Spatial Patterns in Markers of Contaminant Exposure, Glucose And

CBD-00356; No of Pages 13 Comparative Biochemistry and Physiology, Part D 14 (2015) xxx–xxx

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Comparative Biochemistry and Physiology, Part D

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Spatial patterns in markers of contaminant exposure, glucose and glycogen metabolism, and immunological response in juvenile winter ﬂounder (Pseudoplueronectes americanus)

A.E. McElroy a,⁎,1,L.A.Hicea,2, M.G. Frisk a,S.L.Purcellb, N.C. Phillips b,M.D.Fasta,b,1 a School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY, USA b Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada article info abstract

Article history: Inshore winter flounder (Pseudoplueronectes americanus) populations in NY, USA have reached record low num- Received 25 October 2014 bers in recent years, and recruitment into the fishery appears to be limited by survival of post-settlement juvenile Received in revised form 16 January 2015 fish. In order to identify cellular pathways associated with site-specific variation in condition and mortality, we Accepted 26 January 2015 examined differential mRNA expression in juvenile winter flounder collected from six different bays across a gra- Available online 4 February 2015 dient in human population density and sewage inputs. Illumina sequencing of pooled samples of flounder from

Keywords: contrasting degraded sites and less impacted sites was used to guide our choice of targets for qPCR analysis. 253 N mRNA expression transcripts of 100 bp were differentially expressed, with 60% showing strong homology to mostly teleost se- Illuminia quences within the NCBI database. Based on these data, transcripts representing nine genes of interest associated RNAseq with contaminant exposure, immune response and glucose and glycogen metabolism were examined by qPCR in Fish individual flounder from each site. Statistically significant site-specific differences were observed in expression of Long Island, NY all but one gene, although patterns in expression were complex with only one (vitellogenin), demonstrating a west to east gradient consistent with known loadings of municipal sewage effluent. Principal components analysis (PCA) identified relationships among the genes evaluated. Our data indicate that juvenile winter flounder are responding to estrogenic chemicals in more urbanized coastal bays, and suggests potential mechanistic links between immune response, contaminant exposure and energy metabolism. © 2015 Elsevier Inc. All rights reserved.

1. Introduction observed in similar environments from adjacent states including NJ, CT, and RI (Yencho et al., in review). Earlier studies on winter floun- Winter flounder (Pseudoplueronectes americanus) once supported a der from urban bays of Long Island Sound have shown winter flounder dominant commercial and recreational fishery along the Atlantic coast to have reduced fitness in areas with high contaminant loadings of North America from the Gulf of St. Lawrence to Chesapeake Bay. (Nelson et al., 1991; Perry et al., 1991; Black et al., 1988), and more re- Since peaking in the mid-1980s, catches have declined, particularly in cent work has indicated YOY winter flounder and other resident species NY waters (http://www.st.nmfs.noaa.gov/web; Sagarese et al., 2011), from western Long Island bays show evidence of feminization (Mena local stocks are at record lows in abundance (Socrates and Colvin, et al., 2006; McElroy et al., 2006; Duffy et al., 2009). 2006; CTDEP, 2011; Nuttall et al., 2011), and recent data indicates an ex- Winter flounder make a good sentinel species for examining the im- tremely small parental stock contributing to inshore populations pact of environmental change as they tend to have a limited range, with (O'Leary et al., 2013). Likely driving these declines are decades of inshore populations of adults undergoing a seasonal on/off shore migra- overfishing, environmental change, and declining habitat condition. tion to avoid excessively hot summer temperatures, although some Recruitment into the NY fishery appears to be limited by survival of bays may also support resident fish with even more limited ranges post-settlement juveniles (Socrates and Colvin, 2006; Yencho et al., in (Poole, 1966; Sagarese et al., 2011). Spawning of inshore stocks primar- review). Indeed estimates of young-of-the-year (YOY) mortality in ily occurs in winter and early spring with most migrating fish returning two Long Island bays in 2007 and 2008 indicated higher levels than to their natal estuaries to spawn demersal eggs. Juvenile fish are thought to remain in shallow waters for their first couple of years of life (Klein-MacPhee, 2002). Because of their life history, winter flounder ⁎ Corresponding author at: School of Marine & Atmospheric Sciences, Stony Brook embryos and young fish are constantly exposed to sediment associated University, Stony Brook, NY 11794-5000, USA. Tel.: +1 631 632 8488. contaminants, as well as being subject to the highly variable environ- E-mail address: [email protected] (A.E. McElroy). 1 These authors contributed equally to the study. mental conditions of the near shore benthos where dissolved oxygen, 2 Current address: Delaware National Estuarine Research Reserve, Dover, DE, USA. pH, salinity, and temperature vary on daily to decadal time scales.

In this study we used qPCR to evaluate relative gene expression in on levels of common chemical contaminants in sediments. The U.S. En- YOY winter flounder collected from bays representing an urban gradi- vironmental Protection Agency's National Coastal Assessment (http:// ent along Long Island, NY. We hypothesized that environmental factors www.epa.gov/emap/nca/html/data/index.html) provides the most that might be evident along this gradient (such as contaminant expo- comprehensive dataset available for common organic and inorganic sure, temperature, salinity and dissolved oxygen), would cause site- non-nutrient contaminants. A summary of these data evaluating con- specific shifts in metabolic and immunological status of winter flounder. taminant levels in fine (N2% total organic carbon and N10,000 μg/g Fe) This study was part of a larger investigation evaluating a suite of biolog- sediments from the study sites or nearby areas measured between ical, environmental and anthropogenic factors associated with recruit- 2000 and 2005 (the most recent data available) verifies the general ment success in YOY winter flounder populations in south shore bays west to east gradient in contaminant loadings (Supplementary File 1). of Long Island (Frisk et al., 2013). Here we report results of Illumina se- These data not only identify Jamaica Bay as being the most contaminat- quencing on pooled liver samples from more and less degraded sites ed, but also indicate that fish from all sites are likely to experience some and on the site-specific expression of nine genes related to contaminant level of chemical contaminant exposure. It is important to point out that exposure, energy metabolism and immune response in the livers from by only looking at fine sediments, these data represent the high end of individual flounder. general chemical contamination at these sites. Levels of contaminants in sandy sediments would be much lower, and sandy sediments are common, particularly in the more eastern sites. Even considering the 2. Materials and methods worst case scenario of exposure to fine sediments, average contaminant levels generally only exceed Effects Range Low (ERL) values designated 2.1. Site selection by the National Oceanic and Atmospheric Administration (NOAA) at some sites, and the Effects Range Median (ERM) levels are not exceeded Juvenile winter flounder were collected from six bays along the for any contaminants (Long et al., 1995), indicating that most of these south shore of Long Island, NY, USA from May through October during sites are representative of the more wide-spread contamination found 2010 and 2011 (Fig. 1). These study locations represent a west to east in all but remote coastal areas both in the U.S. and world-wide. gradient in urbanization and sewage inputs within a very short geo- graphical range of only 200 km. Based on locally reported census data, 2.2. Fish collection and processing population density differs by a factor of 100 from west to east over the 2 breadth of the study sites, ranging from over 2000/km in the west to Flounder ranging in size from 0.2 to 36 g and 24 to 135 mm total 2 20/km in the east (www.Census.com). An even larger gradient exists length were collected using either a 1 m beam trawl or with 3–60 m 5 in sewage inputs. Jamaica Bay receives about 8 × 10 L of treated efflu- beach seines from May through October in 2010 and 2011. Attempts 2 ent per day, while Hempstead Bay receives only 2 × 10 Lofeffluent per were made to collect fish every other week from each site during the day (IEC, 2009). Moriches Bay receives no municipally treated wastewa- sampling season. All sampling was done during daylight, generally be- ter, although its primary tributary, the Forge River, is known to be signif- tween 9 am and 2 pm. We attempted to collect 10 fish per location icantly impacted by septage leaching in from failing septic systems, and per sampling day, but low flounder abundance at individual sites often this area was historically impacted by excess nutrient loading from duck prevented this. Flounder were collected at three of the sites (Jamaica, farms (Swanson et al., 2010). The three more eastern sites in this study, Moriches, and Shinnecock Bays) in both 2010 and 2011, while Cold Shinnecock Bay, Cold Spring Pond, and Napeague Harbor have both low Spring Pond and Napeague Harbor were only sampled in 2010, and population densities and no reported evidence of impacts from sewage Hempstead Bay was only sampled in 2011. A total of 221 flounder or septage inputs. Very little monitoring data exist in most of these areas were used in these analyses with at least 19 individuals from each location evaluated. Detailed information on the dates of collection and number of fish analyzed from each location is shown in Supplementary File 2. Due to low abundance and high mortality at many of the sites, very few flounder could be caught after mid-August, making analysis of expression patterns as a function of fish age or size problematic. Analysis of year-to-year differences in mRNA expression at the three sites where data were available for both years (Jamaica, Moriches and Shinnecock Bays) indicated no consistent statistical differences between years nor between early and late season fish (data not shown), so all data from both years were pooled for site-specific analysis at these sites. All winter flounder collected were flash frozen immediately upon retrieval from the water between two blocks of dry ice, and main- tained in a −80 °C freezer until processed. Temperature, salinity, and dissolved oxygen were recorded at the time of collection using a YSI Model 85 probe (Yellow Springs, AK). During the 2011 sampling season, continuously recording Hach Hydromet data sondes were moored 0.5 mm off the bottom during June, July and August, providing a contin- uous record of dissolved oxygen, temperature and salinity in Jamaica, Moriches and Shinnecock Bays. Fish collection and processing was conducted in accordance with permits issued to M. Frisk by the New York State Department of Environmental Conservation (#1030 and 1644), and by Stony Brook University's Institutional Animal Care and Use Com- mittee to A. McElroy (IRBNet #260837).

