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l. TITLE OF PROJECT
DISTINGUISHING EXPOSURE FROM EFFECT IN MULTIPLE-STRESSOR SCENARIOS:
EFFECTS OF OCEAN ACIDIFICATION AND METAL-TOXICITY ON MUSSEL LARVAL
DEVELOPMENT
2. PRINCIPAL INVESTIGATOR(S)
Andrew Gracey, Associate Professor of Marine Biology, USC.
3. ASSOCIATE INVESTIGATOR(S)
4. FUNDING REQUESTED
2016-2017 $45,314 Federal/State $22,680 Match
2017-2018 $52,214 Federal/State $26,154 Match
5. STATEMENT OF THE PROBLEM
The regulations governing heavy metal contamination in Southern California’s coastal ocean
must be considered in the context of a changing global ocean. Increased dissolved CO2 levels are
predicted to decrease ocean pH to 7.7 by 2100 as well as significantly lower calcium carbonate
saturation constants, especially in the California Current system. There is growing evidence that
ocean acidification (OA) will have an overall deleterious effect on the health of many organisms.
Furthermore, it is expected that OA will increase toxicity of certain metal contaminants, such as
copper, by reducing the complexation capacity of coastal waters and increasing free metal
concentrations (1). Therefore, understanding the effects of OA on water quality issues remains a
largely unexplored but looming question. This is a particular challenge in urban oceans because
multiple environmental and chemical parameters will vary temporally and spatially due to the
proximity of large sources of urban pollutants. This means that water quality testing approaches
that will be implemented this century will have to be nuanced and capable of analyzing multiple
stressor scenarios in order to predict the impact that contaminants will have on coastal urban
ecosystems.
6. INVESTIGATORY QUESTION
Global climate change will present inhabitants of impacted coastal environments with the
additional stress of OA. This raises the question, will OA exacerbate the deleterious effects of
contaminants on the health of organisms?
We hypothesize that:
multiple-stressor exposures comprising OA and a heavy metal will be more deleterious to
the organism than exposure to either stressor alone
more deleterious exposures will be manifested as increases in abnormal embryo
development and mortality, and concomitant differences in gene expression
molecular biomarkers of contaminant exposure versus effect will be associated with
phenotypic differences among larvae
7. MOTIVATION
Importance of Studying Ocean Acidification Impacts on Metal Bioavailability and Toxicity
2
Global change is progressing at an unprecedented rate, and affecting all global ecosystems.
Ocean acidification in particular is expected to affect marine and estuarine habitats in numerous
ways, most of them negative. Ocean acidification, which is characterized by a shift in carbonate
equilibrium resulting in more bicarbonate, lower calcium carbonate saturation constants, and
lower pH will change ocean chemistry, as well as organismal physiology. Increased dissolved
CO2 levels are predicted to decrease ocean pH to 7.7 by 2100, and lower calcium carbonate
saturation constants significantly as well. The California Current system is expected to
experience particularly strong acidification, as this is already an area where strong upwelling
brings acidified deep water to the surface (2).
The impacts of ocean acidification must also be considered in the context of concurrent changes,
such as rising temperature, higher levels of UV, and increased stratification of the water column
(3). Meanwhile, extant pressures on marine ecosystems, such as chemical pollution and nutrient
loading, will remain and potentially interact with novel factors as well. Thus it will be important
to understand how numerous environmental variables in the ocean affect each other, and leaves
the potential for many unknown interactions. The physiological response of organisms to these
simultaneous changes has been an imperative question for several years now (4-6), and will
ultimately determine biogeographic range shifts, changes in genetic composition of populations,
and alterations in ecosystem function.
Metals, especially iron, copper, cadmium, and zinc are important in coastal ecosystems both
because they serve as micronutrients for all forms of life (with exception of Cd), and because at
higher doses they can become acutely toxic. Our knowledge of metal cycles (bioavailability and
speciation) in marine ecosystems is based on our understanding of extant chemical and physical
ocean parameters, but as these factors change under ocean acidification, many models of metal
biotic and abiotic interactions will have to change as well. Predicted effects of ocean
acidification on metal supply and bioavailability are outlined in recent reviews (1, 7), yet there is
little conclusive proof that numerous predictions will play out as expected. This topic is still
drastically understudied, and more direct studies exposing organisms to realistic future-ocean
scenarios are necessary, especially considering the vital roles that metals play in numerous
biogeochemical cycles.
While a lack of metal micronutrients could pose problems for marine organisms, an excess of
toxins could likewise have significant effects on keystone organisms and marine ecosystems.
The regulations that currently exist for metal contamination, as well as regulations that will be
developed in coming years, must be considered in the context of a changing global ocean (8).
The effects of ocean acidification on metal toxicity is a relatively new field of study, and only
limited research exists on the interactive effects of these two stressors on organismal survival,
reproduction, and physiology.
Several studies have begun to consider the effects of combined metal stress and ocean
acidification on marine invertebrates. Polychaete larvae exposed to both copper and reduced pH
exhibited lower survival than those in either treatment alone (9). In a study by (10), copepods
were exposed to increasing levels of copper under pH regimes representing current and future
ocean conditions. While elevated copper in the presence of CO2 resulted in faster growth of
copepods, it also resulted in lower fecundity, with an ultimately detrimental effect. Another study
3
on benthic isopods found that metal-contaminated sediments have different effects on survival
and DNA damage under acidified and normal pH conditions (8). Only one study has considered
the combined effects of metals and ocean acidification in adult mussels. When exposed to metals
under normal and reduced ocean pH conditions, mussels exhibited altered survival rates (lower,
in most cases), increased immune response, and much higher uptake rates of all metals (11). It is
clear that the combination of ocean acidification and metal exposure results in altered toxicity
patterns, indicating that current contaminant criteria will not apply under different ocean
chemistry conditions. Thus, in areas like southern California where toxic metals are highly
regulated, a detailed investigation of potential toxicity changes is warranted to prove the need for
updating water quality regulations in the coming decades.
Metal Contamination in Southern California
Metal pollution of marine environments has been identified as a persistent problem in urban
areas of southern California. Recommended limits on metal contamination are set by the EPA for
effluent and receiving waters, but the concentrations of several metals still occasionally exceed
limits in some areas along the coast (12). Three metals that still pose a problem in southern
California include copper, cadmium, and zinc (13, 14). Copper and zinc are both micronutrients
required at low concentrations, while cadmium is not necessary for biological function, yet they
are all toxins at concentrations that can occur in coastal waters. Additionally, they all can be
present as divalent cations that are easily absorbed and accumulated by bivalves.
The major sources of metals in this area are storm-water runoff, privately owned treatment works
(POTW) discharge, and anti-fouling paints on the hulls of boats. POTW effluent has been
reduced in overall toxicity over the past 35 years, yet it still contributes 41 and 52% of total
copper and cadmium loads, respectively, to coastal waters. Alternatively, storm-water runoff has
been increasing in volume and toxicity, and contributes a large fraction of cadmium and copper,
as well as most of the zinc, that pollute coastal waters (15). This problem is exacerbated by the
local climate because contaminants accumulate during long dry spells but are then flushed into
coastal waters during the first severe rainfall events. In turn these rain events cause local surges
in the levels of toxic metals and organic contaminants that can temporarily exceed EPA and State
criteria (16-18)
The toxic forms of cadmium, copper, and zinc all form strong complexes with organic ligands,
and thus exist in coastal waters primarily in a complexed, particle-associated form. However,
ocean acidification has the potential to alter the proportion of ligated and free forms of these
metals. The decrease in pH associated with ocean acidification is expected to increase
protonation of negative sites on the organic ligands, thus blocking potential binding locations for
these cations ((1); Hutchins, pers. comm.). This would result in fewer metal ions bound to the
ligands, and more free, toxic (Cd+2, Zn+2, Cu+2) ions in the water. The ability to predict and
anticipate potential changes in toxicity will be important for updating saltwater contaminant
criteria in a timely manner. The results of this research can be used by regulators to pre-empt the
effects of ocean acidification on organisms’ metal tolerance, and thus adjust contaminant limits
accordingly.
