CBD-00356; No of Pages 13
Comparative Biochemistry and Physiology, Part D 14 (2015) xxx–xxx
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part D
journal homepage: www.elsevier.com/locate/cbpd
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. Hice a,2, M.G. Frisk a, S.L. Purcell b, N.C. Phillips b, M.D. Fast a,b,1
a
b
School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY, USA
Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada
a r t i c l e
i n f o
Article history:
Received 25 October 2014
Received in revised form 16 January 2015
Accepted 26 January 2015
Available online 4 February 2015
Keywords:
mRNA expression
Illuminia
RNAseq
Fish
Long Island, NY
a b s t r a c t
Inshore winter flounder (Pseudoplueronectes americanus) populations in NY, USA have reached record low numbers in recent years, and recruitment into the fishery appears to be limited by survival of post-settlement juvenile
fish. In order to identify cellular pathways associated with site-specific variation in condition and mortality, we
examined differential mRNA expression in juvenile winter flounder collected from six different bays across a gradient in human population density and sewage inputs. Illumina sequencing of pooled samples of flounder from
contrasting degraded sites and less impacted sites was used to guide our choice of targets for qPCR analysis. 253
transcripts of N 100 bp were differentially expressed, with 60% showing strong homology to mostly teleost sequences within the NCBI database. Based on these data, transcripts representing nine genes of interest associated
with contaminant exposure, immune response and glucose and glycogen metabolism were examined by qPCR in
individual flounder from each site. Statistically significant site-specific differences were observed in expression of
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
Winter flounder (Pseudoplueronectes americanus) once supported a
dominant commercial and recreational fishery along the Atlantic coast
of North America from the Gulf of St. Lawrence to Chesapeake Bay.
Since peaking in the mid-1980s, catches have declined, particularly in
NY waters (http://www.st.nmfs.noaa.gov/web; Sagarese et al., 2011),
local stocks are at record lows in abundance (Socrates and Colvin,
2006; CTDEP, 2011; Nuttall et al., 2011), and recent data indicates an extremely small parental stock contributing to inshore populations
(O'Leary et al., 2013). Likely driving these declines are decades of
overfishing, environmental change, and declining habitat condition.
Recruitment into the NY fishery appears to be limited by survival of
post-settlement juveniles (Socrates and Colvin, 2006; Yencho et al., in
review). Indeed estimates of young-of-the-year (YOY) mortality in
two Long Island bays in 2007 and 2008 indicated higher levels than
⁎ Corresponding author at: School of Marine & Atmospheric Sciences, Stony Brook
University, Stony Brook, NY 11794-5000, USA. Tel.: +1 631 632 8488.
E-mail address: [email protected] (A.E. McElroy).
1
These authors contributed equally to the study.
2
Current address: Delaware National Estuarine Research Reserve, Dover, DE, USA.
observed in similar environments from adjacent states including
NJ, CT, and RI (Yencho et al., in review). Earlier studies on winter flounder from urban bays of Long Island Sound have shown winter flounder
to have reduced fitness in areas with high contaminant loadings
(Nelson et al., 1991; Perry et al., 1991; Black et al., 1988), and more recent work has indicated YOY winter flounder and other resident species
from western Long Island bays show evidence of feminization (Mena
et al., 2006; McElroy et al., 2006; Duffy et al., 2009).
Winter flounder make a good sentinel species for examining the impact of environmental change as they tend to have a limited range, with
inshore populations of adults undergoing a seasonal on/off shore migration to avoid excessively hot summer temperatures, although some
bays may also support resident fish with even more limited ranges
(Poole, 1966; Sagarese et al., 2011). Spawning of inshore stocks primarily occurs in winter and early spring with most migrating fish returning
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
embryos and young fish are constantly exposed to sediment associated
contaminants, as well as being subject to the highly variable environmental conditions of the near shore benthos where dissolved oxygen,
pH, salinity, and temperature vary on daily to decadal time scales.
http://dx.doi.org/10.1016/j.cbd.2015.01.006
1744-117X/© 2015 Elsevier Inc. All rights reserved.
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
2
A.E. McElroy et al. / Comparative Biochemistry and Physiology, Part D 14 (2015) xxx–xxx
In this study we used qPCR to evaluate relative gene expression in
YOY winter flounder collected from bays representing an urban gradient along Long Island, NY. We hypothesized that environmental factors
that might be evident along this gradient (such as contaminant exposure, temperature, salinity and dissolved oxygen), would cause sitespecific shifts in metabolic and immunological status of winter flounder.
This study was part of a larger investigation evaluating a suite of biological, environmental and anthropogenic factors associated with recruitment success in YOY winter flounder populations in south shore bays
of Long Island (Frisk et al., 2013). Here we report results of Illumina sequencing on pooled liver samples from more and less degraded sites
and on the site-specific expression of nine genes related to contaminant
exposure, energy metabolism and immune response in the livers from
individual flounder.
2. Materials and methods
2.1. Site selection
Juvenile winter flounder were collected from six bays along the
south shore of Long Island, NY, USA from May through October during
2010 and 2011 (Fig. 1). These study locations represent a west to east
gradient in urbanization and sewage inputs within a very short geographical range of only 200 km. Based on locally reported census data,
population density differs by a factor of 100 from west to east over the
breadth of the study sites, ranging from over 2000/km2 in the west to
20/km2 in the east (www.Census.com). An even larger gradient exists
in sewage inputs. Jamaica Bay receives about 8 × 105 L of treated effluent per day, while Hempstead Bay receives only 2 × 102 L of effluent per
day (IEC, 2009). Moriches Bay receives no municipally treated wastewater, although its primary tributary, the Forge River, is known to be significantly impacted by septage leaching in from failing septic systems, and
this area was historically impacted by excess nutrient loading from duck
farms (Swanson et al., 2010). The three more eastern sites in this study,
Shinnecock Bay, Cold Spring Pond, and Napeague Harbor have both low
population densities and no reported evidence of impacts from sewage
or septage inputs. Very little monitoring data exist in most of these areas
on levels of common chemical contaminants in sediments. The U.S. Environmental Protection Agency's National Coastal Assessment (http://
www.epa.gov/emap/nca/html/data/index.html) provides the most
comprehensive dataset available for common organic and inorganic
non-nutrient contaminants. A summary of these data evaluating contaminant levels in fine (N2% total organic carbon and N10,000 μg/g Fe)
sediments from the study sites or nearby areas measured between
2000 and 2005 (the most recent data available) verifies the general
west to east gradient in contaminant loadings (Supplementary File 1).