2.3. RNA isolation, RNA-sequencing and differential analysis

ﬁ Fig. 1. Map of northeast Atlantic coast of North America showing study sites on Long Livers were removed from sh while still frozen and RNA extracted Island, NY. from b130 mg of tissue after homogenization in TRIzol® reagent

(Invitrogen, Carlsbad, CA) following methods supplied by the manufac- sequencing through Macrogen USA (Rockville, MD). Sequences were turer. RNA pellets were dissolved in at least 100 μL of molecular biology Blastn searched against NCBI and the winter flounder transcriptome da- grade water before storage at −80 °C. Total RNA concentration was tabase (generated above) to confirm appropriate product formation. measured using a Thermo NanoDrop-2000 spectrophotometer (Wil- Quantitative PCR was carried out using 12 μL reactions (5 μLof2× mington, DE). Five micrograms of the extracted RNA was then DNase- Promega GoTaq® qPCR MasterMix, 0.5 μLeachof10μMforward/ treated using an Ambion TURBO DNase-freeTM kit (Grand Island, NY) reverse primers, 4 μL of promega nuclease-free water and 2 μL diluted following the manufacturer's protocol. Randomly chosen samples cDNA) on a BioRad CFX Connect System (Mississauga, ON) using a 2- (10%) were then used for RNA quality testing with the BioRad Experion step protocol with a melt curve (95 °C for 2 min followed by 40 cycles system (Mississauga, ON). The RNA Quality Indicator (RQI) of the sam- of 95 °C for 5 s and individual annealing temp for 30 s) and a melt ples ranged from 4.1 to 9.6, with all but two samples N7.0. curve from 65 to 95 °C (0.5 °C increments, 5 s/step). Reference gene sta- Pooled liver RNA samples comprised of 6 fish collected from either bility was confirmed using GeNorm software and relative gene expres- Moriches or Shinnecock Bays were sent to The Centre for Applied Geno- sion was determined using the ΔΔCT method (Livak and Schmittgen, mics (TCAG, Toronto, ON) for paired end (100 bp) Illumina HiSeq2000 2001). Due to the absence of a true reference site, data are expressed instrument analysis. These sites represented the closest locations relative only to ELF1a expression, and were not further normalized. Spe- between the more urbanized western bays and the more rural eastern cifics on names, primer sequences, annealing temperature, PCR efficien- bays analyzed. Individual indexed whole transcriptome libraries were cy and Blastn identity of the mRNA transcripts evaluated are given in constructed with a goal of obtaining N10 gigabases of sequence per Table 1. It should be noted that although some of the transcripts can library in the form of 100 bp Illumina paired-end reads. De novo only be considered as “like” products due to the absence of 100% homol- assembly of transcripts (N100 bp) was performed using SOAPdenovo- ogy to a known product (Table 2), transcripts will hereafter be referred trans (http://soap.genomics.org.cn/SOAPdenovo-Trans.html)(Li et al., to only by their common name or abbreviation in this manuscript. 2010). Reads were mapped back to the assembled transcripts using This project was undertaken to identify pathway marker gene ex- Tophat (Trapnell et al., 2009) and counts coverage for each transcript pression as a proxy for a given pathway's stimulation and subsequent determined. Reads could not be mapped against a reference teleost activity. In doing so we fully realize that there are many posttranscrip- genome due to the lack of an appropriate reference being available. tional events that can affect mRNA stability, ribosomal binding, transla- The number of reads aligning to each assembled transcript provided tion efficiency and eventual protein synthesis and function. While this count data in each case, which were then input to DESeq (Anders and approach has its limitations, and may not always directly translate to Huber, 2010). This program estimates variance–mean dependence in protein function, we considered it to be the best approach for analyzing the data and tests for differential expression based on the negative bino- the small amounts of tissue available from individual juvenile flounder mial distribution. Differential expression was tested at a significance considering the very depressed status of some of these populations. level of p b 0.05 (and fold change N 8) adjusted using the Benjamini– In this study we operationally define gene expression as the “net Hochberg procedure (i.e. to account for 5% false discovery rate). presence” of relative mRNA concentration in a tissue.

