Article
pubs.acs.org/est
Distinctive Metabolite Profiles in In-Migrating Sockeye Salmon
Suggest Sex-Linked Endocrine Perturbation
Jonathan P. Benskin,*,†,‡ Michael G. Ikonomou,‡ Jun Liu,† Nik Veldhoen,§ Cory Dubetz,‡
Caren C. Helbing,§ and John R. Cosgrove†
†
AXYS Analytical Services Ltd. 2045 Mills Road West, Sidney, British Columbia V8L 5X2, Canada
Institute of Ocean Sciences, Fisheries and Oceans Canada, 9860 West Saanich Road, Sidney, British Columbia V8L 4B2, Canada
§
Department of Biochemistry & Microbiology, University of Victoria, P.O. Box 1700 Stn CSC, Victoria, British Columbia V8W 2Y2,
Canada
‡
S Supporting Information
*
ABSTRACT: The health of Skeena River Sockeye salmon
(Onchorhychus nerka) has been of increasing concern due to
declining stock returns over the past decade. In the present work,
in-migrating Sockeye from the 2008 run were evaluated using a
mass spectrometry-based, targeted metabolomics platform. Our
objectives were to (a) investigate natural changes in a subset of the
hepatic metabolome arising from migration-associated changes in
osmoregulation, locomotion, and gametogenesis, and (b) compare
the resultant profiles with animals displaying altered hepatic
vitellogenin A (vtg) expression at the spawning grounds, which
was previously hypothesized as a marker of xenobiotic exposure. Of
203 metabolites monitored, 95 were consistently observed in
Sockeye salmon livers and over half of these changed significantly
during in-migration. Among the most dramatic changes in both sexes were a decrease in concentrations of taurine (a major
organic osmolyte), carnitine (involved in fatty acid transport), and two major polyunsaturated fatty acids (eicosapentaenoic acid
and docosahexaenoic acid). In females, an increase in amino acids was attributed to protein catabolism associated with
vitellogenesis. Animals with atypical vtg mRNA expression demonstrated unusual hepatic amino acid, fatty acid, taurine, and
carnitine profiles. The cause of these molecular perturbations remains unclear, but may include xenobiotic exposure, natural
senescence, and/or interindividual variability. These data provide a benchmark for further investigation into the long-term health
of migrating Skeena Sockeye.
■
INTRODUCTION
km up the Skeena River to the Babine Lake spawning grounds
and, over the course of migration, undergo dramatic
physiological and morphological changes. Sex steroids (e.g.,
estradiol and testosterone), associated with stimulated gametogenesis, are responsible for dramatic alterations in the physical
appearance of the salmon. These changes generally occur over a
period of weeks to months and reach their peaks toward the
end of in-migrations.5
A recent examination of hepatic gene expression profiles in
Sockeye from the Fraser and Skeena Rivers revealed marked
changes in sex-specific gene expression during the 2008 Skeena
spawning migration.6 Among the changes was increased
variability in the expression of mRNA encoding vitellogenin
A (vtg), the main phospholipoglycoprotein used to transport
lipids from storage sites to the oocytes during gonadal
The Skeena River watershed is the second largest freshwater
habitat for Sockeye salmon (Onchorhychus nerka) in British
Columbia (BC), Canada (Figure 1). Within this region, the
spawning grounds of Fulton River and Pinkut Creek on Babine
Lake account for the majority of total Skeena Sockeye
production.1 Installation of spawning channels and flow
controls at these sites in the 1970s produced a rapid increase
in stock returns (catch plus escapement) up until the mid
1990s.2 A concomitant drop in returns and production began in
the Babine Lake system in the early 2000s and has since
continued unabated over the past decade, despite declines in
exploitation.3 In August 2013, Fisheries and Oceans Canada
placed a moratorium on fishing activities along the Skeena
River, including its lakes and tributaries.4
The average Sockeye lifecycle is 4 years, consisting of one or
more years in the ocean followed by a return to natal spawning
grounds. Prior to in-migration, salmon cease feeding and rely
solely on muscle and lipid stores as fuel for locomotion,
gametogenesis, and osmoregulation. Sockeye migrate over 500
© 2014 American Chemical Society
Received:
Revised:
Accepted:
Published:
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September 3, 2014
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molecular perturbation. Taken together, the results form a
baseline of metabolite data against which future stock
assessments can be compared for ongoing management of
the health of Skeena River Sockeye.
■
MATERIALS AND METHODS
Metabolite Targets of Interest. A total of 203 metabolites
were investigated, including 21 amino acids (AAs), 20 biogenic
amines (BAs), 40 acylcarnitines (ACs), 89 phosphatidylcholines (PCs), 15 sphingomyelins (SMs), ∑hexose (Hex), and 17
fatty acids (FAs). This panel represents a diverse set of
metabolites covering multiple systems (a subset of the entire
“metabolome”) suitable for probing a variety of health end
points in a wide range of species. A full list of analytes, internal
standards, and abbreviations is provided in Tables S1−S4 of the
Supporting Information (SI).
Sampling. Sampling of adult Sockeye salmon took place
during the 2008 spawning migration at the mouth of the
Skeena River (July 29−30; n = 41 genotypic males, n = 35
genotypic females; all identified as Fulton stock), and spawning
grounds in Pinkut Creek (September 11; n = 24 genotypic
males and n = 26 genotypic females), and Fulton River
(September 24; n = 23 genotypic males and n = 20 genotypic
females) using capture by gill or seine nets. Fish were killed
immediately following capture by a blow to the head; after
which scales were removed for DNA fingerprint-based stock
identification and liver sections were removed for analysis of
mRNA transcript abundance. Remaining liver samples were
placed in individual Whirl-pak bags on ice and then stored at
−20 °C immediately upon return to the Skeena regional
laboratory. The time between liver removal and placement of
samples into freezers (2 h) was kept relatively constant to
minimize handling-associated effects on metabolite levels across
all sample collection sites. Following shipment to the Institute
of Ocean Sciences, liver samples were stored at −50 °C prior to
analysis.