These data not only identify Jamaica Bay as being the most contaminated, but also indicate that fish from all sites are likely to experience some
level of chemical contaminant exposure. It is important to point out that
by only looking at fine sediments, these data represent the high end of
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
worst case scenario of exposure to fine sediments, average contaminant
levels generally only exceed Effects Range Low (ERL) values designated
by the National Oceanic and Atmospheric Administration (NOAA) at
some sites, and the Effects Range Median (ERM) levels are not exceeded
for any contaminants (Long et al., 1995), indicating that most of these
sites are representative of the more wide-spread contamination found
in all but remote coastal areas both in the U.S. and world-wide.
2.2. Fish collection and processing
Flounder ranging in size from 0.2 to 36 g and 24 to 135 mm total
length were collected using either a 1 m beam trawl or with 3–60 m
beach seines from May through October in 2010 and 2011. Attempts
were made to collect fish every other week from each site during the
sampling season. All sampling was done during daylight, generally between 9 am and 2 pm. We attempted to collect 10 fish per location
per sampling day, but low flounder abundance at individual sites often
prevented this. Flounder were collected at three of the sites (Jamaica,
Moriches, and Shinnecock Bays) in both 2010 and 2011, while Cold
Spring Pond and Napeague Harbor were only sampled in 2010, and
Hempstead Bay was only sampled in 2011. A total of 221 flounder
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 maintained 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 continuous 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 Committee to A. McElroy (IRBNet #260837).
2.3. RNA isolation, RNA-sequencing and differential analysis
Fig. 1. Map of northeast Atlantic coast of North America showing study sites on Long
Island, NY.
Livers were removed from fish while still frozen and RNA extracted
from b130 mg of tissue after homogenization in TRIzol® reagent
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
A.E. McElroy et al. / Comparative Biochemistry and Physiology, Part D 14 (2015) xxx–xxx
3
(Invitrogen, Carlsbad, CA) following methods supplied by the manufacturer. RNA pellets were dissolved in at least 100 μL of molecular biology
grade water before storage at − 80 °C. Total RNA concentration was
measured using a Thermo NanoDrop-2000 spectrophotometer (Wilmington, DE). Five micrograms of the extracted RNA was then DNasetreated using an Ambion TURBO DNase-freeTM kit (Grand Island, NY)
following the manufacturer's protocol. Randomly chosen samples
(10%) were then used for RNA quality testing with the BioRad Experion
system (Mississauga, ON). The RNA Quality Indicator (RQI) of the samples ranged from 4.1 to 9.6, with all but two samples N7.0.
Pooled liver RNA samples comprised of 6 fish collected from either
Moriches or Shinnecock Bays were sent to The Centre for Applied Genomics (TCAG, Toronto, ON) for paired end (100 bp) Illumina HiSeq2000
instrument analysis. These sites represented the closest locations
between the more urbanized western bays and the more rural eastern
bays analyzed. Individual indexed whole transcriptome libraries were
constructed with a goal of obtaining N 10 gigabases of sequence per
library in the form of 100 bp Illumina paired-end reads. De novo
assembly of transcripts (N100 bp) was performed using SOAPdenovotrans (http://soap.genomics.org.cn/SOAPdenovo-Trans.html) (Li et al.,
2010). Reads were mapped back to the assembled transcripts using
Tophat (Trapnell et al., 2009) and counts coverage for each transcript
determined. Reads could not be mapped against a reference teleost
genome due to the lack of an appropriate reference being available.
The number of reads aligning to each assembled transcript provided
count data in each case, which were then input to DESeq (Anders and
Huber, 2010). This program estimates variance–mean dependence in
the data and tests for differential expression based on the negative binomial distribution. Differential expression was tested at a significance
level of p b 0.05 (and fold change N 8) adjusted using the Benjamini–
Hochberg procedure (i.e. to account for 5% false discovery rate).
sequencing through Macrogen USA (Rockville, MD). Sequences were
Blastn searched against NCBI and the winter flounder transcriptome database (generated above) to confirm appropriate product formation.
Quantitative PCR was carried out using 12 μL reactions (5 μL of 2×
Promega GoTaq® qPCR MasterMix, 0.5 μL each of 10 μM forward/
reverse primers, 4 μL of promega nuclease-free water and 2 μL diluted
cDNA) on a BioRad CFX Connect System (Mississauga, ON) using a 2step protocol with a melt curve (95 °C for 2 min followed by 40 cycles
of 95 °C for 5 s and individual annealing temp for 30 s) and a melt
curve from 65 to 95 °C (0.5 °C increments, 5 s/step). Reference gene stability was confirmed using GeNorm software and relative gene expression was determined using the ΔΔCT method (Livak and Schmittgen,
2001). Due to the absence of a true reference site, data are expressed
relative only to ELF1a expression, and were not further normalized. Specifics on names, primer sequences, annealing temperature, PCR efficiency and Blastn identity of the mRNA transcripts evaluated are given in
Table 1. It should be noted that although some of the transcripts can
only be considered as “like” products due to the absence of 100% homology to a known product (Table 2), transcripts will hereafter be referred
to only by their common name or abbreviation in this manuscript.
This project was undertaken to identify pathway marker gene expression as a proxy for a given pathway's stimulation and subsequent
activity. In doing so we fully realize that there are many posttranscriptional events that can affect mRNA stability, ribosomal binding, translation efficiency and eventual protein synthesis and function. While this
approach has its limitations, and may not always directly translate to
protein function, we considered it to be the best approach for analyzing
the small amounts of tissue available from individual juvenile flounder
considering the very depressed status of some of these populations.
In this study we operationally define gene expression as the “net
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
were selected and blasted against the NCBI database using Blastx in
the BLAST2GO software. Blastx was carried out against the NCBI nonredundant (nr) database using default parameters (i.e. minimum Evalue score set to 1.0E−0.6). Blast2GO was used to assign gene ontology terms to each annotated sequence with an annotation cut-off of 55
and GO weight of 5 (Smith et al., 2013). Functional annotation of transcripts was also determined using the Kyoto Encyclopedia of Genes
and Genomes (KEGG) database to further investigate metabolic pathways affected across populations (Kanehisa et al., 2014).
Preliminary analysis indicated that the data were not normally distributed, therefore all expression values were log transformed prior to
analysis. Expression patterns between sites are shown as box plots
where the line is the median, the upper and lower edges of the boxes
show the 25th and 75th percentiles of the data, and the whiskers illustrate either 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 performed with
Tukey's multiple comparisons. A p value of b0.05 was considered to
be statistically significant. Relationships between expression patterns
of all genes were examined using principal components analysis
(PCA). Box plots, PCA analysis and statistical tests were all performed
in R (Crawley, 2012).
