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Vol. I
Issue I
An Oregon State University Publication
An Oregon State University Student Publication
The
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The
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Staff
A letter from the editors:
Editors
“It is the tension between creativity and skepticism that has produced the stunning and
unexpected findings of science.”
-- Carl Sagan, renowned astronomer.
New experiences and perspectives in science are essential to uncovering information
not yet understood in this world. The goal of the Catalyst, as a student scientific
publication, is to share with others the innovation and creativity of young researchers from Oregon State University. Each study included in this issue, from exploring
coral reef fish in the Bahamas to studying RNA in Arabidopsis, strives to expand present knowledge and to inspire new questions. It is with a great deal of pleasure that
we present the innovative research of undergraduate students. The work published
in this journal, represents a small sample of the impressive bank of submissions that
we have received. As editors, we welcome campus-wide submissions for this journal
accepting any work, creative or research based, that promotes a deeper understanding
and appreciation for science. It is our hope that this research compellation inspires
and intrigues you.
Enjoy discovering!
Katy, Megan, and Carly
Megan Cook
Carly Dougher
Katy McHenry
Layout
Tari Tan
Photography
Mark Albins
Advisor
Kevin Ahern
Logo Design
Tari Tan
Now accepting submissions for the next issue of
*Scientific Articles
*Fictional Stories
*Article Reviews
*Poetry
Plus any other ideas you may have!!!
Submit articles to:
Kevin Ahern
ahernk@onid.orst.edu
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Table of Contents
A Survey of Western Oregon Soil Nematodes ............................4
Importance
of
microRNA
for
Seedling
Development
in Arabidopsis.................................................................................6
Zinc Transporter Expression in an Animal Model of
Amyotrophic Lateral Sclerosis.................................................. .8
Effects of a Marine Reserve on a Coral Reef Fish
Community......................................................................................10
Principles, Process and Experiential Learning through a Novel
Marine Science Research Course..............................................13
The Effect of Injectable Selenium on Neutrophil Phagocytosis
and Gene Expression in Adult Sheep with Foot Rot.................16
The Effects of In Ovo Feeding of Vitamin E on Broiler Chicken Hatchability and Chick Tissue Lipids....................................................18
Using RNA Interference Mosaics to Map Mutant Phenotypes.20
Publication of The Catalyst
was made possible by HHMI
grant#52005883 and the University
Honors College at OSU.
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A Survey of Western Oregon Soil Nematodes
Caroline Hilburn and Dee Denver
Department of Zoology
Oregon State University
Introduction
Soil nematodes have been popular biological
models for decades, especially since Caenorhabditis
elegans became the first animal to have its complete
genome sequenced. Unfortunately, little is known
about nematode ecology despite the fact that nematodes are probably the most abundant animals on the
planet. The goal of this project is to gain insight into
the ecology of soil nematodes in western Oregon by
conducting an organized survey across different soil
types and finding new strains of nematodes for use in
the lab. Specifically, nematodes in the Caenorhabditis or Panagrolaimus genus are of interest. Panagrolaimus nematodes have been suggested as a model
species for investigating the evolutionary transitions
between reproductive modes. More Panagrolaimus isolates, however, are necessary to support the
proposed phylogenetic tree of the Panagrolaimus
genus. An organized survey of soil nematodes will
strengthen both Caenorhabditis and Panagrolaimus
nematodes as model systems by identifying natural
populations, strengthening the current phylogenetic
trees, isolating new strains for investigation, and
providing a better understanding of the environmental context their genomes
evolved in.
Methods
Eight sampling locations were chosen in order
to provide as broad a survey as possible within the
bounds of western Oregon.
These sites were Portland,
Salem, Eugene, Corvallis,
Toledo, Sweet Home, Lyons, and Lincoln City. At
each of these locations, five
different soil types were
sampled, based on the locations of known Caenorhabditis natural isolates: natural
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areas, cultivated land, city parks, compost, and rotting fruit. At each site, sixteen samples were collected from the outer layer of soil or from the decaying
leaf litter. The nematodes were extracted from the
soil using modified Baermann tubes. After extraction individual worms were picked onto Paul plates,
a solid medium made with only agar, tap water, and
cholesterol. As a food source for the nematodes, the
plates were seeded with Op 50, an E. coli strain. Individual worms were picked to ensure that each of
the nematode lines started was monogenetic. After DNA had been extracted from each new worm
line, the 18S ribosomal RNA gene was amplified and
sequenced for each strain of worms. The resulting
sequences were then compared to known 18S sequences in Genbank using the BLAST search engine
to identify the nematodes.
Results and Discussion
From the original 352 soil samples, 190 new
worm lines were started, representing 17 species. No
Caenorhabditis sp. nematodes were found, which
might indicate that Caenorhabditis nematodes are
either rare or absent in Western Oregon. However,
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11 new strains of Panagrolaimus sp. nematodes were
isolated from five different locations and three different soil types. In one sample, two different species
of Panagrolaimus were isolated, one parthenogenetic and one gonochoristic. This represents a greater
diversity of Panagrolaimus species in Oregon than
was anticipated.
The total species distribution shows that soil
taken from city parks provided both the greatest total
number of nematodes, and the greatest species diversity. The most abundant nematodes species were the
Cephalobus sp. (Figure 1).
Representative strains from each species isolated
were chosen to be cultivated in the lab for further
testing. The phylogenetic tree of these 37 samples
and their closest related known nematodes has a few
interesting features. One strain, DL0155, part of the
Mesorhabditis genus, is potentially a new species.
One of the Panagrolaimus isolates, DL0117, a gonochoristic strain, is located directly between the gono-
choristic clade and the parthenogenetic clade, supporting the separation of the Panagrolaimus genus at
that point and supporting the use of Panagrolaimus
as a model for studying the evolutionary transitions
between different reproductive modes. (Figure 2)
Acknowledgements
Thank you to the Howard Hughes Medical Institute for providing the funding for this project. Thank
you to Dr. Dee Denver and the entire Denver lab for
their help. Finally, thank you to the many people
who allowed soil samples to be collected from their
property: Portland Parks and Recreation Department,
Cooper Mountain Organic Vineyard, Don Kruger at
Krugerís Farm, Liz Myers at Deerhaven Vineyard,
Vitae Springs Vineyard, and the Oregon State Parks.
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Importance of microRNA for
Seedling Development in Arabidopsis
1
Jennifer Coppersmith1 and Hiro Nonogaki2
Department of Biochemistry and Biophysics, 2Department of Horticulture
Oregon State University
Introduction
Understanding the developmental process of
seeds has many important benefits in both the agricultural and food industries. To gain further insight into the detailed process of seed germination
and seedling establishment, Arabidopsis thaliana
can be used as a good molecular and genetic model,
because its entire genome has been sequenced. For
my project, I focused on a particular gene known as
SQUAMOSA PROMOTER-BINDING PROTEIN13
(SPL13). We are interested in this gene because it
has been found to be expressed in Arabidopsis seeds
and seedlings. The gene SPL13 codes for a transcription factor and is a predicted target of microRNA156/157
(miR156/157). MicroRNA are newly discovered
and play an interesting role in gene regulation at the
mRNA level. MicroRNA are small single stranded
RNA fragments and function by guiding a complex
known as RISC to a complementary target sequence
on the mRNA. The RISC complex then cleaves the
mRNA, which leads to mRNA degradation. My
goal for this project was to determine what effects
SPL13 deregulation from miRNA156 has in the development process of seeds.
