FY 2007-2009 Innovative F&W Program Project Solicitation
Section 10. Narrative
Project ID:
Title: The use of cDNA microarrays to develop biomarkers of environmental stress in
salmonids and other fishes
A. Abstract
The responses of all organisms are controlled by the expression of genes, the
DNA sequences of which encode for specific proteins. These proteins in turn regulate
responses at all levels including cellular, physiological, and behavioral. Microarray
technology is poised to revolutionize the fish and wildlife sciences by revealing the
genetic basis for responses of interest. The spotting of hundreds or thousands of genes on
a single coated glass slide has opened up new possibilities in our ability to investigate and
compare changes in gene expression as a result of exposure to environmental insults.
The technology allows questions to be answered on a scale as coarse as comparative
genomics and as fine as the expression of specific genes responsible for specific proteins.
Whereas previously, microarray technology has been reserved for biomedical research,
recently it has exploded into the field of molecular ecology with great potential for
continued growth. This new advancement is of particular interest to physiologists,
biologists, and managers because of its ability to reliably identify biomarkers for use in
contaminant, climate change, stress, and disease studies. Managers will be able to use
this information to determine the location, timing, and relative severity of environmental
stressors and make more informed decisions regarding remediation or mitigation.
We propose to use cDNA microarrays in two very different types of fish—salmon
and lampreys—to identify reliable biomarkers of environmental stress. For this work, we
will obtain species-specific microarrays, expose fish to relevant environmental stressors
in the laboratory (including contaminants, elevated water temperatures, and physical
stressors or hypoxia), and evaluate the expression of various genes and their potential for
use as biomarkers. Commercially available microarrays exist for salmonids, and we have
successfully used these to investigate differences in gene expression in juvenile Chinook
salmon with different migration histories to uncover the mechanisms of delayed and
latent mortality. A microarray for a non-model organism, such as the Pacific lamprey
Lampetra tridentata, currently does not exist. We plan on developing a microarray for
this species and will conduct parallel studies of salmon and lampreys to elucidate the
effects of common and increasingly serious environmental stressors on their
ecophysiology. We hope to lay a foundation for the use of microarrays as a tool for
monitoring the health and well being of fish populations in an era of increasing
environmental concerns.
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B. Technical and/or scientific background
I. cDNA microarrays
For this work, we will document the genomic responses of juvenile salmonids and
lampreys to common environmental stressors, identify genes that consistently respond to
such stressors, and develop biomarkers of exposure to specific types of stress. The
underlying foundation of this work is based upon cDNA microarrays. Microarrays are
microscopic matrices that allow the organization and study of many genes that are
usually species specific. Hundreds or thousands of segments of DNA are adsorbed onto a
glass microscope slide and each spot of DNA on the slide represents a gene responsible
for the production of a specific bioproduct. The RNA of interest is extracted from tissue
of a test animal, transformed into cloned DNA (cDNA), and tagged with fluorescent
dyes. This tagged cDNA is then hybridized to the corresponding DNA sequence on the
surface of the glass slide. The amount of fluorescent emission for each spot of DNA is
determined with special lasers, and this emission represents the expression level of that
gene in the individual from which the RNA was extracted.
cDNA microarrays have proven to be powerful tools for identifying pathways and
evaluating the transcriptional responses to stress in fish. For example, the 530 base pair
(bp) segment of rainbow trout cDNA, hsp70i, encodes for the inducible form of heat
shock protein 70, a product related to the cellular stress response. Heat and other
stressors will increase the expression of this gene. Thus, by hybridization of RNA from
various tissues on the cDNA microarray slide, one is able to determine which genes, and
therefore which physiological systems, are up or down-regulated during periods of stress
(for a detailed microarray protocol, see Lou et al. 2001). Researchers at the University of
Waterloo reported significant changes in several transcripts known to be affected by
stress in trout after exposure to a standard handling stressor. Further, they noted changes
in transcripts not previously implicated in the stress response, thus confirming the utility
of microarrays for physiological studies (Dr. M. Vijayan, personal communication).
Prodrabsky and Somero (2004) used high density microarray technology to identify
physiological processes that allow an annual killifish Austrofundulus limnaeus to survive
large daily fluctuations in water temperature (20 to 37ºC). They were able to detect
changes in the expression of genes which are responsible for cell growth and
proliferation, metabolic functions (e.g., carbohydrate, intermediary, and nitrogen
metabolism), and immune responses.
Microarray studies allow the discovery of new facets of physiological responses
that are not anticipated by the investigator. Thus, with a single microarray experiment,
established stress-responsive pathways can be monitored, and new stress-responsive
pathways can be identified. In addition, it is highly likely that gene expression
“fingerprints” can be developed for a generalized stress response as well as responses
correlated to very specific stressors such as thermal stress or exposure to environmental
toxicants, thus making microarrays very useful for identifying biomarkers. This trait also
makes microarrays a reliable disease diagnostic tool. In reality, the uses for microarrays
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are growing every day and it is now possible to open up the field of molecular ecology to
the research and management of Columbia River Basin (CRB) fishes.
II. Stress in fish, biomarkers, and microarrays
The literature on the molecular, physiological, and behavioral responses of fish to
environmental stressors is voluminous. Since Hans Selye’s (1950) description of the
general adaptation syndrome (GAS) paradigm describing the stages an organism passes
through in response to stress, there have been a plethora of research articles, several
reviews, and whole books written on the subject of stress in fish. As stated by Barton et
al. (2002), exposure of fish to environmental stressors is a concern to biologists and
managers because of possible detrimental effects on important fish performance metrics,
including metabolism and growth, disease resistance, reproductive capacity, and,
ultimately, the health, condition, and survival of fish populations.