Genotypic and phenotypic anchoring. Stock assessment
via DNA-based fingerprinting was conducted on all animals at
the Pacific Biological Station Molecular Genetics Laboratory
(Nanaimo, BC).12,13 Salmon from the mouth of the Skeena
were confirmed as Fulton Stock. For animals collected at the
spawning grounds, the results of DNA-based stock assessment
matched the site of collection in all instances (i.e., animals
sampled at Fulton were exclusively Fulton stock while animals
sampled at Pinkut were exclusively Pinkut stock). Salmon were
grouped according to genotypic, gametic, and molecular
phenotypic sex using methods described in detail elsewhere.6
Briefly, genotypic sex was assessed via a quantitative real-time
polymerase chain reaction (qPCR) assay using genomic DNA
from each fish with amplification of the male-specific OTY2WSU locus as described in Veldhoen et al. (2010, 2013).6,14
Gametic sex was assessed during collections by visual
confirmation of the presence of milt or roe. Molecular
phenotypic sex was based on relative levels of qPCR-derived
hepatic vtg mRNA abundance, with the female phenotype
displaying vtg transcript levels of ≥100 and the male phenotype
associated with values of <100. These thresholds were
established based on previous analyses of Fraser River during
the 2007 spawning migration in which values of <25 and >13
000 were consistently observed for males and females,
respectively.14 Animals were then grouped using a 3-letter sex
classification system, in which the first two letters denote
genotypic and gametic sex, respectively, and the third letter
Figure 1. Map of Babine lake showing locations of Fulton River and
Pinkut Creek spawning grounds as well as the locations of the
Newman Penninsula (Green Star), and Sterret Island (Red Star)
mines. Inset shows sampling locations within BC relative to Vancouver
and the Fraser River.
development.7 The production of VTG occurs primarily in the
liver and is mediated by estradiol produced in the developing
ovary.8 Sex hormone production is stimulated by pituitary
gonadotropin release, which is in turn controlled by
hypothalamic gonadotropin-releasing hormone.9 While male
fish possess the vtg gene, it is expressed in low abundance under
normal circumstances compared to females, resulting in
relatively higher hepatic VTG concentrations in females when
compared to males. On account of this sexually dimorphic
characteristic, vtg expression in males and juveniles has been
used as a sensitive biomarker of exposure to xenoestrogens
and/or endocrine disrupting compounds (EDCs).10,11 Surprisingly, almost all genetic male Pinkut salmon examined from the
2008 spawning migraton displayed hepatic vtg mRNA levels
consistent with those observed in genetic females. The
underlying causes of this biological perturbation were unclear,
but possible explanations included mobilization of lipophilic
endocrine-disrupting contaminants during migration, in-river
exposure to environmental contaminants, or natural phenomena.6
In the present work, hepatic metabolite profiles of a subset of
animals from the 2008 migration were investigated using a
recently developed, targeted metabolomics platform. Our
objectives were two-fold. First, we sought to characterize
changes in a subset of metabolites within the hepatic
metabolome (this select metabolite panel is referred to herein
as simply “the hepatic metabolome”) over the course of
migration related to osmoregulation, locomotion, and gametogenesis. Second, we were interested in characterizing
biochemical profiles in salmon with altered vtg mRNA
expression, to ultimately determine which metabolic systems
were affected in these fish and suggest possible causes of this
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Figure 2. PLS-DA scores plot (A) and loadings plot (B) of migrating male salmon (Fulton Stock) sampled at the mouth of the Skeena and the
Fulton River spawning grounds. Taurine (i) and fatty acids (iii−v) are circled on the loadings plot. Concentrations of these metabolites changed
significantly during the migration and contributed to the separation of groups observed in the scores plot.
identifies molecular phenotypic sex (e.g., “MMF” describes a
genetic and gametic male with female hepatic vtg expression
levels; SI Figure S1).
Tissue Preparation. Metabolomic analysis was performed
on a subset of animals previously examined by Veldhoen et al.,6
which were of a single sex (e.g., MMM or FFF) or displayed an
altered molecular phenotype with respect to the hepatic
transcriptome (e.g., MMF or FFM). The few salmon within
the remaining sex classification groups were not examined due
to insufficient replicates. Thus, relative to Veldhoen et al.,6 a
total of 156 out of 169 Skeena watershed animals were
examined in the present study.
Prior to metabolite extraction, frozen livers were weighed,
supercooled with liquid nitrogen, and shattered with a pestle.
Several pieces of tissue were ground to a fine powder using a
mortar and pestle and liquid nitrogen. A portion (∼0.5 g of
tissue homogenate) was placed in a precooled 15 mL centrifuge
tube (Corning, NY, U.S.A.). A single liver sample was analyzed
for each animal. Following addition of methanol (MeOH; 6
mL/g tissue), each sample was subjected to a 30 s vortex
followed by 30 s of centrifugation (100g) and collection of the
supernatant. After repeating the sample extraction procedure
twice more, the three MeOH extracts were combined and a
portion (10 μL) was added to a 96-well filter plate (Pall
Corporation, Port Washington, NY, U.S.A.) which had been
preloaded with internal standards. A full list of internal
standards is provided in SI Tables S1 (AAs and BAs), S2
(∑hexose), S3 (ACs, PCs, and SMs), and S4 (FAs). The
extracts were dried; after which AAs and BAs were derivatized
using Edman’s Reagent. After further drying, 250 μL of 5 mM
ammonium acetate in MeOH was added to each well; the plate
was shaken for 30 min and samples eluted into a Nunc 96-deep
well plate (Thermo Scientific, Waltham, MA, U.S.A.) by
centrifugation (100g for 2 min at ambient temperature) using a
Sorvall Legend RT+ centrifuge (Thermo Scientific). Each
sample was subsequently diluted by an equivalent volume of
water prior to analysis.
Method Validation. Ineffective removal of metabolites
from tissue may contribute to underestimates of tissue
metabolite concentrations and increased data variability. To
evaluate the effectiveness of our method for exhaustive
extraction of metabolites, extraction efficiency experiments (n
= 5) were performed. Following extraction using the optimized
protocol (i.e., 3 × MeOH), spent liver tissue was re-extracted
using the same procedure and then a third time using an
orthogonal approach (3 × chloroform). The resulting primary,
secondary, and tertiary extracts were treated as individual
samples. Extraction efficiency (%) was determined as the
concentration of metabolite in the primary extract divided by
the sum of the concentration in the primary, secondary, and
tertiary extracts. Analytical accuracy and precision were also
assessed using spike/recovery experiments, whereby fish liver
extracts were analyzed following fortification with native
standards. A total of 3 different spiking levels were tested
with n = 5 replicates per experiment. The concentration in
unfortified extract was subtracted from the concentration
obtained in the spiked sample, which was then compared to
the theoretical amount.