2.5. cDNA synthesis and qPCR
Complementary DNA (cDNA) synthesis was performed on 1 μg of
DNase-treated total RNA using a Promega Reverse Transcription System
(Madison, WI) and random hexamers, according to the manufacturer's
instructions. Reverse transcriptase-free controls were included to
ensure the absence of genomic DNA. Choice of target genes for qPCR
was primarily based on relative expression analysis from the RNAsequencing dataset.
Representatives of immune signaling and glucose and glycogen
metabolism pathways were chosen for further analysis in individual
YOY winter flounder across the study sites evaluated. Two additional
genes, vitellogenin (VTG), and cytochrome P4501A (CYP1A), although
not differentially expressed between these sites by RNA-seq analysis,
were also analyzed to provide an indication of exposure to estrogenic
compounds associated with sewage inputs (VTG) and a general measure
of exposure to aryl hydrocarbon receptor (AhR) agonists often associated with urban run-off (CYP1A). Primer sets for the reference gene (elongation factor 1a — ELF1a) and genes of interest were developed using
Primer 3 and DNAfold software. Successful amplification of each gene
was verified by gel electrophoresis, product purification and Sanger
3. Results and discussion
3.1. Environmental conditions at the flounder collection sites
Average temperature, salinity and dissolved oxygen measured at the
time of fish collections over the season are shown in Fig. 2, with the average mean, maximum and minimum values for each site between
years shown in Supplementary File 3. Temperature and salinity followed similar seasonal patterns at each site with lower temperature in the
late spring and fall at all sites. Salinity showed less of a seasonal signal,
although Jamaica Bay stands out as having a lower salinity than all
other sites in both years sampled. The depressed salinity observed in
Jamaica Bay, as compared to all other sites, is likely due to the large volume of sewage effluent discharged to this bay. Sewage is the primary
source of freshwater to Jamaica Bay (Swanson et al., 1992). Dissolved
oxygen was highly variable over the season and often between sites,
but average values were highest at Shinnecock Bay as compared to all
other sites (Supplementary File 3). It should be noted that Jamaica Bay
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
4
A.E. McElroy et al. / Comparative Biochemistry and Physiology, Part D 14 (2015) xxx–xxx
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
temperature (°C)
Efficiency
BLAST identity
Elongation factor 1α
F-CGCTCTGTGGAAGTTTGAGA
R-CAGTCAGCCTGAGAGGTTCC
64
0.93
Cytochrome P4501A
F-AATCTGCAGGGTTTCCACTG
R-CCAATGTGATCTGCGGTATG
61.6
0.91
Pleurocidin
F-CCTGCTTATCGCCAAGGTAA
R-CCATCTTCGTCCTCATGGTT
65.3
0.92
Complement C3
F-CAGCGTACGATGTGAATGTGG
R-TGAAATAGTGCGGGCACGTCC
66
0.94
Hepcidin II-like
F-GTCACCAGCAGAGTCAAAGAAC
R-CTCAGGAAAGGTGGCAGAAC
64
0.95
Glutamate decarboxylase-like
F-TCCGTAAAGACCCCAACAAG
R-AACCAAGGATGCTGATCGTC
64
0.97
Phospholipase A2-like
F-GCATAAAGGCGGGAAAGAAG
R-GACAGCCAAACAACCCTGAC
64
1.00
Glucokinase-like
F-GATGTTTTGGCTGCAACTGG
R-CACACTCACGACTGGATGATG
65.3
1.00
Glycerol 3 phosphate dehydrogenase-like
F-AGCCGACATCCTGATCTTTG
R-ATCGATGCCCTTGATGAGAG
64
0.95
Vitellogenin-like
F-TGCAGGAGGTCTTCCTCAGT
R-CCCATCAGCCTTTCCACAGA
65.3
0.99
98% EF-1a
EU561357.1
Hippoglossus hippoglossus
100% CYP1A
HQ659503
Pseudoplueronectes americanus
100% PLEUR
AF301511
Pseudoplueronectes americanus
100% C3
AY225099.1
Pseudoplueronectes americanus
98% HEP II
AY623818.1
Paralichthys olivaceus
83% GAD
JF694446.1
Monopterus albus
85% PLA2
XM_004566948.1
Maylandia zebra
86% GCK
XM_003451020.1
Oreochromis niloticus
91% GDPH
XM_003973087.1
Takifugu rubripes
96% VTG
EF582607.1
Hippoglossus hippoglossus
demonstrated the greatest range between minimum and maximum dissolved oxygen levels of all sites, with minimum dissolved oxygen values
of b3 mg/L in both years. Data obtained from continuously recording
data sondes available in 2011 at Jamaica, Moriches, and Shinnecock
Bays provide a clearer picture of diel patterns in dissolved oxygen and
indicated diminishing frequency of bottom water hypoxia going from
west to east with the percentage of measurements below 2.3 mg/L
being 23, 6, and 0.6% at Jamaica Moriches and Shinnecock Bays, respectively (data not shown).
There unfortunately are not a lot of data available on sediment and
water quality at the collection sites used in this study. The U.S. Environmental Protection Agency's (EPA) National Coastal Assessment provides
the most robust dataset with data available on sediment analyses done
on samples collected near most of our sampling sites over the period
of 2000–2005. A clear trend from west of east is observed for most pollutant chemicals, with Jamaica Bay standing out as being most contaminated, although this is primarily due to high levels observed at one site
(Supplementary File 1). Despite the paucity of sediment chemistry data,
what we do have, and the physical data obtained as part of our sampling
program indicate that in addition to the known gradient in population
density from west to east among our sites, there was also a gradient in
exposure to sediment contaminants and frequency of hypoxia.
3.2. RNA sequencing, differential analysis and ontology of responses
The Illumina HiSeq2000 produced approximately 154 million paired
reads in each pooled sample, with 30% more reads in the Shinnecock sample set. The raw RNA-seq reads have been submitted to the NCBI Short
Read Archive (http://trace.ncbi.nlm.nih.gov: Submission, SUB827557;
BioProject ID, PRNJA275472). After assembly, all small contigs/scaffolds
with a length of b 100 bp were filtered out and a total of 187,354 scaffolds/contigs were taken for further analysis (Supplementary File 4).