Experimental Design
W T S P L 13
S P L 13
AC T2
Figure 1. Quantitative PCR
analysis of SPL 13 mRNA
accumulation. RNA was
extracted
from
3-day
seedlings
of
wild-type
(WT ), and miR156/157resistant SPL 13 (mSP L 13).
ACT 2 is to show equal
loading.
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To be able to characterize the biological function of SPL13
and its regulation by
miR156/157, silent mutations were introduced
in the SPL13 target sequence complementary
to miR156/157. Wildtype Arabidopsis plants
were then transformed
using Agrobacterium,
with the vector containing mutant SPL13
(termed mSPL13). As an outcome from this mutation the mSPL13 mRNA is resistant to miR156/157,
resulting in an overaccumulation of mRNA. I used
RT-PCR (conversion of SPL13 mRNA to cDNA) and
then amplified the cDNA using PCR and was able to
confirm the overaccumulation of mRNA in mSPL13,
which was not seen in the wild-type (Figure1) and
control SPL13 plants. Using the transformed plants,
seeds were harvested and then grown to observe
their phenotypes. The mSPL13 seedlings exhibited
a temporary arrest around the two- to four-leaf stages
(Figure 2) while this phenotype was not observed in
the wild-type plants or the control plants transformed
with vector containing the intact SPL13 (SPL13).
To verify that the phenotype was due to the mutation, a selectable marker, which was inserted along
with the mSPL13 gene, called GUS was used. All of
the delayed (two-leaf) seedlings showed expression
for GUS, whereas the normal (four-leaf) seedlings
Delayed
Normal
Figure 2. Three quarters of seedlings exhibited a
temporal arrest at two-leaf stage (left), when the rest
entered the norm al four-leaf st age.
did not. Therefore, all of the two-leaf seedlings are
mSPL13. From the results of GUS staining, a homozygous line for mSPL13 was determined, and I
was able to check the overaccumulation of SPL13
mRNA by learning northern blot analysis using a
specific SPL13 probe. Once the homozygous mutant
line was established, my objective was to characterize the delayed phenotype that was observed originally in these mSPL13 plants and how the deregula-
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A
H yp o co ty l
R ad i c l
e
B
C o ty l edon s
Shoo t Ap ic al
M er iste m
(S A M)
C
mSPL13 (Figure 3C), therefore suggesting that the
temporary arrest begins at the three-day seedlings. At
four days, there was a significant increase in size of
the primordia of the WT and control SPL13 as compared to mSPL13. At eight days, the adult leaves of
the WT and control SPL13 had fully emerged, which
was not seen in the mSPL13. From my observation three day seedlings are used as the standard day
for all of the functional analysis, because that is the
time-point at which phenotype is observed. Through
this work I was able to characterize the temporary
arrest phenotype of the mutant compared to the wildtype and control.
Summary
WT
m S PL 13
3 day
Figure 3. A. Ti ssu e section stained with toluidine
blue. Left is whole section emb ryo and to the right is
close-up view of shoot apical meristem. B. Schematic
representation showing the position of DIC images in
a wh ole embryo. The i mage shows the SA M region of
0 d -imb ibed wild-type (WT ) emb ryo. C. Differential
Interference Contrast (D IC) microscope images of leaf
primordia in Arabidopsis 3day seedlings. Images of
the SA M regions in WT and mSP L 13 s eedlings
incubated for 3 days sh ow slight increase in size of
primodia of WT comp ared to mSP L 13
tion of microRNA affects the development of these
seeds.
Differential Interference Contrast (DIC) microscopy and tissue sectioning provided good tools
to observe the interior cell structure and organization within tissues. Although tissue sectioning provides more detailed images (Figure 3A); DIC allows
a quick and easy assessment of the different layers
within the tissue by simply clearing the tissue and
using a special microscope to view the different optical planes (Figure 3B). The temporary arrest phenotype observed in the adult leaf (primordia) suggest a
problem in meristem development, in which emerging adult structures arise from. Therefore I utilized
these techniques to analyze the meristem and primordium development in detail in the wild-type, control
SPL13 transgenic and mSPL13 seedlings. Through
DIC I found that at three-day post-imbibition the
wild-type primordia were slightly larger than the
Since a temporary arrest was observed in the
mutant plants, microRNA regulation must play a
significant role in the seed ability to progress from
embryonic stages of development into seedlings.
The data and images I collected from characterizing
the seedling development indicated that the downregulation of SPL13 by miR156/157 is essential to
maintain normal meristem activity during seedling
establishment. Because SPL13 is a transcription factor the next step in the project, which I am currently
working on in understanding the regulation of SPL13
by miR156, is to determine the potential targets of
SPL13. Identification of the target of the SPL13 gene
can provide greater insight into why there is a temporary arrest occurring in the mSPL13 seedlings.
References
1. Cardon, G., Hohmann, S., Klein, J., Nettesheim,
K., Saedler, H., and Huijser, P. (1999). Molecular characterization of the Arabidopsis SBP-box
genes. Gene 237, 91-104.
2. Cardon, G.H., Hohmann, S., Nettesheim, K., Saedler, H., and Huijser, P. (1997). Functional analysis of the Arabidopsis thaliana SBP-box gene
SPL3: a novel gene involved in the floral transition. The Plant Journal 12, 367-377.
Volume 2
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Zinc Transporter Expression in an Animal Model of
Amyotrophic Lateral Sclerosis
Thomas Lew1, Mark Levy2, and Joseph S. Beckman3
Linus Pauling Institute, 2Department of Biochemistry and Biophysics
Oregon State University
1
Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease that is characterized by the loss of motor neurons. The genetic cases of ALS are believed to be linked to a mutant Superoxide Dismutase (SOD) gene. A functional
SOD scavenges the superoxide, but the mutant version facilitates the production of this free radical, allowing the association of
superoxide with nitrous oxide to form peroxynitrite. Oxidative stress by peroxoxynitrite is the suspected mechanism of motor
neuron death. Mutant SOD in ALS rats have been shown to be zinc-deficient. Here we investigate the role of zinc transporters
as a possible mechanism of this zinc deficiency. Using RT-PCR, we characterized the levels of zinc transporters, ZnT1, ZnT3,
and ZnT4 in ALS rat spinal cords. Only ZnT3 shows a significant difference in expression, indicating a possible dysregulation in
zinc transport function.
Background
Amyotrophic lateral sclerosis (ALS) is a devastating, progressive neurological disease that is characterized by the destruction of motor neurons. Made
famous by the baseball player, Lou Gehrig (and
hence ALS is also known as Lou Gehrigís Disease),
ALS has an incidence of 30,000 in the US alone. The causes of ALS are not well understood, but
the symptoms have been well documented. As a consequence of motorneuron degeneration, ALS causes
muscle atrophy, paralysis, and eventually death within 2 to 5 years of symptom onset; age of onset usually occurs around 55 years. The majority of cases
are sporadic, but 10% of all ALS incidents are genetically linked. Of that group, 20-70% of the individuals inherit a mutation in the gene for superoxide
dismutase (SOD1).