2.4. Gene ontology and KEGG analysis 2.6. Statistical analysis

Significantly regulated (up or down) transcripts across populations Preliminary analysis indicated that the data were not normally dis- were selected and blasted against the NCBI database using Blastx in tributed, therefore all expression values were log transformed prior to the BLAST2GO software. Blastx was carried out against the NCBI non- analysis. Expression patterns between sites are shown as box plots redundant (nr) database using default parameters (i.e. minimum E- where the line is the median, the upper and lower edges of the boxes value score set to 1.0E−0.6). Blast2GO was used to assign gene ontolo- show the 25th and 75th percentiles of the data, and the whiskers illus- gy terms to each annotated sequence with an annotation cut-off of 55 trate either two standard deviations from the mean, or the maximum and GO weight of 5 (Smith et al., 2013). Functional annotation of tran- and minimum values (whichever is smaller). Differences in expression scripts was also determined using the Kyoto Encyclopedia of Genes between sites were assessed by one-way ANOVAs performed with and Genomes (KEGG) database to further investigate metabolic path- Tukey's multiple comparisons. A p value of b0.05 was considered to ways affected across populations (Kanehisa et al., 2014). be statistically significant. Relationships between expression patterns of all genes were examined using principal components analysis 2.5. cDNA synthesis and qPCR (PCA). Box plots, PCA analysis and statistical tests were all performed in R (Crawley, 2012). Complementary DNA (cDNA) synthesis was performed on 1 μgof DNase-treated total RNA using a Promega Reverse Transcription System 3. Results and discussion (Madison, WI) and random hexamers, according to the manufacturer's instructions. Reverse transcriptase-free controls were included to 3.1. Environmental conditions at the flounder collection sites ensure the absence of genomic DNA. Choice of target genes for qPCR was primarily based on relative expression analysis from the RNA- Average temperature, salinity and dissolved oxygen measured at the sequencing dataset. time of fish collections over the season are shown in Fig. 2, with the av- Representatives of immune signaling and glucose and glycogen erage mean, maximum and minimum values for each site between metabolism pathways were chosen for further analysis in individual years shown in Supplementary File 3. Temperature and salinity follow- YOY winter flounder across the study sites evaluated. Two additional ed similar seasonal patterns at each site with lower temperature in the genes, vitellogenin (VTG), and cytochrome P4501A (CYP1A), although late spring and fall at all sites. Salinity showed less of a seasonal signal, not differentially expressed between these sites by RNA-seq analysis, although Jamaica Bay stands out as having a lower salinity than all were also analyzed to provide an indication of exposure to estrogenic other sites in both years sampled. The depressed salinity observed in compounds associated with sewage inputs (VTG) and a general measure Jamaica Bay, as compared to all other sites, is likely due to the large vol- of exposure to aryl hydrocarbon receptor (AhR) agonists often associat- ume of sewage effluent discharged to this bay. Sewage is the primary ed with urban run-off (CYP1A). Primer sets for the reference gene (elon- source of freshwater to Jamaica Bay (Swanson et al., 1992). Dissolved gation factor 1a — ELF1a) and genes of interest were developed using oxygen was highly variable over the season and often between sites, Primer 3 and DNAfold software. Successful amplification of each gene but average values were highest at Shinnecock Bay as compared to all was verified by gel electrophoresis, product purification and Sanger other sites (Supplementary File 3). It should be noted that Jamaica Bay

Table 1 Gene and putative gene primer sequences, annealing temperatures and efficiencies for qPCR analysis. BLAST identities are shown from the hit with the highest E-value and % identity and confirmed through sequencing PCR product and RNA-seq database for winter flounder.

Primer sequences Annealing Efﬁciency BLAST identity temperature (°C)

Elongation factor 1α F-CGCTCTGTGGAAGTTTGAGA 64 0.93 98% EF-1a R-CAGTCAGCCTGAGAGGTTCC EU561357.1 Hippoglossus hippoglossus Cytochrome P4501A F-AATCTGCAGGGTTTCCACTG 61.6 0.91 100% CYP1A R-CCAATGTGATCTGCGGTATG HQ659503 Pseudoplueronectes americanus Pleurocidin F-CCTGCTTATCGCCAAGGTAA 65.3 0.92 100% PLEUR R-CCATCTTCGTCCTCATGGTT AF301511 Pseudoplueronectes americanus Complement C3 F-CAGCGTACGATGTGAATGTGG 66 0.94 100% C3 R-TGAAATAGTGCGGGCACGTCC AY225099.1 Pseudoplueronectes americanus Hepcidin II-like F-GTCACCAGCAGAGTCAAAGAAC 64 0.95 98% HEP II R-CTCAGGAAAGGTGGCAGAAC AY623818.1 Paralichthys olivaceus Glutamate decarboxylase-like F-TCCGTAAAGACCCCAACAAG 64 0.97 83% GAD R-AACCAAGGATGCTGATCGTC JF694446.1 Monopterus albus Phospholipase A2-like F-GCATAAAGGCGGGAAAGAAG 64 1.00 85% PLA2 R-GACAGCCAAACAACCCTGAC XM_004566948.1 Maylandia zebra Glucokinase-like F-GATGTTTTGGCTGCAACTGG 65.3 1.00 86% GCK R-CACACTCACGACTGGATGATG XM_003451020.1 Oreochromis niloticus Glycerol 3 phosphate dehydrogenase-like F-AGCCGACATCCTGATCTTTG 64 0.95 91% GDPH R-ATCGATGCCCTTGATGAGAG XM_003973087.1 Takifugu rubripes Vitellogenin-like F-TGCAGGAGGTCTTCCTCAGT 65.3 0.99 96% VTG R-CCCATCAGCCTTTCCACAGA EF582607.1 Hippoglossus hippoglossus

demonstrated the greatest range between minimum and maximum dis- winter flounder juveniles: 73 over-expressed in Shinnecock Bay as solved oxygen levels of all sites, with minimum dissolved oxygen values compared to Moriches, and 180 overexpressed in Moriches as com- of b3 mg/L in both years. Data obtained from continuously recording pared to Shinnecock Bay (Supplementary File 5). Based on the non- data sondes available in 2011 at Jamaica, Moriches, and Shinnecock redundant annotation and the E-value distribution, further analysis of Bays provide a clearer picture of diel patterns in dissolved oxygen and these transcripts revealed that 73% had significant hits within NCBI indicated diminishing frequency of bottom water hypoxia going from megablast, and 83% of these showed strong homology (E-value b 10−20). west to east with the percentage of measurements below 2.3 mg/L 20). Most of the strong homologous sequences were within teleost ge- being 23, 6, and 0.6% at Jamaica Moriches and Shinnecock Bays, respec- nomes, the most common being the Nile tilapia, Oreochromis niloticus tively (data not shown). (Supplementary File 6). These were further characterized into three There unfortunately are not a lot of data available on sediment and gene ontology categories: cellular component (29% of unique tran- water quality at the collection sites used in this study. The U.S. Environ- scripts; Fig. 3A), biological process (32% of unique transcripts; Fig. 3B), mental Protection Agency's (EPA) National Coastal Assessment provides and molecular function (39% of unique transcripts; Fig. 3C). the most robust dataset with data available on sediment analyses done Another more quantitative way of looking at the differential tran- on samples collected near most of our sampling sites over the period script ontologies was used whereby the expression changes observed of 2000–2005. A clear trend from west of east is observed for most pol- in the RNA-seq data were incorporated in weighting ontologies rather lutant chemicals, with Jamaica Bay standing out as being most contam- than ‘word counts’ of descriptive data. In this case, non-redundant fold inated, although this is primarily due to high levels observed at one site change sums were created based on the cumulative fold changes within (Supplementary File 1). Despite the paucity of sediment chemistry data, a process or grouping as illustrated by Fig. 3D (Supplementary File 7). what we do have, and the physical data obtained as part of our sampling In this case the importance of catalytic and structural genes, many of program indicate that in addition to the known gradient in population which were in the glucose metabolism and extracellular matrix/ density from west to east among our sites, there was also a gradient in wound healing components, respectively, were strongly represented exposure to sediment contaminants and frequency of hypoxia. in the dataset. Important metabolic pathways were further supported by the KEGG analysis where, nitrogen metabolism (3.5%), oxidative 3.2. RNA sequencing, differential analysis and ontology of responses phosphorylation (3.2%), glycolysis/gluconeogenesis (1.6%), glycero- phospholipid metabolism (1.4%) and arginine and proline metabolism The Illumina HiSeq2000 produced approximately 154 million paired (1.4%) of unique transcripts, were the strongest enriched pathways reads in each pooled sample, with 30% more reads in the Shinnecock sam- (Supplementary File 8). ple set. The raw RNA-seq reads have been submitted to the NCBI Short Based on these data, several markers in the metabolic and Read Archive (http://trace.ncbi.nlm.nih.gov: Submission, SUB827557; inflammatory-immune pathways identified were chosen not only to BioProject ID, PRNJA275472). After assembly, all small contigs/scaffolds validate the RNA-seq data on two populations of flounder, but also to in- with a length of b100 bp were filtered out and a total of 187,354 scaf- vestigate their use as markers for health in YOY winter flounder from folds/contigs were taken for further analysis (Supplementary File 4). multiple populations in Long Island, NY. RNA samples that were pooled The longest scaffold was 21,319 bp in length and the mean size was for RNA-seq analysis were individually analyzed using qPCR and mean 579. There were 253 differentially expressed transcripts in the liver of fold changes between populations compared with the RNA-seq results

