1

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

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f em

bry

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wit

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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

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f em

bry

os

that

su

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ort

alit

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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.

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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 gracey@usc.edu

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

steveb@sccwrp.org

!

!

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!dahutch@usc.edu!