SOD1 is an enzyme that functions to scavenge
the reactive free radical, superoxide, which is made
naturally in the body. Superoxide can cause cellular
damage and massive oxidative stress, but SOD1 is
able to transfer the extra electron from the free radical and produce molecular oxygen and hydrogen
peroxide. There are over 100 known mutations in the
SOD1 gene that have been found in ALS patients.
The toxicity cannot be directly caused by the loss of
function, since many of these mutant proteins can efficiently scavenge peroxynitrite.
It has been proposed that the toxicity of mutant
SOD is due to its reduced affinity for zinc. Without zinc, the copper ion within SOD becomes much
more reactive and the redox properties of the enzyme
change profoundly. Although Zn-deficient SOD still
can scavenge superoxide at a slightly slower rate,
it has gained the propensity to steal electrons from
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cellular antioxidants and transfer these electrons to
oxygen to produce superoxide. This production of
superoxide by Zn-deficient SOD is not toxic by itself because remaining CuñZn SOD present in a cell
recaptures superoxide. However, the molecule nitric
oxide reacts so rapidly with superoxide to produce
peroxynitrite that it can effectively compete with
SOD for superoxide. It is peroxynitrite, which can
nitrate tyrosine residues, that is harmful to motor
neurons.
Recently it has been discovered that there is
quantitatively more zinc deficient SOD in the ventral gray matter of the spinal cord than in the dorsal
region. This has led us to hypothesize that levels of
zinc-deficient SOD are increased in the ventral grey
matter as a consequence of zinc transporter dysregulation. In this study, we used an animal model of ALS
to examine the expression level of genes involved
in zinc transport within the CNS. In particular, we
focused on three members of the Zinc Transporter
(ZnT) family: ZnT1, which functions to export zinc
from the cytosol to the extracellular space; ZnT3
which transfers zinc from the cytosol to synaptic
vesicles, and ZnT4 which facilitates zinc transport
from the cytosol to the endoplasmic reticulum. The
expression level of these genes was then and compared between the ventral (disease-affected) and the
dorsal (unaffected) area of the spinal cord.
Methods
We utilized the technique of quantitative polymerase chain reaction (Q-PCR) to characterize the
expression level of ZnT-1, ZnT-3 and ZnT-4. In
brief, RNA was isolated separately from the dorsal
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and ventral spinal cord grey matter of rats expressing the G93A mutation of SOD. cDNA was then
synthesized from the RNA template using reverse
transcription, and the expression level of each gene
was then determined through real-time PCR using
gene specific primers. Bacterial vectors containing
the gene of interest were cloned and used to create
standard curves.
The C(t) value, defined as the number of PCR
cycles required to elevate product signal above background, was used to determine sample expression
level by comparing it to a standard curve derived
from serial dilutions of gene specific bacterial vectors. SYBR green, which fluoresces when intercalated between double stranded DNA, was used to
measure the PCR product.
Results and Discussion
Through the use of Q-PCR, we have quantified
the expression of three members of the ZnT family
of zinc transporters, namely ZnT-1, ZnT-2 and ZnT3, in the dorsal and ventral grey matter of the spinal cord. Our data indicate that ZnT-1 expression
is upregulated in ventral grey matter, in both nontransgenic as well as transgenic animals. However,
when compared to non-transgenic controls, the level
of induction was significantly lower in animals carrying the G93A SOD mutation (what was the ratio).
Subsequently, we have shown that ZnT-4 expression
is significantly upregulated in both dorsal and ventral
grey matter of rats carrying the G93A mutation when
compared to non-transgenic controls. Each of these
observations are consistent with the hypothesis that
a dysregulation in zinc transport may account for the
increased levels of zinc deficient SOD in the ventral
grey matter of G93A rats. (you can be more specific
here)
If Znt-1 is lower in G93A that nTg, then one
might consider the demand for SODbinding more
zinc such that less is available for export. Notably,
there was no difference in the level of expression of
the ZnT-3 transporter, which is responsible for the
transfer of zinc into synaptic vesicles. As such, it
is reasonable to postulate that the dysregulation in
zinc transport is not systemic, but rather the result of
(kind of stops here)local mechanisms.
This study examined G93A rats at 40-days old,
to study zinc transporter function at early time points.
The next step is to look further down the time-course
Fig 1: (a) Q-PCR of ZnT-3, comparing gene expression in 40-day old nontransgenic to transgenic mice in the dorsal and ventral regions of the spinal
cord. No significant differences were found in the ratio of dorsal to ventral of
Ntgto ALS rats. (b) Q-PCR of ZnT-4, comparing gene expression in 40-day
old nontransgenic to transgenic mice in the dorsal and ventral regions of the
spinal cord. No significant differences were found in the ratio of dorsal to
ventral of Ntg to ALS rats. (c) Q-PCR of ZnT-3, comparing gene expression in 40-day old nontransgenic to transgenic mice in the dorsal and ventral
regions of the spinal cord. A difference was found in the ratio of dorsal to
ventral of Ntg to ALS rats, indicating a possible dysregulation.
of ALS disease progression and investigate zinc
transporter function at (would not bother with this
section. 70-days and 100-days.
Acknowledgements
I thank Dr. Joe Beckman and the Beckman lab
for graciously hosting me this summer and Dr. Mark
Levy for taking me under his wing. I’d also like to
acknowledge the Howard Hughes Medical Institute and Oregon State University for putting on the
HHMI program.
Volume 2
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Effects of a Marine Reserve on a Coral Reef Fish Community
Robert Lamb and Mark Hixon
Zoology Department
Oregon State University
Introduction
In the summer of 2007, I worked with the research team headed by Dr. Mark Hixon of the OSU
department of Zoology studying the ecology of coral
reef fishes in the Bahamas. The site for my project,
the Exuma Cays Land and Sea Park, is a no-take reserve that has been fully protected since the 1980s.
This means that no extractive activities of any kind,
be it sport or commercial fishing, or other destructive activities, are allowed inside the boundaries of
the Park. This protection potentially has significant
impacts on the structure of the local marine ecosystem, because the removal of target fishery species is
known to affect the relative abundances of other species in coral reef fish communities (McClanahan and
Nyawira, 1998). For the most part, target fish species are large, top-level predators, such as groupers.
This means that they are at the top of the food web,
and through their interactions with other fish species,
specifically by predation and competition, they can
regulate many aspects of the reef community.
When a large number of the naturally existing
top-level predators are removed, an effect called a
trophic cascade can occur (Frank et al. 2005). A
ìtrophic levelî is a species or group of species at a
particular level within a food web. When large predatory fish, which are at the highest trophic level of
a food web, are removed through fishing activities,
species below that trophic level are also affected.
What can result is a shift in the proportional abundance of the trophic levels of the entire food web.
Without the top predators present to eat the middle
predators, these second-level fish species proliferate,
putting more pressure on herbivores and other lowerlevel species.
With this background in mind, I sought to answer
the following question: Is the fish community structure on protected (unfished) reefs inside the marine
reserve different from that on unprotected (fished)
reefs outside the reserve?