Perhaps because of their high economic, aesthetic, and ecological value, much of
the research on stress in fish has focused on members of the family Salmonidae.
Although much of this work was initially directed at understanding the responses of
salmonids to aquaculture-related stressors, research topics have broadened over the years.
For example, recent studies on stress in salmonids have addressed topics such as
contaminant exposure, dam passage, exposure to predators, tagging effects, and thermal
insults. Along with new subjects being addressed, new tools have been developed to
allow unprecedented insight into the effects of stress on fish. Gone are the days of
simply measuring plasma glucose or cortisol levels to assess the stressful experiences of
fish. Today, researchers can use a variety of molecular, cellular, physiological, and
behavioral indicators to assess the effects of stress on fish and develop relevant
biomarkers (see Adams 2002 for a detailed review).
It is perhaps axiomatic to state that the management and restoration of salmonids
in the CRB have benefited tremendously from research on stress. Understanding how
fish respond to stressors provides insight into the relative seriousness of different
stressors and leads to innovations in methods or equipment to minimize the effects of
stress. Certainly the aquaculture industry in the CRB, which is largely dedicated to
mitigation of losses of wild fish due to development of the Columbia Basin hydroelectric
system, has refined husbandry techniques based in large part on our knowledge of stress
in salmonids. For example, common aquaculture techniques such as handling and
crowding are known to elicit severe physiological responses in salmonids and can lead to
immunosuppression and poor health (Maule et al. 1987, 1989). Thus, personnel at
modern hatcheries take great care to minimize these types of procedures on their fish.
Information on the responses of fish to environmental stressors has also be useful for
such things as modifying and improving routes of passage at dams (Maule et al. 1988),
refining fish transportation techniques (Wagner and Congleton 2004), and conducting
survival and tagging studies (Martinelli et al. 1998; Mesa et al. 2003). Many of the
methods used today by biologists to capture, handle, tag, transport, or in any other way
work with salmonids in the CRB endeavor to minimize the effects of stress on fish. For
example, biologists conducting surgeries on fish to implant various tags have made use of
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our knowledge of stress in fish to fully appreciate the need to minimize the time fish
spend in anesthesia and on the surgical table. Many other approaches to the research and
management of salmonids, including reducing densities of barged and transported fish,
not mixing fish of different sizes or species during transportation, minimizing handling
wherever possible, and the use of mild doses of anesthetics for the transport, have
resulted from our extensive knowledge of stress in salmonids.
Surprisingly, little is known about the responses, physiological or otherwise, of
lampreys to common environmental stressors. Part of the reason for our poor
understanding of the stress response in lampreys is because lampreys, to most people, do
not rank high in economic, ecological, or aesthetic value and thus receive relatively little
funding for research. However, this notion is changing because populations of lampreys
in the CRB are in decline. The ecological, economic, and cultural significance of
lampreys has been underestimated by most people and actions are currently being
considered for their recovery in the CRB (Close et al. 1995). Several recent events
indicate that the conservation and restoration of lampreys in the CRB is increasing in
importance, including: (1) the January 2003 petitioning of four species of lamprey
(Pacific lamprey, brook lamprey L. richardsoni; river lamprey L. ayresi; and the Kern
River lamprey L. hubbsi) for listing under the Endangered Species Act; (2) the formation
of a regional Lamprey Technical Work Group charged with providing technical review,
guidance, and recommendations for activities related to lamprey conservation and
restoration; and (3) the holding of a lamprey summit in 2004 that brought together
biologists and managers from tribal and government agencies to discuss lamprey issues in
the CRB. Today, all indications suggest that lampreys will command increasing attention
by managers and biologists in the future.
Another reason for the paucity of information on stress in lampreys is that limited
research so far indicates that lampreys do not respond to stressors in a manner similar to
teleosts. There exist no consistent, reliable, or specific indicators of stress in lampreys
that could serve as biomarkers. Katz et al. (1980) documented changes in the hormone
androstenedione in response to surgery in the sea lamprey Petromyzon marinus. The
authors suggested that androstenedione may be a putative stress hormone in lampreys,
but further work to confirm this notion does not exist. Several studies have reported
hyperglycemia in lampreys after exposure to stress (Leibson and Plisetskaya 1969;
Larsen 1976; Close et al. 2003; Mesa et al. 2003), but glucose is a general, non-specific
indicator of stress in fish. Recently, Mesa et al. (2003) reported elevations of plasma
lactate after exhaustive exercise in the Pacific lamprey, but, like glucose, such indicators
of stress have limited utility as biomarkers. No endocrine system products—for example,
something akin to cortisol in teleosts—have been identified that could serve as stress
hormones in lampreys, although research is currently underway (Dave Close, Michigan
State University, personal communication). Complicating the issue is the possibility that
putative stress hormones in lampreys differ from those typically seen in more advanced
fishes. For example, recent studies suggest that the sex hormones in lampreys differ from
those in teleosts by having an extra hydroxyl group at the C15 position (Kime and Rafter
1981; Kime and Callard 1982; Lowartz et al. 2003; Bryan et al. 2003). All of this leads
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to the conclusion that to understand how these fish respond to environmental stressors
may require studies at the genomic level.