Sample Analysis. Metabolite analysis was carried out using
an Agilent 1100 high performance liquid chromatography
(HPLC) system coupled to an API4000 triple quadrupole mass
spectrometer (Applied Biosystems/Sciex, Concord, ON,
Canada). AAs and BAs were analyzed as phenylthiocarbamyl
derivatives by HPLC−tandem mass spectrometry (HPLC−
MS/MS). ∑Hexose and FAs were analyzed separately by
HPLC−MS/MS. All analytes were quantified by isotope
dilution using authentic native standards and either identical
or homologous isotopically labeled internal standards and a
quadratic calibration curve with 1/x weighting. ACs, SMs, and
PCs were analyzed by flow-injection tandem mass spectrometry
(FI−MS/MS), and following deconvolution of overlapping
isotopic peaks,15 were quantified relative to an internal standard
(i.e., single point internal quantification). Detection limits were
defined as the concentration at a signal-to-noise ratio of 3 for
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Figure 3. PLS-DA scores plot (A) and loadings plot (B) of migrating female salmon (Fulton Stock) sampled at the mouth of the Skeena and the
Fulton River spawning grounds. Amino acids (i), taurine (ii) and fatty acids (iii and iv) are circled on the loadings plot. Concentrations of these
metabolites changed significantly during the migration and contributed to the separation of groups observed in the scores plot.
HPLC−MS/MS data or the mean concentration of the blank
+3σ for FI-MS/MS data.
Data Handling and Statistical Analyses. Targets which
were detected at ≤50% frequency in all sex/location subgroups
were not included in multivariate statistical analysis. To the
remaining 95 targets, which included 26 AAs and BAs, 14 FAs,
∑hexose, 4 ACs, 45 PCs, and 5 SMs, values below limits of
detection (LODs) were replaced with a randomly generated
number between 0 and the detection limit as described
elsewhere.16 Not all data were normally distributed, thus
statistical significance (i.e., p < 0.05) was evaluated among sex/
location subgroups using the Mann−Whitney U test. Principle
Components Analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) models were applied to data which
had been mean-centered and divided by the square root of
standard deviation of each variable (Pareto scaling). Ten-fold
cross validation of the PLS-DA models produced accuracy, R2
and Q2 values of 0.98, 0.87, and 0.80 and 0.96, 0.90, and 0.83
for males and females, respectively, in 2 components (SI
Figures S2A and S3A). Permutation testing for both models
produced p-statistics of <0.01 (SI Figures S2B and S3B). All
statistical analyses were carried out using MetaboAnalyst 2.0
(www.metaboanalyst.ca).17
method still displayed good precision and were therefore
included for comparison on a qualitative basis. Considering the
number and diversity of substances examined in the present
work, the method was highly effective for reproducible and
accurate quantification of metabolites in salmon liver.
Metabolomic Changes during in-Migration. To
elucidate natural changes in metabolite profile over the course
of migration, the hepatic metabolome of Fulton River stock
collected at the mouth of the Skeena River prior to in-migration
was compared to that of the same fish stock sampled 9 weeks
later at the spawning grounds (SI Tables S13 and S14). We
limited our comparisons to only salmon displaying consistent
genetic and phenotypic sex assignments (i.e., MMM and FFF).
PLS-DA scores and loadings plots for migrating males and
females are provided in Figures 2 and 3, respectively (see SI
Figures S4 and S5 for corresponding PCA plots). Not
surprisingly, metabolite profiles in salmon from the mouth of
the Skeena highly contrasted those at the spawning grounds,
resulting in two clear groupings in the scores plots. These
differences are consistent with physiological changes associated
with osmoregulation, reproduction, and translocation.
A total of 54 metabolites in females (FFF) and 57
metabolites in males (MMM) were significantly (p < 0.05)
different between the Skeena River mouth and the Fulton River
spawning grounds. Among metabolites displaying the most
dramatic change over the course of migration was taurine (see
loadings plots in Figures 2 and 3), which was present at similar
concentrations in males and females at the mouth of the Skeena
(1666 ± 33.3 and 1712 ± 32.4 μg/g, respectively), but
decreased (p < 0.001) 3−4 fold at the Fulton River spawning
grounds (395 ± 15.8 and 597 ± 55.9 μg/g, respectively) (SI
Tables S13 and S14). Taurine is important in various biological
processes, including osmoregulation, immunomodulation, and
bile acid formation.18,19 As an osmolyte, it modulates cell
volume in response to changes in extracellular osmolarity,
which is particularly important given the metabolic sensitivity of
■
RESULTS AND DISCUSSION
Method Validation. Results are provided of spike/recovery
(SI Tables S5−S8) and extraction efficiency (SI Tables S9−
S12) experiments along with a detailed discussion of targetspecific method. With only a few exceptions (e.g., spermine,
spermidine, and putricine), validation experiments indicated
that metabolites were exhaustively extracted using the
optimized, 3 × MeOH extraction procedure. Spiked samples
were typically within 20% of the expected values with % RSDs
of <20%, regardless of fortification level, indicating good
accuracy and precision of the method. The few targets which
displayed sub-optimal extraction efficiency using the optimized
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fish liver cells to changes in cell volume.20 Taurine therefore
tends to be present at much higher concentrations in marine
organisms compared to their freshwater counterparts.21,22 The
significant reduction in hepatic taurine from the mouth of the
Skeena to the spawning grounds reflects such a role in animals
migrating from a highly saline to freshwater environment. A
similar finding was noted in Tilapia (Oreochromis mossambicus)
exposed to freshwater, salt water, and 200% saltwater, wherein
the lowest hepatic taurine concentrations were observed in
freshwater-exposed fish.23
Among hepatic FAs measured in Sockeye, docosaheptaenoic
acid (DHA; FA C22:6), eicosapentaenoic acid (EPA; FA
C20:5), and hexadecanoic acid (palmitic acid; FA C16:0) were
consistently present at the highest concentrations in both males
and females, regardless of location (SI Tables S13 and S14).