Table 2 Metabolic and immunological marker ontologies from Illumina RNA sequencing of Pleuronectes americanus livers. Query IDs are the sequence assembly IDs from this experiment, whereas homologous IDs are the proposed gene function, annotated to the subject ID from the species' subject with highest % identity and greatest alignment length. The log2 fold change is the log2 fold expression difference of this transcript in Moriches Bay ﬁsh livers compared to Shinnecock Bay (i.e. negative values exist where Shinnecock N Moriches), and associated adjusted p- value for multiple test correction.

Query IDs Subject IDs % Homologous ID Species Alignment E value Log2 fold p adjusted identity length change

C423218_154 XM_003450016.1 75.97 Complement C3-like (C3) Oreochromis niloticus 154 2.E−21 −3.40 1.0E−02 scaffold26635_2082 XM_003451020.1 86.26 Glucokinase (hexokinase 4) (GCK) O. niloticus 1376 0.E+00 5.17 1.2E−06 scaffold26338_1956 XM_003438134.1 83.28 Glutamate decarboxylase (GAD) O. niloticus 1962 0.E+00 3.13 2.3E−02 scaffold9339_1434 XM_003456095.1 85.65 Glycerol-3-phosphate dehydrogenase (GPDH) O. niloticus 1073 0.E+00 4.47 2.4E−02 C440357_168 AY623818.1 91 Hepcidin II (HEP) Paralichthys olivaceus 163 2.E−51 2.96 4.7E−02 scaffold343_966 AY282499.1 78 Pleurocidin 8/9 pseudogenes Pleuronectes americanus 251 4.E−43 9.68 2.3E−15 C748475_3618 XM_008287354.1 81 Secretory phospholipase A2-like (PLA2) Stegastes partitus 647 2.E−133 4.72 1.6E−05

Binding and catalytic activity scaffold20893_1576 XM_003449047.1 75.7 Alkaline phosphatase-like O. niloticus 1457 0.E+00 3.06 4.3E−02 C721205_1264 XM_003457440.1 86.08 Calsequestrin-1-like O. niloticus 1286 0.E+00 5.43 1.1E−03 C721519_1272 XM_003448136.1 75.25 Cathepsin E-A-like O. niloticus 1204 0.E+00 −4.62 1.6E−03 C701927_909 XM_003460428.1 72.12 Cathepsin-L O. niloticus 911 5.E−131 3.23 1.5E−02 scaffold17663_3267 XM_003437684.1 82.1 Synaptotagmin-4-like O. niloticus 1536 0.E+00 4.46 2.6E−04 scaffold13886_2131 XM_003459563.1 79.29 Zinc ﬁnger protein RFP-like O. niloticus 1125 0.E+00 3.79 2.7E−02

Energy metabolism scaffold9980_1947 XM_003446761.1| 86.27 Glycogen phosphorylase O. niloticus 1653 0.E+00 5.31 7.4E−04 C750565_5150 XM_003449226.1 79.74 Hyaluronidase-4 O. niloticus 1540 0.E+00 −3.47 3.9E−02 scaffold7234_2088 XM_003458534.1 78.49 Metalloreductase STEAP4-like O. niloticus 437 6.E−98 3.83 1.3E−03 C728959_1508 XR_134092.1 71.11 NADH dehydrogenase subunit 3 (nad3) gene Nasonia vitripennis 1350 8.E−179 9.31 1.9E−11 scaffold28121_1979 XM_003443869.1 88.13 Pyruvate kinase Oreochromis niloticus 1634 0.E+00 4.32 6.1E−03 C744201_2626 XM_003447041.1 76.52 Transglutaminase Oreochromis niloticus 2027 0.E+00 3.05 4.4E−02 C749493_4122 XM_003443868.1 78.48 UDP-glucuronosyltransferase 2A1-like Oreochromis niloticus 1603 0.E+00 −5.89 1.2E−07

Extracellular matrix and inﬂammation C723821_1339 XM_003437714.1 84.17 ADAM metallopeptidase (adamts18) Oreochromis niloticus 575 0.E+00 4.70 4.9E−02 scaffold24229_5713 XM_003446398.1 87.34 Collagen alpha-1(I) chain-like Oreochromis niloticus 4035 0.E+00 3.13 2.5E−02 scaffold6366_2027 XM_003445004.1 75.78 Collagen alpha-1(VII) Oreochromis niloticus 801 6.E−162 5.93 2.6E−04 scaffold27582_3183 XM_003437929.1 76.39 Collagen alpha-3(IV) Oreochromis niloticus 1241 0.E+00 4.42 8.3E−04 scaffold25650_1779 XM_003437930.1 73.98 Collagen, type IV, alpha 6 (COL4A6) Oreochromis niloticus 1299 0.E+00 3.81 2.5E−02 scaffold2476_4343 XM_003456559.1 77.57 Fibroblast growth factor 19-like Oreochromis niloticus 321 1.E−63 −2.98 4.5E−02 C654905_532 XM_003459122.1 75.35 Galectin-4-like Oreochromis niloticus 495 7.E−91 −5.55 4.9E−02