If one accepts the notion—like that presented earlier for salmonids—that
knowledge of the stress physiology of an animal is useful for its conservation,
management, and restoration, then directed research on the stress responses of lampreys
is long overdue. We propose to explore the genetic basis of the stress response in
lampreys exposed to some common environmental stressors and to identify genomic
markers of stress exposure. To this end, we will create a microarray containing several
thousand cDNA’s isolated from the tissues of L. tridentata. This microarray will be used
to profile changes in gene expression associated with stressors such as elevated
temperatures, exposure to contaminants, and hypoxia.
In summary, although traditional biochemical indicators have been the basis for
many of the previous studies evaluating stress in salmonids, microarrays will provide a
genomic look at the stress response in these fish and offer great potential for identifying
reliable biomarkers of stress. Because of the current lack of understanding of the stress
response in lampreys, the high probability that microarrays will identify reliable
biomarkers of stress, and the high likelihood of poorly-understood complexities in their
stress responses, we propose that lampreys are also ideal subjects for stress assessments
using cDNA microarrays. Parallel studies of this ancient fish—with its unique
evolutionary standing—and salmonids present a unique opportunity to significantly
advance our knowledge of stress in fish. We are confident that microarrays can fill in the
gaps of information concerning lampreys and salmon and will benefit their conservation
and management in the future. Using this approach, we will be able to detect up- or
down-regulated expression in classes of genes in a population—genes that are related to
the function of various physiological systems. The genomic understanding of the stress
response in fish and the identification of biomarkers of exposure to stressors is important
now and will become more so in the future. As humans struggle with serious
environmental concerns such as climate change, contaminants, and habitat degradation,
having tools to monitor the health and well being of animal populations will be critical to
understanding how our natural resources are dealing with environmental change.
III. Statement of the problem and objectives
The world is entering an era of unprecedented environmental concerns. Climate
change, contaminants, and physical habitat degradation are some of the most serious
environmental problems facing humans today. Understanding how fish—including
economically and spiritually valuable species like salmon and evolutionarily significant
fish like lampreys—respond to these environmental insults will be critical to their
conservation and management. We propose to explore the efficacy of cDNA microarrays
for the study of stress physiology in salmon and lampreys. This work will result in the
identification of biomarkers of environmental stress that can be used to monitor the
health and well being of populations in the wild, not only for these species, but for others
as well. Specifically, the objectives of this research are: (1) develop a lamprey-specific
cDNA microarray for use in physiological studies (a microarray for salmonids already
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exists); (2) document the genomic responses of juvenile salmonids and lampreys to a
variety of environmental stressors, including, but not limited to, elevated temperatures,
contaminant exposure, and hypoxia; (3) identify genes that consistently respond to
specific stressors; and (4) collate specific genes into “DNA fingerprints” for use as
biomarkers of exposure to specific types of stress.
C. Rationale and significance to regional programs
I. Salmonids
This proposal pertains to the following recommended proposed actions outlined in
the 2000 FCRPS Biological Opinion: (1) Action 46 (page 9-78, Section 9.6.1.3.3)—“The
Corps and BPA shall…evaluate the effects of transportation on summer-migrating
subyearling Snake River Chinook salmon…”; (2) Action 141 (page 9-126, Section
9.6.1.6.2.)—“The Action Agencies shall evaluate juvenile fish condition due to disease in
relation to high temperature impacts during critical migration periods. This evaluation
should include monitoring summer migrants at lower Columbia and lower Snake River
dams to clarify the possible link between temperature and fish disease and mortality.
This information will be used to assess the long-term impacts of water temperature on
juvenile fish survival.” This proposal is also relevant to the following strategies
discussed in the Mainstem Amendment: (1) meet state and federal water quality standards
under the Clean Water Act; (2) implement actions to reduce toxic contaminants in the
water; and (3) maintain water temperatures within the tolerance range of native fish
species.
Our proposal will provide innovative molecular tools to address the above
mentioned RPAs and Mainstem Amendments. Also, new life history information will
most certainly be obtained from our work. Action 190 of the BiOp clearly states that “the
Action Agencies shall continue to fund studies that monitor survival, growth, and other
early life history attributes of Snake River wild juvenile fall Chinook.” Our research with
juvenile Chinook salmon will help identify factors influencing the survival of Snake
River Chinook salmon. Finally, microarrays can help in identifying cause-and-effect
relations as mentioned in Action 155.
II. Lampreys
This research directly addresses a high ranking critical uncertainty for
anadromous and resident lampreys in the CRB as described in the 2005 report of the
Lamprey Technical Work Group (LTWG) entitled “Critical uncertainties for lamprey in
the Columbia River Basin: results from a strategic planning retreat of the Columbia
River Lamprey Technical Workgroup”. This group, which is a subcommittee of the
Anadromous Fish Committee of the Columbia Basin Fish and Wildlife Authority
(CBFWA), was created to provide technical review, guidance, and recommendations for
activities related to lamprey conservation and restoration. The LTWG has been charged
with identifying critical uncertainties regarding lamprey conservation, establishing
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lamprey research, monitoring, and evaluation needs, prioritizing research, reviewing new
proposals and existing projects, and disseminating technical information.
Because of their cultural and ecological importance, lampreys are commonly
mentioned as focal species, species of concern, or taxa of interest in many subbasin plans
and the Fish and Wildlife Program. The subbasin plans specifically addressing lamprey
issues include the Deschutes River, John Day River, Lower Columbia River, Fifteenmile
Creek, Hood River, Lower mid-Columbia River, Umatilla/Willow Rivers, Walla Walla
River, Willamette River, and the Yakima River. Information needs discussed in these
plans include abundance and distribution, general life history, limiting factors, passage
issues, contaminants, and artificial production and captive breeding.