This observation is consistent with the major fatty acid
composition in plasma phospholipids in migrating Sockeye
reported by Magnoni et al.24 In that work, fatty acid
compositions in plasma phospholipids remained fairly constant
over the course of in-migration but a significant decrease in
plasma DHA (FA C22:6) was observed at the spawning site.
Our current observations demonstrate a decrease (p < 0.001) in
hepatic DHA (FA C22:6) and EPA (FA C20:5) over the course
of the spawning migration by 1.8 and 3.1 fold, respectively, in
males, and 1.6 and 1.8 fold, respectively, in females (SI Tables
S13 and S14). Palmitic acid was only observed to decrease in
males (p = 0.01) (SI Tables S13 and S14). Consistent with this
result was the decrease (p < 0.001) in carnitine levels from the
mouth of the Skeena to the Fulton River spawning grounds (SI
Tables S13 and S14). In males, carnitine levels decreased from
30.1 ± 1.55 μg/g to 8.37 ± 0.70 μg/g, while in females,
concentrations decreased from 32.1 ± 1.25 μg/g to 10.6 ± 1.55
μg/g. FAs are transported by carnitine from the cytosol into
mitochondria, where they undergo β-oxidation for use as
energy. Therefore, declines in carnitine are consistent with
declining hepatic fatty acid levels and diminished postmigratory
lipid reserves.
Lipids are used for a number of physiological processes
during in-migration of Sockeye, including gonad/gamete
development (∼6% of total mobilized lipids) and locomotion
(94% of mobilized lipids).25,26 Out of 45 detectable PCs, up to
24 were observed to change during migration in either sex (see
loadings plots in Figures 2 and 3 and concentrations provided
in SI Tables S13 and S14). In females, a decrease (p < 0.05)
was observed for 10 diacyl-, 6 acyl-alkyl, and 2 lyso PCs, while
only 3 diacyl- and 1 acyl-alkyl PCs increased significantly at the
spawning grounds. In contrast, decreases (p < 0.05) in 5 diacyl,
7 acyl-alkyl, and 3 lyso PCs and increases in 4 diacyl, 2 acylalkyl, and 3 lyso PCs were observed over the course of
migration in males. Concentrations of all 5 detected SMs were
not significantly different (p > 0.05) between males and females
at the mouth of the Skeena River, and SM C24:1 was always
the major species detected in both sexes, regardless of location.
In males, SM C24:1 and SM C26:1 increased at the spawning
grounds (p < 0.05) while in females, SM C16:0, SM C18:0, SM
C24:0, and SM C24:1, were all observed to increase
significantly (p < 0.05) postmigration. SMs play an important
role in signal transduction and apoptosis. The reason for an
increase in this class of lipid over the course of migration is
unclear; however, SMs and PCs were previously identified as
the major phospholipids in carotenoid-carrying lipoproteins
(CCLs) isolated from the high-density lipoprotein fraction of
serum in in-migrating male Chum salmon (Oncorhynchus
keta).27 CCLs are involved in transporting carotenoid pigments
(the most common in salmon being astaxanthin) from the
muscle to the integument and gonads during migration. The
dramatic change in skin pigmentation during breeding may be
explained by carotenoid mobilization to peripheral tissues.28
Not all metabolite concentrations changed significantly over
the course of migration. ∑hexose levels, for example, remained
constant regardless of location but markedly differed between
males and females at both the Skeena River mouth and Fulton
spawning grounds (6070 ± 430 μg/g in males versus 1500 ±
210 μg/g in females at the Skeena River mouth and 5180 ± 287
μg/g in males versus 1300 ± 160 μg/g in females at the Fulton
spawning grounds) (SI Table S13 and S14). While there are
few prior data on hepatic hexose concentrations in migrating
Sockeye salmon, a previous study reported higher hepatic
glycogen levels in male Fraser River Sockeye compared to
females throughout in-river migration.29 In that work, lower
hepatic glycogen in females was associated with a greater
increase in weight of the ovaries,30 and possibly also related to a
greater estrogenic hormone level produced during the rapid
sexual development associated with spawning migration.29 Our
present observations suggest normal maintenance of hexose
homeostasis over the course of the Skeena in-migration.
Perhaps the most remarkable change over the course of the
Sockeye migration, highlighted by PLS-DA analysis in Figures 2
and 3, involved AAs. In males (Figure 2), concentrations of
alanine, arginine, asparagine, citrulline, glycine, histidine,
isoleucine, leucine, methionine, proline, threonine, tyrosine,
and valine and all decreased (p < 0.05, Mann−Whitney U test,
SI Figure S6) between entry into the Skeena River and the
spawning grounds. Females (Figure 3), however, displayed
much higher amino acid concentrations at the spawning
grounds compared to commencement of in-migration. In fact, a
total of 16 AAs increased significantly over the course of
migration in females (p < 0.05, Mann−Whitney U test, SI
Figure S6), including aspartate, glycine, histidine, isoleucine,
leucine, lysine, methionine, ornithine, phenylalanine, proline,
serine, threonine, tryptophan, tyrosine, and valine. Glutamate,
which was present at the highest concentration of all AAs in
both males and females, increased (p < 0.01) in both sexes
during migration. Interestingly, this amino acid was previously
reported as the major amino acid in CCLs and apolipoproteins
in Chum27 and Pink (Oncorhynchus gorbuscha)31 salmon during
the spawning migration.
In addition to energy demands required for locomotion are
biosynthetic requirements for completion of oogenesis and
vitellogenesis; processes resulting in a 6-fold increase in gonadal
weight coincident with cessation of feeding until arrival at the
spawning grounds.32 The increase in hepatic amino acid
concentrations in females is consistent with previous studies,
which have described preferential utilization of protein-derived
AAs for oxidation and gluconeogensis to support gonad
maturation and oogenesis.32 Indeed, it has been estimated
that approximately half of a Sockeye’s white muscle (1200 g of
a 4000 g fish) is mobilized over the course of in-migration.32
Comparison of Sex-Based Differences in Hepatic
Metabolome in Fulton River and Pinkut Creek Stocks.