(Fig. 4). Here we see the qPCR (normalized either to one reference gene species, the horneyhead turbot (Pleuronichthys verticalis) collected in (EF1a) or to total RNA) data showed good agreement in the magnitude southern California in areas receiving sewage inputs, strong upregula- and direction of differences with the RNA-seq expression data. Further, tion of CYP1A in liver tissue was observed by microarray analysis, but 5 out of 6 (C3 was only one that did not) genes showed significant qPCR analysis of individual fish indicated high variability (Baker et al., differences between populations in both the qPCR and RNA-seq 2013). Elevated CYP1A activity (as measured by ethoxyresorufinO- datasets, and 3 out of 3 genes which were not significantly different in deethylase (EROD) activity) and gene expression have been reported the RNA-seq dataset were also not different in the qPCR analysis. previously for adult winter flounder from Jamaica Bay as compared to Shinnecock Bay (Mena et al., 2006), and increased expression of 3.3. Measures of exposure to environmental contaminants CYP1A mRNA has been reported in pooled samples of adult flounder from Raritan Bay, NJ as compared to flounder collected from the less CYPIA and VTG mRNA expression were evaluated to provide bio- urban NJ Atlantic coast (Straub et al., 2004). However, earlier work eval- markers of exposure to environmental contaminants. Both are well uating EROD activity and CYP1A protein levels in adult winter flounder known to respond to a variety of contaminants in both laboratory and collected from sites around the northeastern Atlantic coast of the U.S. field assessments (Schlenk et al., 2008a). Expression of both genes var- reported uninduced flounder could only be found at the remote area ied significantly among the sites sampled (Fig. 5). of Georges Bank off the New England coast or Northern ME, while coast- CYP1A is a biomarker commonly used to evaluate exposure to planar al flounder, particularly flounder sampled from Long Island Sound, aromatic hydrocarbons that act via the AhR, such as chlorinated hydro- showed highly variable, elevated levels (Monosson and Stegeman, carbons like PCBs, dioxins and furans, as well as polycyclic aromatic 1994). A previous study evaluating CYP1A and PLEUR expression in hydrocarbons associated with urban runoff (Stegeman and Hahn, YOY winter flounder collected from six Long Island bays including two 1994). Induction of CYP1A is usually considered a biomarker of expo- that were investigated in this present study (Jamaica and Shinnecock sure to AhR agonists, but due to the cellular and genotoxicity of activat- Bays) also did not find site specific differences in hepatic CYP1A expres- ed metabolites produced and expression of other downstream gene sion in YOY winter flounder (Romany et al., 2015). Data from the products from some AhR agonists that are recalcitrant to metabolism, USEPA's National Coastal Assessment indicates that PCBs are found in upregulation of CYP1A can also be considered a biomarker of toxic effect somewhat elevated levels in sediments from all of the sites examined (Schlenk et al., 2008a). CYP1A expression was significantly depressed in (Supplementary File 1), therefore elevated and variable expression of flounder from Hempstead Bay as compared to all other sites (Fig. 5A). CYP1A in YOY flounder from throughout the area is not surprising. Although median CYP1A levels were highest in flounder from the most The depressed expression of CYP1A in flounder from Hempstead Bay urban and westernmost site, Jamaica Bay, due to the large variation in was unexpected. Increased expression of CYP1A is commonly found in among individual fish, expression levels were not significantly different studies evaluating exposure to oil pollution and chlorinated aromatic from the more eastern sites. In recent papers evaluating another flatfish hydrocarbons (Schlenk et al., 2008a), but there have been reports

Fig. 2. YSI data for dissolved oxygen, salinity and temperature for all sites and sampling days in 2010 and 2011. Data plotted as means and SD for each trawl or seine taken on the same day. where expression patterns do not follow this standard paradigm. Maes Expression of VTG mRNA showed the most consistent trends with et al. (2013) conducted a study examining condition status of resident respect to the urban gradient on Long Island, with values generally European eel (Anguilla anguilla) chronically exposed to a suite of decreasing from west to east (Fig. 5B). Median levels were highest in metal and organic pollutants in three Belgian river basins evaluating Jamaica Bay in the west and lowest in Shinnecock Bay, with expression condition factor and lipid reserves, expression of CYP1A and metallo- at Jamaica Bay being significantly elevated as compared to both thionein (MT), and bioaccumulation of metals, PCBs and organochlorine Shinnecock Bay and Napeague Harbor. However, median expression pesticides (DDTs), comparing the environmentally exposed river eels to levels in flounder from Cold Spring Pond were higher than the other eels raised under clean conditions in the laboratory. Although expres- two eastern sites, making expression levels at this site not significantly sion levels of CYP1A were significantly elevated in eels from all river lo- different from the three western sites. Further work should attempt to cations as compared to the eels raised in captivity, variability was identify agents inducing VTG in flounder from this site. extremely high in the wild eels, with fish from some of the most pollut- Elevated VTG in the more urban sites is consistent with sewage ined sites showing the lowest levels of CYP1A expression. In fact PCA anal- puts and agrees well with elevated levels of sewage tracer compounds ysis revealed a negative correlation between PCB concentrations and measured in the sediments of Jamaica and Hempstead Bays (Doherty, expression of CYP1A in the liver of the eels examined. Co-exposure to 2013), and extremely high levels (10 s of ppm) of nonylphenol previ- AhR antagonists such as cadmium and tributyltin and even some ously reported in Jamaica Bay sediments (Ferguson et al., 2001). It is PAHs such as fluoranthene and 2-aminoanthracene can diminish re- also consistent with previous studies showing elevated expression of sponse to AhR agonists (Schlenk et al., 2008b). Development of resis- VTG protein in YOY winter flounder from Jamaica Bay as compared to tance to model AhR agonists has also been reported in several species Shinnecock Bay (McElroy et al., 2006) as well as female biased sex ratios (Elskus, 2001), and can confound interpretation of gene expression observed in another local fish, the Atlantic silverside (Menidia menidia) data (Hoffmann and Willi, 2008; Oleksiak, 2010). It is possible that the from more urban bays around Long Island (Duffy et al., 2009). Straub et flounder from Hempstead Bay are less sensitive to inducers, or possibly al. (2004) previously reported elevated VTG expression in pooled sam- are responding to other confounding factors such as the presence of AhR ples of adult winter flounder from a more urban area of NJ as compared antagonists as has been observed in other studies. Further work will be to a less contaminated site in southern NJ. Both Vtg1 and Vtg2 as well as needed to identify the causes of the relative CYP1A depression at this several other estrogen responsive transcripts were also found to be up- site. regulated in the horneyhead turbot study of southern California sewage Vitellogenin is an egg yolk precursor protein that is synthesized by exposed flatfish (Baker et al., 2013). the liver during oocyte development under control of the estrogen re- A number of environmental factors in addition to exposure to ceptor. Elevated expression of VTG in male or juvenile fish is a common- chemical contaminants have been shown to influence both CYP1A and ly used biomarker of exposure to estrogen or chemicals that mimic VTG expression. Hypoxia has been shown to down regulate CYP1A in estrogen in wild caught fish (Heppell et al., 1995; Tyler et al., 1998). zebrafish (Prasch et al., 2004), and Rahman and Thomas (2012) have

Fig. 3. Distribution of annotated transcripts assigned to cellular components: A) biological processes, B) molecular function, and C) according to gene ontology association (Blast2Go). D) Non-redundant fold-change sums for ontological groups associated with differentially expressed transcripts. As detailed in Supplemental File 7.

recently demonstrated that hypoxia downregulates hepatic CYP1A may also be limiting the range of response observed in these variables. expression in Atlantic croaker through alterations of nitric oxide and For both these genes, mRNA expression represents recent exposure to oxidant status through a pathway involving interleukin 1ß and hypoxia inducers (Kloepper-Sams and Stegeman, 1987; Hemmer et al., 2002). inducible factor (HiF). Hypoxia has also been shown to act as an anti- Including an assessment of plasma VTG protein expression and catalytic estrogen, masculinizing fish through downregulation of aromatase activity of CYP1A would provide a more complete picture of chronic ex- activity that converts androgens to estrogen (Thomas and Rahman posure in YOY winter flounder. Analysis of contaminant levels in sedi- 2009). Alternatively, increased temperature has been found to increase ments at the site of collection and in fish tissues would also provide VTG expression in response to exogenous estrogens in the mummichog additional valuable information on exposure, but does not diminish Fundulus heteroclitus (Chandra et al., 2012). Temperature may have en- the importance of field data on biological responses, which account for hanced VTG response to environmental estrogens in flounder from both factors affecting bioavailability as well as the possibility of detecting re- Jamaica Bay and Cold Spring Pond. The relative young age of these fish sponses from rapidly metabolized compounds such as PAHs, and many estrogenic contaminants such as hormones and hormone mimics. Also, several laboratory studies evaluating exposure to sewage effluents have not shown these biomarkers to link directly to dose (Vidal-Dorsch et al., 2014). Further work will be needed to evaluate what combinations of environment factors (chemical exposure, temperature, hypoxia) may be contributing to the patterns in CYP1A and VTG expression we observed in juvenile flounder.