Rationale for Mytilus Embryo-Larval Development Toxicity Testing Model
4
The genus Mytilus was reported to be the most sensitive genus to copper toxicity in an EPA
survey of genus mean acute values (19). As a result, Mytilus larvae are among the most
important test organisms used in marine metal toxicity assessment in the United States and the
criteria for many priority pollutants, such as Cu, are based on Mytilus larvae EC50 data (see
(20)and refs. therein). In the standard US EPA embryo-larval development test, Mytilus larvae
are incubated in a given water sample for 48 hr and then data on larval mortality and abnormal
development are collected (Fig. 1).
Figure 1. Toxicity of Cu2+ to Mytilus californianus larvae reared for 48 hr in Catalina Island seawater with
increasing concentrations of copper. (A) The proportion of embryos exhibiting abnormal development and the rate
of larval mortality were both elevated significantly at and above 6 g/L Cu2+ (* indicates p<0.01). The data are
presented as the mean standard deviation (n=5) of the proportion of larvae exhibiting abnormal development or
larval mortality relative to control cultures to which Cu was not added. (B) Images of larvae exhibiting normal
versus abnormal development in US EPA toxicity test at 48 hrs.
Mytilus larvae are a particularly appropriate study system in the context of ocean acidification,
because acidification is expected to have an especially large effect on calcifying marine
invertebrates. Indeed, mussels have been the subjects of several key ocean acidification
experiments. Some of the primary effects that have been observed include: developmental
abnormalities ultimately resulting in death, delayed larval development (21), reduced immunity
(22), reduced tissue mass, thinner shells (23) and alteration of shell structural integrity (24),
weaker byssal threads (25), and in oyster larvae increased metabolic costs (26). Some of these
effects may also increase time that the larvae spend in the plankton resulting in greater predation
and lowered settlement rates (23). Shell integrity is of special concern for Mytilus because their
shells are partially composed of the calcium carbonate crystal form aragonite (27). Of aragonite
and calcite, the two crystal forms of calcium carbonate, aragonite has a lower saturation constant,
and is thus expected to dissolve at a higher pH than calcite. Therefore it is likely that Mytilus will
Cu2+ (g/L)
Pro
po
rtio
n o
f em
bry
os
wit
h n
orm
al
dev
elo
pm
ent
0
0.2
0.4
0.6
0.8
1
1.2
0
0.2
0.4
0.6
0.8
1
1.2
0 5 10 15 20 25
Pro
po
rtio
n o
f em
bry
os
that
su
ffer
m
ort
alit
y re
lati
ve t
o c
on
tro
l cu
ltu
res
Development
Mortality
*
* ** *
*
B
A
Normal Abnormal
5
have to increase shell maintenance sooner than other calcifying marine organisms that precipitate
solely calcite.
Toxicity testing in the 21st century
Recent advances in ecotoxicology have called for the inclusion of molecular data in assessing the
response of test organisms to contaminants (28, 29). The rationale for the inclusion of molecular
data in establishing testing toxicity criteria is that changes in cell state are invariably linked to
changes in gene expression (30), and for this reason gene expression profiling is showing
increasing promise in the context of environmental monitoring (31). Integration of classic
measurements of toxicity, gross morphological and mortality effects, and molecular responses,
provides a rich source of data yielding crucial insights into the effects of contaminants at
concentrations that are below those that give rise to the visible changes in survival and
development. This more comprehensive approach is necessary because the actual mechanisms of
toxicity are poorly understood at low concentrations and in the context of multiple stressor
scenarios. Indeed, the EPA has called for the development of “Adverse Outcome Pathway”
perspective on toxicity (28), which seeks to provide a more mechanistic representation of
toxicological events which lead to an adverse outcome (32, 33). Incorporation of molecular data
is an important step towards achieving this goal because it provides a more nuanced overview of
how toxicants exert their effects at the cellular level, and helps to inform the interpretation of
observed shifts in survival and adverse health effects under different exposure scenarios.
One of the promises of molecular approaches is that they will identify biomarkers such as gene
transcripts or proteins, which can be used to monitor the presence of a chemical in the body,
biological responses, or adverse health effects (29). Biomarkers are often grouped into
biomarkers of exposure versus those of effect, with biomarkers of exposure serving as a measure
of the amount of toxicant that the organism has been exposed to, whereas biomarkers of effect
serve as indicators of a change in biological function in response to toxicant exposure (34).
Molecular biomarkers of exposure are normally going to be surrogates, representing a
physiological response to the toxicant, but providing little to no information regarding the effects
on the health of the organism. In contrast, biomarkers of effect indicate physiological changes
that are linked to the adverse health effects of the toxicant. One of the challenges of developing
molecular biomarkers is distinguishing between responses linked to simple exposure versus
those that are linked to the deleterious effects of the exposure and adverse outcomes to the
organism’s health (28). A report by the National Research Council (NRC) of the U.S. National
Academy of Sciences (35) envisions the delineation of ‘‘toxicity pathways’’ defined as ‘‘cellular
response pathways that, when sufficiently perturbed in an intact animal, are expected to result in
adverse health effects’’ as a goal in the future of toxicology. Delineation of these pathways is
important because they differentiate between adaptive responses that are activated upon exposure
to low levels of a contaminant and which serve to defend the physiology of the organism, from
stress responses that indicate that defense measures have been overwhelmed and damage has
occurred (36). Characterization of these cell stress response pathways is important because these
pathways are extensively networked and their activation can lead to programmed cell death and
developmental consequences, thus linking their activation to adverse consequences (36, 37).
8. GOALS AND OBJECTIVES
6
Our overarching research goal is to use mussel embryo-larvae as a model to test the hypothesis
that OA will affect metal toxicity. To achieve this goal we will undertake an integrative analysis
of toxicity by simultaneously monitoring mortality, developmental abnormality, and global
changes in gene expression.
Objective 1) To determine the interactions of metals and elevated pCO2 in mussel embryo-larval
toxicity assays
The combined effects of ocean acidification and metal contaminants have the potential to act
additively, synergistically, or antagonistically on the physiology and health of marine organisms.
We will use mussel embryo-larval toxicity assays to study the effect of increasing metal
concentrations under current and future CO2 concentrations and seawater pH. Specifically, we
will test the effects of copper, cadmium, and zinc, all still pollutants of concern in southern
California (see sources listed above).
Objective 2) To complement the toxicity assay results with molecular data
In line with the EPA’s call to develop more mechanistic rather than end-point assessments of
toxicity, we will complement the standard mussel embryo-larval toxicity assay with gene
expression data. This approach will identify genes and pathways which exhibit a concentration-
response to metal/OA exposure and which can be correlated with US EPA-approved markers of
larval toxicity. This phenotype-to-molecular relationship will provide a scaffold upon which we
can investigate the molecular signatures and mechanisms responsible for differences in toxicity
under changing conditions of OA.
Objective 3) To dissect the molecular signatures associated with the normal versus abnormal
developmental phenotypes that arise in toxicity assays
One metric of toxicity in embryo-larval toxicity assays is the proportion of normal and abnormal
larvae in a given sample (Fig. 1B). Abnormal development occurs naturally but an increase in
the incidence of abnormal larvae is considered to be a marker of the adverse effects of chemical
exposure. All the larvae in the assay have received the same level of exposure but only the
abnormal individuals are exhibiting morphological evidence of adverse effect, thus these
phenotypically distinct larvae may yield biomarkers that can help to differentiate exposure from
effect. In this objective we will leverage the fact that a given assay will contain these
phenotypically distinct larvae to develop a novel stratified sub-sampling technique that will
identify the molecular signatures belonging to larvae that are exhibiting toxicant-induced
abnormal development from those that develop normally. This sub-sampling approach has the
potential to untangle the complex interactions that occur when multiple variables are employed
in water quality testing protocols, and to dissect the transcriptional contribution of the normal
and abnormal larvae to the bulk molecular data collected in objective 2. These data will also
serve to corroborate transcriptional patterns observed in the concentration-response results.