The longest scaffold was 21,319 bp in length and the mean size was
579. There were 253 differentially expressed transcripts in the liver of
winter flounder juveniles: 73 over-expressed in Shinnecock Bay as
compared to Moriches, and 180 overexpressed in Moriches as compared to Shinnecock Bay (Supplementary File 5). Based on the nonredundant annotation and the E-value distribution, further analysis of
these transcripts revealed that 73% had significant hits within NCBI
megablast, and 83% of these showed strong homology (E-value b 10−20).
20). Most of the strong homologous sequences were within teleost genomes, the most common being the Nile tilapia, Oreochromis niloticus
(Supplementary File 6). These were further characterized into three
gene ontology categories: cellular component (29% of unique transcripts; Fig. 3A), biological process (32% of unique transcripts; Fig. 3B),
and molecular function (39% of unique transcripts; Fig. 3C).
Another more quantitative way of looking at the differential transcript ontologies was used whereby the expression changes observed
in the RNA-seq data were incorporated in weighting ontologies rather
than ‘word counts’ of descriptive data. In this case, non-redundant fold
change sums were created based on the cumulative fold changes within
a process or grouping as illustrated by Fig. 3D (Supplementary File 7).
In this case the importance of catalytic and structural genes, many of
which were in the glucose metabolism and extracellular matrix/
wound healing components, respectively, were strongly represented
in the dataset. Important metabolic pathways were further supported
by the KEGG analysis where, nitrogen metabolism (3.5%), oxidative
phosphorylation (3.2%), glycolysis/gluconeogenesis (1.6%), glycerophospholipid metabolism (1.4%) and arginine and proline metabolism
(1.4%) of unique transcripts, were the strongest enriched pathways
(Supplementary File 8).
Based on these data, several markers in the metabolic and
inflammatory-immune pathways identified were chosen not only to
validate the RNA-seq data on two populations of flounder, but also to investigate their use as markers for health in YOY winter flounder from
multiple populations in Long Island, NY. RNA samples that were pooled
for RNA-seq analysis were individually analyzed using qPCR and mean
fold changes between populations compared with the RNA-seq results
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
A.E. McElroy et al. / Comparative Biochemistry and Physiology, Part D 14 (2015) xxx–xxx
5
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 pvalue for multiple test correction.
Query IDs
Subject IDs
%
identity
Homologous ID
Species
Alignment
length
E value
Log2 fold
change
p adjusted
C423218_154
scaffold26635_2082
scaffold26338_1956
scaffold9339_1434
C440357_168
scaffold343_966
C748475_3618
XM_003450016.1
XM_003451020.1
XM_003438134.1
XM_003456095.1
AY623818.1
AY282499.1
XM_008287354.1
75.97
86.26
83.28
85.65
91
78
81
Complement C3-like (C3)
Glucokinase (hexokinase 4) (GCK)
Glutamate decarboxylase (GAD)
Glycerol-3-phosphate dehydrogenase (GPDH)
Hepcidin II (HEP)
Pleurocidin 8/9 pseudogenes
Secretory phospholipase A2-like (PLA2)
Oreochromis niloticus
O. niloticus
O. niloticus
O. niloticus
Paralichthys olivaceus
Pleuronectes americanus
Stegastes partitus
154
1376
1962
1073
163
251
647
2.E−21
0.E+00
0.E+00
0.E+00
2.E−51
4.E−43
2.E−133
−3.40
5.17
3.13
4.47
2.96
9.68
4.72
1.0E−02
1.2E−06
2.3E−02
2.4E−02
4.7E−02
2.3E−15
1.6E−05
Binding and catalytic activity
scaffold20893_1576 XM_003449047.1
C721205_1264
XM_003457440.1
C721519_1272
XM_003448136.1
C701927_909
XM_003460428.1
scaffold17663_3267 XM_003437684.1
scaffold13886_2131 XM_003459563.1
75.7
86.08
75.25
72.12
82.1
79.29
Alkaline phosphatase-like
Calsequestrin-1-like
Cathepsin E-A-like
Cathepsin-L
Synaptotagmin-4-like
Zinc finger protein RFP-like
O.
O.
O.
O.
O.
O.
1457
1286
1204
911
1536
1125
0.E+00
0.E+00
0.E+00
5.E−131
0.E+00
0.E+00
3.06
5.43
−4.62
3.23
4.46
3.79
4.3E−02
1.1E−03
1.6E−03
1.5E−02
2.6E−04
2.7E−02
Energy metabolism
scaffold9980_1947
C750565_5150
scaffold7234_2088
C728959_1508
scaffold28121_1979
C744201_2626
C749493_4122
XM_003446761.1|
XM_003449226.1
XM_003458534.1
XR_134092.1
XM_003443869.1
XM_003447041.1
XM_003443868.1
86.27
79.74
78.49
71.11
88.13
76.52
78.48
Glycogen phosphorylase
Hyaluronidase-4
Metalloreductase STEAP4-like
NADH dehydrogenase subunit 3 (nad3) gene
Pyruvate kinase
Transglutaminase
UDP-glucuronosyltransferase 2A1-like
O. niloticus
O. niloticus
O. niloticus
Nasonia vitripennis
Oreochromis niloticus
Oreochromis niloticus
Oreochromis niloticus
1653
1540
437
1350
1634
2027
1603
0.E+00
0.E+00
6.E−98
8.E−179
0.E+00
0.E+00
0.E+00
5.31
−3.47
3.83
9.31
4.32
3.05
−5.89
7.4E−04
3.9E−02
1.3E−03
1.9E−11
6.1E−03
4.4E−02
1.2E−07
Extracellular matrix and inflammation
C723821_1339
XM_003437714.1
scaffold24229_5713 XM_003446398.1
scaffold6366_2027
XM_003445004.1
scaffold27582_3183 XM_003437929.1
scaffold25650_1779 XM_003437930.1
scaffold2476_4343
XM_003456559.1
C654905_532
XM_003459122.1
84.17
87.34
75.78
76.39
73.98
77.57
75.35
ADAM metallopeptidase (adamts18)
Collagen alpha-1(I) chain-like
Collagen alpha-1(VII)
Collagen alpha-3(IV)
Collagen, type IV, alpha 6 (COL4A6)
Fibroblast growth factor 19-like
Galectin-4-like
Oreochromis niloticus
Oreochromis niloticus
Oreochromis niloticus
Oreochromis niloticus
Oreochromis niloticus
Oreochromis niloticus
Oreochromis niloticus
575
4035
801
1241
1299
321
495
0.E+00
0.E+00
6.E−162
0.E+00
0.E+00
1.E−63
7.E−91
4.70
3.13
5.93
4.42
3.81
−2.98
−5.55
4.9E−02
2.5E−02
2.6E−04
8.3E−04
2.5E−02
4.5E−02
4.9E−02
(Fig. 4). Here we see the qPCR (normalized either to one reference gene
(EF1a) or to total RNA) data showed good agreement in the magnitude
and direction of differences with the RNA-seq expression data. Further,
5 out of 6 (C3 was only one that did not) genes showed significant
differences between populations in both the qPCR and RNA-seq
datasets, and 3 out of 3 genes which were not significantly different in
the RNA-seq dataset were also not different in the qPCR analysis.