Methods
To answer my question, I surveyed five sites
Volume 2
within the marine reserve, and five sites near Lee
Stocking Island, about 50 kilometers to the south
and outside of the protection of the marine reserve.
I chose the sites to cover as broad a range of coral
reef habitats as possible. Also, to control for differences in normal fish community structure potentially
caused by differences in habitat, I paired structurally
similar reefs between the two regions (protected and
unprotected).
I performed underwater surveys at each site using SCUBA (self-contained underwater breathing
apparatus) and recorded the information on plastic
slates. As a sampling unit for my data, I used a tool
common in ecological fieldwork called a ìtransectî.
My transects were 25 meters long, which is about
the distance one can see with clarity in the Bahamian
ocean, and 5 meters wide. Within these transects,
I visually estimated and recorded the species and
length of every fish that I encountered.
Transect placement was assigned haphazardly,
and I also tried to control for the effects of my presence as an observer by identifying and measuring
larger, more mobile species on the first swimming
pass as I lay down the transect line. This approach
ensured that I recorded larger species before my
presence spooked them away. On the second pass,
I proceeded closer in to the reef structure, looking
for smaller, more cryptic species. I also measured
the bottom contour to further compare the structural
similarity of the paired study reefs. I performed six
transects at each of the five reefs inside the protection of the marine reserve, and six transects at each
of the five reefs in unprotected waters.
Results
My preliminary data analysis consisted of three
parts: average number of species per unit area; average sizes of representative large carnivore species;
average densities of representative predatory species; and relative distribution of species by trophic
category.
The difference in average predatory fish sizes
between protected sites and unprotected sites varied
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with trophic level (Figure 1). The two top predators,
great barracuda and Nassau grouper, were larger, on
average, inside protected areas. Some second-level
predators, including yellowtail snapper, saucereye
porgy, and schoolmaster were also larger, on average,
inside protected areas. Other second-level predators,
including the bar jack and graysby grouper, were
larger, on average, in unprotected areas.
The difference in average numbers of fish per
transect between protected sites and unprotected sites
was also variable (Figure 2). The Nassau grouper,
yellowtail snapper, saucereye porgy, schoolmaster,
and graysby grouper were all found in greater densities in protected areas. The great barracuda and bar
jack were found in greater abundance in unprotected
areas.
The distribution of all individuals larger than
30cm total length (as measured from the tip of the
mouth to the tip of the extended tail) when separated
into trophic categories was similar between protected
and unprotected sites (Figure 3). The largest discrepancies existed in herbivores (protected = 25%, unprotected = 12%) and omnivores (protected = 11%,
unprotected = 22%).
Inside the marine reserve, the larger predatory
fish species, such as the Nassau grouper and great
barracuda, grow to a much greater size, and, more
importantly, to a much greater size relative to the
smaller, lower-level predators such as the graysby
and schoolmaster. In contrast, in the unprotected
areas, the difference in size between top-level and
middle-level predators is much smaller. This corresponds to previous research, which has shown that
in coral reef systems, larger predatory fish actually
impede the success and growth of smaller predatory fish (Stallings 2007). Thus it is possible that
the larger, more abundant top-level predatory fish
in the protected areas are maintaining lower numbers and smaller sizes in the middle-level predatory
fish group. Also, many large-bodied predators were
only found in protected areas, and so no comparison
of size was possible. However, these species, such
as the black grouper, Caribbean reef shark, and tiger grouper are all major predators in the coral reef
ecosystem, and their total absence from transects in
unprotected areas is a strong testament to the direct
effects of fishing pressures.
The overall distribution of fish larger than 30cm
total length by trophic level was not very different
between protected and unprotected areas. However,
the proportion of omnivores was twice as large in
unprotected sites than in protected sites, and the proportion of herbivores was twice as large in protected
sites. If the predatory fish are selectively targeting
omnivores, and herbivores and omnivores compete,
than the decrease in size and abundance of predators in unprotected areas may account for these two
changes.
For the most part, these results indicate that the
Exuma Cays Land and Sea Park exhibits a strong influence on the average distribution, size, and numbers
of individuals of coral reef fish species. This adds to
Figure 1: Average sizes of seven different representative species of pretory fish. Each species has two columns: one for
average size found in protected sites, and one for average size
found in unprotected sites.
Figure 2: Average densities in number of fish per transect
of the seven representative species of predatory fish. Each
species has two columns: one for average density in protected
sites, and one for density in unprotected sites.
Discussion
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Figure 3: Distribution of species by number of individuals larger than 30cm total length in each of five trophic categories:
planktivores (suspended particulate matter feeders), herbivores (algae-eating fish), omnivores (eat a mixture of algae and
animals), mixed carnivores (eat a variety of animals including fish, mollusks, arthropods, etc.), and piscivorous carnivores (eat
only other fish).
the growing number of reports showing the negative
effects of fishing on marine ecosystems. As shown
in a similar study on the Great Barrier Reef (Graham
et al. 2003), the effects can be far-reaching, and the
removal of even a few of the top-level predatory species can drastically alter the coral reef community
structure. In the future, assessments of the success of
marine reserves must measure indirect effects such
as trophic cascades for a clear picture of the impacts
on the marine ecosystem.
References
1. Frank, K.T., Petrie, B., Choi, J.S., Leggett, W.C.
2005. Trophic cascades in a formerly cod-domi-
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nated ecosystem. Science. 308: 1621-1623
2. Graham, N.A.J., Evans, R.D., Russ, G.R. 2003.
The effects of marine reserve protection on the
trophic relationships of reef fishes on the Great
Barrier Reef. Environmental Conservation. 30:
200-208
3. McClanahan, T.R., Nyawira, A.M. 1998. Changes in Kenyan coral reef community structure and
function due to exploitation. Hydrobiologia.
166:269-276
4. Stallings, C.D. 2007. Indirect effects of fishing
on predators and their prey. Doctoral dissertation. Oregon State University.
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Principles, Process and Experiential Learning through a
Novel Marine Science Research Course
Jay Well
The Science and Math Investigative Learning Experiences (SMILE) Program
Oregon State University
In mid-August, five incoming, first-year OSU
students who just ten weeks prior participated in
their high school graduations, stood before an audience of 60 public school teachers from across the
state of Oregon as competent researchers, having already completed a rigorous college science course.
Ruby Canchola, Emily Escobedo, Bianca Quinones,
Rafael Marquez, and Aurturo Valdivia calmly and articulately explained, in detail, their research project,
“Distribution of Clam Species in Relation to the Area’s Tidal Zones at Hatfield Marine Science Center
(HMSC) Mud Flats.” This culminating presentation
centered around a new, eight-week, inquiry-based,
summer marine science course that incorporated a
variety of engaging teaching formats and resources
from university partners. The formats, which included hands-on experiments, open discussion, field trips
and student-driven research projects, afforded the
summer bridge students a variety of opportunities to
explore marine science and develop meaningful personal learning.
