The Effects of Oxalic Acid on Caenorhabditis elegans Chemotaxis

By: Jenna Gearhart

Abstract
In the wild C. elegans uses chemotaxis to locate its food source of lactic acid bacteria. Lactic
acid bacteria are often found among rotting fruits and vegetables with a popular by-product of
this bacteria being diacetyl. In previous studies, diacetyl has been shown to be a strong
chemoattractant to C. elegans in the wild due to its association with C. elegans food source.
However, the effects of other natural by-products of rotting fruits and vegetation, such as oxalic
acid, on C. elegans chemotaxis have not been looked into. In this study between 80-120 C.
elegans were put on plates with a control (ethanol) and test variables (oxalic acid, diacetyl) in
order to measure C. elegans chemotactic index to each of these chemoattractants. The results of
this study show that oxalic acid has a higher chemotactic index to C. elegans than diacetyl, and
now shed more light on the inner mechanisms of C. elegans chemotaxis.
Introduction
Just as an animal predator can track prey using their keen sense of smell, the nematode
Caenorhabditis elegans employs sensitive olfactory sensory neurons that express vertebrate-like
odor receptors to locate bacteria (Choi et al., 2016). Chemotaxis of C. elegans is an important
function that involves the movement the organism in the direction corresponding to a gradient of
increasing or decreasing concentration of a particular substance (Troemel et al., 1997). In the
wild, C. elegans can be found in the soil with recent studies having shown that they gather in
large numbers in rotten fruit and vegetation (Barriere and Felix, 2005). Found in these fruits and
veggies are lactic acid bacteria, these are a group of gram-positive bacteria that produce lactic
acid as an end product of carbohydrate fermentation, and are found in fermenting vegetative
matter and milk (Marshall, 1987; Smit et al., 2005). Using a combination of food microbiology
and genetics, a possible predator-prey relationship between C. elegans and lactic acid bacteria in
rotting citrus fruit was found (Choi et al., 2016).
A common by product that can be found in the soil among rotting fruits and vegetables is
diacetyl. Diacetyl is an oxidized by product of fermentation that is often found in the presence of
citrate (Choi et al., 2016). Along with this, diacetyl is also known for the strong chemotaxis
attraction that C. elegans has for it (Bargmann et al., 1993). This attraction is likely due to C.
elegans ability to sense, show attraction to and remember certain odors, in order to better find
their food source (Choi et al., 2016). Although diacetyl is a naturally known chemoattractant of
C. elegans, there are still many organic compounds whose effects remain largely unclear on the
chemotaxis effects of C. elegans.
Among the common compounds that occur naturally in
plants oxalic acid is the simplest dicarboxylic acid (Oxalic).
Along with its simple chemical makeup, oxalic acid also Diacetyl
shares similarities in structure with diacetyl (Fig 1.) The most
notable difference between diacetyl and oxalic acids structures is that diacetyl is made up of
more carbons and hydrogens while oxalic acid contains more oxygen’s. These two compounds
similarity in structure, along with them both being found naturally in plants, makes oxalic acid a
prime candidate to test its effects on C. elegans chemotaxis. In this study, C. elegans chemotaxis,
showed a significantly greater response to oxalic acid compared to diacetyl. This newly
identified chemoattractant helps strengthen our understanding of the basis of chemotaxis in C.
elegans.

Materials and methods

The following lab methodology was provided by Margie et al., 2013.
Preparing Chemotaxis plates
A 5cm petri dish was divided into four quadrants, with each quadrant being
designated as either a test (T) or control ( C ) area. The inner circle of the
four quadrants is an area in which the C. elegans movement was not scored
as to prevent the results from being skewed by immobile worms (Fig. 2).
Running the Chemotaxis Assay
Fig.2. Petri dish setup
To mix the test solution 25 ul of 1% diacetyl and 25 ul of 500 mM sodium (Margie et al., 2013 )
azide were combined. The sodium azide was used as to keep the worms
from moving once they’ve reached a quadrant. The control solution was then prepared by mixing
25 ul of 100% ethanol with 25 ul of 500 mM sodium azide. Then 10 ul of the C. elegans solution
was pipetted from the pellet onto the origin of the assay plate. This was doen with a P100
Pipettor with the end of the plastic tip cut off to create a slightly larger opeing. Immediately after
this 2 ul of the test solution (diacetyl) was placed onto the “T” sites. Then the same amount of
the control solution was pipetted onto the two “C” sites. Once the worm and chemoattractant
drops are absorbed in the agar, the lids should be replaced and the plates inverted. The worms
should then be let sit for 60 minutes at room temperature. Repeat this process again to test the
effects of oxalic acid instead of diacetyl. Set up 9 plates, three with 1% diacetyl, three with 1%
oxalic acid, and three with all control drops (ethanol).
Scoring the Chemotactic Assay
In order to score the results the number of worms in each quadrant that complelety crossed the
inner circle were recorded. The Chemotaxis index was then calculated using the equation:
Chemotaxis Index = (# Worms in Both Test Quadrants - # Worms in Both Control Quadrants)/(Total
# of Scored Worms)
This yielded a chemotactic index between -1 and +1. A +1 score indicated maximal attraction
towards the target and represented 100% of the worms arriving in the quadrant containing the
chemical target. An index of -1 is evidence of max repulsion. Once done the mean and standard
error of the mean of the CI was calculated for each of the two conditions, and a graph was made
and a one way ANOVA test ran on the results.