A microarray for lampreys in the CRB will be an invaluable tool to any number of
other researchers and research interests in the basin. For example, this array can be used
to study gene expression during development in lampreys, may be useful in helping to
determine unique populations of lampreys, or distinguishing between anadromous and
resident forms. Further, due to the close evolutionary relations between all species of the
CRB lampreys, a microarray produced for Pacific lamprey will almost certainly be useful
for all other species.
III. Summary
Molecular ecology allows for the linkage of the results from scientific
experiments and management objectives. As mentioned in the NPCC’s 2000 Fish and
Wildlife Program description: “…management actions must be taken in an adaptive,
experimental manner because ecosystems are inherently variable and highly complex.
This includes using experimental designs and techniques as part of management actions,
and integrating monitoring and research with those management actions to evaluate their
effects on the ecosystem.” The long-term goal of this project is to develop biomarkers as
tools that can detect stress exposure and response in all fishes in the CRB. The final
result is an ideal implementation of integrating monitoring, research, and management
actions.
D. Relationships to other projects
This study would continue along the lines of work we are conducting for the U. S.
Army Corps of Engineers (USACE) on the mechanisms of delayed mortality in juvenile
salmon in the Columbia River Basin. We have been using cDNA microarrays to assess
the health and physiological condition of juvenile spring Chinook salmon that have been
barged or remained in-river during their migration. This study has established the
baseline and technology development for the use of cDNA microarrays in the basin. We
are also familiar with other research funded by the USACE evaluating the disease
resistance of juvenile salmon with different migration histories, which should be relevant
to the work proposed here. This proposed project will be related to any other lamprey
projects funded by BPA because the information gained should be useful to many
activities ongoing in the basin. In particular, this proposal is related to an objective of
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Project # 199402600 “Pacific Lamprey Research and Restoration Project”. David Close,
a principal investigator on this project, is conducting research to identify stress hormones
in lampreys. We have spoken to Dave about this project and will collaborate and share
information with him whenever possible.
E. Project history (for ongoing projects)
This is a new project.
F. Proposal biological objectives, work elements, and methods
Objective 1. Create a cDNA microarray for assessing stress in Pacific lampreys.
Task 1.1. Collect adult Pacific lampreys from the wild
Adult Pacific lampreys will be collected from a trap in the north fish ladder at
Bonneville Dam during October, 2007. We will attempt to collect all fish (N = 50)
within a short period and will exclude any fish showing secondary sexual characteristics
or coloration or are in poor condition. Fish will be transported to the Columbia River
Research Laboratory and placed in several 1.5-m-diameter circular tanks lined with
cobble and gravel substrates and receiving water from the Little White Salmon River.
Water temperature and photoperiod in the holding tanks will mimic ambient Columbia
River conditions. Lighting will be provided by overhead incandescent lights and timers
that will produce a gradual intensity dusk and dawn. Fish will be held under laboratory
conditions for four weeks prior to conducting initial stress exposures for developing the
microarray.
Task 1.2. Expose lampreys to struggling, elevated temperatures, and hypoxia in the
laboratory.
The usefulness of a cDNA library is largely dependent on the genes that are
expressed in fish at the time of sacrifice. Thus, in order to ensure that stress-responsive
genes are represented on our lamprey microarray, a pilot study will be conducted in
which fish are exposed to presumably serious stressors under laboratory conditions. A
subsample of the fish from Task 1.1 will be netted from their tank, placed in empty,
rectangular basins, and allowed to struggle and be exposed to hypoxia for 5 min. Also,
we will expose a subsample of fish to a slow (over a few hours), chronic warming of their
holding water from about 10°C to 25°C. Previous work by us revealed that temperatures
in the mid-twenties elicited altered oxygen consumption in Pacific lampreys. After the
stressors, all fish will be placed back in their tanks (at normal temperatures) and, at
selected time points, will be sacrificed and tissues removed and frozen in liquid nitrogen.
We will sample six fish at 1, 4, 12, 24, and 72 h post-stressor. By first exposing fish to
these serious stressors and then sampling various tissues (see below), we will maximize
the likelihood that genes with a stress-specific expression pattern are identified in later
experiments using the microarray.
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Task 1.3. Prepare cDNA libraries and construct the cDNA microarray.
Preparation of cDNA libraries and construction of a lamprey-specific cDNA
microarray will follow procedures described in Podrabsky and Somero (2004). Briefly,
livers will be dissected from fish after cervical transection and total RNA extracted using
Trizol according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA).
The pooled sample of total RNA from all fish will be used to prepare cDNA for the
production of the microarray and to profile changes in gene expression during stress. A
cDNA library will be prepared from the total RNA by creating a cDNA copy of each
mRNA using reverse transcriptase. The single-stranded cDNA will then be amplified via
long distance polymerase chain reaction (LD-PCR; Cheng et al.1994) and ligated into a
cloning vector. Competent bacterial cells will then be transformed with the ligated
plasmids via electroporation. The transformed cells will be selected for presence of a
cDNA insert and relevant clones isolated into microtiter plates. The plates will be
replicated and stored at –80°C.
The cDNA inserts from each isolated clone in the cDNA library will be amplified
using polymerase chain reaction (PCR). Microarrays will be printed onto glass slides
coated with poly-L-lysine according to standard protocols (see www.microarray.org).