Inspired by weekend experiential marine science
courses offered at HMSC during the academic year,
The SMILE (Science and Math Investigative Learning Experiences) Program and the HMSC Academic
Program developed an introductory research-based
science class that would give students in SMILE’s
summer bridge program a broad introduction to scientific thought, a contextual use of research methods,
and an engaging introduction to university-level science. A member of the SMILE team responsible for
designing the course, Ryan Collay, adds, “Part of our
goal in partnering to create this course was the need
to give our summer bridge students a real world/
contextual research based experience. We knew this
would require a number of partners who would help
create a course that would focus on the student experience, getting them excited not just about the scientific content of the class, but in the different ways
that content can be used and applied. We were looking for a course that did not exist among the relatively limited summer term classes so we decided to
make up our own.”
Such a multidisciplinary course required an
equally multidisciplinary subject matter, marine science. Collay continued, “Given our interests and success with partnerships through the Hatfield Marine
Science Center (HMSC), we knew we would be able
to not only find the research partnership but also capable educators with a history of engaging students.”
Working with Itchung Cheung, a biology instructor
and HMSC Academic Programs Coordinator, the
course started to take shape, “Developing and teaching this course to lower division undergraduates, es-
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The
Catalyst
ing Assistant (GTA) that designed and conducted the
on-campus labs and served as one of the instructors
on the field trips. The three GTAs were Sarah Mikulak in Marine Resource Management, Bryan Rebar in Environmental Studies, and Becca Baldwin
in Fisheries and Wildlife. The first module, “A Drop
of Water,” explored the physical, chemical and biological characteristics of sea water. At HMSC the
students studied salinity fluctuations and plankton
diversity in the estuary. Module two, “In the Neighborhood,” provided an overview of marine biology
and ecology. During the trip to HMSC students explored different marine habitats (such as the estuary,
intertidal zone and the sandy shore) along the Oregon coast and at the Oregon Coast Aquarium. The
last module, “What’s For Dinner,” examined marine
resources and the policies that govern them. Led on a
local commercial fishing dock walk, students gained
pecially the bridge students was very rewarding and
to see how much the students gained by the end of the
course - Wow! Lower division high-impact courses
that engage through hands-on experiential inquiry of
the marine environment are what we [HMSC] are all
about...”
The course consisted of lectures and lab activities held each week at OSU and fieldwork every other
week at HMSC. Students began the first week of the
course learning about the history of marine exploration, current areas in marine research and concluded
with students presenting tsunami science activities
during HMSC’s “SeaFest” open house. The next six
weeks of the course were divided into three twoweek modules that were linked to Friday-Saturday
trips to HMSC. Each module had a Graduate Teach-
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first hand knowledge of the ins and outs of a working fishing fleet from Oregon Sea Grant’s Extension
Agent, Jeff Feldner, and local commercial fishermen.
In addition students went on a behind-the-scenes tour
of the Molluscan Broodstock Program, an aquaculture research program at HMSC.
The course encouraged students to explore marine science topics they found interesting. To link to
the science content and research methods, the students
made presentations on self-selected, peer-reviewed
journal articles of current research in marine science. The students also gained a critical understanding of how different media presents marine science
to the public and learned how to critically analyze
these messages to discover biases. As GTA Bryan
The
Catalyst
Rebar explains it, “Recognizing and understanding
how science is communicated in different settings
is an invaluable skill that the students can draw on
throughout their lives. Science is a human endeavor
and, therefore, must not be taken for granted without
thoughtful reflection and interpretation whether it’s
presented in peer reviewed articles, newspaper stories, or YouTube videos.”
The student-driven group research project was
an opportunity for the students to answer a challenging scientific question involving marine science
while exploring how research questions are developed. Early in the course, the students were able to
choose a topic of interest and devise a research question. “Coming up with a research question was not
as easy as it seemed… There is not just one part to a
question; you have to think of all the other variables
that go along with it as well. It was really frustrating!” remembers Bianca Quinones about the hours
the group spent defining their research question.
With the research question defined, the students
began the experimental design phase. While in the
field at HMSC students were able to test out their
experimental design and, similar to the work of published researchers, the students continually made
subtle tweaks until they had a protocol that was effective. They collected their data during week seven,
allowing the students just one week to complete the
analysis and put together their final report. The students also prepared individual posters focused on a
specific area of interest which were on display during their formal research presentation to 60 SMILE
teachers at SMILE’s August Teacher’s Workshop.
Quinones, when asked how it felt to complete such
a challenging project, remarked, “After we finished
our research and presentation it felt so good, and
through the experience I developed a new appreciation for researchers…and how long it takes to make
discoveries.”
Given its initial success, this new course will be
offered again next summer with some minor modifications that will allow up to 20 students to participate. Collay notes, “From a program perspective,
the partnerships have been a wonderful experience
and we will continue to work to support these kinds
of experiences for broader audiences.” Classes such
as the one described in this article are increasingly
important if OSU is to realize its goal of preparing
students with content knowledge in the sciences and
the skills to critically assess scientific questions, to
engage in research and make informed decisions.
Aurturo Valdivia expressed the impact this class had
on him this way, “The GS 199 class gave me the opportunity to not only learn about marine science but
also engage in scientific research. Although the process was challenging it reassured me of my decision to go into a science major.” Involving students
in research-based content is the key to engagement.
When students see the need, understand the context,
develop their own questions and research strategies,
they see science as more than just content - they see
science as a necessary component of their academic
experience.
********************
Jay Well is faculty with The SMILE Program at
OSU and is program lead for Study Techniques, Academics and Research, and Research Skills (STARS)
Program, SMILE’s Summer bridge program, a project funded through the Howard Hughes Medical Institute (HHMI).
Volume 2
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The
Catalyst
The Effect of Injectable Selenium on Neutrophil Phagocytosis
and Gene Expression in Adult Sheep with Foot Rot
Rachel S. Sendek and Jean A. Hall
Department of Animal Science
Oregon State University
Background
Insufficient intake of selenium is common in
sheep in the Northwest, and may contribute to increased susceptibility to infectious diseases such as
foot rot.
Hypothesis/Objective
To determine the clinical effectiveness of selenium supplementation on selected immune function
studies and the incidence and severity of foot rot in
sheep over a 15-month period.
Methods
(diameter and thickness (ear tip) of skin reactions)
were taken at 30 min, and at 24, 48, 72, and 96 hr.
The humoral immune response was assessed at 0, 2,
and 4 weeks after KLH immunization by measuring
antibody response to KLH. Neutrophils were studied at 15 months to assess phagocytosis and relative
abundance of selected mRNAs specific for neutrophil
migration or killing functions (L-selectin, IL-8R, and
myeloperoxidase).
Neutrophils are the most numerous and important cellular component of innate immunity. They are
highly specialized for their primary functions, which
are phagocytosis and destruction of microorganisms.
To measure gene expression of selected mRNA, neutrophils were isolated using a Percoll gradient technique, resuspended in RPMI 1640 media, and counted
using a Coulter counter. Small aliquots were frozen
at -80°C until total RNA could be extracted (Qiagen
RNeasy kit) following manufacturerís instructions.
These preparations were frozen in dry ice and subsequently used to make cDNA for qRT-PCR. To assess
gene expression of a marker specific for neutrophil
killing activity, we measured the relative abundance
of mRNA for myeloperoxidase (MPO) by qRT-PCR.