Of the 20 genetic females collected at the Fulton spawning
grounds, 8 animals (40%) displayed lower vtg mRNA levels
characteristic of genetic/gametic males based upon sexassociated abundance profiles for Sockeye previously established during the 2007 Fraser River spawning migration.6 This
is in contrast to genetic males (n = 23) at this location, none of
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Figure 4. Concentrations of amino acids in Sockeye from Fulton River (A) and Pinkut Creek (B). A statistically significant difference (p < 0.05;
Mann−Whitney U test) between FFF and FFM animals or between MMM and MMF animals is indicated by an asterisk (*) and circle (deg),
respectively.
which displayed induced vtg expression. In Pinkut, 4 of the 26
genetic females (15%) collected displayed male-like vtg mRNA
levels, while 20 of the 24 genetic males (83%) displayed a
female-like vtg transcript abundance profile.6 A subset of fish
collected at the spawning grounds (84 in total) was assessed for
hepatic metabolome profile including sexually consistent
animals (n = 16 MMM and n = 12 FFF from Fulton; n = 4
MMM and n = 22 FFF from Pinkut) and atypical vtg status (n
= 6 FFM from Fulton; n = 4 FFM and n = 20 MMF from
Pinkut) individuals.
From the present investigation of metabolomic changes
arising during in-migration, we hypothesized that alterations in
the concentration of some metabolites at the spawning grounds
are the result of normal physiological changes associated with
gamete production (e.g., increased amino acid concentrations
in females and production of VTG). Given this posit, we
expected salmon with altered sex-associated hepatic vtg mRNA
levels to display unusual metabolite profiles (in particular AAs)
compared to stock-matched animals exhibiting canonical vtg
expression profiles (i.e., MMM or FFF). On examination of
animals at the spawning grounds, this hypothesis appears to be
confirmed: a comparison of FFF versus FFM females from the
Fulton River spawning grounds revealed differences (p < 0.05)
in 23 metabolites, including 2 ACs, 2 FAs, 10 PCs, 1 SM, 6 AAs
(phenylalanine, isoleucine, aspartate, tyrosine, serine, leucine),
and 2 biogenic amines (taurine and methionine sulfoxide;
Figure 4 and SI Figure S3, Tables S13 and S15). Moreover,
concentrations of many of these metabolites in FFM females
aligned with MMM males at this site (SI Table S9). In fact, a
comparison of AA concentrations between Fulton River FFM
females and MMM males revealed only glutamate to be
significantly (p < 0.05) different, despite the fact that FFF
females contained significantly higher concentrations of nearly
half the AAs measured compared to their male counterparts
(Figure 4, SI Table S15).
While these results point to masculinisation of some Fulton
females, concentrations of several highly sexually dimorphic
metabolites remained unchanged in sex-skewed animals.
Hexose, for example, was among the most sexually dimorphic
metabolites at all locations (SI Figure S7), yet concentrations of
this metabolite were not significantly different between MMF
and MMM males. Other metabolites in animals displaying
atypical vtg status, such as carnitine, did not align with MMM
or FFF animals (SI Figure S7).
Similar variability, albeit not nearly as dramatic, was observed
at Pinkut Creek. A total of 9 metabolites had statistically
different levels between MMM and MMF animals, including
palmitic acid (FA C16:0), DPA isomer 1 (FA C22:5n3c1),
alanine, asparagine and 3 PCs and 2 SMs; while comparison of
FFF and FFM animals demonstrated 4 metabolites (lysine,
stearic acid [FA C18:0], DPA isomer 1 [FA C22:5n3c1], and
PC AA C36:4) that differed significantly (SI Tables S13 and
S16). There was additional evidence of decreased sex
phenotypic definition at this site. For example, while 10 AAs
were significantly different between MMM vs FFF animals
sampled from Pinkut only 2 AAs were dissimilar in abundance
between MMM and FFM animals and only 3 AAs were
different between FFF and MMF animals at this site (Figure 4
and SI Table S16). Interestingly, concentrations of most AAs in
Pinkut salmon livers were >50% lower than those from Fulton,
regardless of sex (Figure 4). The concentration profiles of other
metabolites in Pinkut animals demonstrating skewed sex
phenotype, such as carnitine, appeared distinct from MMM
or FFF animals (consistent with Fulton stock), although
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following the spawn, the sexual definition of the animal
deteriorates. While there were insufficient data on spawning
status to rigorously test this hypothesis, we noted that for the
few Pinkut Sockeye to which spawning status was evaluated,
17/22 FFF and 2/4 FFM animals had spawned. If the decrease
in sexual differentiation was triggered by spawning, then we
would expect to see masculanization in a large proportion of
these animals. These data are suggestive that natural senescence
cannot explain the observed variability in metabolite concentrations or vtg expression.
Exposure to anthropogenic substances has also been
suggested as a potential explanation for endocrine disruption
in migrating Sockeye salmon. 6 This could arise from
mobilization of lipophilic contaminants during in-river
migration gluconeogenesis40 or alternatively, from environmental exposure. A recent Fisheries and Oceans Canada report
highlighted resource extraction as a possible contributing factor
to changes in Sockeye production in this region. While the
Skeena River watershed is sparsely populated (<70 000 people),
extensive logging has occurred on Babine Lake since the mid
1920s, with most activity around Pinkut and Fulton watersheds
occurring in the1970s. The effects of logging in this area are
unclear, but one study noted that such activities could hinder
fish access to spawning sites.41 Historial mining operations have
also taken place on Babine Lake. The Bell Porphyry Copper−
Gold mine operated from 1977 to 1992 on Newman
Penninsula, while the Granisle Porphyry Copper−Gold Mine
operated on Sterret Island from 1966 to 1982 (Figure 1).42
Since their closure, tailings ponds from these mines have
discharged directly into Babine Lake.3
Experiments conducted by Fisheries and Oceans Canada in
1977 to investigate the potential hazard of copper to Babine
Lake Sockeye (fry, fingerlings and smolts), indicated that the
low levels of copper in the lake at the time (4−44 μg/L Cu2+)
posed no acute toxicity threat, but that 109−154 μg Cu2+/L in
Babine Lake may lead to disruption in osmoregulation.43 It was
suggested that the complexing capacity of the sediments would
maintain water-borne copper concentrations well below these
levels.43 Nonetheless, longer term studies involving salmonids
have reported adverse effects at around concentrations in
Babine lake in the 1970s. For example, a 22-month exposure
study involving Brook trout (Salvelinus fontinalis) to 1.9−32.5
μg/L copper reported reduced gamete viability and hatching
success44 while exposure of juvenile salmon to 5−20 μg/L
waterborne copper for 7 days impaired sensory physiology and
predator avoidance.45,46 Babine Lake water quality data for the
past decade are not publicly available, making connections
between copper exposure and the molecular perturbations
observed here difficult. Future water quality monitoring is
recommended to rule out such an exposure.