3.4. Measures of inﬂammation/immune response

Stress triggers a complex set of endocrine control responses leading to the release of stress hormones (i.e. cortisol). These lead to secondary responses such as elevated blood sugar and diuresis if the stressor persists. If the stressor persists over the longer term, tertiary responses can ensue such as reductions in growth, immunocompetence, reproductive success and survival (Wedemeyer et al., 1990). In fish, the effects of long-term exposure to stress and chronically elevated levels of cortisol are well known to depress inflammation, immune responses and dis- Fig. 4. Log2 fold changes in relative expression between Moriches and Shinnecock Bays. White bars show RNA-seq data; black bars show qPCR data relative to EF-1; gray bars ease resistance, and increase morbidity (Pickering and Pottinger, show raw qPCR data relative to total RNA for cytochrome P4501A (CYP1A), pleurocidin 1989; Bols et al., 2001; Fast et al., 2008). In particular, exposure to sew- (PLEUR), complement C3 (C3), hepcidin (HEP), vitellogenin (VTG), glutamate decarboxyl- age or contaminated water sources is known to have immunosuppres- ase (GAD), and phospholipase A2 (PLA2). Glucokinase (GCK), and glycerol 3-phosphate sive activity (Kennedy and Farrell, 2008). Four genes associated with dehydrogenase (GPDH). Asterisk denotes significant differences between populations in inflammation/immune response were evaluated in this study, and all all three groups. CYP1A, PLEUR and VTG also show agreement between all three groups fi of data in that there were no significant differences in relative expression irrespective of but pleurocidin (PLEUR), showed signi cantly different patterns in ex- method of analysis. pression among sites (Fig. 6).

Fig. 5. Box plots of mRNA expression of contaminant response genes: A) cytochrome P4501A (CYP1A), and B) vitellogenin (VTG), plotted for each site, where the line is the median, the upper and lower edges of the boxes show the 25th and 75% percentiles of the data and the whiskers illustrate either roughly two standard deviations from the mean or the maximum and minimum values (whichever is smaller). Differences in expression between sites were assessed by one way ANOVAs (p value presented on figure) performed with Tukey's multiple comparisons. Sites identified by different letters are significantly different from each other (p b 0.05).

The PLEUR gene codes for the antimicrobial peptide pleurocidin, an (Choi et al., 2007). Interestingly, Jamaica Bay with greatest incidence of innate molecule effective at killing Gram positive and negative bacteria hypoxia showed the lowest expression of HEP. (Cole et al., 1997; Patrzykat et al., 2003). In YOY winter flounder expres- Transcripts of another acute phase protein, complement C3 (C3), sion in the liver is much lower than that observed in fintissue(Romany also differed in their expression significantly among sites (Fig. 6C), et al. 2015). As with CYP1A, Romany also did not see site related differ- with expression levels being significantly lower at two of the three ences in her study of YOY winter flounder from different bays of Long more eastern sites (Shinnecock and Napeague) as compared to all Island (Romany et al., 2015). PLEUR was also not found to be differen- three western sites and Cold Spring Pond in the east, similar to patterns tially expressed in the RNAseq analysis comparing pooled samples observed in VTG expression. Elevated C3 expression could indicate a from Moriches and Shinnecock Bays, though pleurocidin (PL) 8–9, a po- generally higher level of pathogen exposure and/or inflammation in tential pseudogene was only observed in the Moriches population and flounder from more urban areas. Straub et al. (2004) also reported ele- not the Shinnecock population (Table 2). vated expression of C3 in pooled samples of adult winter flounder from In contrast to PLEUR, hepcidin II (HEP), a peptide hormone produced Raritan Bay, as compared to a reference site in NJ. Relative expression of by the liver, known to have antimicrobial activity and be involved in C3 was much higher than CYP1A, VTG and PLEUR in this study. Similar re- regulating iron homeostasis, was differentially expressed between sults have been reported in the Senegalese sole, (Solea senegalensis), sites (Fig. 6B), although not in a manner associated with a west to east where high expression of hepatic C3 was observed, especially in com- urban gradient. Hepcidin, in particular its iron sequestration role, is im- parison to the kidney (N5000×; Prieto-Alamo et al., 2009) which is portant in vertebrate responses to combat bacterial infection and deal one of the primary sites of hematopoiesis and immunological function with septicaemia. Hepcidin is consistently produced by cells that are in teleosts (Kibenge et al., 2012). Strong hepatic C3 upregulation in crucial to the generation of an effective immune response against response to lipopolysaccharide (LPS) challenge was also reported for acute infection and HEP can affect disease pathogenesis (Armitage the Senegalese sole, while other flatfish, salmonids and even zebrafish et al., 2011; Ba Sow et al., 2008; Frazier et al., 2011). Highest levels of ex- have shown differential expression of C3 (among other complement pression were observed in flounder from Hempstead and Moriches proteins: C3a, C2b, etc.) in immune related organs after immunization Bays from the west, and Cold Spring Pond from the east. Lowest levels against rhabdoviral (viral hemorrhagic septicemia virus and infectious of expression were observed at the two ends of the sampling area, hematopoietic necrosis virus) infection (Byon et al., 2006; MacKenzie Jamaica in the west, and Napeague in the east. Straub et al. (2004) et al., 2008; Encinas et al., 2010). These studies and others speak to also reported upregulation of HEP in flounder from a polluted site in the importance of complement C3 as a central mediator in classical NJ. Hepcidin is an acute phase reactant and anti-microbial peptide that and alternative complement activation and the complement cascade, disrupts microbial membranes. Induction of hepcidin in response to an integral part of innate immunity (Whyte, 2007). inflammatory stimuli is also a mechanism by which the animal can se- The fourth inflammatory/immune responsive gene assessed quester and control iron availability to bacteria or other pathogens was phospholipase A2 (PLA2). Significant site-specific differences were that have initiated the inflammatory stimuli (Weinstein et al., 2002; also noted in the expression of this gene (Fig. 6D), yet expression Nemeth et al., 2003). Anemia, often observed in chronic inflammatory was only significantly elevated in flounder from Moriches Bay as states, can be a result of hepcidin-induced sequestration of iron in the compared to flounder collected from all other sites. PLA2 is an impor- macrophage. HEP transcription can be affected by hypoxia, since the tant catalyst within the arachidonic acid pathway, leading to the forma- promoter region of HEP has a hypoxia-inducible transcription factor tion of inflammatory and thrombogenic compounds (Pruzanski and (Hif-1a) binding site (Peyssonnaux et al., 2007). Hypoxia stress has Vadas, 1991). High levels of hepatic expression of PLA2, C3 and HEP been shown in other teleosts to downregulate inflammatory responses in Moriches Bay flounder may indicate that these fish have enhanced

Fig. 6. Box plots of mRNA expression of immune response genes: A) pleurocidin (PLEUR), B) complement C3 (C3), C) hepcidin (HEP), and D) phospholipase A2 (PLA2) plotted for each site as described in Fig. 5. ability to ward off infection as compared to flounder from Jamaica and to identify factors associated with both up and downregulation of Hempstead Bays where even more degraded conditions may lead to immune responsive genes in juvenile winter flounder collected from immunosuppression. urban estuaries. While environmental factors such as hypoxia may af- Comparing data for all four immune response genes, HEP showed fect these genes, pathogen exposure will as well, and both stressors the most discriminating power between the sites, and PLEUR the least. may change seasonally and annually at different time scales across PLEUR expression has been described as increasing with age in winter sites, making identification of clear and consistent patterns difficult. Fu- flounder (Douglas et al., 2001), and work by Romany et al. (2015), ture work should evaluate how expression patterns in these immune showed increased expression of this gene in fin tissue of larger winter response genes are associated with either pathogen load or susceptibil- flounder compared to YOY winter flounder from a north shore Long ity to pathogen exposure. Island bay population as well. The absence of site-specific variation in PLEUR observed in these studies may reflect an immature and poorly 3.5. Measures of glucose and glycogen metabolism responsive state of this anti-microbial peptide in YOY flounder, possibly due to an incompletely developed immune system at this life stage. HEP The majority of differentially regulated genes identified by Illumina on the other hand, showing the most significant differences among differential expression from Moriches and Shinnecock Bays were associ- sites, may be more responsive to environment factors. HEPII (the tran- ated with glycolysis and glucose metabolism. These data supported the script measured in our work) was also found to be significantly upregu- decision to evaluate several key genes in these pathways using qPCR lated in sewage impacted flatfish in the horneyhead turbot study (Baker analysis of individual flounder from all six study areas. Statistically sig- et al. 2013). Further work should be focused on process-oriented studies nificant differences in expression among the sites were observed for