3.3. Measures of exposure to environmental contaminants
CYPIA and VTG mRNA expression were evaluated to provide biomarkers of exposure to environmental contaminants. Both are well
known to respond to a variety of contaminants in both laboratory and
field assessments (Schlenk et al., 2008a). Expression of both genes varied significantly among the sites sampled (Fig. 5).
CYP1A is a biomarker commonly used to evaluate exposure to planar
aromatic hydrocarbons that act via the AhR, such as chlorinated hydrocarbons like PCBs, dioxins and furans, as well as polycyclic aromatic
hydrocarbons associated with urban runoff (Stegeman and Hahn,
1994). Induction of CYP1A is usually considered a biomarker of exposure to AhR agonists, but due to the cellular and genotoxicity of activated metabolites produced and expression of other downstream gene
products from some AhR agonists that are recalcitrant to metabolism,
upregulation of CYP1A can also be considered a biomarker of toxic effect
(Schlenk et al., 2008a). CYP1A expression was significantly depressed in
flounder from Hempstead Bay as compared to all other sites (Fig. 5A).
Although median CYP1A levels were highest in flounder from the most
urban and westernmost site, Jamaica Bay, due to the large variation in
among individual fish, expression levels were not significantly different
from the more eastern sites. In recent papers evaluating another flatfish
niloticus
niloticus
niloticus
niloticus
niloticus
niloticus
species, the horneyhead turbot (Pleuronichthys verticalis) collected in
southern California in areas receiving sewage inputs, strong upregulation of CYP1A in liver tissue was observed by microarray analysis, but
qPCR analysis of individual fish indicated high variability (Baker et al.,
2013). Elevated CYP1A activity (as measured by ethoxyresorufin Odeethylase (EROD) activity) and gene expression have been reported
previously for adult winter flounder from Jamaica Bay as compared to
Shinnecock Bay (Mena et al., 2006), and increased expression of
CYP1A mRNA has been reported in pooled samples of adult flounder
from Raritan Bay, NJ as compared to flounder collected from the less
urban NJ Atlantic coast (Straub et al., 2004). However, earlier work evaluating EROD activity and CYP1A protein levels in adult winter flounder
collected from sites around the northeastern Atlantic coast of the U.S.
reported uninduced flounder could only be found at the remote area
of Georges Bank off the New England coast or Northern ME, while coastal flounder, particularly flounder sampled from Long Island Sound,
showed highly variable, elevated levels (Monosson and Stegeman,
1994). A previous study evaluating CYP1A and PLEUR expression in
YOY winter flounder collected from six Long Island bays including two
that were investigated in this present study (Jamaica and Shinnecock
Bays) also did not find site specific differences in hepatic CYP1A expression in YOY winter flounder (Romany et al., 2015). Data from the
USEPA's National Coastal Assessment indicates that PCBs are found in
somewhat elevated levels in sediments from all of the sites examined
(Supplementary File 1), therefore elevated and variable expression of
CYP1A in YOY flounder from throughout the area is not surprising.
The depressed expression of CYP1A in flounder from Hempstead Bay
was unexpected. Increased expression of CYP1A is commonly found in
studies evaluating exposure to oil pollution and chlorinated aromatic
hydrocarbons (Schlenk et al., 2008a), but there have been reports
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
6
A.E. McElroy et al. / Comparative Biochemistry and Physiology, Part D 14 (2015) xxx–xxx
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
et al. (2013) conducted a study examining condition status of resident
European eel (Anguilla anguilla) chronically exposed to a suite of
metal and organic pollutants in three Belgian river basins evaluating
condition factor and lipid reserves, expression of CYP1A and metallothionein (MT), and bioaccumulation of metals, PCBs and organochlorine
pesticides (DDTs), comparing the environmentally exposed river eels to
eels raised under clean conditions in the laboratory. Although expression levels of CYP1A were significantly elevated in eels from all river locations as compared to the eels raised in captivity, variability was
extremely high in the wild eels, with fish from some of the most polluted sites showing the lowest levels of CYP1A expression. In fact PCA analysis revealed a negative correlation between PCB concentrations and
expression of CYP1A in the liver of the eels examined. Co-exposure to
AhR antagonists such as cadmium and tributyltin and even some
PAHs such as fluoranthene and 2-aminoanthracene can diminish response to AhR agonists (Schlenk et al., 2008b). Development of resistance to model AhR agonists has also been reported in several species
(Elskus, 2001), and can confound interpretation of gene expression
data (Hoffmann and Willi, 2008; Oleksiak, 2010). It is possible that the
flounder from Hempstead Bay are less sensitive to inducers, or possibly
are responding to other confounding factors such as the presence of AhR
antagonists as has been observed in other studies. Further work will be
needed to identify the causes of the relative CYP1A depression at this
site.
Vitellogenin is an egg yolk precursor protein that is synthesized by
the liver during oocyte development under control of the estrogen receptor. Elevated expression of VTG in male or juvenile fish is a commonly used biomarker of exposure to estrogen or chemicals that mimic
estrogen in wild caught fish (Heppell et al., 1995; Tyler et al., 1998).
Expression of VTG mRNA showed the most consistent trends with
respect to the urban gradient on Long Island, with values generally
decreasing from west to east (Fig. 5B). Median levels were highest in
Jamaica Bay in the west and lowest in Shinnecock Bay, with expression
at Jamaica Bay being significantly elevated as compared to both
Shinnecock Bay and Napeague Harbor. However, median expression
levels in flounder from Cold Spring Pond were higher than the other
two eastern sites, making expression levels at this site not significantly
different from the three western sites. Further work should attempt to
identify agents inducing VTG in flounder from this site.