Poly-L-lysine coated microscope slides will be produced using Fisher Scientific
(Hampton, NH, USA) Gold Seal microscope slides and Sigma Chemical brand poly-Llysine solution. The slides will be stored in a desiccator for 3 weeks to allow the poly-Llysine coat to age properly. The microarrays will be printed using a Genemachines
OmniGrid Accent arraying robot with a 16 pin configuration (TeleChem Stealth pins,
Telechem ArrayIt, Sunnyvale, CA, USA) and printing an 18x18 spot grid pattern with a
spacing of 240·mm between spots for each pin. Microarrays will be post-processed
according to standard procedures (see below).
Objective 2. Document the genomic responses of juvenile salmonids and lampreys to a
variety of environmental stressors
Task 2.1. Expose juvenile Chinook salmon and adult lampreys to some common
environmental stressors in a laboratory setting.
For these experiments, we will obtain juvenile salmon from local hatcheries as
needed and collect adult lampreys from Bonneville Dam or from a trap in the fish ladder
at Willamette Falls Dam. Fish will be handled and transported as described earlier and
held for at least three weeks prior to experiments. In separate experiments, fish will be
exposed to the following stressors:
Elevated temperature—This experiment will explore the effects of chronic
rearing of fish at temperatures moderately and severely above what might be considered
normal. This work addresses the real-life scenario of fish living for a time in waters with
elevated temperatures and should provide insight into how these fish might deal with
various climate change scenarios. Briefly, for each species, the experimental design will
involve rearing three groups of fish at different ranges of temperature over a period of 1-2
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months. Initially, all fish will be reared for at least two weeks at the control temperature
range, from 10-12°C. After this, we will collect a baseline sample of fish, as described
above. Then, fish in one group will have the temperature in their tanks raised 1-2°C per
day until they reach a range of 15-17°C. The third group of fish will experience the same
rate increase, but their final temperature range will be 20-22°C. Fish will be sampled
daily during the temperature increase to monitor shifts in gene expression as the cells
respond to the changing environmental conditions. After fish have reached their final
temperature range, we will collect a sample of 6 fish. Thereafter, we will sample all
groups once per week for 4-6 weeks.
Contaminant exposure—This experiment will explore the effects of exposure to
toxicants that are common in the CRB. Briefly, we will subject fish to chronic exposures
of sublethal levels of contaminants of concern in the CRB. Exposures would include, but
would not be limited to, dichlorodiphenyltrichloroethane (DDT) and its metabolites (e.g.,
dichlorodiphenylethylene [DDE]), polychlorinated biphenyls (PCB’s), and mercury.
Groups of fish would be held in water containing a sublethal dose of the toxicant of
interest for several weeks or months. Fish held in fresh, clean water would serve as
controls. Groups will be sampled at selected time intervals for analysis of gene
expression.
Exposure to hypoxia—This experiment will explore the effects of degraded water
quality, such as might be caused by pollution, on gene expression. Basically, we will
expose groups of fish to normoxic (e.g., 10 mg/L dissolved oxygen [DO]) and two levels
of hypoxic (e.g., 6-7 mg/L and 3-4 mg/L DO) water. Fish will be held in different water
conditions for several weeks and sampled at selected time intervals to evaluate gene
expression.
Task 2.2. Collect and process relevant tissues from test fish after exposure to stress.
All fish sampled for the experiments under Objective 2 will be rapidly netted
from their tank and placed in a lethal dose of buffered tricaine (MS-222; 300 mg/L).
After breathing has ceased, we will remove fish from the anesthetic, collect a blood
sample from the caudal vasculature, and excise the liver, brain, and kidney. Also, we will
remove a small piece of gill tissue and fin tissue to evaluate the possibility of non-lethal
sampling for gene expression. Blood samples will be centrifuged to obtain plasma,
which will be transferred to another tube and stored at -80°C for later analysis. Tissues
will be placed in an appropriate volume of Trizol and homogenized. The samples will be
stored in this condition at -20°C until processing as described above.
Task 2.3. Assay tissues for relative level of gene expression using the cDNA microarray
developed under Objective 1 and the GRASP array for salmon.
Tissues from each fish will be assayed for relative level of gene expression using
the lamprey-specific cDNA microarray and the array produced by the consortium
Genomics Research for All Salmon, or GRASP. This array is produced at the University
of Victoria, Canada, contains over 15,000 genes, and is described in detail at
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http://web.uvic.ca/cbr/grasp/array.html. Assay procedures will follow those described by
Podrabsky and Somero (2004) and at www.microarray.com.
Objective 3. Identify genes that consistently respond to specific stressors.
Task 3.1. Compare levels of gene expression between unstressed control fish and those
exposed to different stressors.
Relative levels of gene expression will be determined at each time point by
comparing the amount of mRNA transcript present in the experimental sample (pooled
from six fish at each time point) compared to a reference sample (pooled from all the fish
used in the experiment). The use of a reference sample will allow comparison of the
relative amount of each transcript at each time point to a common sample. The data will
be normalized for each fish and expressed as the percent-fold change in the unknown
sample relative to the reference sample. All fish will be normalized in exactly the same
way (to the same reference sample), samples within a time period will be pooled, and
differences between groups will be analyzed using Analysis of Variance procedures.
Task 3.2. Determine the nucleotide sequence of any microarray spots showing
interesting expression patterns, indicating relatively high or low gene expression.
Microarray spots with interesting expression patterns will be identified by
sequencing the cDNA insert isolated from plasmid DNA from the clone of interest.
Sequencing will be accomplished using an ABI 373 sequencer with dye terminator
reaction mix. Plasmid-specific primers will be used for the sequencing reactions. All
cDNAs of interest will be sequenced from the 5´ end to maximize the possibility of
identifying the transcript. Sequences will be identified by homology to known sequences
using an NCBI Blastx search of the GenBank database.