A sheep flock in southern Oregon was chosen for
this study based on a high incidence of foot rot and
the ownerís willingness to participate in a long-term
clinical trial. A placebo-controlled clinical trial was
completed in which foot rot-affected sheep were randomly divided into 2 groups of 19 sheep each. An
additional control group of 19 sheep without foot rot
were identified. Sheep feet were examined, trimmed,
and scored for foot rot using a scale of 0 (no foot
rot) to 4 (extensive). Half the foot rot-affected sheep
were treated with 5 mg injectable selenium
at 1-month intervals for the duration of the
study; the other half were given saline injections. Controls received no treatment. Sheep
feet were reexamined, trimmed and rescored
at 3, 6, 9, and 15 months. Sheep were also
bled at time 0 and then at 3, 6, and 15 months
to assess whole-blood selenium levels. To
assess cell-mediated immunity and humoral
immunity, after 3 months of selenium supplementation all sheep were immunized twice,
2-weeks apart with keyhole limpet hemocyanin (KLH), a novel protein. The day following the second injection, a DTH skin test
was performed. Intradermal injections of Figure 1: Healthy control sheep demonstrated the highest % bacterial killing. There was no
difference in % bacterial foot rot-affected sheep supplemented with saline or selenium. Each
KLH were administered at the ear tip and in box represents the middle 50% of the data, with the median indicated by a horizontal line in
2 wool-free sites on the ventro-lateral abdo- the box. The whiskers extend 1.5 times the interquartile range above and below the 75th and
25th percentiles. Boxes with different letters above them are significantly different (P<0.05).
men. Measurements for the DTH skin test
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(CFU). Internal control wells without neutrophils were used to determine baseline bacterial counts at the
assay endpoints. Percent survival of
bacteria was calculated as [(CFU/mL
experimental well)/(CFU/mL control
well)] x 100%. Data was analyzed
using ANOVA to compare bacterial
survival in neutrophil killing assays.
Results
Although no differences in percent bacterial killing was detected
between foot rot-affected sheep
Figure 2: qRT-PCR data suggests that neutrophil mRNA for IL-8R may be
supplemented with saline and those
up-regulated in foot rot-affected sheep supplemented with selenium vs. saline
supplemented with selenium, control
(compared with controls), indicating selenium supplementation could increase a
sheep without foot rot were shown
mRNA marker associated with neutrophil migration.
to have higher bacterial killing ability in an ex vivo phagocytosis assay
Levels of mRNA were determined for L-selectin
and IL-8R to evaluate gene expression of markers than sheep affected with foot rot. Thus, other facinvolved in neutrophil migration, adherence, and ac- tors besides selenium status, such as genetics, may
tivation. Levels of β-actin mRNA were measured to be involved in protecting these sheep from infectious
normalize input template cDNA. Data was analyzed agents.
The qRT-PCR data suggest that the neutrophil
using a 2-sample t-test to compare fold increases in
mRNA (compared with healthy control sheep) in markers MPO and IL-8R may be up-regulated in
foot rot-affected sheep treated with selenium vs foot- sheep supplemented with selenium vs. saline (when
compared with controls), indicating selenium supplerot affected sheep treated with saline.
To assess neutrophil phagocytosis, neutrophils mentation could increase mRNA markers associated
were isolated as above and seeded into triplicate with neutrophil functions in sheep.
wells of a 96-well tissue culture plate, previously
coated with poly-D-lysine, at 2 x 105 cells/well.
Neutrophils were activated for 10 minutes with 25
nM PMA (diluted in DMSO; <1% final solution).
Logarithmic phase Lactococcus lactis
(MG1363) bacteria containing an erythromycin-resistant plasmid were grown
in M17 + glucose (M17G) + erythromycin media and diluted to the desired
concentration in RPMI media + 2%
heat-inactivated FBS. Bacteria were
added to the neutrophils at a multiplicity of infection (MOI) of 0.01. After 1
h incubation at 37°C in 5% CO2, media
in the wells containing neutrophils and
bacteria was serially diluted and plated
onto M17G medium containing eryth3: qRT-PCR data suggests that neutrophil mRNA for myeloperoxidase (MPO) may be upromycin for overnight incubation and Figure
regulated in foot rot-affected sheep supplemented with selenium vs saline (compared with controls),
enumeration of colony forming units indicating selenium supplementation could increase a mRNA marker associated with phagocytosis.
Volume 2
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18
The
Catalyst
The Effects of In Ovo Feeding of Vitamin E on Broiler Chicken
Hatchability and Chick Tissue Lipids
Travis Schaal and Gita Cherian
Department of Animal Science
Oregon State University
Introduction
Methods
Administration of vitamin E was facilitated
through a modified Noor, et al. (1995) method of inovo injection. Treatments of fertile Cobb 500 broiler
eggs were purchased from a commercial hatchery
(Tangent, OR). All eggs were set in the same tray in
the same incubator. A total of 100 eggs were placed in
treatments of 25 eggs. Two treatments were injected
in-ovo with vitamin E (10 IU and 20 IU, respectively) at day 14 of incubation. Two treatments of fifty
eggs remained as controls (injection of vegetable oil,
and no injection). Incubation conditions were constant at 37.5°C dry and 28.3°C wet bulb until hatching when the dry bulb temperature was reduced to
36.3°C and the wet bulb temperature was increased
to 30.2°C. Hatching was monitored, and tissues were
collected from hatched chicks (n=6 from each treatment) including: heart, brain, liver, yolk sack, and
bursa. Blood was also collected for isolation of plasma. Weights of chicks and all collected tissues were
recorded. Tissues were frozen for assays including:
lipid extraction, gas chromatography, and high pressure liquid chromatography to characterize fatty acids and to determine the concentration of lipids and
Hatchability of fertile eggs (%)
Avian incubation is a fascinating process and
very important to the poultry industry. Eggs for commercial broilers (meat chickens) are placed in large
scale hatcheries where computers monitor conditions
and highly regulate the environment for developing
chicks. Recent advancements in management, nutrition and genetic selection have resulted in the production of wholesome poultry products in a short
period of time. Each year, an increasing number of
broilers and turkeys are raised for consumption and
following this trend, there has also been an increase
in the number of eggs set in commercial hatcheries
each year. Over the past twenty years, hatchability
of broiler eggs has only reached 82% (nearly 20% of
all eggs incubated do not hatch). Modern advancements in nutrition, genetic selection and management
of broiler flocks have developed fast growing birds
(with increased muscle protein accretion) and low
feed conversion ratios. Even with so many advances,
there has been no increase in hatchability of broiler
eggs. The economic loss associated with the lack of
improved hatchability in the year 2005 was in excess
of 460 million dollars (Schaal and Cherian, 2007).
A chicken egg contains 5.5-6g of fat which is the
only source of fatty acids available to the growing
embryo throughout embryogenesis and hatchability.
During the 21-day incubation period of a chicken
egg, over 80% of yolk fatty acids are absorbed by the
developing chick embryo for energy production
90
and structural membrane synthesis. During the
85
third week of incubation, there is an intense in80
crease in the uptake of fatty acids by the develop75
ing embryo. Long chain fatty acids are the major
70
fuel of the heart in that fatty acid oxidation pro65
60
vides over 70 percent of the energy requirements
55
for the chick embryo. The use of low quality
50
(low antioxidant, low essential fatty acids) fats in
breeder chicken diets provides subsequent eggs
and growing embryos (offspring) with a low
quality fat source during the important stages
of development. The objective of this research is to
understand the contribution of an exogenous supply
of antioxidants (vitamin E) during avian embryonic
development on hatchability.