Elevated VTG in the more urban sites is consistent with sewage inputs and agrees well with elevated levels of sewage tracer compounds
measured in the sediments of Jamaica and Hempstead Bays (Doherty,
2013), and extremely high levels (10 s of ppm) of nonylphenol previously reported in Jamaica Bay sediments (Ferguson et al., 2001). It is
also consistent with previous studies showing elevated expression of
VTG protein in YOY winter flounder from Jamaica Bay as compared to
Shinnecock Bay (McElroy et al., 2006) as well as female biased sex ratios
observed in another local fish, the Atlantic silverside (Menidia menidia)
from more urban bays around Long Island (Duffy et al., 2009). Straub et
al. (2004) previously reported elevated VTG expression in pooled samples of adult winter flounder from a more urban area of NJ as compared
to a less contaminated site in southern NJ. Both Vtg1 and Vtg2 as well as
several other estrogen responsive transcripts were also found to be upregulated in the horneyhead turbot study of southern California sewage
exposed flatfish (Baker et al., 2013).
A number of environmental factors in addition to exposure to
chemical contaminants have been shown to influence both CYP1A and
VTG expression. Hypoxia has been shown to down regulate CYP1A in
zebrafish (Prasch et al., 2004), and Rahman and Thomas (2012) have
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
A.E. McElroy et al. / Comparative Biochemistry and Physiology, Part D 14 (2015) xxx–xxx
A
B
C
D
7
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
expression in Atlantic croaker through alterations of nitric oxide and
oxidant status through a pathway involving interleukin 1ß and hypoxia
inducible factor (HiF). Hypoxia has also been shown to act as an antiestrogen, masculinizing fish through downregulation of aromatase
activity that converts androgens to estrogen (Thomas and Rahman
2009). Alternatively, increased temperature has been found to increase
VTG expression in response to exogenous estrogens in the mummichog
Fundulus heteroclitus (Chandra et al., 2012). Temperature may have enhanced VTG response to environmental estrogens in flounder from both
Jamaica Bay and Cold Spring Pond. The relative young age of these fish
may also be limiting the range of response observed in these variables.
For both these genes, mRNA expression represents recent exposure to
inducers (Kloepper-Sams and Stegeman, 1987; Hemmer et al., 2002).
Including an assessment of plasma VTG protein expression and catalytic
activity of CYP1A would provide a more complete picture of chronic exposure in YOY winter flounder. Analysis of contaminant levels in sediments at the site of collection and in fish tissues would also provide
additional valuable information on exposure, but does not diminish
the importance of field data on biological responses, which account for
factors affecting bioavailability as well as the possibility of detecting responses 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
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
show raw qPCR data relative to total RNA for cytochrome P4501A (CYP1A), pleurocidin
(PLEUR), complement C3 (C3), hepcidin (HEP), vitellogenin (VTG), glutamate decarboxylase (GAD), and phospholipase A2 (PLA2). Glucokinase (GCK), and glycerol 3-phosphate
dehydrogenase (GPDH). Asterisk denotes significant differences between populations in
all three groups. CYP1A, PLEUR and VTG also show agreement between all three groups
of data in that there were no significant differences in relative expression irrespective of
method of analysis.
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 disease resistance, and increase morbidity (Pickering and Pottinger,
1989; Bols et al., 2001; Fast et al., 2008). In particular, exposure to sewage or contaminated water sources is known to have immunosuppressive activity (Kennedy and Farrell, 2008). Four genes associated with
inflammation/immune response were evaluated in this study, and all
but pleurocidin (PLEUR), showed significantly different patterns in expression among sites (Fig. 6).
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
8
A.E. McElroy et al. / Comparative Biochemistry and Physiology, Part D 14 (2015) xxx–xxx
A
B
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
innate molecule effective at killing Gram positive and negative bacteria
(Cole et al., 1997; Patrzykat et al., 2003). In YOY winter flounder expression in the liver is much lower than that observed in fin tissue (Romany
et al. 2015). As with CYP1A, Romany also did not see site related differences in her study of YOY winter flounder from different bays of Long
Island (Romany et al., 2015). PLEUR was also not found to be differentially expressed in the RNAseq analysis comparing pooled samples
from Moriches and Shinnecock Bays, though pleurocidin (PL) 8–9, a potential pseudogene was only observed in the Moriches population and
not the Shinnecock population (Table 2).
In contrast to PLEUR, hepcidin II (HEP), a peptide hormone produced
by the liver, known to have antimicrobial activity and be involved in
regulating iron homeostasis, was differentially expressed between
sites (Fig. 6B), although not in a manner associated with a west to east
urban gradient. Hepcidin, in particular its iron sequestration role, is important in vertebrate responses to combat bacterial infection and deal
with septicaemia. Hepcidin is consistently produced by cells that are
crucial to the generation of an effective immune response against
acute infection and HEP can affect disease pathogenesis (Armitage
et al., 2011; Ba Sow et al., 2008; Frazier et al., 2011). Highest levels of expression were observed in flounder from Hempstead and Moriches
Bays from the west, and Cold Spring Pond from the east. Lowest levels
of expression were observed at the two ends of the sampling area,
Jamaica in the west, and Napeague in the east. Straub et al. (2004)
also reported upregulation of HEP in flounder from a polluted site in
NJ. Hepcidin is an acute phase reactant and anti-microbial peptide that
disrupts microbial membranes. Induction of hepcidin in response to
inflammatory stimuli is also a mechanism by which the animal can sequester and control iron availability to bacteria or other pathogens
that have initiated the inflammatory stimuli (Weinstein et al., 2002;
Nemeth et al., 2003). Anemia, often observed in chronic inflammatory
states, can be a result of hepcidin-induced sequestration of iron in the
macrophage. HEP transcription can be affected by hypoxia, since the
promoter region of HEP has a hypoxia-inducible transcription factor
(Hif-1a) binding site (Peyssonnaux et al., 2007). Hypoxia stress has
been shown in other teleosts to downregulate inflammatory responses
(Choi et al., 2007). Interestingly, Jamaica Bay with greatest incidence of
hypoxia showed the lowest expression of HEP.
