxidases ( cox4i2 , 
 cox7a ) in the liver and kidney , which have been linked with 
 oxidative stress generation and response , but are also associated 
 with metal - induced metabolic changes ( see below ) .
70 kd heat shock 
 protein ( hsp70 ) , which temporarily binds and stabilizes 
 damaged proteins was also significantly up - regulated in the gill and 
 gut , while other heat shock proteins ( hsp40 , hsp27 ) were differentially expressed in the liver and gut , respectively . 

overall , this suggests a modest response of the antioxidant system 
 of hayle trout , particularly in the gill .
this is perhaps because 
 after uptake from the environment and before being bound and/or transported 
 elsewhere , the concentration of toxic free ions is likely to be higher 
 in the gills compared to other tissues , leaving it more susceptible 
 to oxidative stress .
however , the response of the antioxidant system 
 as a whole is much less pronounced than expected to occur following 
 acute metal exposures , suggesting that other mechanisms of tolerance 
 are likely to play a key role in reducing oxidative stress caused 
 by metal ions in the hayle brown trout population .
these mechanisms 
 may include increased synthesis of mts , which have a high affinity 
 for ros and are therefore powerful cellular antioxidants and may offer 
 significant protection against oxidative stress .
this mechanism is likely to be a consistent and significant 
 contributor to the metal tolerance of this brown trout population
. 
 corresponding with this , we found no evidence of changes in pathways 
 related to cellular repair mechanisms or evidence of cellular toxicity , 
 such as changes in expression of apoptotic gene markers . a large proportion of the genes 
 differentially expressed in the gut are related to digestion .
this 
 is particularly obvious from the go term analysis ; various peptidases dominate molecular function over - representation 
 and proteolysis is the only over - represented biological 
 process in this organ .
protein metabolism , including proteolysis specifically , 
 has been shown to be altered in response to both short - term and chronic 
 metal exposures .
furthermore , it has been suggested that this facilitates 
 enhanced protein turnover which is important in the replacement of 
 damaged proteins and proteins involved in metal detoxification , repair 
 and storage .
however , in the present 
 study differentially expressed genes related to proteolysis were predominantly 
 proteases , carboxypeptidases , chymotrypsins and elastases , which are 
 more specifically associated with digestion and almost exclusively 
 occurred in the gut .
dietary differences between the hayle 
 and teign fish may be influenced by many environmental variables , 
 although may not be entirely unrelated to metal contamination because 
 metal exposure is well - known to alter food webs through changes in 
 river species assemblage [ e.g. , ref ( 55 ) ]
. the mechanisms of metal tolerance employed 
 by the hayle trout are likely to result in increased energetic demand . 

metal exposure has induced metabolic changes that have been suggested 
 to be compensatory , in order to facilitate metal tolerance and detoxification . 

for example , chronically exposed wild yellow perch show some evidence 
 of enhanced aerobic respiration , potentially increasing atp production .
conversely , metal exposure has also been associated with down - regulation 
 of various metabolic processes , due to the energetic demands of metal 
 detoxification and/or through impairment of metabolic enzymes . in 

chronically exposed wild yellow perch there is evidence of lower aerobic 
 capacity ( 3 ) and down - regulation of various 
 components of lipid synthesis and transport , which have been associated 
 with depletion of lipid reserves leading to adverse impacts on fish 
 health and condition . in the hayle trout
, there 
 was some evidence of changes in aerobic 
 respiration , in particular down - regulation of genes with a role in 
 oxidative phosphorylation including down - regulation in all tissues 
 of several nadh dehydrogenases ( mt - nd1 , mt - nd1b , mt - nd4 , mt - nd5 ) and up - regulation of cytochrome c oxidases ( cox4i2 , cox7a ) in the liver and kidney respectively .
with regard to other metabolic 
 processes , in particular there were changes in expression of a number 
 of genes involved in lipid , fatty acid and steroid synthesis and transport , 
 mainly in the liver and kidney .
these include peroxisome proliferator - activated 
 receptor alpha , low - density lipoprotein receptor , lysophosphatidylglycerol 
 acyltransferase , cyp46 , stearoyl - coa desaturase and five apolipoproteins . 

in contrast to previous studies , these genes were almost exclusively 
 up - regulated .
a likely explanation for the up - regulation of lipid 
 metabolism in the hayle trout is that the sample population of five 
 fish consisted of three reproductively mature females and two immature 
 fish , while the teign population sample was dominated by reproductively 
 immature fish with no maturing females .
lipid metabolic pathways are 
 essential for the synthesis of the egg yolk precursor protein vitellogenin 
 and other egg shell proteins , in the liver of females as they undergo 
 gonadal development and maturation .
the energetic costs of reproductive 
 maturation are also likely to influence other metabolic processes 
 ( i.e. , energy generation through aerobic respiration and diversion 
 from proteolysis ) , therefore the differences in metabolic processes 
 observed can by no means be exclusively attributed to metal exposure . 