9. METHODS
Preparation of mussel embryos
M. californianus broodstock will be collected from jetties in northern Santa Monica Bay, and
held in pristine water (collected from mid San Pedro Channel, https://dornsife.usc.edu/spot/) at
the Wrigley Marine Science Center on Catalina Island for 4 weeks. Spawning will be induced
using standard protocols that employ a mild thermal shock. Spawning male and female mussels
7
(judged by type of gametes released) will be placed into separate containers. Once spawning is
complete, good quality eggs will be inoculated with sperm at a density of ~5 sperm/egg. When
the majority of eggs are fertilized, evidenced by the production of a polar body, embryo density
will be determined and embryos will be stocked at a density of 10 embryos/ml into 1 L vessels
containing the experimental seawater samples.
In our proposed program of work we will use larvae of the California mussel, Mytilus
californianus, as the bioindicator organism. One of the reasons for this decision is that M.
californianus is the only remaining native mussel in southern California and is ecologically
relevant because it dominates rocky intertidal habitats on exposed coasts. The other native
mussel was a bay mussel, M. trossulus, but it has been displaced by an invasion of M.
galloprovincialis from the Mediterranean (38). While M. edulis and M. galloprovincialis are the
mussels that have been most widely used in contaminant testing, their deployment presents
challenges because they are almost impossible to distinguish morphologically and can hybridize
with one another. Recent reports indicate that incorrect identification of bay mussel species can
confound efforts to standardize regulatory criteria and have called for data collected in M.
californianus to be added to toxicity databases (39). Moreover, as an open water species, M.
californianus may be particularly sensitive to Cu and therefore an appropriate species for the
development of criteria.
Experimental water sample preparation
The experimental treatments will be set up in a factorial cross that tests control and elevated CO2
against increasing concentrations of heavy metals. Mussel embryo-larval cultures will be
incubated at two CO2 concentrations, reflecting current (400 ppm) and future (year 2100
prediction - 800 ppm) conditions in the ocean. For each CO2 concentration, six metal
concentrations (3-20 ppb Cu, 3-30 ppb Cd, 100-250 ppb Zn) will be tested, in addition to a set of
heavy metal-free controls. Each metal by CO2 concentration combination will be assayed across
3 replicate containers, resulting in 21 containers assayed under current pCO2 (ie. 3 metal-free
control replicates, and 3 replicates for each of the 6 metal concentrations), and a similar 21
containers assayed under conditions of elevated pCO2.
The acidification and confirmation of carbonate chemistry will be conducted under the guidance
of the Hutchin’s lab at USC. pH and dissolved inorganic carbon (DIC) will be measured at the
beginning and end of the experiment (T = 0 hrs and T = 48 hrs). Samples for pH will be
collected in 50 mL conical tubes, and samples for DIC will be collected in 25 mL borosilicate
glass vials. The samples used for pH readings will be stored at -20C for later metal chemistry
analysis.
Analysis of metals in the preserved samples will be assayed using the ICP-mass spectrometer in
the laboratory of Prof. James Moffett (USC), using their established methods (40). Prior to the
initiation of the experiments, the starting water samples will be titrated by anodic stripping
voltammetry (ASV) to assess the complexation capacity (41, 42). This will serve to verify that
the sample is not anomalous due to inadvertent contamination by organics.
Mussel embryo-larval toxicity assays
8
Mussel embryo-larval toxicity assays will be performed according to standard EPA protocols
(43). The cultures will be incubated for 48 hrs and then harvested by filtration through a 20 m
sieve, followed by resuspension of the larvae in a known volume of seawater (typically 50 ml).
Then 5x 1 ml sub-samples of the larval suspension will be removed, centrifuged, resuspended in
60% EtOH, and stored for later microscopic analysis of counts for mortality and frequency of
abnormally developing larvae. The count and larval abnormality data will be analyzed by
ANOVA to compare survival and development under normal versus elevated CO2 conditions
across increasing metal exposure regimes. The remaining larval suspension will be centrifuged,
resuspended in RNAlater, and stored at 4C for future molecular analysis. About 50% of the
RNAlater preserved sample will be used for bulk analysis of gene expression and the remainder
used for the picking of normal and abnormal pools of larvae.
RNA sequencing of combined metal and OA stressor experiments
Poly A+ RNA will be isolated from a fraction of the RNAlater-preserved samples that was
equivalent to 500ml of each replicate experimental culture (this has proved to be sufficient larval
material for library construction, Fig. 3). Each experiment will yield 42 samples (2 CO2
concentrations x 7 metal concentrations x 3 replicates). The RNA samples will be fragmented,
reverse-transcribed into cDNA and converted into bar-coded Illumina DNA libraries (44) using
an optimized in-house protocol that is used extensively by the Gracey laboratory. Our standard
RNASeq protocol is to index-barcode each library and to sequence pools of 8-12 libraries per
lane of the Illumina flow cell which yields ~25-16 million reads per library. The 42 libraries per
metal experiment will be pooled and sequenced at the USC Epigenome Facility as single-end 50
bp reads across 5 lanes on the Illumina HiSeq 2000 platform, yielding ~21 million reads per
sample.
The reads will be mapped to our existing M. californianus reference transcriptome using Bowtie
(45) and read-counts per sample calculated using RSEM (46). Differentially expressed genes will
be identified using DESeq (47) employing the reads counts from the 3 replicate cultures as the
biological replicates. The Sigmoidal Dose Response Search (SDRS) grid-based algorithm (48)
will be used to identify a subset of the differentially expressed transcripts whose expression is
correlated with increasing metal exposure over some range of concentrations, as well as the
minimum concentration of metal required to induce differential expression. Other correlations
between gene expression and metal or CO2 concentration, or mortality and developmental
abnormality, will be explored using a variety of emerging data mining procedures such as the
MINE algorithm (49).
Stratified sub-sampling of normal versus abnormal larvae
For each metal assayed under current and elevated CO2 conditions, we will select the metal
concentration that yielded =>40% incidence of abnormal development in both CO2 conditions
(this will ensure that an adequate number of abnormal larvae are represented in the samples from
which larvae will be picked). From each of the replicate samples, we will pick 100 larvae
exhibiting either normal or abnormal development. The larvae will be picked manually using a
microscope, a 20 l micropipette, and an aspirator tube assembly (~60 larvae can be picked per
hour). Corresponding sets of normal and abnormal embryos will also be picked from metal-free
control samples. This will yield a total of 24 samples (3 normal and 3 abnormal larval replicates
x 4 conditions (control/current CO2, control/OA, metal/current CO2, metal/OA)).
9
Total RNA will be isolated from the picked samples and converted into bar-coded Illumina DNA
libraries using a template-switching cDNA synthesis protocol that was developed for single-cell
RNA sequencing (50), with the inclusion of index-barcodes during the final PCR amplification
step. The 24 libraries arising from each experiment collected from each spawn will be pooled
and sequenced as single-end 50 bp Illumina reads across 2 lanes (yielding ~17 million reads per
library). The reads will be mapped and DESeq statistical analysis will be used to identify genes
that differentiate normal versus abnormal larvae as candidate biomarkers of toxic effect and not
just exposure.
Special attention will be placed on genes and pathways that are implicated in the cell stress
response as these may be biomarkers of the deleterious toxic effects of exposure, cellular
damage, and the onset of pathology (37). There will be several ways to interpret these data.