Results

0.9
0.8
CI averages of of C. elegans

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Ethanol Diacetyl Oxalic Acid
-0.1
Chemoattractant

Fig. 3. Average Chemotactic Index (CI) of C. elegans under different chemo attractants. The CI averages
of between 80-120 C. elegans were calculated with the use of varying attractants (ethanol, diacetyl, oxalic
acid). The group that demonstrated the highest CI index was oxalic acid while the group that
demonstrated the lowest was Ethanol (control). The values are expressed as the mean +/-S.E.M.

Table 1. One way ANOVA of Chemotactic Index’s between C. elegans under different chemo attractants

To test the chemotactic effects of oxalic acid on C. elegans, chemoattractants such as diacetyl
and ethanol were used in order to observe the chemotactic index that C. elegans would
demonstrate for oxalic acid. Ethanol was used as a control on the C. elegans, while diacetyl and
oxalic acid were used as test variables. C. elegans, along with 2 ul of the test solution (1%
diacetyl) was placed onto the “T” sites of three of the Petri dishes. The same amount of the
control solution was then pipetted onto the two “C” sites. Once the worm and chemoattractant
drops were absorbed in the agar, the lids were replaced and the plates inverted. The worms then
sat for 60 minutes at room temperature. This process was repeated with 1% oxalic acid and
100% ethanol (negative control). After performing this, oxalic acid was shown to have a
significantly higher chemotactic index in C. elegans compared to diacetyl (Fig. 3). As the
treatment groups had a p-value equal to p<0.000 after a one way ANOVA was performed on the
averages of the chemotactic indexes of each treatment. These results indicate that oxalic acid is a
significantly stronger chemoattractant to C. elegans than diacetyl and helps strengthen our
understanding on the basis of chemotaxis in these nematodes.
Discussion
In this study the chemotactic responses and index’s of C. elegans were measured among
different chemoattractants, both known and unknown (diacetyl, oxalic acid). This experiment
differed from a similar study (Choi et al., 2016) in that it’s main focus was on the effects of other
chemoattractants on C. elegans chemotaxis response. While Choi’s study focused solely on the
chemoattractant diacetyl as diacetyl is a known by-product of fermentation. Diacetyl was the
main focus in Choi’s experiment so that they could obtain a better understanding of the inner
mechanisms of C. elegans olfactory behavior and ecological relationship between the microbial
and metazoan worlds. With them finding that C. elegans response to diacetyl was meditated by
the diacetyl odor receptor ODR-10.
The results from this experiment showed a significantly higher chemotactic index among
C. elegans and oxalic acid compared to C. elegans and diacetyl. With there being a p-value of
p<0.000 between the groups. In the wild, both oxalic acid and diacetyl show up in fermenting
fruits and vegetation. Due to these two chemoattranctants having a similar structure and being
found in similar places in nature it was initally theorized that diacetyl and oxalic acid would have
similar chemotactic indexes. However, the findings of this study showed that oxalic acid had a
much higher chemotactic index compared to diacetyl’s. A theory as to why oxalic acid’s
chemotactic index was higher than diacetyl’s involves the slight differences in these chemical’s
strucutre’s, as seen in Figure 1. Oxalic acids chemical structure is composed of more oxygens,
while diacetyl’s is composed of more carbons and hydrogens. Due to this, it could be theorized
that C. elegans was signficantly more attracted to oxalic acid because of the extra oxygens in the
chemicals makeup that attracted the worms. In this study diacetyl still showed itself to be a
reliable chemoattractant, such as in Choi et al., 2016 study, it just wasn’t as strong of a
chemoattractant as oxalic acid demonstrated itself to be. Further testing on lactic acid bacteria
and their chemoattractants should be looked into in order to gain a fuller understanding on C.
elegans chemotactic abilities.
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