Graduate Student Awards for Marine Science Research
AY 2012-2013 Application Form
Application Deadline: Monday October 22 2012, 5:00 p.m. PDT
Student Applicant Information
First Name:
Jeffrey
Email:
Last Name:
Sharick
Phone:
Student ID#:
[email protected]
GPA in Major
Courses:
CSU Campus:
Sonoma State University
Department
or Degree
Program:
Biology department
Anticipated
graduation date
(mm/yyyy):
12/2013
Degree
Sought (e.g.,
MS, PhD):
MS
Thesis-based?
(Y/N):
Yes
Have you previously received a COAST Research Award? (Y/N)
No
If yes, please provide year of award:
Faculty Advisor Information
First Name:
Dan
Last Name:
Crocker
CSU Campus:
Sonoma State University
Department:
Biology
Research
Project Title:
Oxidative Stress: A potential cost of breeding in adult male
and female northern elephant seals (Mirounga angustirostris
Project Keywords (5-7 keywords related to your
project):
Position/Title:
Email:
Phone:
Associate Professor
[email protected]
(707) 664-2995
Oxidative stress, Cost of breeding, Fasting,
Pinnipeds, Northern elephant seals
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Please refer to the Award announcement for detailed instructions on the information required
for each of the following sections.
Project Description (50 points)-1500 word maximum
Introduction
Evolutionary explanations for life history patterns assume that successful reproduction carries
potential fitness costs to future survival and reproduction (Sterns 1992). In capital breeders,
the trade-off between energetic investment in current reproduction and growth is distinct as
reproductive effort is supported exclusively from body reserves (Jornsson 1997; Bonnet et al.
1998; Stephens et al. 2009). However, the mechanisms underlying fitness costs to
reproduction are not well understood. Air-breathing marine predators face unique constraints
while breeding due to the temporal separation of marine foraging and terrestrial breeding.
Several groups of animals, including pinnipeds, seals and sea lions, fast for highly variable
periods during breeding (Costa 1991). Fasting is stressful (Mitsui et al., 2002; Sorensen et al.
2006).and the ability to preserve physiological function when nutrient limited may be a key
mechanism mediating fitness costs of reproduction in these species.
Extended fasting is associated with activation of several different stress hormone axes
which alter physiological function including metabolism, fluid and electrolyte balance and
cardiovascular function (Munck et al., 1984; Sapolsky et al., 2000). Several stress hormones,
including glucocorticoids and angiotensin II are also direct promoters of oxidative stress.
Fasting directly promotes the production of hydrogen peroxide and lipid hydroperoxides that
promote depletion of anti-oxidant defenses, oxidative damage and inflammation
(Crescimanno et al., 1989; Di Simplicio et al., 1997; Grattagliano et al., 2000). Fasting
induced reactive oxygen species (ROS) can induce oxidative damage to proteins, lipids and
DNA (Alonza-Alverz et al. 2004; Speakman 2008). Chronic elevation of ROS production and
oxidative stress can lead to cardiovascular diseases, organ failure and many other
complications (Mancini et al., 1996; Kimi and Iwao, 2000; McFarlane et al., 2001; Cooper et
al., 2007). Recently, evolutionary ecologists have focused on oxidative stress as a universal
constraint on the life history of all animals (Dowling and Simmons, 2009; Monaghan et al.,
2009; Metcalfe and Alonso-Alvarez, 2010; Isaksson et al., 2011) suggesting that energetic
trade-offs between reproductive effort and maintenance may impact fitness costs through
oxidative insult. Because oxidative stress is also promoted by fasting, capital breeders may be
especially prone to fitness costs from current reproductive effort. Numerous studies have
shown that body reserves influence reproductive effort in pinnipeds and those individuals
maintain high rates of energy expenditure to breed successfully despite extended fasting
durations (Crocker et al. 2012). Further, impacts of ocean climate on foraging success result
in strong annual variation in the body reserves available for breeding effort (Crocker et al.
2006). Together, these features suggest that pinnipeds may be an ideal study system in which
to identify associations between variation in breeding effort and success and evidence for
systemic oxidative damage.
Northern elephant seals (Mirounga angustirostris) are naturally adapted to undertake
long (up to 3 months) bouts of fasting which coincide with breeding (Crocker et al. 2012).