Volume 2
Non-injected
Control
10IU VE
Treatment
Figure 1
20IU VE
The
Catalyst
Results
Overall, a trend for increased hatchability of fertile eggs was observed in the experimental treatments
in this trial. As seen in Figure 1, both experimental
treatments with vitamin E injections had increased
hatchability over the control treatments. Chicks that
received injection of 10 IU or 20 IU vitamin E injection during incubation had a hatch rate of 88%
compared to non-injected with a hatch rate of 85%,
and control injection treatment with a hatchability of
80%. No difference was observed in body weight,
liver, heart, or yolk sac weight of chicks throughout
the treatments. The brain weight as percent of body
weight was higher in vitamin E injected chicks compared to non-injected chicks (p<0.05). Injection of
vitamin E increased the brain total lipids and vitamin
E compared to control chicks (p<0.05). Levels of vitamin E in blood plasma were also increased in the
injected treatments, compared with the control injected treatment. Brain total lipids were increased in
all three injected chicks compared with non-injected
chicks (p<0.05). Figure 2 depicts the trend for increased vitamin E in brain tissue among experimental
treatments compared with the non injected group. No
difference was found between the total lipid content
of liver, heart or yolk sac in all treatments (p>0.05).
Conclusions
The use of antioxidants may reduce oxidative
stress while the developing embryo begins utilizing lipids during incubation. The data suggest that
in-ovo administration of antioxidants may increase
hatchability of broiler eggs; however replication of
the trial must be conducted to perform statistical
analysis and determine statistical significance. Concentrations of vitamin E in tissues and blood plasma
were increased in injected treatments as compared to
control groups, in addition to yolk sac tissue. Lipid
content of brain tissue and vitamin E levels in the
brain were also increased in the injected treatments.
The injection of antioxidants or exogenous fat may
play a role in protecting brain tissue from lipid per-
oxidation and prevent diseases such affecting brain
tissue such as encephalomalacia. Decreasing damage
due to free radicals with injection of vitamin E may
play a role in preventing embryo mortality or produce
a healthier hatchling. Further investigations on the
affects of exogenous antioxidants for the developing
embryo will be necessary to have conclusive results,
but the data collected so far proves promising.
Acknowledgements
I would like to express my thanks to the Howard
Hughes Medical Institute for the opportunity to participate in the summer research program. The experience I have gained in the lab working with the scientific method, has allowed me to develop and refine
skills of research in an area that is of great interest to
me. I also want to thank Dr. Gita Cherian for allow0.4
Microgram vitamin E per gram of brain
tissue (ug/gm)
vitamin E in each tissue/blood plasma. Eggs that did
not hatch were broken open to determine the embryo
status (fertile, early or late dead). Data was analyzed
using SAS with a one way analysis of variance and
means compared using Duncan’s multiple comparison with level of significance p < 0.05.
a
0.35
a,b
0.3
0.25
c
b,c
0.2
0.15
0.1
0.05
0
Control
Non-injected
10IU VE
20IU VE
Treatment
Figure 2
ing me to work in her lab. I greatly appreciate her
guidance and enthusiasm.
References
1. Schaal, T., and G. Cherian. 2007. A survey on the
hatchability of broiler and turkey eggs in the
United States from 1985 through 2005. Poult.
Sci.86: 598-600.
2. Noor, S. M., A. J. Husband and P. R. Widders
(1995). “In Ovo Oral Vaccination with Campylobacter-Jejuni Establishes Early Development
of Intestinal Immunity in Chickens.” British
Poultry Science 36(4): 563-573.
Volume 2
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The
Catalyst
Using RNA Interference Mosaics to Map Mutant Phenotypes
Megan Kelly and Barbara Taylor
Department of Zoology
Oregon State University
Introduction
In the summer of 2007 under the mentorship
of Dr. Barbara Taylor I participated in a project using RNA interference mosaics to map retained(retn)
mutant phenotypes in female fruit flies. Dr. Taylor’s
lab is currently exploring what genes are expressed
in the making of the female fruit fly reproductive
tract. It is known that the retn gene plays a role in
the development of the female fruit fly reproductive
tract as well as in eye and behavioral development.
Retn mutant females are sterile, which might be due
to either behavioral abnormality, such as a failure to
copulate, or from a physical abnormality in the reproductive track causing egg retention. This summer
I altered retn gene expression in just a subset of cells
in the female reproductive tract and found that these
females did not lay eggs, replicating part of the retn
mutant phenotype.
Hypothesis
My hypothesis was if I made females in which
only the reproductive tract was without retn expression then I could determine whether the female sterility was due to retn function in the reproductive
tract or whether it might be caused outside the tract
such as in the nervous system where retn is also expressed. My goal was to map the location of cells
responsible for female sterility by comparing the retn
mutant flies to retnRNA-interference
(retn-RNAi) mosaics. In the mosaic I
was able to choose s the cell types that
express the retn gene and those that
will not express the retn gene by using
a system where we can turn off retn expression in the reproductive tract only.
I focused on two mutant alleles that
disrupt retn gene expression leading to
sterilization and the failure to lay eggs.
The first retn mutation called deadringer is caused by a transposable insertion; this mutation prevents the gene
from transcribing resulting in an embryonic lethal phenotype in mutant
homozygotes. The second mutation
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named retnz2-428 is a mis-sense mutation caused by a
chemical and is a weaker mutation that is adult viable but results in female sterility and abnormalities in
courtship behavior when homozygous. I crossed flies
from these two strains and brooded the adult viable
sterile female progeny. The sterility was thought to
come either from behavioral abnormalities or from a
physical abnormality in the reproductive tracking.
Strategy
In order to turn retn expression off in the reproductive tract, I had to use a specially created genetic
system. This genetic system used a gene expressed
in the female reproductive track called homothorax,
which was chosen because it also plays a role in
the development of the female reproductive tract. A
strain of flies available for this project was hthGal4.
This strain had the Gal4 gene inserted in the DNA
upstream of the gene homothorax. This allowed the
gal4 gene to fall under homothorax regulatory control and therefore be expressed in the homothorax
pattern in the reproductive tract in females. Because
the Gal4 gene encodes a transcription factor that
binds to a DNA sequences called upstream regulatory sequences (UAS), I used the expression of Gal4
to cause other genes to be expressed.
I used another strain of flies, which had an insertion of a UAS retn –RNAinterference gene, or retn
Figure 1
The
Catalyst
-RNAi that, when transcribed, made a small double
stranded RNA leading to the degradation of normally
expressed retn transcripts. For this project, I selected
three separate UAS retn-RNAi strains to use as a
way of broadeningthe data. Two strains were made
from the same construct but inserted into different
chromosomes; one was placed on chromosome 1
Figure 2
named UAS retn-RNAi I and the other was placed
on chromosome 3 and named UAS retn-RNAi III.