consistent with this , genes associated with reproductive development 
 in females were strongly up - regulated , including of genes encoding 
 vitellogenin and egg outer membrane zona pellucida proteins in the 
 liver of hayle fish .
impairment of immune 
 function has been 
 extensively demonstrated in previous studies to be associated with 
 exposure to metals .
this effect has been attributed , in part , to disruption 
 of energy budget as well as to a direct inhibition of immune function 
 and commonly results in increased susceptibility to bacterial and 
 viral infection .
gene expression analysis revealed a predominant down - regulation 
 of a number of components of the complement system in the liver , gut 
 and kidney of hayle trout .
these include complement components 1qa , 
 1qb , 1q2l , 3 , 4 and 8 ; complement factors h , h1 and d and fanconi 
 anemia , complementation group c. this suggests an impairment of the 
 immune system and is consistent with the findings of pierron et al . and reynders et al . who found inhibited expression of genes in the immune system in 
 fish exposed to metals , especially those involved in the complement 
 system .
we have sequenced , assembled and annotated a transcriptome 
 for the brown trout , providing a useful resource for further research 
 in this species . using this information
, we investigated the molecular 
 mechanisms of tolerance to metals in a brown trout population exposed 
 to significantly elevated concentrations of multiple metals in the 
 river hayle , u.k . and anchored our data to metal accumulation in tissues 
 and river water concentrations .
tissue metal accumulation patterns 
 indicate the gill represents the major route of metal uptake in hayle 
 fish , while the accumulation in the kidney and liver reflects their 
 important role in metal storage and detoxification .
the considerable 
 tissue metal accumulation observed in the absence of overt toxicity 
 confirms the metal tolerance of hayle brown trout . global gene expression 
 profiling revealed that the two broad strategies likely to contribute 
 to the metal tolerance of this population are the regulation of metal 
 homeostasis pathways and ion homeostasis pathways , with up - regulation 
 of the antioxidant system playing a relatively minor role . within 
 these
these include 
 increased synthesis of metallothionein , the prominence of the kidney 
 in regulating ion balance and the putative role of iron - handling pathways 
 in wider metal homeostasis .
although our data set highlighted some 
 potential mechanisms of metal toxicity , particularly inhibition of 
 the immune system , there is little to suggest that the brown trout 
 inhabiting the river hayle are incurring adverse health effects as 
 a result of the presence of toxic concentrations of metals in their 
 environment .
this contrasts with more extensive evidence of metal 
 toxicity from studies on yellow perch , potentially causing adverse 
 impact at both the individual and population levels .
a possible explanation 
 for this is that metal contamination in the river hayle has been present 
 for a greater length of time , perhaps leading to a greater degree 
 of metal tolerance in this population .
whether this is a result of 
 an inherent genetic plasticity , allowing individual acclimation , or 
 local population adaptation , leading to inherited metal - tolerance , 
 is unclear and would require further research to be elucidated .
 worldwide , 
 a number of viable populations of fish are found in 
 environments heavily contaminated with metals , including brown trout 
 ( salmo trutta ) inhabiting the river hayle in south - west 
 of england . 
 this population is chronically exposed to a water - borne 
 mixture of metals , including copper and zinc , at concentrations lethal 
 to nave fish . 
 we aimed to investigate the molecular mechanisms 
 employed by the river hayle brown trout to tolerate high metal concentrations . 
 to achieve this , we combined tissue metal analysis with whole - transcriptome 
 profiling using rna - seq on an illumina platform . 
 metal concentrations 
 in the hayle trout , compared to fish from a relatively unimpacted 
 river , were significantly increased in the gills , liver and kidney 
 ( 63- , 34- and 19-fold respectively ) , but not the gut . 
 this confirms 
 that these fish can tolerate considerable metal accumulation , highlighting 
 the importance of these tissues in metal uptake ( gill ) , storage and 
 detoxification ( liver , kidney ) . 
 we sequenced , assembled and annotated 
 the brown trout transcriptome using a de novo approach . 
 
 subsequent gene expression analysis identified 998 differentially 
 expressed transcripts and functional analysis revealed that metal- 
 and ion - homeostasis pathways are likely to be the most important mechanisms 
 contributing to the metal tolerance exhibited by this population . 
Introduction
Materials and Methods
Results and Discussion