Transcripts of another acute phase protein, complement C3 (C3),
also differed in their expression significantly among sites (Fig. 6C),
with expression levels being significantly lower at two of the three
more eastern sites (Shinnecock and Napeague) as compared to all
three western sites and Cold Spring Pond in the east, similar to patterns
observed in VTG expression. Elevated C3 expression could indicate a
generally higher level of pathogen exposure and/or inflammation in
flounder from more urban areas. Straub et al. (2004) also reported elevated expression of C3 in pooled samples of adult winter flounder from
Raritan Bay, as compared to a reference site in NJ. Relative expression of
C3 was much higher than CYP1A, VTG and PLEUR in this study. Similar results have been reported in the Senegalese sole, (Solea senegalensis),
where high expression of hepatic C3 was observed, especially in comparison to the kidney (N 5000 ×; Prieto-Alamo et al., 2009) which is
one of the primary sites of hematopoiesis and immunological function
in teleosts (Kibenge et al., 2012). Strong hepatic C3 upregulation in
response to lipopolysaccharide (LPS) challenge was also reported for
the Senegalese sole, while other flatfish, salmonids and even zebrafish
have shown differential expression of C3 (among other complement
proteins: C3a, C2b, etc.) in immune related organs after immunization
against rhabdoviral (viral hemorrhagic septicemia virus and infectious
hematopoietic necrosis virus) infection (Byon et al., 2006; MacKenzie
et al., 2008; Encinas et al., 2010). These studies and others speak to
the importance of complement C3 as a central mediator in classical
and alternative complement activation and the complement cascade,
an integral part of innate immunity (Whyte, 2007).
The fourth inflammatory/immune responsive gene assessed
was phospholipase A2 (PLA2). Significant site-specific differences were
also noted in the expression of this gene (Fig. 6D), yet expression
was only significantly elevated in flounder from Moriches Bay as
compared to flounder collected from all other sites. PLA2 is an important catalyst within the arachidonic acid pathway, leading to the formation of inflammatory and thrombogenic compounds (Pruzanski and
Vadas, 1991). High levels of hepatic expression of PLA2, C3 and HEP
in Moriches Bay flounder may indicate that these fish have enhanced
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
A.E. McElroy et al. / Comparative Biochemistry and Physiology, Part D 14 (2015) xxx–xxx
9
A
B
C
D
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
Hempstead Bays where even more degraded conditions may lead to
immunosuppression.
Comparing data for all four immune response genes, HEP showed
the most discriminating power between the sites, and PLEUR the least.
PLEUR expression has been described as increasing with age in winter
flounder (Douglas et al., 2001), and work by Romany et al. (2015),
showed increased expression of this gene in fin tissue of larger winter
flounder compared to YOY winter flounder from a north shore Long
Island bay population as well. The absence of site-specific variation in
PLEUR observed in these studies may reflect an immature and poorly
responsive state of this anti-microbial peptide in YOY flounder, possibly
due to an incompletely developed immune system at this life stage. HEP
on the other hand, showing the most significant differences among
sites, may be more responsive to environment factors. HEPII (the transcript measured in our work) was also found to be significantly upregulated in sewage impacted flatfish in the horneyhead turbot study (Baker
et al. 2013). Further work should be focused on process-oriented studies
to identify factors associated with both up and downregulation of
immune responsive genes in juvenile winter flounder collected from
urban estuaries. While environmental factors such as hypoxia may affect these genes, pathogen exposure will as well, and both stressors
may change seasonally and annually at different time scales across
sites, making identification of clear and consistent patterns difficult. Future work should evaluate how expression patterns in these immune
response genes are associated with either pathogen load or susceptibility to pathogen exposure.
3.5. Measures of glucose and glycogen metabolism
The majority of differentially regulated genes identified by Illumina
differential expression from Moriches and Shinnecock Bays were associated with glycolysis and glucose metabolism. These data supported the
decision to evaluate several key genes in these pathways using qPCR
analysis of individual flounder from all six study areas. Statistically significant differences in expression among the sites were observed for
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
10
A.E. McElroy et al. / Comparative Biochemistry and Physiology, Part D 14 (2015) xxx–xxx
A
B
C
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
studied, expression patterns did not consistently vary along the west
to east gradient examined. Glucokinase (GCK) gene expression was significantly elevated at Moriches as compared to all other sites (Fig. 7A).
Similar to HEP, GCK also has a Hif binding site within its promoter region; however it is upregulated in response to hypoxia (Barker et al.,
2012; Roth et al., 2004) potentially to increase anaerobic glucose metabolism. For glutamate decarboxylase (GAD) expression, although the
difference between Moriches and Shinnecock was statistically significant, expression in flounder from Moriches was not significantly
different from expression levels in flounder from the rest of the sites
(Fig. 7B). Glycerol 3-phosphate dehydrogenase (GPDH) gene expression
was depressed in flounder from both Shinnecock Bay and Napeague
Harbor as compared to flounder from both Jamaica and Moriches
Bays, but not Hempstead Bay or Cold Spring Pond (Fig. 7C). VidalDoresch et al. (2013) also found GPDH to be mildly depressed in
horneyhead turbot exposed to 5% sewage effluent in the laboratory. It
should be noted that GAD was expressed at much higher levels than
either GCK or GPDH, and that GAD is the rate-limiting enzyme in
gamma-aminobutyric acid synthesis, (a neurotransmitter with a primary signaling role in the brain, but also found in the pancreas). In flounders and other members of Osteichthyes, a diffuse pancreas develops
in postlarvae and branches along veins running to the liver and the hepatic portal vein in the parenchymal tissue of the liver (Kurokawa and
Suzuki, 1995). Thus the presence of pancreatic tissue can be expected
in the liver samples collected from these flounder. The high levels of
GAD mRNA in flounder may therefore be linked to expression of high
levels of this enzyme in the liver and pancreas, as has been reported in
mammals (MacDonnell and Greengard, 1975). Hypoxia has also been
shown to enhance GAD activity of both isoforms 65 and 67 in mammals
(Kobayashi and Millhorn, 2001). As with the other gene families evaluated in this study, further experimental work will be needed to identify
specific factors driving expression of the glucose and glycogen metabolism genes evaluated.
3.6. Expression patterns among all the genes evaluated
Principal components analysis (PCA) was used to assess patterns in
expression of all the genes in individual flounder sampled. Principal
components (PC) 1 and 2 explained 62% of the variance in the data. As
can be seen in Fig. 8, where arrows show the direction and the relative
magnitudes of the loadings of individual variables on PC1 and PC2
(provided in Table 3), expression of all genes primarily project in
three directions. Along PC1, only CYP1A is positive, albeit with a relatively small loading of 0.09. The three variables with the greatest loadings
were HEP, GCK, and PLA2 which had the largest absolute values of loadings in both PC1 and PC2. The immune response gene HEP stands out as
being most positive on PC1 (loading = 0.69), but another immune response gene, C3, also plots on this axis. CYP1A plots opposite the axis
dominated by HEP indicating a negative correlation between CYP1A
and HEP expression. The other two immune response genes (PLEUR
and PLA2) and all the glucose and glycogen metabolism genes (GCK,
GAD, and GPDH) plot approximately 90° to the axis defined by HEP,
with GCK being most negative on PC2 (loading = −0.56). Also plotting
along this axis with fairly large loading values are the other two immune
response genes, PLA2 and PLEUR (loadings = −0.31 and −0.16 respectively). It should be noted that although the PCA displays the relationship in expression between these genes in individual flounder, it
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
A.E. McElroy et al. / Comparative Biochemistry and Physiology, Part D 14 (2015) xxx–xxx
11
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
at higher levels of biological organization such as physiological or population level responses.