The third strain was made from a different construct
which inserted in the ARID region, which is an AT
rich region of the retn DNA, this was named UAS
retn-RNAi ARID. I then crossed the hthgal4 strain
of flies with the three retn-RNAi strains. (When I
crossed the hthGal4 strain to the UAS retnRNAi strains, the female offspring would express the double stranded retn-RNAi, which
would seek out and degrade retn transcripts
in the cell, only in the cells that expressed
both homothorax and retn genes which occurs only in cells of the reproductive tract).
With these mosaics I then performed
three different behavioral experiments to examine if there was any change in mating behavior compared to retn mutant or wild-type
females. The behavioral tests were carried
out in a small chamber with one wild-type
male and either a wild-type, retn mutant, or
mosaic female. Over a span of 10 minutes,
I recorded and videotaped the amount of
time it took for the male to initiate courtship with
the female and the duration of courtship. If the pair
copulated I recorded the copulation duration of the
couple. Traditionally the average time from initiation
of the male to the beginning of courtship, called latency to courtship, is between 1-2 minutes for wildtype flies. The average courtship duration, or latency
to copulation, is 3-5 minutes and the
average copulation duration is between
18-30 minutes. I also included mosaic
controls, which consisted of females
that had either the hthgal4 or the three
different RNAi constructs heterozygous
to a wild-type chromosome. I also included a retn control, which had one of
the mutant alleles over a wild-type allele on a balancer chromosome, (CyO),
which causes curling of the wings.
The results showed that the average latency to courtship (Figure 1) was
around a minute. I ran an ANOVA statistical test and found that there were no
behavioral differences due to genotype.
Thus, all females were equally attractive
and were courted quickly. The average
latency to copulation (Figure 2) was between 3-5 minutes with the exception of the retn/CyO
controls, which had much longer courtship duration.
Even so there were no differences due to genotype
by a one-way ANOVA. Thus, the mutant, mosaic and
wild-type had similar courtship characteristics.
In the last behavioral test, the average time of
the copulation duration (Figure 3) was between 18-
Figure 3
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Catalyst
Figure 4A (left) and 4B (right)
22 minutes for the mutant and mosaics, thus these
values fall in the range of the wild-type and control
flies average. As seen all genotypes had similar durations and analysis of variance also concluded that
there were no behavioral differences in copulation between strains of flies. In summary, these
data support my hypothesis that retn based sterility is not due to behavioral faults caused by
the retn degradation in the mutant flies.
The flies that were used in the behavioral
experiments were also tested for their fertility.
Fertility tests involve taking a wild-type male
and either a wild-type, retn mutant, or mosaic
female and adding them to a small glass tube
containing nutrient agar.
The fertility subjects were placed in a 29C
incubator for an average of 48 hours after which
the number of eggs were counted and recorded.
All of the wild-type, mosaic controls, and the
retn control laid eggs. In the retn mutants there
were no egg-laying females out of the 23 tested. Only 1 out of the 19 mosaic hthgal4; UAS
retn-RNAi ARID females laid 20 eggs. Of the
hthGal4; UAS retn-RNAi I females, 3 of 20 laid
2, 3, and 30 eggs, respectively. Finally, only 2
of the 22 hthgal4; UAS retn-RNAi III females
laid eggs counted at 14 and 15. This data helped
support my hypothesis that retn sterility is due
not to behavioral defects but egg retention in the
reproductive tract. However, it also shows that
that either I cannot fully turn off retn function
in the mosaic strains or there is another aspect
causing some fertility to leak through I have not
been able to locate and degrade.
Females used in fertility testing were also
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used in my last procedure, which
was the dissection and examination
of the physical makeup of the reproductive tract of the retn mutant compared to the mosaics. Females were
dissected and their reproductive
tracts were mounted and examined
under a microscope.
In a wild-type, the female reproductive system (Figure 4A) is made
up of two ovaries which attach to
lateral oviducts leading down and attaching to a common oviduct which
then leads on to the uterus. The retn
mutants did not have a common oviduct, instead their lateral oviducts continued on to
the uterus where they attached. Figure 4B is of one
of our dissected retn mutants, we can clearly see that
Figures 5A (top) and 5B (bottom)
The
Catalyst
she lacks a common oviduct with the lateral oviducts
very visible.
Unlike the retn mutant reproductive tracts,
which have two lateral oviducts (double arrows, Figure 5A), the mosaic females dissected and inspected
were found to have a common oviduct (arrow) and
two lateral oviducts (double arrows, Figure 5B).
The mosaic clearly has a common oviduct, although this picture does not contain a uterus as it was
removed during dissection. In summary, these results
suggest that the egg retention and loss of egg laying
behavior in retn mutant females is the result of absence of retn expression in the reproductive tract. It
is interesting that the egg laying defects are still present even though the reproductive tracts look normal
and have a common oviduct.
Learning the process of gene expression of the
female fruit fly reproductive tract brings science one
step closer to understanding the process of cell expression in every animal including ourselves. This
summer’s internship has opened my eyes to a different way of exploring how mutations can be used to
benefit science and aid in exploring how genes work
in creating structure and function. The use of muta-
tion in comparison benefits science by allowing the
ability to knock out gene function in one area of the
body and stabilizing its expression in other areas as
an efficient way to better learn about what role, if
any, that particular gene plays in the development of
that area.
References
1. Casares, M. and Mann, R.S. (2001). The Ground
State of the Ventral Appendage in Drosophilia.
Science Vol:293, 1477-1480
2. Christiansen, A.E. et al. (2002) Sex comes in
from the cold: the integration of sex and pattern.
TRENDS in Genetics Vol:18 Issue:10, 510-516
3. Foronda, D. et al.(2005) Requirement of abnormalA and Abnormal-B in the developing genitalia of
Drosophilia breaks the posterior downregulation
rule. Development 133, 117-127
4. Villella, A. et al. (2006) Defective transfer of
seminal-fluid materials during matings of semifertile fruitless mutants in Drosophilia. Journal
of comparative physiology A. Vol:192 Issue:12,
1253-1269
HHMI at OSU
OSU’s Summer Undergraduate Research Program, sponsored by grant #52005883 from the Howard
Hughes Medical Institute (HHMI) is grateful for generous support from many sources. They include
HHMI (http://www.hhmi.org/)
University Honors College (http://oregonstate.edu/dept/honors/)
OSU’s Undergraduate Research, Innovation, Scholarship, Creativity (URISC) Program
(http://oregonstate.edu/research/incentive/awards.htm#URISC)
Ernest and Pauline Jaworski Fund
(http://www.science.oregonstate.edu/bpp/ernest_and_pauline_jaworski_fund.htm)
DeLoach Undergraduate Research Fund in the University Honors College
Ray, Frances, and Dale Cripps Scholarship Fund in the College of Science
The HHMI Web site (http://oregonstate.edu/dept/biochem/hhmi/summerresearch.html) features additional
information about the program, including PowerPoints, streaming videos of student presentations and
downloadable versions of Catalyst issues. Applications for the 2009 program will be available for download from the site in late January, 2009.
Volume 2
23
The
Catalyst
Now accepting submissions for the next issue of
*Scientific Articles
*Fictional Stories
*Article Reviews
*Poetry
Plus any other ideas you may have!!!
Submit articles to:
Volume 2
Kevin Ahern
ahernk@onid.orst.edu