Adult male elephant seals arrive at the beach and compete to establish dominance hierarchies
used to control access to estrus females (Le Boeuf and Laws 1994). Females give birth to a
single pup and provision it with one of the highest energy density milks found in nature for
~25 days (Crocker et al. 2001). For both sexes, body reserves directly impact energy
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expenditure while breeding and successful males maintain metabolic rates near the values
suggested as metabolic ceilings for sustained energy expenditure, despite fasting completely
from food and water (Crocker et al. 2001, 2012). I propose to examine oxidative stress
mechanisms in breeding elephant seals in order to better understand the relationship of
oxidative stress to body reserves and mating success. I will combine measures of pro-oxidant
stress, systemic and macromolecule-specific measures of oxidative damage and anti-oxidant
defenses to understand how stress, defense mechanisms and oxidative damage vary with
fasting duration in breeding adult elephant seals. To measure whether breeding seals show an
increase in oxidative damage, markers of lipid oxidation (8-isoprostane, TBARS and 4Hydroxynonenal), protein oxidation (Nitrotyrosine) and DNA oxidation (8-hydroxy-2-deoxy)
will be measured along and markers of inflammation (TNF-α and C-reactive protein). I will
measure plasma levels of enzymes that serve as pro-oxidants (Xanthine Oxidase) or
antioxidants (Superoxide Dismutase, GPx and catalase). Specifically, I will answer the
following questions: Do these metabolically active capital breeders incur oxidative damage
across the breeding fast? Is oxidative stress a cost of increased breeding effort? Do antioxidant defenses up-regulate in response to breeding and help prevent oxidative damage.
Materials and Methods
Study subjects and site.
This study will be conducted at Año Nuevo state reserve, San Mateo County, CA,
during the elephant seal breeding season. 30 breeding adult males and 40 breeding adult
females will be sampled across the 1-3 month breeding fast between January and March. 15
males will be sampled early (1-2 week fasted) and late (9-10 weeks fasted), while 20 females
will be sampled early (1 week fasted) and late (4 weeks fasted) in lactation. During the initial
procedure, seals will be marked with hair dye (Stamford, Conn.), and given a green
numbered plastic flipper tag (Dalton jumbo Roto-tags, Oxon, England) for future
identification.
Sample collection and processing
An initial intramuscular injection of Telazol (teletamine/zolazepam HCl) at a dose of
~0.3mg/kg for the males and ~ 1 mg/kg for the females will be used to immobilize each seal.
To maintain immobilization, 100mg boluses of ketamine and 5mg of diazepam will be given
as needed (all drugs from Fort Dodge Laboratories, Ft. Dodge, IA). All seals will be kept
awake and eupnic throughout all procedures. Upon immobilization, an intravenously set
spinal needle will be used to collect blood samples in chilled, EDTA vacutainers. Once blood
samples are collected, protease inhibitor cocktail and an anti-oxidant (BHT) will be used to
preserve molecules associated with oxidative stress. Immediately after sampling, samples will
be centrifuged for 15 minutes at 3,000 RPM on the beach and plasma will be frozen on dry
ice until transferred to a -80oc freezer. Muscle samples will be collected by first cleaning the
biopsy site with both isopropyl alcohol and betadine, followed by a subcutaneous injection of
3ml lidocaine. After the lidocaine has taken affect (~10 minutes), a small incision will be
made with a sterile scalpel. From the incision a muscle sample will be taken using a sterile
biopsy punch. Upon collection, the muscle will be immediately place in a cryogenic vial and
frozen on dry ice until transfer to a-80oc freezer. Standard and total curve length of each
animal will be measured. Girth and ultrasound measurements of blubber thickness will be
taken at seven specific points along the body. These measurements will be used to calculate
3
body mass of the 30 adult males used in this study (Le Boeuf et al. 2000). For the 40 females,
mass will be determined using a tripod and scale (MSI tension dynamometer, ±2 kg; Seattle,
WA) connected to a canvas sling. Weaning success will be monitored for all females. Males
will be observed daily to establish dominance hierarchies, observe fights and their outcomes
and estimate mating success.