Under one scenario, one could argue that the abnormal larvae will express more markers of stress
because they are exhibiting gross phenotypic evidence of the deleterious effects of the toxicant
exposure, ie. abnormal amorphous development. On the other hand, one can argue that the larvae
that are exhibiting normal development will be the pool which will be expressing cell stress
markers because they have successfully mounted a stress response and are resisting the
deleterious effects of the exposure. Examples of such interpretations have recently been reported
in studies of the response of mature and larval corals to heat stress (51, 52). Thus focus will be
placed on identifying genes and pathways which may serve to differentiate the stress response
from those associated with substantial cellular damage.
Data integration
The statistical outputs of the SDRS algorithm analysis will provide a framework upon which
shifts in the concentration-response kinetics of individual genes can be related to the actual
toxicity of heavy metals under current and OA scenarios as reported by the embryo-larval
toxicity assay. Our prediction is that the transcriptional concentration-response curves reflect
metal toxicity, and that OA will shift these concentration-response curves to the left - that is to
say that concentration of metal that induces a transcriptional response will be lowered. Thus,
genes with concentration-response curves that shift to the left may reveal genes and pathways
that are more sensitive to the metal contaminant under future OA scenarios, and clues as to the
nature of the synergistic effects that occur between heavy metals and OA.
The provision of lists of candidate biomarkers of effect provides a layer of information across
which the concentration-response results can be interpreted. For example, characterization of the
minimum metal concentration at which biomarkers of effect are differentially expressed would
provide insights into the concentration at which the deleterious effects of exposure are first felt
by the organism. The table below depicts some interpretations that can be derived from the
comparison of expression in normal versus abnormal larvae, with asterisks indicating the relative
expression of genes across the samples.
10
Cu free Control Normal
Cu free Control
Abnormal
Cu + Control Normal
Cu + Control
Abnormal
Cu + OA
Normal
Cu + OA
Abnormal Interpretation
* *** * *** * *** Genes associated with
natural abnormal development
* * * *** * *** Genes associated with toxicity-linked abnormal
development
* * *** *** *** *** Genes associated with Cu
exposure
* * * * *** *** Genes associated with OA
exposure
Collaboration at USC
We will collaborate on this project with two other scientists at USC. Prof. David Hutchins is an
ocean chemist with expertise in ocean acidification (53). He has experience designing and
advising multiple experiments in which accurate preparation and measurement of pH and other
carbonate chemistry parameters are integral (5, 6, 54, 55). Please see the attached letter of
support for more detail. Our other long-term collaborator, and co-advisor of the nominated
trainee, Prof. James Moffett, will assist in the analysis of metal concentrations in the treatment
water samples. Research space will be provided by the Wrigley Marine Science Center on
Catalina Island.
10. RELATED RESEARCH
Toxicity responses of adult mussels
We investigated the molecular changes associated with exposure of adult M. californianus to
heavy metals in a USC SeaGrant project titled ‘Contaminant stressor response in Mytilus using
genomics: mussel monitoring for the new millennium’. Toxicity assays in other disciplines such
as drug discovery rely heavily on the study of concentration-response curves to characterize the
activities of a particular compound on the biology of the test organism. Concentration-response
approaches are invaluable because they can define the level of the compound required to produce
a response, as well as the levels associated with the half-maximal and maximal responses. We
adopted a similar approach to Cu toxicity testing, working on the hypothesis that increasing Cu
exposure will be associated with increasing levels of cytotoxicity which in turn would give rise
to ever more complex transcriptional responses.
11
Figure 2. Transcriptional profiling of the concentration response of adult M. californianus to copper.
Heatmaps of 572 induced (A) and 323 repressed (B) genes that exhibited a statistically significant sigmoidal
transcriptional response. Each row corresponds to a single gene and each column corresponds to a particular
concentration of copper. The average expression of each gene at each concentration is represented by a color, with
yellow or blue indicating that the gene is up-regulated or down-regulated relative to the expression observed in
control mussels. The genes are ranked and grouped according to their observed expression half maximal induction
value, with more responsive transcripts located at the top of each heatmap. Selected genes whose expression may be
pertinent to the response to copper are indicated next to each grouping. (C) Example of the transcriptional dose
response profile of Zinc transporter ZIP12 indicating the lowest dose of copper that induced the expression of this
transcript (Low), and the dose that induced the half-maximal induction of the transcript (HalfMax). The data are
presented as the median expression standard deviation of the transcript from microarray data collected from 3
replicate mussels at each concentration. (D) Example profiles of other transcripts that exhibited sigmoidal
concentration-response profiles. (E) Histogram of induced and repressed transcripts showing the frequency of
minimum and HalfMax Cu concentrations at 5 g/L intervals.
Copper (g/L)
0 20 40 60 80 100 120
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
0 20 40 60 80 100 120
-1
0
1
2
3
4
5
0 20 40 60 80 100 120
-1
0
1
2
3
4
HalfMax
20-29
30-39
40-49
50-59
60-69
70-79
80-89
1-19
20-29
40-49
30-39
50-59
60-69
70-79
80-89
0 3 6 9 15
30
60
90
120
A
B
>2
Relative expression
<0.5 1
Transcription intermediary factor α
Transcription intermediary factor β
Apoptosis inducing factor 3
Programmed cell death protein 4
Transcription intermediary factor 1-β
Centriolin
Caspase 7
Adenosine deaminase
Adenylate kinase 5
Adenylosuccinate synthetase isozyme 2
Nicotinamidephosphoribosyltransferase
Transcription intermediary factor 1-α
G1/S-specific cyclin-D2
Centrosome associated protein CEP25
Centrosomal protein of 135KDa
Caspase 3
Myc proto-onco gene
Pim-1 proto-onco gene
DNAJ homolog C12
Baculoviral IAP repeat-containing protein 2
Baculoviral IAP repeat-containing protein 3
Inositol-3-phosphate synthase
DNA damage-regulated autophagy modulator protein 2
Growth arrest and DNA damage-inducible protein GADD45
Thioredoxin reductase
Cell division control protein 42 homolog
Cyclic AMP-responsive element-binding protein 1
Cell division cycle protein 123 homolog
Zinc transporter ZIP12
Heat shock factor protein 1
Glutaredoxin-1
T-complex protein 1 subunit alpha
Glutathione S-transferase Mu 4
Growth arrest and DNA damage-inducible protein GADD45
T-complex protein 1 subunit delta
T-complex protein 1 subunit eta
Ferric-chelate reductase 1
Sequestosome-1
Stress-induced-phosphoprotein 1
Peroxiredoxin-1
Hsp70-binding protein 1
Glutamate--cysteine ligase catalytic subunit
10 kDa heat shock protein, mitochondrial
T-complex protein 1 subunit beta
Cyclic AMP-dependent transcription factor ATF-3
Glutathione S-transferase omega-1
Alpha-crystallin A chain
Alpha-crystallin B chain
Glutathione S-transferase A2
Glutamate--cysteine ligase regulatory subunit
Tubulin beta-2B chain
Tubulin beta-2C chain
Tubulin alpha-3C/D chain
D
Hsp70B2
Low=37 g/L
HalfMax=79 g/L
Sequestosome 1
Low=43 g/L
HalfMax=116 g/L
C
0 20 40 60 80 100 120
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Zinc transporter ZIP12
Low=19 g/L
HalfMax=63 g/L
Copper (g/L)
Rela
tive
mR
NA
exp
ressio
n (
log
2)
0 20 40 60 80 100 120
-1.2
-1.0
-0.8
-0.6
-0.4
-0.2
0.0
0.2
0.4
0.6G1/S-specific cyclin-D2
Low=15 g/L
HalfMax=29 g/L
Adenylosuccinate
synthetase 2
Low=31 g/L
HalfMax=69 g/L
E
Rela
tive
mR
NA
exp
ressio
n (
log
2)
Copper (g/L)
Peroxisome proliferator-activated receptor alpha
T-complex protein 1 subunit epsilon
T-complex protein 1 subunit theta
S-adenosylmethionine synthase 1
Peroxisomal acyl-coenzyme A oxidase 1
Multidrug resistance protein 1
cAMP-responsive element-binding protein-like 2
Heat shock-related 70kDa protein 2
90-99
0
20
40
60
80
100
120
Tra
nscri
pt fr
eq
ue
ncy
0
10
20
30
40
50
60
70
Bin center (g/L Cu)
2.5
7.5
12.5
17.5
22.5
27.5
32.5
37.5
42.5
47.5
52.5
57.5
62.5
67.5
72.5
77.5
82.5
87.5
92.5
Low dose
HalfMax
Induced
Repressed
12
Adult mussels were exposed for 24 hrs to increasing concentration of Cu in 2L containers. To
ensure that Cu concentrations were not depleted during the experiment, the water was replaced
every 4 hrs with fresh seawater adjusted to the appropriate Cu concentration. After 24 hrs total
RNA from gill tissue was isolated from 3 mussels incubated at each dose, converted to amplified
RNA, and hybridized to M. californianus cDNA microarrays. ANOVA identified 1,012 genes
that show a show statistically significant difference in expression between doses (p<0.01, FDR
corrected). In total, 413 genes were down-regulated in response to copper and 599 were up-
regulated. Transcripts that exhibited a concentration-response relationship were identified with
the SDRS algorithm (48), revealing that 95% (572/599) of the induced transcripts, and 78%
(323/413) of the repressed transcripts, exhibited an expression profile that fitted a sigmoidal
concentration-response curve to copper. Ranking the transcripts according to the effective
concentration of copper that elicited a half-maximal induction of the transcript serves to
highlight the range of responses of individual transcripts to copper (Figs. 2A & 2B). Inspection
of the concentration-response profiles of individual genes further illustrates the sigmoidal nature
of the transcriptional response to copper (Figs. 2C & 2D). An important conclusion that can be
drawn from these data is that exposure to Cu induces proportional changes in the transcriptome,
and that the abundance of specific transcripts is a function of the concentration of Cu.
