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Ana L. Heully
Biology 4915
22 February 2016
The effects of caffeine on the longevity of Caenorhabditis elegans
Purpose and Objectives
Caffeine is not only found in various medications and treatments, but is also part of daily
diets in the form of coffee and teas. The purpose of this study is to find the effects of caffeine on
the longevity of Caenorhabditis elegans in a homologous pathway to humans. In order to
determine these effects lab work will need to be done on C. elegans that have been treated with
caffeine. This study will aim to find if caffeine allows for a mutation in the daf-2 gene, and in
turn increasing the longevity of C. elegans.
Background
Daf-2 allows for a gain-of-function mutation, meaning that when it is mutated the gene
still has a function, which in this case is increasing the lifespan of C. elegans. When daf-2 is
inhibited, the cell will activate a series of stress-related pathways and antimicrobial genes to
better protect the cell (Golden & Melov, 2007). Many studies have been attempting to find a
correlation between caffeine and any affects it may have on the daf-2 gene. One study found that
different amounts of caffeine have various affects on the lifespans of C. elegans by studying the
changes in population on plates treated with caffeine and the expression of daf-16, which is
responsible for protecting the cell and normally inhibits daf-2 (Sutphin et al., 2012).
Caffeine has been widely examined in its role in aging- associated disorders, with C.
elegans being used as the model. It has already been found that caffeine has a variety of effects
on C. elegans, both positive and negative (Al-Amin, Kawasaki, Gong, & Shim, 2016). One study

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found that when caffeine is dosed correctly it can increase the lifespan of C. elegans, delay their
larval development, and reduce reproduction and body length (Birdi et al., 2015). The gene that
is responsible for reproduction and overall growth of C. elegans is daf-2 (Pierce et el., 2016).
Another study determined that high-doses of caffeine affects the stress-response pathways in C.
elegans and puts the nematode in developmental arrest, which will then lead to apoptosis (AlAmin, Kawasaki, Gong, & Shim, 2016).
The daf pathway is primarily observed in the relationship between caffeine and the
lifespan of C. elegans because of its similarities to the insulin/IGF-1 signaling pathway in
humans. It has been found that the daf-2 sequence is 35% identical to the human insulin receptor
that regulates metabolism (Gami & Minaxi, 2006). The daf pathway plays a lot of rolls in C.
elegans, having a better understanding of this pathway can only aid in future research in the
insulin/IGF-1 signaling pathway in humans.
Hypothesis
The hypothesis is that caffeine allows for a mutation in the daf-2 gene that leads to an
increased lifespan in C. elegans.
Experimental Design/Budget
In order to conduct the study, first all of the reagents must be acquired. Medium to grow
the C. elegans, a refrigerator for storing, PCR machine, caffeine powder, and primers to detect
mutation in the daf-2 gene will be needed. The only things that I do not expect to already be
available in the lab are caffeine powder, which can be purchased from Sigma-Aldrich for $35.50
and Type-it Mutation Detect Kit from Qiagen for $95.80. C. elegans will be grown until there are
enough eggs to populate five plates; the mature C. elegans will be washed away while the eggs
are cared for. Each plate will be dosed with a different amount of caffeine and the C. elegans will

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be observed until they enter the dauer stage and die. Then, their DNA will be extracted and ran
through PCR in order to detect a mutation.
The observations made throughout the experiment should show that lower doses of
caffeine will have more positive results than those with higher doses. It is expected for the C.
elegans that are kept in the plates with lower doses will enter a developmental arrest during the
larval stage and will show a higher rate of reproduction than those that are kept at higher doses.
The C. elegans are expected to live roughly two weeks, hopefully the nematodes kept in at a
lower dose will outlive this expectancy and assist in partially proving the hypothesis. The
hypothesis would be fully proven, if through PCR and the use of mutation detection PCR kit
there is in fact a mutation in the daf-2 gene, which could be held responsible for the increased
lifespan of the C. elegans.
Potential Results
Through this study, it is expected to find that the C. elegans that survived the longest
throughout the study were kept on a low dose of caffeine and also had a mutation to the daf-2
gene. It is possible that the increased amounts of caffeine inhibit any mutation to the daf-2 gene
and allow daf-16 gene to protect the cell. This will then allow the C. elegans to develop normally
and survive for their normal expected lifespan of two weeks.
It is possible for the opposite to occur, that instead of caffeine promoting the mutation, it
inhibits a mutation in the daf-2 gene. This would then allow daf-16 to activate the stress
pathways of the cell and protect the cell. Another possibility would be that the cell will only be
affected by high levels of caffeine instead of lower levels, as I predicted. It is possible for the cell
to only express any changes when exposed to large amounts of caffeine.

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Impact
The results of this study could influence a large variety of future studies. Caffeine is
frequently being studied because it is used in the treatment of Alzheimers Disease. It is
important to see how caffeine affects other pathways in patients being treated with Alzheimers.
For instance, the pathway affected in C. elegans that is similar to humans is the daf pathway and
it is most similar to the insulin pathway. It is possible to find that the results shown from this
study are negative when compared to the insulin pathway in humans, meaning that different
amounts of caffeine, while aiding in the progression of Alzheimers, impeding the patient in
another way.

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References
Al-Amin, M., Kawasaki, I., Gong, J., & Shim, Y. H. (2016). Caffeine induces the stress response
and up-regulates heat shock proteins in Caenorhabditis elegans. Molecules and Cells,
39(2), 1-6. http://dx.doi.org/10.14348/molcells.2016.2298.
Birdi, J. C., Barros, A. G. A., Sampaio, L. R., Ferreira, J. C. D., Soares, F. A. A., & RomanoSilva, M. A. (2015). Lifespan extension induced by caffeine in Caenorhabditis elegans
is partially dependent on adenosine signaling. Frontiers in Aging Neuroscience, 7(220),
1-10. http://doi.org/10.3389/fnagi.2015.00220.
Golden, T.R. and Melov, S. Gene expression changes associated with aging in C. elegans
(January 2, 2007), WormBook, ed. The C. elegans Research Community, WormBook,
http://doi/10.1895/wormbook.1.127.1.
Kenyon, C. (2001). Regulation of longevity by insulin/igf-1 signaling, sensory neurons and the
germline in the nematode C. elegans. TheScientificWorldJOURNAL, 1(S3), 139-145.
Pierce, S. B., Costa, M. Wisotzkey, R., Devadhar, S., Homburger, S., Buchman, A. R.,
Ruvkun, G. (2016). Regulation of daf-2 receptor signaling by human insulin and ins-1, a
member of the unusually large and diverse C. elegans insulin family. Genes &
Development, 15, 672-686. http://doi:10.1101/gad.867301
Sutphin, G. L., Bishop, E., Yanos, M. E., Moller, R. M., & Kaeberlein, M. (2012). Caffeine
extends life span, improves healthspan, and delays age-associated pathology in
Caenorhabditis elegans. Longevity & Healthspan Longevity Healthspan, 1(9).
Gami, M. S. & Wolkow, C. A. (2006). Studies of Caenorhabditis elegans DAF-2/insulin
signaling reveal targets for pharmacological manipulation of lifespan. Aging Cell, 5, 3137. http://doi: 10.1111/j.1474-9726.2005.00188.x

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