Objective 4. Collate specific genes into “DNA fingerprints” for use as biomarkers of
exposure to specific types of stress.
Task 4.1. All of the genes that responded significantly to a specific stressor in Tasks 3.1
and 3.2 will be collated and classified as the DNA fingerprint for that species and stressor
combination. Such fingerprints could be compared to data collected from fish in the wild
to document exposure to certain types of stressors. Examples of successful microarray
biomarkers in an ecological context come from Tabuchi et al. (2006), who correlated
thyroid hormone gene expression with PCB exposure in harbor seals; Hook et al. (2006),
who linked specific gene expression profiles in rainbow trout to exposure to model
toxicants; and Roberge et al. (2007), who reported differential gene expression in Atlantic
salmon Salmo salar infected with Saprolegniasis relative to healthy fish. .
Task 4.2. We will prepare, as needed, annual and final reports of research for
submission to the BPA. Also, we will prepare articles for submission to peer-reviewed
journals. Finally, all of our data will be uploaded onto the internet in a searchable public
database such as NCBI’s Gene Expression Omnibus (GEO). The MIAME (Minimum
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Information About a Microarray Experiment) guidelines followed by most journals
require this. Details on MIAME can be found at
http://www.mged.org/Workgroups/MIAME/miame.html.
G. Facilities and equipment
Experiments for this study will be conducted at the Columbia River Research
Laboratory (CRRL). This facility is well supplied with all the modern equipment, wet
laboratory, computers, and analysis software necessary to complete this research. The
CRRL in Cook, WA, is part of the USGS’s Western Fisheries Research Center. The CRRL
has three state-of-the-technology analytical laboratories dedicated to enzymology,
immunology and cell culture, and general physiology. In addition to standard equipment
such as centrifuges, pH meters, and balances, the laboratories are equipped with VIS-UV
and reflectance spectrophotometers, enzyme-linked-immunosorbent assay (ELISA) plate
readers, flame photometers, and ultracold freezers. The laboratory is staffed with trained
technicians and biologists proficient in these techniques with backgrounds in fish behavior,
immunology, physiology, and endocrinology. The 1500 sq. ft. wet lab facility is adequate to
conduct a variety of studies, including investigations into fish development or behavior,
disease resistance, reproduction, predator avoidance, thermal preference, osmoregulation,
swimming performance and bioenergetics. The CRRL has a modern computer network that
services over 100 users at the facility and a GIS laboratory. Computer software available for
data analyses includes SAS, Excel, SigmaPlot, Statgraphics, and a variety of other word
processing and data management software. The CRRL has its own T-1 line for fast Internet
access.
The Podrabsky lab at Portland State University (PSU) is well equipped for the
production and analysis of microarrays. Dr. Podrabsky has unlimited access to the PSU
Keck Genomics Facility which houses a Genemachines Omnigrid Accent arraying robot, a
bank of four, 96-well plate format thermocylers for PCR amplifications, and an Axon
Instruments GenePix 4000B microarray scanner for the acquisition and analysis of array
data. The Keck genomics facility is housed in a 600 sq. ft. lab space. In addition, Dr.
Podrabsky has an 800 sq. ft. research laboratory that is geared for the production and
screening of cDNA libraries from fish. Thus, the equipment necessary to create the array
and the expertise required to generate an array with maximal utility are already in place.
H. References
Reference (include web address if available online)
Adams, S. M. 2002. Biological indicators of aquatic ecosystem stress: introduction
and overview. Pages 1-12 in S. M. Adams, editor. Biological indicators of
aquatic ecosystem stress. American Fisheries Society, Bethesda, Maryland.
Barton, B. A., J. D. Morgan, and M. M. Vijayan. 2002. Physiological and conditionrelated indicators of environmental stress in fish. Pages 111-148 in S. M.
Adams, editor. Biological indicators of aquatic ecosystem stress. American
Fisheries Society, Bethesda, Maryland.
FY 2007-09 Project Selection, Section 10
12
Reference (include web address if available online)
Bryan, M. B., A. P. Scott, I. Cemy, S. S. Yun, W. Li. 2003. 15-Hydroxytestosterone
produced in vitro and in vivo in the sea lamprey. General and Comparative
Endocrinology 132:418-426.
Close, D. A., M. Fitzpatrick, H. Li, B. Parker, D. Hatch, and G. James. 1995. Status
report of the Pacific lamprey (Lampetra tridentata) in the Columbia River
Basin. Report (Contract No. 95BI39067) to Bonneville Power
Administration, Portland, Oregon.
Close, D. A., M. S. Fitzpatrick, C. M. Lorion, H. W. Li, and C. B. Schreck. 2003.
Effects of intraperitoneally implanted radio transmitters on the swimming
performance and physiology of Pacific lamprey. North American Journal of
Fisheries Management 23:1184-1192.
Gracey, A. Y., and A. R. Cossins. 2003. Application of microarray technology in
environmental and comparative physiology. Annual Review of Physiology
65:231-259.
Hook, S. E., A. D. Skillman, J. A. Small, and I. R. Schultz. 2006. Gene expression
patterns in rainbow trout, Oncorhynchus mykiss, exposed to a suite of model
toxicants. Aquatic Toxicology 77:372-385.
Katz, Y., L. Dashow, and A. Epple. 1982. Circulating steroid hormones of
anadromous sea lampreys under various experimental conditions. General
and Comparative Endocrinology. 48:261-268.
Kime, D. E., and C. V. Callard. 1982. Formation of 15-hydroxylated androgens by
the testis and other tissues of the sea lamprey, Petromyzon marinus. General
and Comparative Endocrinology 4:267-270.