Of particular relevance to toxicity testing, the SDRS algorithm reports the minimum Cu
concentration that induced a transcriptional response for each gene. A plot of the frequency
distribution of the minimum Cu concentration for both induced and repressed transcripts (Fig.
2E) showed that transcript repression tends to occur at lower concentrations of copper than
transcript induction. For example, the mode for the minimum concentration of copper required to
reduce transcript levels occurred at 12.5 g/L, whereas the mode for induced transcripts occurred
at 22.5 g/L. The list of Cu-induced transcripts included many genes associated with the
oxidative stress response such as Thioredoxin reductase 3, Peroxiredoxin, Glutaredoxin, and 3
isoforms of Glutathione S-transferase, consistent with the known deleterious effects of copper on
mitochondrial function (56, 57). We observed evidence of increasing proteotoxic stress as dose is
increased with T-complex protein chaperones induced at 22 g/L, Heat shock protein 70
isoforms induced at 35 g/L, and Sequestosome 1 induction at 41 g/L. Detection of hierarchical
series of stress responses provides compelling insights into toxicity and damage thresholds.
Toxicity responses of embryo-larval mussels
It should be noted that adult mussels are far more tolerant to copper than their larvae and that the
EPA Cu testing criteria is based upon toxicity to larvae (43). Therefore, Megan Hall, the
graduate student who is the nominated trainee for this proposal, has conducted a series of Cu
toxicity assays reproducing conditions of the standard EPA embryo-larval development assay.
Larvae were exposed to increasing concentrations of Cu and at the end of the 48 hr exposure
period, larval survival and development were quantified. Survival and normal development
declined at similar Cu concentrations to those in assays by EPA testing facilities (Fig. 1).
Increases in amorphous, deformed larvae, which characterize an “abnormally developed” animal,
are easily detected via visual microscopic analysis. This experiment was conducted in triplicate,
with a different pair of parents contributing the larvae to each trial.
Megan also collected additional samples of larvae from these experiments for gene expression
analysis. Samples from the first two trials were processed for barcoded RNAseq analysis and
13
were sequenced across two lanes of an Illumina HiSeq 2000 instrument. Analysis of read count
expression data revealed that gene expression profiles were closely correlated to Cu exposure
(Fig. 3A), with some genes induced or repressed at low concentrations (~2-3.1 g/L), and others
induced or repressed at higher concentrations (~10-25 g/L). The majority of genes showed
similar patterns of differential expression across the two trials (Fig. 3B). Megan is currently
writing a thesis chapter and an accompanying manuscript that compares and contrasts the
concentration-response kinetics of the adult and embryo-larval molecular response to Cu
exposure.
Figure 3. Transcriptional profiling of the concentration response of larval M. californianus to copper. (A)
Example of a heat map of copper-responsive genes collected under US EPA embryo-larval toxicity assay conditions.
Increasing intensity of yellow or blue color indicates transcripts whose abundance increased or decreased
respectively in response to Cu exposure. Columns represent increasing Cu concentrations (g/L), and each row
represents a different gene.. B) Examples of consistent gene expression profiles between two replicate trials of the
assay. Solid lines represent the first trial, and dashed lines represent the second trial, and represent data collected
from independent spawns.
Mussel embryo-larval toxicity assays under OA
This proposal is a natural progression of a 1 year student thesis project, “Predicting the Effects of
Copper Toxicity and Ocean Acidification on Marine Invertebrates”, funded by CA SeaGrant
(02/01/15-01/31/16), which will effectively prime the activities in the project proposed here. This
project is funding a pilot transcriptional profile of a standard mussel embryo-larval toxicity assay
of copper under elevated CO2 concentrations, followed by corroboration in ‘real-world’ samples,
achieved by running toxicity assays on Marina Del Rey Harbor water samples under acidified
conditions. It will also investigate the utility of extended toxicity assays for providing long-term
insights into the effects of copper and OA on larval health.
Histone Deacetylase Zinc Finger; C2H2-type
Sequestosome Glycoside hydrolase
HSP 70 Proteasome
0 2 3.1 4 6 8 10 15 20 25
-2
0
2
4
6
8
10
0 5 10 15 20
Fold
Cha
nge
in g
ene
expr
essi
on (L
og 2
)
-1
-0.5
0
0.5
1
1.5
2
2.5
3
0 5 10 15 20
Cu +2 (μg/L)-2
0
2
4
6
8
10
12
14
0 5 10 15 20
Fold
Cha
nge
in g
ene
expr
essi
on (L
og 2
)
Cu +2 (μg/L)
-5
-4
-3
-2
-1
0
1
0 5 10 15 20
-2
0
2
4
6
8
10
0 5 10 15 20
Fold
Ch
an
ge
in g
en
e
expr
essi
on (L
og 2
)A B
-4
-3
-2
-1
0
1
0 5 10 15 20
14
11. BUDGET RELATED INFORMATION
A. Budget Explanation/Justification.
SALARIES:
As project leader, the PI will be heavily involved in the day-to-day operation of this project.
Gracey will spend 1.4 and 1.6 months of effort on this project in years 1 and 2, and requests 0.25
months of summer salary support per year from USC SeaGrant. One Sea Grant Research Trainee
is requested for the 2 year duration of this project to work exclusively on all aspects of this
project, which will contribute towards her doctoral dissertation. The trainee proposed is Megan
Hall, a 4th year graduate student in the Gracey lab, to be the student associated with this project.
She has generated all of the supporting larval toxicity data for this proposal.