Finally, while it is tempting to ascribe variations in hepatic
molecular end points that are under hormone control to a
possible exposure event during in-migration involving xenobiotics, another important consideration is that the subgroups
of MMF and FFM animals evaluated at spawning grounds
within the Skeena River watershed are simply demonstrating
natural variation in physiological condition that results in
differential patterns within the hepatic transcriptome and
metabolome. Longitudinal data on spawning Sockeye to
confirm the appropriate baseline profiles within the liver
metabolome are lacking and the present initiative begins to
establish such information. With respect to the hepatic
transcriptome, analysis of in-migrating Sockeye during 2007
differences were not significant at Pinkut (SI Figure S7 and
Tables S3 and S16). While differences in location make direct
comparisons between Pinkut and Fulton animals difficult, the
combined metabolomic and molecular phenotypic results point
to decreased definition of certain sexually dimorphic parameters
at the molecular level following in-migration.
Combined changes in vtg mRNA levels and certain AAs at
the two spawning ground sites are suggestive of disruption not
only in the signaling pathway involved in hepatic vtg expression,
but also encompassing processes involved in protein catabolism. Among the major proteolytic enzyme families in
vertebrates (including calpains, proteasomes, and lysosomes),
the lysosomal system is thought to be responsible for most
protein degradation during in-migration of Sockeye. Specifically, lysosomal cathepsin D, an aspartic protease, is proposed
to play a central role in fish muscle proteolysis. Targets for this
enzyme include myosin heavy chain, actin, and tropmyosin.33
Induction of cathepsin D has been reported in migrating
Sockeye32 and Chum,34 spawning Atlantic salmon (Salmo
salar)35 and spawning Ayu (Plecoglossus altivelis).36
While it is well-known that vtg mRNA expression is mediated
by estradiol, there is also evidence that this steroid, as well as
known environmental chemical contaminants, can play a role in
facilitating protein catabolism with the cathepsin D gene (ctsd)
containing an estrogen-responsive promoter element. For
example, Cavailles et al. demonstrated that transcription of
ctsd mRNA is up-regulated by estradiol in breast cancer cells,37
while β-naphthoflavone, an agonist of the AhR receptor
decreased cathepsin D activity and expression of ctsd in black
gobi (Gobius niger).38 Carnevali and Maradonna (2003) also
observed a signficant increase in expression of cathepsin D
following exposure of black gobi to nonylphenol (a known
estrogen-mimicking xenobiotic)38 while Kaivarainen et al.
noted increased cathepsin D activity following lab- and fieldbased exposures of a variety of aquatic species (European
whitefish larvae [Coregollus laI’Qretus], rainbow trout larvae
[Salmo gairdlleri R.], pike [Esox lucius), roach [Rutilus rutilus],
and perch [Perea f lul’iatilis] to ore-processing effluent.39
Other signficant molecular changes in animals with an altered
hepatic transcriptome included carnitine levels at Fulton and
FAs abundance at both sites (SI Tables S13, S15, and S16),
may indicate modulation in fatty acid oxidation. Taurine
concentrations (which we previously hypothesized were
associated with osmoregulation) also displayed statistically
signficant differences in fish with atypical vtg expression at the
spawning grounds. Interestingly, osmoregulation in teleosts is
dependent on the interplay of a variety of hormones including
cortisol, and pituitary hormones, prolactin and growth
hormone.23 The cascade of biochemical signals leading to
changes in amino acid concentrations originates in altered
hypothalamo-pituitary activity.9 Modulation in the concentration of this organic osmolyte may, therefore, reflect a
common modulation in hypothalomo-pituitary action that also
manifests in the observed changes in hepatic vtg expression,
protein catabolism, and fatty acid oxidation.
Potential Sources of Variation in Molecular Profiles.
Our observations in fish returning to the spawning grounds
during the Skeena River in-migration of 2008 demonstrate
considerable variation in the biological status of stock-matched
animals of both sexes. It remains unclear whether this variability
arises from natural senescence, an environmental exposure,
heritable trait diversity, or some combination thereof. At first
glance, the former explanation appears most plausible; that is,
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(9) Jones, P. D.; De Coen, W. M.; Tremblay, L.; Giesy, J. P.
Vitellogenin as a biomarker for environmental estrogens. Water Sci.
Technol. 2000, 42, 1−14.
(10) Henry, T. B.; McPherson, J. T.; Rogers, E. D.; Heah, T. P.;
Hawkins, S. A.; Layton, A. C.; Sayler, G. S. Changes in the relative
expression pattern of multiple vitellogenin genes in adult male and
larval zebrafish exposed to exogenous estrogens. Comp. Biochem.
Physiol. A Mol. Integr. Physiol. 2009, 154, 119−126.
(11) Chow, W. S.; Chan, W. K.; Chan, K. M. Toxicity assessment and
vitellogenin expression in zebrafish (Danio rerio) embryos and larvae
acutely exposed to bisphenol A, endosulfan, heptachlor, methoxychlor
and tetrabromobisphenol A. J. Appl. Toxicol. 2013, 33, 670−678.
(12) Beacham, T. D.; Candy, J. R.; Supernault, K. J.; Wetklo, M.;
Deagle, B.; Labaree, K.; Irvine, J. R.; Miller, K. M.; Nelson, R. J.;
Withler, R. E. Evaluation and application of microsatellites for
population identification of Fraser River chinook salmon (Oncorhynchus tshawytscha). Fish. Bull. 2003, 101, 243−259.
(13) Beacham, T. D.; Lapointe, M.; Candy, J. R.; McIntosh, B.;
MacConnachie, C.; Tabata, A.; Kaukinen, K.; Deng, L.; Miller, K. M.;
Withler, R. E. Stock identification of Fraser River Sockeye salmon
(Oncorhynchus nerka) using microsatellites and major histocompatibility complex variation. Trans. Am. Fish. Soc. 2004, 133, 1117−1137.