Kime, D. E., and J. J. Rafter. 1981. Biosynthesis of 15-hydroxylated steroids by
gonads of the river lamprey, Lampetra fluviatilis. General and Compartive
Endocrinology 44:69-76.
Larsen, L. O. 1976. Blood glucose levels in intact and hypophysectomized river
lampreys (Lampetra fluviatilis L.) treated with insulin, ‘stress’, or glucose,
before and during the period of sexual maturation. General and Comparative
Endocrinology 29:1-13.
Leibson, L. G., and E. M. Plisetskaya. 1969. Hormonal control of blood sugar level
in cyclostomes. General and Comparative Endocrinology 2:528-534.
Lou, X. J., M. Schena, F. T. Horrigan, R. M. Lawn, and R. W. Davis. 2001.
Expression monitoring using cDNA microarrays: a general protocol.
Methods in Molecular Biology 175:323-340.
FY 2007-09 Project Selection, Section 10
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Reference (include web address if available online)
Lowartz, S., R. Petkam, R. Renaud, F. W. H. Beamis, D. E. Kime, J. Raeside, and J.
F. Leatherland. 2003. Blood steroid profile and in vitro steroidogenesis by
ovarian follicles and testis fragments of adult sea lamprey, Petromyzon
marinus. Compartive Biochemistry and Physiology A 134:365-376.
Martinelli, T.L., H.C. Hansel, and R.S. Shively. 1998. Growth and physiological
responses to surgical and gastric transmitter implantation techniques in
subyearling chinook salmon (Oncorhynchus tshawytscha). Hydrobiologia
371/372:79-87.
Maule, A.G., C.B. Schreck, and S.L. Kaattari. 1987. Changes in the immune
system of coho salmon (Oncorhynchus kisutch) during the parr-to-smolt
transformation and after implantation with cortisol. Canadian Journal of
Fisheries and Aquatic Sciences 44:161-166.
Maule, A.G., C.B. Schreck, C.S. Bradford, and B.A. Barton. 1988. The
physiological effects of the collection and transportation of emigrating
juvenile chinook salmon past dams on the Columbia River. Transactions
of the American Fisheries Society 117:245-261.
Maule, A.G., R.A. Tripp, S.L. Kaattari, and C.B. Schreck. 1989. Stress alters
immune function and disease resistance in chinook salmon (Oncorhynchus
tshawytscha). Journal of Endocrinology 120:135-142.
Mesa, M. G., J. L. Bayer, and J. G. Seelye. 2003. Swimming performance and
physiological responses to exhaustive exercise in radio-tagged and untagged
Pacific lampreys. Transactions of the American Fisheries Society 132:482493.
Podrabsky, J. E., and G. N. Somero. 2004. Changes in gene expression associated
with acclimation to constant temperature and fluctuating daily temperatures in
an annual killifish Astrtofundulus limnaeus. Journal of Experimental Biology
207:2237-2254.
Roberge, C., D. J. P´aez, O. Rossignol, H. Guderley, J. Dodson, and L. Bernatchez.
2007. Genome-wide survey of the gene expression response to saprolegniasis
in Atlantic salmon. Molecular Immunology 44:1374-1383.
Selye, H. 1950. Stress and the general adaptation syndrome. British Medical
Journal 1(4667):1383-1392.
Tabuchi M., N. Veldhoen, N. Dangerfield, S. Jeffries, C. C. Helbing, and P.S. Ross.
2006. PCB-Related Alteration of Thyroid Hormones and Thyroid Hormone
Receptor Gene Expression in Free-Ranging Harbor Seals (Phoca vitulina).
Environmental Health Perspectives 114:1024–1031
FY 2007-09 Project Selection, Section 10
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Reference (include web address if available online)
Wagner, T. and J.L. Congleton. 2004. Blood-chemistry correlates of nutritional
condition, tissue damage, and stress in migrating juvenile Chinook salmon
Oncorhynchus tshawytscha. Canadian Journal of Fisheries and Aquatic
Sciences 61:1066-1074.
I. Key personnel
INDIVIDUAL VITAE
1. Name: Matthew Gilbert Mesa
2. Present position:
Research Fishery Biologist
U.S. Geological Survey, Columbia River Research Laboratory
5501A Cook-Underwood Road
Cook, Washington 98605
Phone: 509-538-2299, ext. 246
Fax: 509-538-2843
E-mail: matt_mesa@usgs.gov
3. Education:
B. S. in Natural Resources Mgt., 1984, California Polytechnic State University at San Luis Obispo
M. S. in Fisheries Science, 1989, Oregon State University
Ph.D. in Fisheries Science, 1999, Oregon State University
4. Research specialties:





Stress and general physiology of fishes
Fish behavior
Performance capacity of fishes
Predator-prey interactions
Fish-ecosystem interactions
5. Publications, reports, and other public expression:
a. Publications: over 20 peer reviewed journal publications
b. Agency reports and presentations: over 50
c. Representative publications:
Mesa, M.G. and C.B. Schreck. 1989. Electrofishing mark-recapture and depletion methodologies evoke behavioral
and physiological changes in cutthroat trout. Transactions of the American Fisheries Society 118:644-658.
Mesa, M.G. 1994. Effects of multiple acute stressors on the predator avoidance ability and physiology of juvenile
Chinook salmon. Transactions of the American Fisheries Society 123:786-793.
Mesa, M.G., T.P. Poe, D.M. Gadomski, and J.H. Petersen. 1994. Are all prey created equal? A review and
synthesis of differential predation on prey in substandard condition. Journal of Fish Biology 45 (Supplement
A):81-96.