EXPENDABLE SUPPLIES AND EQUIPMENT:
RNA sequencing
Using our optimized protocols, each RNA sample costs about $50 to process through to a
quality-checked and titrated Illumina sequencing library. RNA sequencing will be performed at
the USC Epigenome Center Data Production Facility at an institutional discount rate of $1,300
per lane of 50 bp of single-end reads. With accurate titration we consistently obtain >200 million
reads per lane. For each metal we will sequence 42 libraries from the concentration-response/OA
experiment and 24 samples of normal/abnormal picked larvae, across 7 Illumina lanes. Therefore
the cost for expression analysis per metal is ($1,300 x 7) + ($50 x 66) = $12,400, x 3 metals =
$37,200
Bottled gases
We request $500 per year to cover the cost of compressed gases and other expendable supplies
for ICP-MS analysis, and the defined CO2 gas mixtures for larva-embryo toxicity assay.
Laboratory supplies
Funds of $1,000 per year are requested for laboratory supplies and chemicals necessary to
undertake the molecular biology and toxicity testing components of this project (plastic
consumables @ $500, gases, chemicals & reagents @ $250, oligonucleotides and qRT-PCR
reagents @ $250)
TRAVEL:
Funds of $500 per year are requested to cover the costs of the collection of water samples,
mussel broodstock, and local outreach activities. Funds of $3,000 in year 2 are requested towards
travel, accommodation, and registration for Gracey and the Sea Grant Trainee to attend one
Northern American Society of Environmental Toxicology and Chemistry (SETAC) meeting. The
trainee will present a poster and Gracey will communicate our results in an oral presentation.
PUBLICATION COSTS AND DATA DISTRIBUTION:
We request $2,000 to cover page charges associated with publishing our research in high quality
scientific journals in both years of the project. We request $1,000 to cover the cost to hosting
1Terabyte of data at the USC Digital Repository.
B. Matching Funds.
Gracey will spend 1.15 and 1.35 months of effort on this project in each academic year supported
on his academic salary and this will be contributed by USC as cost sharing. This constitutes
$22,680 and $26,154 in years 1 and 2 respectively.
12. ANTICIPATED BENEFITS
The proposed research will be timely and important for addressing several key issues facing
coastal ecosystems of California. Ocean acidification is expected to have notable effects on
15
contaminant speciation and bioavailability, so it will likely influence the concentration of metals
that trigger toxic responses. This information will be vital to policy-makers, scientists, and the
general public. From a biological perspective, it is invaluable to gain insight into the
physiological and ecological effects of ocean acidification and contaminant metals in marine
ecosystems. Data will contribute to the growing body of research on biological responses to
multiple stressors, and inform future studies that attempt to investigate other issues concerning
metal toxicity and ocean acidification. Most importantly, policy-makers will be able to set limits
for metal pollution that protect sensitive members of local marine ecosystems under dynamic
ocean conditions. Regulators must set contaminant limits that account for other important aspects
of water chemistry. According to the results of our study, and needed investigations into other
contaminants of concern, regulatory limits can be adjusted appropriately and in a timely fashion
to meet the challenges that a changing global ocean presents.
The research described here focuses on contaminants and sites in southern California, but the
approach could be applied to any receiving waters and any contaminant nationwide. The
proposed work strives to more accurately assess coastal water quality, and will thus result in an
improved ability to manage contaminant levels effectively. Reconsidering water quality criteria
is especially important in a changing global ocean, where we must measure and define toxicity
when there are numerous dynamic parameters at play. In order to ultimately predict how multiple
stressors may interact, we need to understand the fundamental effects of stressors by interpreting
their effects on specific biochemical pathways. By examining toxicity in the presence of multiple
stressors, and analyzing the fundamental physiological changes that occur, this research will
facilitate this understanding.
The benefit of incorporating gene expression measurements—a relatively new technology—into
standard toxicity testing could be remarkable. This technology has the potential to make water
quality toxicity testing faster, cheaper, and more reliable, as the laborious process of identifying
morphological abnormalities and counting larvae would be unnecessary, and individual
subjectivity in counts would be more or less eliminated. We have already entered discussions
with other non-academic research groups and environmental consulting agencies in the southern
California area that are interested in incorporating transcriptional profiling into their toxicity
testing. We intend to continue these talks and branch out to other parties, such as regional water
quality control boards, who may benefit from using this kind of tool. Ultimately, transcriptional
profiling could allow us to retrieve a specific gene expression profile, and be able to determine
which contaminants (or combination of contaminants) an organism has been exposed to, and
how severe that exposure has been. This kind of tool would provide substantial power for rapidly
identifying toxins in coastal ecosystems, and thus quickly disseminating warnings for fisheries,
shellfish farmers, and the general public.
Locally, we will work closely with researchers at the Southern California Coastal Water
Research Project (SCCWRP) – see attached letter of support. They play a pivotal role in advising
water quality management issues in southern California and are heavily involved in the ongoing
discussions regarding the future cleanup of copper from Marina del Rey Harbor. We will share
our findings with them and will assist in the molecular characterization of our identified
biomarkers in their collected Marina del Rey harbor water samples.
16
13. COMMUNICATION OF RESULTS
We will present our findings at both the local and national Society of Environmental Toxicology
and Chemistry (SETAC) meetings. These are important forums at which academic as well as
environmental and governmental agencies are represented. To reach the broader public in LA,
we propose to participate in the Aquarium of the Pacific’s “Urban Ocean Festival”, as a venue
for informing the public of some of the unanticipated challenges which climate change poses to
impacted coastal ecosystems. We will work with Dave Bader of the aquarium to devise an
appropriate educational output of our results. We will also explore a similar effort at the
California Science Center that is located adjacent to USC.
The trainee will also pursue a range of outreach initiatives. First, she will organize a mini
seminar series with the Long Beach Marina Boat Owners Association and the Del Rey yacht
club to discuss the costs and benefits of switching to copper-free non-toxic antifouling paints.
She will also use this as an opportunity to poll boaters on how they view the copper problem in
Marina del Rey and other California harbors, and try to develop cost-effective solutions with
them. She will also collaborate with a citizen science group associated with LA Makerspace that
she has worked with previously. With this group she will organize a quarterly (every 3 months)
toxicology workshop, and bring citizen scientists to the field to experience a day in the life of a
marine toxicologist. They will collect water samples, and even spawn mussels to go through the
assay set-up. This exercise will also be documented by video recording on Periscope, a live
streaming app for smartphones and computers. This will allow any viewers following her on
Twitter and Facebook to access the link, and get to witness the citizen science activities in action.