(14) Veldhoen, N.; Ikonomou, M. G.; Dubetz, C.; Macpherson, N.;
Sampson, T.; Kelly, B. C.; Helbing, C. C. Gene expression profiling
and environmental contaminant assessment of migrating Pacific
salmon in the Fraser River watershed of British Columbia. Aquat.
Toxicol. 2010, 97, 212−225.
(15) Liebisch, G.; Lieser, B.; Rathenberg, J.; Drobnik, W.; Schmitz, G.
High-throughput quantification of phosphatidylcholine and sphingomyelin by electrospray ionization tandem mass spectrometry coupled
with isotope correction algorithm. Biochim. Biophys. Acta 2004, 1686
(1−2), 108−117.
(16) Antweiler, R. C.; Taylor, H. E. Evaluation of statistical
treatments of left-censored environmental data using coincident
uncensored data sets: I. Summary statistics. Environ. Sci. Technol.
2008, 42, 3732−3738.
(17) Xia, J. M. R.; Sinelnikov, I.; Broadhurst, D.; Wishart, D. S.
MetaboAnalyst 2.0A comprehensive server for metabolomic data
analysis. Nucleic Acids Res. 2012, 40, W127−33.
(18) Della Corte, L. C.; Duburs, G.; Nolan, K.; Tipton, K. F.; Tirzitis,
G.; Ward, R. J. The use of taurine analogues to investigate taurine
functions and their potential therapeutic applications. Amino Acids
2002, 23, 367−379.
(19) Militante, J. D.; Lombardini, J. B. Dietary taurine supplementation: Hypolipineric and antiatherogenic effects. Nutr. Res. (N.Y.) 2004,
24, 787−801.
(20) Hallgren, N. K.; Busby, E. R.; Mommsen, T. P. Cell volume
effects glycogen phosphorylase activity in fish hepatocytes. J. Comp.
Physiol. B 2003, 173, 591−599.
(21) Awapara, J. Free Amino Acids in Invertebrates: A Comparative
Study of Their Distribution and Metabolism. In Amino Acid Pools:
Distribution, Formation and Function of Free Amino Acids; Holden, J.T.,
Ed.; Elsevier: Amsterdam, The Netherlands, 1962; p 158−175.
(22) Schaarschmidt, Th.; Meyer, E.; Jürss, K. A comparison of
transport-related gill enzyme activities and tissue specific free amino
acid concentrations of Baltic Sea (brackish water) and freshwater
threespine sticklebacks, Gasterosteus aculeatus, after salinity and
temperature acclimation. Mar. Biol. 1999, 135, 689−697.
(23) Fiess, J. C.; Kunkel-Patterson, A.; Mathias, L.; Riley, L. G.;
Yancey, P. H.; Hirano, T.; Grau, E. G. Effects of environmental salinity
and temperature on osmoregulatory ability, organic osmolytes, and
plasma hormone profiles in the Mozambique tilapia (Oreochromis
mossambicus). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 2007, 146,
252−264.
(24) Magnoni, L. J.; Patterson, D. A.; Farrell, A. P.; Weber, J.-M.
Effects of long-distance migration on circulating lipids of Sockeye
salmon (Oncorhynchus nerka). Can. J. Fish. Aquat. Sci. 2006, 63, 1822−
1829.
at spawning grounds within the Fraser River, BC watershed
(Figure 1) failed to demonstrate such a marked range in vtg
transcript abundance within each sex7 suggesting either a
regional contributory variable or stock-specific factor is at play
within the Skeena River Sockeye populations. Continued
monitoring of stock returns with inclusion of molecular end
point assessment will help further elucidate the cause-effect
relationship.
■
ASSOCIATED CONTENT
* Supporting Information
S
Further details on method validation, genotype-phenotype sex
classification, metabolite abreviations and concentrations, and
statistical analyses. This material is available free of charge via
the Internet at http://pubs.acs.org/.
■
AUTHOR INFORMATION
Corresponding Author
*Phone: +1 (250) 655-5800; fax: +1 (250) 655-5811; e-mail:
[email protected].
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
Mike Jakubowski, Brad Thompson, Steven Cox-Rogers, George
Chandler, Doug Lofthouse, Peter Hall, and Brenda Donas as
well as other personnel associated with Fisheries and Oceans
Canada Skeena test fishery program are thanked for their
assistance. We also thank Jayme Hills for technical assistance
and Fisheries and Oceans Canada Pacific Region contaminants
analysis program. David Patterson (Fisheries and Oceans
Canada) is thanked for helpful feedback on this manuscript.
■
REFERENCES
(1) Fisheries and Oceans Canada 2003. Skeena River Sockeye
Salmon (update) DFO. Can. Sci. Advis. Sec. Stock Status Rep. 2003/
047.
(2) West, C. J. 1978. A review of the Babine Lake Development
Project 1961−1977. Fisheries Marine Service Technical Report 812.
(3) Cox-Rogers, S., and Spilsted, B. 2012. Update assessment of
Sockeye salmon production from Babine Lake, British Columbia. Can.
Tech. Rep. Fish. Aquat. Sci. 2956: ix + 65 p.
(4) Fisheries and Oceans Canada Fishery Notice FN0697-Recreational-Salmon-Sockeye-Region 6-Skeena River: All Lakes and Tributaries-Closure to sockeye salmon fishing. Accessed January 21, 2014
from: http://www-ops2.pac.dfo-mpo.gc.ca/fns-sap/index-eng.cfm?pg=
view_notice&lang=en&DOC_ID=152824&ID=all.
(5) Cook, K. V.; McConnachie, S. H.; Gilmour, K. M.; Hinch, S. G.;
Cooke, S. J. Fitness and behavioral correlates of pre-stress and stressinduced plasma cortisol titers in pink salmon (Oncorhynchus
gorbuscha) upon arrival at spawning grounds. Horm. Behav. 2011,
60, 489−497.
(6) Veldhoen, N.; Ikonomou, M. G.; Rehaume, V.; Dubetz, C.;
Patterson, D. A.; Helbing, C. C. Evidence of disruption in estrogenassociated signaling in the liver transcriptome of in-migrating Sockeye
salmon of British Columbia, Canada. Comp. Biochem. Physiol. C
Toxicol. Pharmacol. 2013, 157, 150−161.