Interactions between inflammation/innate immunity and glucose
and glycogen metabolism have been well described in mammalian
models. Starved rats have shown both significantly increased and reduced PLA2 levels, but more importantly PLA2 alterations may contribute to the diminished insulin secretory response of islets from starved
rats to relatively low concentrations of glucose. Interleukin-1, another
pro-inflammatory marker, has also shown to decrease GAD activity
and mRNA expression when administered with glucose to rat islet
cells (Velloso et al., 1994). These data in rats suggest that active inflammatory responses within the same tissues may co-regulate these glucose and glycogen metabolism genes. To the authors' knowledge the
current study is the first evidence that this co-regulation may be occurring in fish tissues.
Additional information can be gleaned from site-specific patterns in
the distribution of individual flounder on the biplot (Fig. 8). Flounder
from Moriches Bay are grouped in the left two quadrants. Flounder
from the other locations primarily project on the right two quadrants
with flounder from Napeague Harbor grouped in the lower right quadrant. The PCA analysis indicates that expression patterns of the genes
investigated in this study can at least partially differentiate the subpopulations studied along the south shore bays of Long Island, and supports
our hypothesis that mRNA expression profiles in the flounder from the
more urban areas differ from those from less impacted eastern habitats.
These results also support the inclusion of both immune responsive and
intermediary metabolism genes in expression studies evaluating population level responses in gene expression.
The results of this study are consistent with data emerging as part
of the European Union (EU) funded multinational study GENIPOL initiative on the European flounder Platichthys flesus (Williams et al., 2008;
Falciani et al., 2008; Williams et al., 2011). As described by Williams et
Table 3
Loadings from all gene PCA.
Genes
PC1
PC2
CYP1A
VTG
PLEUR
C3
HEP
PLA2
GCK
GAD
GPDH
0.0928
−0.0973
−0.1226
−0.1677
−0.6410
−0.4007
−0.5812
−0.1114
−0.1309
−0.2240
0.0241
−0.1571
0.1254
0.6945
−0.3171
−0.5626
−0.0411
−0.0879
al. (2011), levels of VTG and CYP1A protein were highly variable
among sites, and were not generally different among the more contaminated sites, despite large differences in contaminant exposure. However taking a systems biology approach looking at network analysis, they
found significant associations between measures of liver histopathology, altered metabolism and toxicology. In earlier work by this same
group examining gene expression patterns by microarray, the ability
to distinguish patterns among sites was significantly improved when
data from field caught flounder was combined with responses observed
in controlled laboratory exposures (Falciani et al., 2008). More recent
work coming out of the Southern California Coastal Water Research Project (SCCWRP) on another flatfish, adult horneyhead turbot (Baker
et al., 2009, 2013; Vidal-Dorsch et al., 2013, 2014) also reveals complex
patterns in gene expression, with many of the same transcripts examined in this study (CYP1A, VTG, HEP, GPDH) being differentially regulated
in field caught turbot from sewage impacted areas of southern California and in turbot exposed to sewage effluent in the laboratory. However
in the SCCWRP studies the direction of response was not always the
same, with the laboratory exposed fish generally showing downregulation as compared to controls, while many of these transcripts were
upregulated in field caught organisms. These data indicate that additional environmental variables may be at play, or that less persistent
components of the sewage effluent (that field collected fish might not
see) may also be influencing expression of these genes, or that some
level of adaptation has occurred in the field caught fish. Indeed the
lack of large differences in expression among fish collected from the different sites in our study could be interpreted as supporting some level of
adaptation in these populations, and that conditions between these
bays are not so different. In our case the extremely small effective
stock size contributing to local populations of winter flounder
(O'Leary et al., 2013) may have already selected for individuals better
adapted to degraded habitats at some of the sites investigated. Across
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
this life stage, which in itself may be the most impactful finding of the
study. However, the specific stressors that are driving these responses
within sites, be they changes in temperature, hypoxia, salinity, contaminants or likely different combinations of these, requires targeted analyses at transcriptional, translational and functional levels.
4. Conclusions
This is to our knowledge the most comprehensive quantitative analysis of gene expression in wild caught winter flounder conducted to
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
tool for biomarker identification and provides a rich dataset that can be
utilized in future work on winter flounder and other related species. Despite high inter-individual variability in all genes investigated, statistically significant, site-specific differences were observed in expression
of all but one gene evaluated. However patterns in expression were
complex, with only VTG demonstrating a strong west to east gradient
consistent with known loadings of municipal sewage effluent and/or
groundwater septage. Gene expression patterns observed suggest that
contaminant exposure or stimuli driving CYP1A has a greater opposing
effect on immunological status than other factors influencing glucose
or glycogen metabolism. The links between genes associated with glucose and glycogen metabolism and some genes associated with immune
response suggest that common environmental factors may be influencing both systems. Now that this first step has been taken to identify
major pathways impacting YOY flounder in these environments and
biomarkers associated with them, follow-up work should be done to examine mechanistic links between biomarker expression, physiological
responses and survival in this species. A subsequent modeling study
evaluating the associations between gene expression evaluated here,
several measures of condition and otolith microgrowth increments
revealed statistical associations between growth and condition and
CYP1A, PLEUR, and GPDH indicating gene expression patterns observed
here link to higher order ecologically significant responses (Gallagher
et al. in press). Through the use of interdisciplinary approaches such
as these we can identify factors associated with reduced survival in
YOY winter flounder survival, and hopefully begin to address the
population's recovery.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.cbd.2015.01.006.
Acknowledgments
Funding for this project was provided by the National Marine Fisheries Service Saltonstall Kennedy Program (award #NA10NMF4270202)
to M. Frisk, M. Fast, and A. McElroy, and an award from the NY State Department of State to A. McElroy and M. Frisk, and the Novartis Chair at
PEI University to M. Fast. Sampling assistance was generously provided
by the Towns of East Hampton and Hempstead, the New York Department of Environmental Conservation, and students and staff of the
School of Marine and Atmospheric Sciences at Stony Brook University.
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