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PROJECTED WORK SCHEDULE Project Title: DISTINGUISHING EXPOSURE FROM EFFECT IN MULTIPLE-STRESSOR SCENARIOS: EFFECTS OF OCEAN ACIDIFICATION AND METAL-TOXICITY ON MUSSEL LARVAL DEVELOPMENT
Activities 2016-2017 F M A M J J A S O N D J
Metal 1 Embryo-larval toxicity assay
X X X
Metal 1 Gene expression analysis
X X X
Metal 1 Normal versus abnormal larvae analysis
X X X
Metal 2 Embryo-larval toxicity assay
X X X
Metal 2 Gene expression analysis
X X
Metal 2 Normal versus abnormal larvae analysis
Data analysis and integration and writeup
X X X
Page 1
Activities 2017-2018 F M A M J J A S O N D J
Metal 2 Gene expression analysis
X
Metal 2 Normal versis abnormal larvae analysis
X X X
Metal 3 Embryo-larval toxicity assay
X X X
Metal 3 Gene expression analysis
X X X
Metal 3 Normal versus abnormal larvae analysis
X X
Data analysis and integration and writeup
X X X x x x x x x
OMB Control No. 0648-0362
Expiration Date 1/31/2018
SEA GRANT BUDGET FORM 90-4
GRANTEE: USC SeaGrant GRANT/PROJECT NO.:
DURATION (months 2402/01/2016 - 01/31/2017
12 months 1 Yr.A. SALARIES AND WAGES: man-months
1. Senior PersonnelNo. of People
Amount of Effort Sea Grant Funds Matching Funds
a. (Co) Principal Investigator: 1 1.4 3,736 17,300b. Associates (Faculty or Staff):
Sub Total: 1 1.4 3,736 17,300
2. Other Personnela. Professionals:b. Research Associates:c. Res. Asst./Grad Students:d. Prof. School Students:e. Pre-Bachelor Student(s):f. Secretarial-Clerical:g. Technicians:h. Other:
Total Salaries and Wages: 1 1.4 3,736 17,300
B. FRINGE BENEFITS: 31.1% 1,162 5,380Total Personnel (A and B): 4,898 22,680
C. PERMANENT EQUIPMENT:
D. EXPENDABLE SUPPLIES AND EQUIPMENT: 20,100
E. TRAVEL:1. Domestic 5002. International
Total Travel: 500 0
F. PUBLICATION AND DOCUMENTATION COSTS: 2,000
G. OTHER COSTS:1234567
Total Other Costs: 0 0
TOTAL DIRECT COST (A through G): 27,498 22,680
INDIRECT COST (On campus 65% ): 2291.4913 17,816 0INDIRECT COST (Off campus % of $ ):
Total Indirect Cost: 17,816 0
TOTAL COSTS: 45,314 22,680
PRINCIPAL INVESTIGATOR: Andrew Gracey
BRIEF TITLE: OCEAN ACIDIFICATION AND METAL TOXICITY IN MUSSEL
OMB Control No. 0648-0362
Expiration Date 1/31/2018
SEA GRANT BUDGET FORM 90-4
GRANTEE: USC SeaGrant GRANT/PROJECT NO.:
DURATION (months 2402/01/2017 - 01/31/2018
12 months 1 Yr.A. SALARIES AND WAGES: man-months
1. Senior PersonnelNo. of People
Amount of Effort Sea Grant Funds Matching Funds
a. (Co) Principal Investigator: 1 1.6 3,848 19,950b. Associates (Faculty or Staff):
Sub Total: 1 1.6 3,848 19,950
2. Other Personnela. Professionals:b. Research Associates:c. Res. Asst./Grad Students:d. Prof. School Students:e. Pre-Bachelor Student(s):f. Secretarial-Clerical:g. Technicians:h. Other:
Total Salaries and Wages: 1 1.6 3,848 19,950
B. FRINGE BENEFITS: 31% 1,197 6,204Total Personnel (A and B): 5,045 26,154
C. PERMANENT EQUIPMENT:
D. EXPENDABLE SUPPLIES AND EQUIPMENT: 20,100
E. TRAVEL:1. Domestic 3,5002. International
Total Travel: 3,500 0
F. PUBLICATION AND DOCUMENTATION COSTS: 2,000
G. OTHER COSTS:1 Data storage 1,000234567
Total Other Costs: 1,000 0
TOTAL DIRECT COST (A through G): 31,645 26,154
INDIRECT COST (On campus 65%): 65% 20,569 0INDIRECT COST (Off campus of $ ):
Total Indirect Cost: 20,569 0
TOTAL COSTS: 52,214 26,154
PRINCIPAL INVESTIGATOR: Andrew Gracey
BRIEF TITLE: OCEAN ACIDIFICATION AND METAL TOXICITY IN MUSSEL
BRIEF CURRICULUM VITAE
NAME Andrew Y. Gracey
ADDRESS Marine Environmental Biology, University of Southern California, 3616
Trousdale Parkway #107, Los Angeles, CA 90089
PHONE (work) 213-740 2288 (cell) 408-425 9397 EMAIL [email protected]
EDUCATION
University of Liverpool, UK: B.Sc. with Honors in Marine Biology—1991
University of Liverpool, UK: Ph.D. in Comparative & Molecular Physiology—1996
International Institute of Genetics & Biophysics, Naples, Italy: Postdoctoral study—1995-1996
Stanford University: Postdoctoral study: physiology—1997-2000
University of Liverpool: Postdoctoral study: physiology—2000-2002
POSITIONS HELD
Stanford University: Research Associate professor: physiology—2002-July 2005
University of Southern California: Assistant professor, Biological Sciences—Aug 2005-Apr
2012
University of Southern California: Associate professor, Biological Sciences—Apr 2012-present
SELECTED PUBLICATIONS
1. Tiku, P.E., Gracey, A.Y., Macartney, A.I., Beynon, R.B. and Cossins, A.R. (1996) Cold-
inducible expression of desaturase by transcriptional and post-translational mechanisms.
Science, 271: 815-818.
2. Gracey, A.Y., Troll, J. and Somero, G.N. (2001) Hypoxia-induced expression profiling
in the euryoxic fish Gillichthys mirabilis. Proc. Natl. Acad. Sci. USA, 94: 1993-1998.
3. Gracey, A.Y. and Cossins, A.R. (2003) Application of microarray technology in
environmental and comparative physiology. Annu. Rev. Physiol., 65: 231-59.
4. Gracey, A.Y., Fraser, E. J., Li, W., Fang, Y., Taylor, R. R., Rogers, J., Brass, A. and
Cossins, A. R. (2004) Coping with cold: An integrative, multitissue analysis of the
transcriptome of a poikilothermic vertebrate. Proc. Natl. Acad. Sci. USA, 101: 16970-
16975.
5. Williams, D., Epperson, L., Li, W., Hughes, M., Taylor, R. R., Rogers, J., Martin, S.,
Cossins, A. R. and Gracey, A.Y. (2005) Seasonally hibernating phenotype assessed
through transcript screening. Physiol. Genomics, 24: 13-22.
6. Fraser, E. J., Vieira de Mello, L. Ward, D., Rees, H., Williams, D., Fang, Y., Brass, A.,
Gracey, A.Y. and Cossins, A.R. (2006) Hypoxia-inducible myoglobin expression in non-
muscle tissues. Proc. Natl. Acad. Sci. USA, 103: 2977-2981.
7. Murray, P., Hayward, S.A., Govan, G.G., Gracey, A.Y. and Cossins, A.R. (2007) An
explicit test of the phospholipid saturation hypothesis of acquired cold tolerance in
Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA, 104: 5489-5494.
8. Gracey, A.Y., Chaney, M. L., Boomhower, J., Tyburczy, W., Connor, K. and Somero, G.
N. (2008) Rhythms of gene expression in a fluctuating intertidal environment. Current
Biol., 18, 1501-1507.
9. Chaney, M. L. and Gracey, A.Y. (2011) Mass mortality in Pacific oysters is associated
with a specific gene expression signature. Mol. Ecol., 20, 2942-2954.
10. Gracey, A.Y., Lee, B., Higashi, R. and Fan, T (2011) Hypoxia-induced mobilization of
triglycerides in the euryoxic goby, Gillichthys mirabilis. J. Exp. Biol., 214, 3005-3012.
11. Connor, K.M. and Gracey, A.Y. (2011) Circadian cycles are the dominant transcriptional
rhythm in the intertidal mussel Mytilus californianus. Proc. Natl. Acad. Sci. USA, 108,
16110-16115.
12. Connor, K. M. and Gracey, A.Y. (2012) High resolution analysis of metabolic cycles in
the intertidal mussel Mytilus californianus. Am. J. Phys-Reg. I. 302(1), R103-11.
13. Nydam, M.L., Netuschil, N., Sanders, E., Langenbacher, A., Lewis, D.D., Taketa, D.A.,
Marimuthu, A., Gracey, A.Y. and De Tomaso, A.W. (2013) The candidate
histocompatibility locus of a basal chordate encodes two highly polymorphic proteins.
PLoS One, 8 (6):e65980.
14. Mandic, M., Ramon, M.L., Gracey, A.Y. and Richards, J.G. (2014) Divergent
transcriptional patterns are related to differences in hypoxia tolerance between the
intertidal and the subtidal sculpins. Mol. Ecol., 24, 6091-103.
