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Effects of glyphosate

Lenard N , Lambert T, Bethay K .


Our Lady of the Lake College
Introduction
In previous research, it was discovered that
underdeveloped countries are using glyphosate as
a pesticide for their crops. There has been an
increase of birth defects in these countries, and we
believe that glyphosate may have a correlation to
these birth defects. This gave us the rationale to
conduct this specific research. We are conducting
independent research on C. elegans at the
Biological Learning and Research facility at Our
Lady of the Lake College. These are nonhazardous, non-infectious, non-pathogenic and
non-parasitic worms. We specifically wanted to
focus on the developmental effects of the chemical
glyphosate. Glyphosate is the active ingredient in
the product RoundUp, which is a commonly used
weed killer and pesticide. C. elegans are
microscopic nematodes which are found in soil and
have direct contact with this chemical if it is used by
a consumer. We suspect that glyphosate will cause
the over expression of the gene ced-3 which
controls the organisms ability to perform apoptosis
on unwanted cells. This could link glyphosate to
birth defects.

Objectives
To measure gene expression of the Ced-3
before and after treatments with glyphosate.
We will use this data to determine if the gene
is being overexpressed, under expressed, or
not changing. The Ced-3 gene is responsible
for programmed cell death (apoptosis) in C.
elegans. .

Methods

Our methods were based on previous research


and coupled with a bit of our own design. We are
using a series of chemicals to isolate RNA and
convert into cDNA, gel electrophoresis to
separate cDNA bands, NanoDrop for DNA
concentration, ChemiDoc Imager for DNA
visualization, and qPCR (real-time polymerase
chain reaction) to amplify the cDNA so that gene
expression can be quantified. We have also
designed primers (DNA sequences) to bind
specifically to the Ced-3 gene, so we can isolate
and measure expression of the gene in question.
Our specific primers target an mRNA sequence
that code for the Ced-3 gene and are about 20
base pairs long

Results

Discussion

After a few trial runs we were able to have some


clean results. The last test run gave us results in
our controls that were acceptable, but the results
we saw did not match our hypothesis. The control
worms had an expression of more than twice as
much as the Glyphosate. This means that there
was less Ced-3 DNA present in the worms treated
with 0.1% Glyphosate than the control worms.

References

Analysis
To quantify data a technique called real time
PCR was used. This techniques allows one to
replicate specific DNA sequences. With every
cycle of the machine our specific sequence
was replicated, and tagged with a fluorescence
(SYBR green). The real time PCR machine is
able to measure the abundance of this
fluorescence after each cycle, and give real
time feed back. That is how we were able to
see if our gene was up regulated in the worms
treated with glyphosate vs. the control (treated
with water).

Our hypothesis was that glyphosate would cause


an overexpression of the apoptotic gene Ced-3.
The results showed that the nematodes treated
with 0.1% glyphosate had lower expression of the
gene in question than the nematodes treated with
distilled water. This is significant because while it
doesnt confirm our hypothesis it does show that
the glyphosate had some effect on the nematodes.
It is important to note that a reduction in normal
apoptosis could still lead to birth defects. More
tests will be run to see how different concentrations
of glyphosate will affect ced-3 gene expression,
and if it does then we can see what effects the
concentration in Roundup will have on these
organisms. Future research could include testing
the ced-9 gene ,which is one of the genes that
regulates ced-3, to see how glyphosate affects its
expression.

1. Stiernagle, T. (1999) C. elegans: A practical approach pp.


51-67.
2. iTaq Universal SYBR Green Supermix. Bio-rad. 25
August 2014.
3. iScript cDNA Synthesis Kit. Bio-rad. 25 August 2014.

Figure 1: This table shows the comparison of Glyphosate treated


nematodes vs. nematodes treated with water, and their ced-3
expression normalized to Actin expression. We have a P-value of
0.8462,NS. n= 3 for each

Acknowledgments: This work was supported by the Louisiana Board of Regents


Sr. M. Edana Corcoran Endowed Professorship. The C. elegans strains were
provided by the CGC, which is funded by NIH Office of Research Infrastructure
Programs (P40 OD010440). Special thanks to Dr. Natalie Lenard for her
indispensable guidance.