Sample analysis
Pro-oxidants and Antioxidants: Xanthine Oxidase activity (Pro-oxidant) will be quantified by
using a commercially available florometric EIA assay (Cayman Chemical, Ann Arbor, MI,
USA). The levels of plasma antioxidants produced will be quantified by measuring SOD
(Superoxide Dismutase), GPx (Glutathione peroxides) and Catalase using EIA assay kits from
Cayman Chemical. Inflammatory markers and oxidative damage: Inflammation will be
quantified by EIA assay kits used to measure plasma level TNF-α (Cayman Chemical) and Creactive protein(CRP) (Cayman Chemical). 8-isoprostane (8-iso-PGF2α) (Cayman Chemical),
TBARS (Thiobarbituric acid reactive substances; Cayman Chemical) and 4-hydroxynonenal
(Antibodies online GmBh, GE) EIA assay kits will be used to quantify plasma level lipid peroxidation. EIA assay kits will be used to measure 8-hydroxy-2-deoxy Guanosine (Cayman
Chemical) and Nitrotyrosine (Alpxo, NH) to quantify DNA and protein per-oxidation
respectively. Cortisol levels will be measured using radioimmunoassy (Seimens). Blood urea
nitrogen, ketones and non-esterified fatty acids will be measured using colorimetric assays
(Cayman Chemical; Wako Diagnostic, Richmond, VA)
Changes across the breeding fasts in males and females will be compared using linear mixed
models. Relationships between oxidative stress markers, hormones and metabolites will be
evaluated using mixed model regression.
Significance
Despite being a critical assumption of most theoretical structures for the evolution of life
history patterns, evidence for mechanisms underlying fitness costs to reproduction are rare.
Capital breeders are the group most likely to exhibit these costs and are therefore key study
systems for examining physiological fitness costs to reproduction. Air-breathing marine
predators that have maintained terrestrial reproduction are particularly susceptible to fitness
constraints on reproductive effort. Using a model pinniped system, the proposed work will
provide a novel insight into how fitness costs may manifest and the ability of individuals to
mediate these costs through defense mechanisms. Understanding reproductive costs is key to
not only understanding life-history theory but also to predicting how variation in body
reserves influences survival impacts of breeding. Thus these data will provide critical insight
into how variability in foraging success due to changing ocean climate (e.g. ENSO, PDO or
climate change) may influence reproduction and survival and have demographic impacts on
population of marine apex predators.
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Relation to COAST goals (20 points)-250 word maximum
The proposed project will help elicit how this model pinniped system can provide insight into
the effect of changing ocean climate (e.g. ENSO, PDO or climate change) on foraging
success, reproduction fitness and survival of many of California’s costal marine mammals. By
looking at oxidative stress in this model capital breeder, we can measure the cost and
consequences of current reproductive effort and how that can vary with body reserves.
Breeding is a very costly ecological challenge that all mammals must undertake to sustain
their personal fitness and the fitness of their species. This project will provide a new insight
into how individual marine mammals can defend against oxidative stress, a dangerous
physiological process, which has been hypothesized as a major component of the cost of
breeding.
This project will be conducted at one of California’s state funded marine reserves. Año Nuevo
state reserve is home to one of the California northern elephant seal rookeries. Every year
northern elephant seals congregate here to undergo a very intensive breeding fast that is
observed by the public in large numbers. Understanding the costs of breeding in these animals
can lead to better management of this and other marine mammal rookeries across California.
It will also provide more knowledge and understanding of the life history behind northern
elephant seals, and marine mammals in general, to the public who visit these rookeries each
year.
Need for Support (10 points)-250 word maximum plus timeline
For this project, there will be a need for field supplies, which will cost approximately $120$150 per procedure, and lab physiology assay kits. Although my adviser will be funding all of
the field work costs, I am in great need of support for the purchase of these many assay kits.
Many other projects that have look at oxidative stress and its consequences only measure one
or two of the molecules or enzymes I am measuring. Being awarded this grant money will
allow me to expand the range of molecules, enzymes and metabolites measured to elicit a
more in depth and informed answer to my proposed hypotheses.
For the lab work I will need a duplicate of each of the assays kits I will be running (1 for the
set of males, and 1 one for the set of females). For pro-oxidant and antioxidants I need kits for
Xanthian oxidase, SOD, GPx and catalase. For oxidative damage and inflammation I need
kits for TNF-α, CRP, 8-isoprostane, TBARS, 4-hydroxynonenal, 8-hydroxy-2-deoxy
Guanosine and Nitrotyrosine. For other stress markers I need kits for Cortisol levels, Blood
urea nitrogen, ketones and non-esterified fatty acids. To complete these measurements, the
physiological assay kits will cost approximately $300-$500 each.
This grant will be a huge help by giving me the financial capability to get these desired
measurements analyzed and included in this project.
Timeline:
Sample collection - January-March -> Sample processing - April-June -> Paper writing and
submission - July-November – > Graduation - December
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Bonnet, X., D. Bradshaw, and R. Shine. 1998. Capital versus income breeding: an
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Costa, D.P. 1991 Reproductive and foraging energetics of high latitude penguins, albatrosses
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