15. Rodriguez, D., Sanders, E.N., Farell, K., Langenbacher, A.D., Taketa, D.A., Hopper,
M.R., Kennedy, M., Gracey, A.Y. and De Tomaso, A.W. (2014) Analysis of the basal
chordate Botryllus schlosseri reveals a set of genes associated with fertility. BMC
Genomics, 15(1):1183. [Epub ahead of print]
SUMMARY PROPOSAL FORM PROJECT TITLE: DISTINGUISHING EXPOSURE FROM EFFECT IN MULTIPLE-STRESSOR SCENARIOS: EFFECTS OF OCEAN ACIDIFICATION AND METAL-TOXICITY ON MUSSEL LARVAL DEVELOPMENT OBJECTIVE: Our overarching research goal is to use mussels as a model to test the hypothesis that ocean acidification (OA) will affect metal toxicity. Mussels are model organisms in the fields of both environmental toxicity and OA research. Copper criteria are determined by embryo-larval toxicity testing of the genus Mytilus, and as calcifying marine invertebrates mussels have become a key model for predicting the effects of OA. A long-standing problem in environmental toxicology has been to distinguish biomarkers of exposure from those of effect, which has led the EPA to call for the development of “Adverse Outcome Pathway” markers of toxicity. To address this challenge, we will combine classic embryo-larval testing protocols with a novel stratified sub-sampling technique that can distinguish biomarkers of exposure from those that are markers of adverse effect. This stratified sampling approach has the potential to untangle the complex interactions that occur when multiple variables are employed in water quality testing protocols. METHODOLOGY: Mussel embryo-larval toxicity assays will be performed according to standard EPA protocols (US EPA 1995). Reference water samples will be prepared with a range of environmentally relevant metal doses, and the effects of increasing metal contaminants will be tested at present day CO2 concentrations (400ppm), and predicted future concentrations (800ppm). Seawater pH and carbonate chemistry assays will be conducted in collaboration with the Hutchins lab at USC. Mortality and the proportion of abnormally developing embryos under each set of conditions will be quantified after 48 hr. Biomarkers will be developed using a stratified sub-sampling regime that leverages the fact that all the embryos in the vessel received the same contaminant exposure but only a sub-population will exhibit an adverse morphological outcome. Thus, for each treatment we will select 100 embryos that exhibit either normal or abnormal development, with abnormal development being a visual marker of the adverse effects of the particular conditions. Next-generation RNA sequencing will be used to identify transcripts that distinguish contaminant exposure from those linked to the onset of adversely effects. RATIONALE: The regulations governing copper and other metal contamination in Southern California’s coastal ocean must be considered in the context of a changing global ocean. Increased dissolved CO2 levels are predicted to decrease ocean pH to 7.7
by 2100 as well as significantly lower calcium carbonate saturation constants, especially in the California Current system. It is expected that OA will increase copper toxicity by reducing the complexation capacity of coastal waters and increasing free copper (Cu+2) concentrations. Understanding the effects of OA on water quality issues remains a
largely unexplored but looming question. This is a particular challenge in urban oceans because multiple environmental and chemical parameters will vary temporally and spatially due the proximity of large sources of urban pollutants. This means that water quality testing approaches that will be implemented this century will have to be nuanced and capable of analyzing multiple stressor scenarios in order to predict the impact that contaminants will have on coastal urban ecosystems. DATA SHARING PLAN: We will make all data visible and accessible to the wider academic community, government agencies, and public as both raw data (to encourage independent review), and as summarized findings in formats appropriate for academic users and in formats that will be understandable by the general public and educators. These data will be hosted by the USC Digital Repository which will ensure efficient access to these large datasets. Next generation sequences will be deposited in the public Short Read Archive hosted by the NCBI.
July 3, 2015
Dr. Andrew Gracey
Marine Environmental Biology
University of Southern California
3616 Trousdale Parkway #107
Los Angeles, CA 90089
Re: Letter of Support for proposed project on metal toxicity and ocean acidification
Dear Dr. Gracey,
The Southern California Coastal Water Research Project (SCCWRP) strongly supports your
proposed research project on metal toxicity and ocean acidification. The proposed research
addresses two important issues related to the assessment and management of water quality in
coastal urban areas: development of improved tools for assessing contamination impacts, and
adapting management programs to the impacts of climate change (e.g., ocean acidification).
This research will have nationwide applicability and value, as these are issues of concern for
many organizations.
SCCWRP is a public research institute focusing on the coastal ecosystems of Southern
California, from watersheds to the ocean. A primary focus of our activities is to improve the
ability of water quality managers to assess and protect water quality, through the incorporation of
state of the art research and technology in monitoring and policy. As the head of SCCWRP's
toxicology department, I am conducting several research projects focused on metal toxicity and
the development of genomic tools to improve environmental monitoring.
The goal of this proposal, to understand the potential impacts of ocean acidification on metal
toxicity, is highly relevant to SCCWRP’s mission and the concerns of our governing
Commission, which includes the key coastal water quality management agencies in southern
California. I am currently conducting research on copper toxicity in Marina del Rey Harbor that
includes testing with the same type of organism proposed in your project (mussel embryos). Our
research will use traditional toxicity test methods to investigate the influence of variations in
harbor water quality characteristics on copper toxicity. Your proposed research project is
complementary to our research and provides an excellent opportunity for collaboration and
expanding the application of our research.
SCCWRP would like to collaborate with you on your ocean acidification/metal toxicity research.
We will be conducting harbor water sampling, toxicity testing, and chemical analyses during
2015/2016 and can provide you with samples and data from our analyses. Collaboration will
increase the relevance of your work to ongoing regulatory studies in Marina del Rey Harbor,
facilitate communication of your results to water quality managers, and enable SCCWRP to
extend our research findings to future scenarios influenced by ocean acidification.
Please contact me if you would like to further develop a collaboration on our projects.
Sincerely,
Steven Bay, Principal Scientist
Toxicology Department
!
!
Biological Sciences Marine and Environmental Biology
Professor David A. Hutchins
!
University of Southern California 3616 Trousdale Parkway, Los Angeles, California 90089-0371 • Tel: 213 821-5779 • Fax: 213 740 8123
! ! ! ! ! ! ! ! 7/1/15!Dear!Andy!and!Megan:!!I!am!happy!to!support!your!pending!California!Sea!Grant!proposal!entitled!“Distinguishing exposure from effect in multiple-stressor scenarios: effects of ocean acidification and metal-toxicity on mussel larval development”.!!The!results!of!your!proposed!work!will!provide!important!and!novel!insights!into!the!how!future!ocean!acidification!could!affect!ecologically!and!economically!important!species!of!bivalves.!!Accumulation!of!toxic!copper!in!mussels!is!a!long!standing!problem!in!many!areas!along!the!California!coast!that!have!been!contaminated!by!copperLbased!antiLfouling!paint,!and!ocean!acidification!has!the!potentially!to!greatly!increase!these!already!problematic!toxicity!effects.! !I’ll!be!happy!to!support!your!project!by!helping!to!oversee!the!ocean!acidification!aspects!of!your!experiments.!!!My!laboratory!is!well!set!up!to!manipulate!and!analyze!the!seawater!carbonate!buffer!system,!as!ocean!acidification!has!been!a!longLstanding!area!of!research!in!our!group!supported!by!both!Sea!Grant!and!NSF!awards.!!I!can!also!provide!you!with!advice!and!oversight!of!your!experimental!designs,!in!order!to!make!sure!they!meet!the!highest!standards!for!global!change!manipulative!studies.!!I!am!very!enthusiastic!about!having!the!chance!to!collaborate!with!you!on!this!work!that!will!significantly!advance!our!knowledge!of!the!impacts!of!a!changing!coastal!ocean!on!key!species!of!commercially!important!shellfish.!!!
Best!of!luck!with!your!proposal,!
David A. Hutchins David!A!Hutchins!Professor!and!Section!Head!Marine!Environmental!Biology!University!of!Southern!California!3616!Trousdale!Parkway!Los!Angeles,!CA!90089!phone!213!740!5616,[email protected]!