(7) Babin, P. J.; Vernier, J. M. Plasma lipoproteins in fish. J. Lipid Res.
1989, 30, 467−489.
(8) Anderson, M. J.; Olsen, H.; Matsumura, F.; Hinton, D. E. In vivo
modulation of 17 β-estradiol-induced vitellogenin synthesis and
estrogen receptor in rainbow trout (Oncorhynchus mykiss) liver cells
by β-naphthoflavone. Toxicol. Appl. Pharmacol. 1996, 137, 210−218.
11677
dx.doi.org/10.1021/es503266x | Environ. Sci. Technol. 2014, 48, 11670−11678
Environmental Science & Technology
Article
(25) Idler, D. R. and Clemens, W. A.The energy expenditures of the
Fraser River Sockeye salmon during the spawning migration to Chilko
and Stuart Lakes. Int. Pacific Salmon Fish. Comm. Progr. Rep. No. 6;
New Westminster, B.C., 1959; 80 pp.
(26) Brett, J. R. Energy Expenditure of Sockeye salmon,
Oncorhynchus nerka, during sustaned performance. J. Fish. Res. Board
Can. 1973, 30, 1799−1809.
(27) Ando, S.; Hatano, M. Isolation of apolipoproteins from
carotenoid-carrying lipoprotein in the serum of Chum salmon
Oncorhynchus keta. J. Lipid Res. 1988, 29, 1264−1271.
(28) García-Chavarría, M.; Lara-Flores, M. The use of carotenoid in
aquaculture. Res. J. Fish Hydrobiol. 2013, 8, 38−49.
(29) Chang, V. M.; Idler, D. R. Biochemical studies on Sockeye
salmon during spawning migration. XII. Liver glycogen. Can. J.
Biochem. Physiol. 1960, 38, 553−558.
(30) Idler, D. R.; Tsuyuki, H. Biochemical studies on Sockeye salmon
during spawning migration. I. Physical measurements, plasma
cholesterol, and electrolyte levels. Can. J. Biochem. Physiol. 1958, 36,
783−791.
(31) Nelson, G. J.; Shore, V. G. Characterization of the serum high
density lipoproteins and apolipoproteins of pink salmon. J. Biol. Chem.
1974, 249, 536−542.
(32) Mommsen, T. P. Salmon spawning migration and muscle
protein metabolism: The August Krogh principle at work. Comp.
Biochem. Physiol. B 2004, 139, 383−400.
(33) Nielsen, L. B.; Nielsen, H. H. Purification and characterization
of cathepsin D from herring muscle (Clupea harengus). Comp. Biochem.
Physiol. B 2001, 128, 351−363.
(34) Ando, S.; Hatano, M.; Zama, K. Protein degradation and
protease activity of Chum salmon (Oncorhynchus keta) muscle during
spawning migration. Fish Physiol. Biochem. 1986, 1, 17−26.
(35) von der Decken, A. Physiological changes in skeletal muscle by
maturation-spawning of non-migrating female atlantic salmon, Salmo
salar. Comp. Biochem. Physiol. B 1992, 101, 299−301.
(36) Toyohara, H.; Ito, K.; Ando, M.; Kinoshita, M.; Shimizu, Y.;
Sakaguchi, M. Effect of maturation on activities of various proteases
and protease inhibitors in the muscle of Ayu (Plecoglossus altivelis).
Comp. Biochem. Physiol. B 1991, 99, 419−424.
(37) Cavailles, V.; Augereau, P.; Rochefort, H. Cathepsin D gene is
controlled by a mixed promoter, and estrogens stimulate only TATAdependent transcription in breast cancer cells. Proc. Natl. Acad. Sci. U.
S. A. 1993, 90, 203−207.
(38) Carnevali, O.; Maradonna, F. Exposure to xenobiotic
compounds: Looking for new biomarkers. Gen. Comp. Endrocrinol.
2003, 131, 203−208.
(39) Kaivarainen, E. I. N.; Krupnova, M. Y.; Bondareva, L. A. The
effect of toxic factors on intracellular proteinase activity in feshwater
fish. Acta. Veterinaria Brno 1998, 67, 8.
(40) Kelly, B. C.; Gray, S. L.; Ikonomou, M. G.; Macdonald, J. S.;
Bandiera, S. M.; Hrycay, E. G. Lipid reserve dynamics and
magnification of persistent organic pollutants in spawning Sockeye
salmon (Oncorhynchus nerka) from the Fraser River, British Columbia.
Environ. Sci. Technol. 2007, 41, 3083−3089.
(41) Gottesfeld, A.; Rabnett, K. A. Skeena River Fish and their
Habitat; Ecotrust: Portland, OR, 2008; p 341.
(42) Astorius Resources Ltd. Fact Sheet, March 2013. Accessed April
26, 2014 from: http://www.astoriusresources.com/i/pdf/ASQ-FactSheet-March-2014.pdf.
(43) Davis, J. C. S. Acute and sublethal copper sensitivity, growth and
saltwater survival in young Babine Lake Sockeye salmon. Fish. Mar.
Servo Technol. Rep. 1978, 847, 55.
(44) McKim, J. M.; Benoit, D. A. Effects of long-term exposures to
copper on survival, growth, and reproduction of brook trout
(Salvelinus fontinalis). J. Fish. Res. Board Can. 1971, 28, 655−662.
(45) Hecht, S. A.; Baldwin, D. H.; Mebane, C. A.; Hawkes, T.; Gross,
S. J.; Scholz, N. L. An overview of sensory effects on juvenile salmonids
exposed to dissolved copper: Applying a benchmark concentration
approach to evaluate sublethal neurobehavioral toxicity. U.S. Dept.
Commer. NOAA Tech. Memo.; 2007, NMFS-NWFSC-83, p 39.
(46) Sandahl, J. F.; Baldwin, D. H.; Jenkins, J. J.; Scholz, N. L. Odorevoked field potentials as indicators of sublethal neurotoxicity in
juvenile coho salmon (Oncorhynchus kisutch) exposed to copper,
chlorpyrifos, or esfenvalerate. Can. J. Fish. Aquat. Sci. 2004, 61, 404−
413.
11678
dx.doi.org/10.1021/es503266x | Environ. Sci. Technol. 2014, 48, 11670−11678