FY 2007-09 Project Selection, Section 10
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Mesa, M.G., L.K. Weiland, and P. Wagner. 2002. Effects of acute thermal stress on the survival, predator
avoidance, and physiology of juvenile fall Chinook salmon. Northwest Science 76:118-128
Mesa, M.G., J.M. Bayer, and J.G. Seelye. 2003. Swimming performance and physiological responses to exhaustive
exercise in radio-tagged and untagged Pacific lampreys. Transactions of the American Fisheries Society
132:483-492.
INDIVIDUAL VITAE
1. Name: Jason Podrabsky
2. Present position:
Assistant Professor
Department of Biology
Portland State University
Room 344, Science Building 2
(503) 725-5772
(503) 725-3888
podrabsj@pdx.edu
3. Education:
B.S. in Biology, 1993, Oregon State University
Ph.D. in Biology, 1999, University of Colorado at Boulder
4. Research specialties:



Physiological adaptation of organisms to their environment
Stress physiology
cDNA microarrays
5. Publications, reports, and other public expression:
a. Publications: over 10 peer reviewed journal publications
b. Presentations: over 12
c. Representative publications:
Hand, S.C., Podrabsky, J.E., Eads, B.D. and F. van Breukelen. (2001). Interrupted development in aquatic
organisms:Ecological context and physiological mechanisms. In Animal Developmental Ecology. (ed. D.
Atkinson and M. Thorndyke). Oxford: BIOS Scientific Publishers Ltd. (in press).
Podrabsky, J.E., Carpenter, J.F. and S.C. Hand. (2001). Survival of water stress by annual killifish embryos:
Dehydration avoidance and amyloid fibrils in the egg envelope. Am. J. Physiol. Regulatory Integrative Comp.
Physiol. 280: R123-R131.
Podrabsky, J.E. and S.C. Hand. (2000). Depression of protein synthesis during diapause in embryos of the annual
killifish, Austrofundulus limnaeus. Physiol. Biochem. Zool. 73: 799-808.
Podrabsky, J.E., Javillonar, C., Hand, S.C. and D.L. Crawford. (2000). Intraspecific variation in aerobic metabolism
and glycolytic enzyme expression in heart ventricles from Fundulus heteroclitus. Am. J. Physiol. Regulatory
Integrative Comp. Physiol. 279: R2344-R2348.
Podrabsky, J.E. and S.C. Hand. (1999). The bioenergetics of embryonic diapause in an annual killifish, Austrofundulus
limnaeus. J. Exp. Biol. 202: 2567-2580.
INDIVIDUAL VITAE
1. Name: Alec G. Maule
FY 2007-09 Project Selection, Section 10
16
2. Present position:
Supervisory Research Physiologist and Leader—Ecology & Environmental Physiology Section
USGS-BRD, Columbia River Research Laboratory
5501A Cook-Underwood Rd.
Cook WA 98605
(509) 538-2299 x 239; (509) 538-2843 (FAX)
alec_maule@usgs.gov.
3. Education:
B.A. in Psychology, 1969, University of California, Riverside
B.S. in Natural Resources Mgt. 1979, California Polytechnic University, San Luis Obispo
M.S. in Fisheries Science, 1982, Oregon State University
Ph.D. in Fisheries Science, 1989, Oregon State University
4. Research specialties:



Stress physiology of fishes
Immune function of fishes
Effects of contaminants on organisms
5. Publications, reports, and other public expression:
a. Publications: over 50 peer reviewed journal publications
b. Presentations: over 70
c. Representative publications:
Sauter, S.T., L.Crawshaw, and A.G. Maule. 2001. Behavioral thermoregulation in spring and fall Chinook salmon,
Oncorhynchus tshawytscha, during smoltification. Environmental Biology of Fishes 61:295-394.
Jørgensen, E.E., M.M. Vijayan, N. Aluru, and A. G. Maule. 2002. Fasting modifies Aroclor 1254 impact on plasma
cortisol, glucose and lactate responses to a handling disturbance in Arctic charr. Comparative Biochemistry and
Physiology, Part C 132: 235-245.
Gale, W.L., R. Patiño, and A.G. Maule. 2004. Interaction of xenobiotics with estrogen receptors alpha and beta and a
putative plasma sex hormone binding globulin from channel catfish (Ictalurus punctatus). General and
Comparative Endocrinology 136:338-345.
Aluru, N., E.H. Jorgensen, A.G. Maule, and M. M. Vijayan. 2004. PCB disruption of the hypothalamus-pituitaryinterrenal axis involves brain glucocorticoid receptor downregulation in anadromous Arctic charr. American
Journal of Physiology: Regulatory, Integrated and Comparative Physiology 287: R787-793.
Maule, A.G., E. H. Jorgensen, M.M. Vijayan, and J.-E. A. Killie. 2005. Aroclor 1254 exposure reduces disease
resistance and innate immune responses in fasted Arctic charr. Environmental Toxicology and Chemistry 24:117124.
Beeman, J.W. and A.G. Maule. 2006. Migration Depths of Juvenile Chinook salmon and Steelhead relative to total
dissolved gas supersaturation in a Columbia River reservoir. Transactions of the American Fisheries Society
135:584-594.
Maule, A.G., A.L. Gannam, and J.W. Davis. 2007. Chemical contaminants in fish feeds used in federal salmonid
hatcheries in the USA. Chemosphere 67:1308-1315.
FY 2007-09 Project Selection, Section 10
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