Fig. 7. Box plots of mRNA expression of glucose and glycogen metabolism genes: A) glycerol 3-phosphate dehydrogenase (GPDH), B) glucokinase (GCK), and C) glutamate decarboxylase (GAD) plotted for each site as described in Fig. 5. all genes evaluated (Fig. 7). However, as with most of the other genes shown to enhance GAD activity of both isoforms 65 and 67 in mammals studied, expression patterns did not consistently vary along the west (Kobayashi and Millhorn, 2001). As with the other gene families evalu- to east gradient examined. Glucokinase (GCK) gene expression was sig- ated in this study, further experimental work will be needed to identify nificantly elevated at Moriches as compared to all other sites (Fig. 7A). specific factors driving expression of the glucose and glycogen metabo- Similar to HEP, GCK also has a Hif binding site within its promoter re- lism genes evaluated. gion; however it is upregulated in response to hypoxia (Barker et al., 2012; Roth et al., 2004) potentially to increase anaerobic glucose metab- 3.6. Expression patterns among all the genes evaluated olism. For glutamate decarboxylase (GAD) expression, although the difference between Moriches and Shinnecock was statistically sig- Principal components analysis (PCA) was used to assess patterns in nificant, expression in flounder from Moriches was not significantly expression of all the genes in individual flounder sampled. Principal different from expression levels in flounder from the rest of the sites components (PC) 1 and 2 explained 62% of the variance in the data. As (Fig. 7B). Glycerol 3-phosphate dehydrogenase (GPDH) gene expression can be seen in Fig. 8, where arrows show the direction and the relative was depressed in flounder from both Shinnecock Bay and Napeague magnitudes of the loadings of individual variables on PC1 and PC2 Harbor as compared to flounder from both Jamaica and Moriches (provided in Table 3), expression of all genes primarily project in Bays, but not Hempstead Bay or Cold Spring Pond (Fig. 7C). Vidal- three directions. Along PC1, only CYP1A is positive, albeit with a relative- Doresch et al. (2013) also found GPDH to be mildly depressed in ly small loading of 0.09. The three variables with the greatest loadings horneyhead turbot exposed to 5% sewage effluent in the laboratory. It were HEP, GCK,andPLA2 which had the largest absolute values of load- should be noted that GAD was expressed at much higher levels than ings in both PC1 and PC2. The immune response gene HEP stands out as either GCK or GPDH, and that GAD is the rate-limiting enzyme in being most positive on PC1 (loading = 0.69), but another immune re- gamma-aminobutyric acid synthesis, (a neurotransmitter with a prima- sponse gene, C3, also plots on this axis. CYP1A plots opposite the axis ry signaling role in the brain, but also found in the pancreas). In floun- dominated by HEP indicating a negative correlation between CYP1A ders and other members of Osteichthyes, a diffuse pancreas develops and HEP expression. The other two immune response genes (PLEUR in postlarvae and branches along veins running to the liver and the he- and PLA2) and all the glucose and glycogen metabolism genes (GCK, patic portal vein in the parenchymal tissue of the liver (Kurokawa and GAD,andGPDH) plot approximately 90° to the axis defined by HEP, Suzuki, 1995). Thus the presence of pancreatic tissue can be expected with GCK being most negative on PC2 (loading = −0.56). Also plotting in the liver samples collected from these flounder. The high levels of along this axis with fairly large loading values are the other two immune GAD mRNA in flounder may therefore be linked to expression of high response genes, PLA2 and PLEUR (loadings = −0.31 and −0.16 respec- levels of this enzyme in the liver and pancreas, as has been reported in tively). It should be noted that although the PCA displays the relation- mammals (MacDonnell and Greengard, 1975). Hypoxia has also been ship in expression between these genes in individual flounder, it

Fig. 8. Relationships between expression of all genes examined by principal components analysis (PCA). Arrows show the direction and the relative magnitudes of the loadings of individual variables on PC1 and PC2 as shown in Table 2. provides no direct information on how these genes are related to effects al. (2011), levels of VTG and CYP1A protein were highly variable at higher levels of biological organization such as physiological or pop- among sites, and were not generally different among the more contam- ulation level responses. inated sites, despite large differences in contaminant exposure. Howev- Interactions between inflammation/innate immunity and glucose er taking a systems biology approach looking at network analysis, they and glycogen metabolism have been well described in mammalian found significant associations between measures of liver histopatholo- models. Starved rats have shown both significantly increased and re- gy, altered metabolism and toxicology. In earlier work by this same duced PLA2 levels, but more importantly PLA2 alterations may contrib- group examining gene expression patterns by microarray, the ability ute to the diminished insulin secretory response of islets from starved to distinguish patterns among sites was significantly improved when rats to relatively low concentrations of glucose. Interleukin-1, another data from field caught flounder was combined with responses observed pro-inflammatory marker, has also shown to decrease GAD activity in controlled laboratory exposures (Falciani et al., 2008). More recent and mRNA expression when administered with glucose to rat islet work coming out of the Southern California Coastal Water Research Pro- cells (Velloso et al., 1994). These data in rats suggest that active inflam- ject (SCCWRP) on another flatfish, adult horneyhead turbot (Baker matory responses within the same tissues may co-regulate these glu- et al., 2009, 2013; Vidal-Dorsch et al., 2013, 2014) also reveals complex cose and glycogen metabolism genes. To the authors' knowledge the patterns in gene expression, with many of the same transcripts exam- current study is the first evidence that this co-regulation may be occur- ined in this study (CYP1A, VTG, HEP, GPDH) being differentially regulated ring in fish tissues. in field caught turbot from sewage impacted areas of southern Califor- Additional information can be gleaned from site-specific patterns in nia and in turbot exposed to sewage effluent in the laboratory. However the distribution of individual flounder on the biplot (Fig. 8). Flounder in the SCCWRP studies the direction of response was not always the from Moriches Bay are grouped in the left two quadrants. Flounder same, with the laboratory exposed fish generally showing downregula- from the other locations primarily project on the right two quadrants tion as compared to controls, while many of these transcripts were with flounder from Napeague Harbor grouped in the lower right quad- upregulated in field caught organisms. These data indicate that addi- rant. The PCA analysis indicates that expression patterns of the genes tional environmental variables may be at play, or that less persistent investigated in this study can at least partially differentiate the subpop- components of the sewage effluent (that field collected fish might not ulations studied along the south shore bays of Long Island, and supports see) may also be influencing expression of these genes, or that some our hypothesis that mRNA expression profiles in the flounder from the level of adaptation has occurred in the field caught fish. Indeed the more urban areas differ from those from less impacted eastern habitats. lack of large differences in expression among fish collected from the dif- These results also support the inclusion of both immune responsive and ferent sites in our study could be interpreted as supporting some level of intermediary metabolism genes in expression studies evaluating popu- adaptation in these populations, and that conditions between these lation level responses in gene expression. bays are not so different. In our case the extremely small effective The results of this study are consistent with data emerging as part stock size contributing to local populations of winter flounder of the European Union (EU) funded multinational study GENIPOL initia- (O'Leary et al., 2013) may have already selected for individuals better tive on the European flounder Platichthys flesus (Williams et al., 2008; adapted to degraded habitats at some of the sites investigated. Across Falciani et al., 2008; Williams et al., 2011). As described by Williams et the entire dataset, differential regulation of secondary stress response pathways such as glucose metabolism, inflammation and immune response, are indicative of primary stressors affecting winter flounder of Table 3 this life stage, which in itself may be the most impactful finding of the Loadings from all gene PCA. study. However, the specific stressors that are driving these responses Genes PC1 PC2 within sites, be they changes in temperature, hypoxia, salinity, contam- CYP1A 0.0928 −0.2240 inants or likely different combinations of these, requires targeted analy- VTG −0.0973 0.0241 ses at transcriptional, translational and functional levels. PLEUR −0.1226 −0.1571 C3 −0.1677 0.1254 HEP −0.6410 0.6945 4. Conclusions PLA2 −0.4007 −0.3171 GCK −0.5812 −0.5626 This is to our knowledge the most comprehensive quantitative anal- GAD −0.1114 −0.0411 ysis of gene expression in wild caught winter flounder conducted to GPDH −0.1309 −0.0879 date. The Illumina sequence data showed strong agreement with the

Please cite this article as: McElroy, A.E., et al., Spatial patterns in markers of contaminant exposure, glucose and glycogen metabolism, and immunological response in juvenile winte..., Comp. Biochem. Physiol., D (2015), http://dx.doi.org/10.1016/j.cbd.2015.01.006 12 A.E. McElroy et al. / Comparative Biochemistry and Physiology, Part D 14 (2015) xxx–xxx individual qPCR data, thereby validating its use as a sensitive screening virus (VHSV) recombinant glycoprotein and DNA vaccine using a microarray analysis. – fi Vaccine 24, 921 930. tool for biomarker identi cation and provides a rich dataset that can be Chandra, K., Bosker, T., Hogan, N., Lisler, A., MacLatchy, D., Currie, S., 2012. Sustained high utilized in future work on winter flounder and other related species. De- temperature increases the vitellogenin response to 17α-ethynylestradiol in mummi- spite high inter-individual variability in all genes investigated, statisti- chog (Fundulus heteroclitus). Aquat. 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Federal Aid in Sport contaminant exposure or stimuli driving CYP1A has a greater opposing Fish Restoration F54-R-31 Annual Performance Report. effect on immunological status than other factors influencing glucose Doherty, A.C., 2013. The Distribution, Fate and Application as Tracers of Quaternary Am- monium Compounds (QACs) in Sewage Impacted Estuaries. (Ph.D. Dissertation). or glycogen metabolism. The links between genes associated with glu- Stony Brook University, Stony Brook, NY. cose and glycogen metabolism and some genes associated with immune Douglas, S.E., Gallant, J.W., Gong, Z., Hew, C., 2001. Cloning and developmental expression response suggest that common environmental factors may be influenc- of a family of pleurocidin-like antimicrobial peptides from winter flounder, – fi Pleuronectes americanus (Walbaum). Dev. Comp. Immunol. 25, 137 147. ing both systems. Now that this rst step has been taken to identify Duffy, T.A., McElroy, A.E., Conover, D.O., 2009. Variable susceptibility and response to es- major pathways impacting YOY flounder in these environments and trogenic chemicals in Menidia menidia. Mar. Ecol. Prog. Ser. 380, 245–254. biomarkers associated with them, follow-up work should be done to ex- Elskus, A.A., 2001. Toxicant resistance in wildlife: fish populations. In: Robsertson, L.W., Hansen, L.G. (Eds.), PCBs: Recent Advances in Environmental Toxicology and Health amine mechanistic links between biomarker expression, physiological Effects. University Press of Kentucky, Lexington, KY, pp. 273–276. responses and survival in this species. A subsequent modeling study Encinas, P., Rodriguez-Milla, M., Novoa, B., Estepa, A., Figueras, A., Coll, J., 2010. Zebrafish fin evaluating the associations between gene expression evaluated here, immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach. BMC Genomics 11, 518. several measures of condition and otolith microgrowth increments Falciani, F., Diab, A.M., Sabine, V., Williams, T.D., Ortega, F., George, S.G., Chipman, J.K., revealed statistical associations between growth and condition and 2008. Hepatic transcriptomic profiles of European flounder (Platichthys flesus)from CYP1A, PLEUR, and GPDH indicating gene expression patterns observed field sites and computational approaches to predict site from stress gene responses – here link to higher order ecologically significant responses (Gallagher following exposure to model toxicants. Aquat. Toxicol. 90, 92 101. Fast, M.D., Hosoya, S., Johnson, S.C., Afonso, L.O.S., 2008. Cortisol response and immune- et al. in press). Through the use of interdisciplinary approaches such related effects of Atlantic salmon (Salmo salar Linnaeus) subjected to short- and as these we can identify factors associated with reduced survival in long-term stress. Fish Shellfish Immunol. 24, 194–204. YOY winter flounder survival, and hopefully begin to address the Ferguson, P.L., Iden, C.R., Brownawell, B.J., 2001. Distribution and fate of neutral alkylphenol ethoxylate metabolites in a sewage-impacted urban estuary. Environ. population's recovery. Sci. Technol. 35, 2428–2435. Supplementary data to this article can be found online at http://dx. Frazier, M.D., Mamo, L.B., Ghio, A.J., Turi, J.L., 2011. Hepcidin expression in human airway doi.org/10.1016/j.cbd.2015.01.006. epithelial cells is regulated by interferon-γ. Respir. Res. 12, 100. Frisk, M.G., McElroy, A.E., Fast, M.D., Hice, L.A., 2013. Restoring Long Island's winter flounder fishery: influence of natural and anthropogenic factors on health, fitness and recruitment success. Final Report. Award #NA10NMF4270202, National Marine Acknowledgments Fisheries Service. Gallagher, B.K., Hice, L.A., McElroy, A.E., Cerrato, R.M., Frisk, M.G., 2015. Factors influencing fl Funding for this project was provided by the National Marine Fisher- daily growth in young-of-the-year winter ounder along an urbangradient revealed using hierarchical linear models. Mar. Coast. Fish: Dyn. Manage. Ecosys. Sci. ies Service Saltonstall Kennedy Program (award #NA10NMF4270202) (in press). to M. Frisk, M. Fast, and A. McElroy, and an award from the NY State De- Hemmer, M.J., Bowman, C.J., Hemmer, B.L., Friedman, S.D., Marcovich, D., Kroll, K.J., partment of State to A. McElroy and M. Frisk, and the Novartis Chair at Denslow, N.D., 2002. Vitellogenin mRNA regulation and plasma clearance in male sheepshead minnow (Cyprinodon variegatus) after cessation of exposure to 17ß- PEI University to M. Fast. Sampling assistance was generously provided estradiol and P-nonylphenol. Aquat. Toxicol. 58, 99–112. by the Towns of East Hampton and Hempstead, the New York Depart- Heppell, S.A., Denslow, N.D., Folmar, L.C., Sullivan, C.V., 1995. 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