Effects of CO2 on aphid-plant interaction

THE EFFECTS OF ELEVATED CO2 LEVELS ON THE
INTERACTION BETWEEN MYZUS PERSICAE AND
ARABIDOPSIS THALIANA
The Ace-phids
Monish Ahluwalia, Bronwyn Barker, Elsie Loukiantchenko,
Urszula Sitarz, Elias Vitali
October 23, 2016
ISCI 2A18
1

PAIx Group 9 Manuscript Page 2
Abstract1
The effects of the increase in atmospheric CO2 are not completely clear in terms of plant-animal2
interactions. This study measures the effects of elevated CO2 concentrations on Myzus persicae3
and Arabidopsis thaliana in a closed system. M. persicae are herbivorous and have a preference4
for elevated carbon dioxide concentrations, while the elevated CO2 improves photosynthetic5
efficiency. Therefore, we hypothesize that M. persicae per-capita growth rate will increase6
unless a plateau is reached. Four chambers, with six pots of A. thaliana and initially two7
M. persicae each, were manipulated with dry ice to have increased CO2 levels. M. persicae8
population growth, A. thaliana stem height, and CO2 concentration were measured to study9
CO2 effects on aphid-plant interactions. Results show that aphid population was significantly10
increased under medium CO2 environments and the potential for stems sprouting significantly11
decreased under high CO2 environments. It was concluded that due to factors such as soil12
quality, aphid adaptation, and adjusted plant response, the net effect of increased CO2 on the13
interaction is small. This study has applications in ecosystems as increased aphid populations14
can have negative impacts on other insect species and plants. Similarly, the results of this study15
can provide insight into the changing ecosystems as the CO2 concentrations rise.16
Keywords17
Myzus persicae, Arabidopsis thaliana, aphids, climate change, CO2 , performance, herbivory,18
proliferate19

Introduction20
Atmospheric CO2 levels have nearly doubled, from 280 to over 400 ppm, since the 1800s due to21
the industrial revolution and the subsequent increase in anthropogenic CO2 production (Sun,22
Guo, and Ge, 2016). Aside from many large-scale environmental concerns, such as increasing23
global temperatures, the increase in atmospheric CO2 has been anticipated to directly affect24
the health, positively or negatively, of all organisms. The effects could be the result of CO225
asphyxiation, aiding biological processes that require CO2 , aiding or inhibiting other processes26
that interact with CO2 , as well as inter-organism interactions (Sun, Guo, and Ge, 2016).27
CO2 is a vital component of photosynthesis (Zhang et al., 2007). Therefore, it is reasonable28
to assume that an increase in atmospheric CO2 levels would be helpful to plant species since29
diffusion is a major mechanism whereby C3 plants obtain CO2 . A study by the Ontario Ministry30
of Agriculture, Food, and Rural Affairs found that C3 plants have an increase in efficiency of31
photosynthesis in higher CO2 conditions, up to 1000 ppm (Ontario Ministry of Agriculture,32
Food, and Rural Affairs, 2016). However, this increase in photosynthetic efficiency has also33
been found to decrease the nutritional value of the plants, which decreases the palatability to34
herbivores, including humans (Myers et al., 2014).35
The opposite generalisation does not apply to all animals; one cannot say that high CO2 levels36
are harmful for all species in the animal kingdom. Previous studies on Myzus persicae, more37
commonly known as the green peach aphid, have had variable results on the effects of increased38
CO2 conditions (Robinson, Ryan, and Newman, 2012; Holopainen, 2002; Bezemer, Knight,39
Newington and Jones, 1999). However, with atmospheric CO2 rising, these results is are of40
reasonable concern.41
This study focuses on the effects increased atmospheric carbon dioxide has on the growth42
of aphid populations and the growth of Arabidopsis thaliana, a small flowering weed of the43

cabbage family. These two species have a predator-prey interaction, in which the aphids prey44
on the A. thaliana through sucking out phloem from the stem, which can affect plant growth,45
water reservation, and wilting, while also potentially infecting the plant with carried viruses46
(Jaouannet et al., 2014). The speciﬁc research questions it sets out to answer are: How does the47
concentration of CO2 in the atmosphere affect the growth rate of A. thaliana and the per capita48
growth rate of aphids? What concentration will be too high to support aphid populations despite49
potentially increased plant performance?50
The null hypothesis for the experiment is that an increase in atmospheric carbon dioxide levels51
has no statistically signiﬁcant effect on aphid per capita growth rate. The alternate hypotheses52
are that the aphid per capita growth rate will increase as CO2 concentrations are increased due53
to increased plant photosynthesis and aphid preference, the aphid per capita growth rate will54
decrease as CO2 concentrations are increased due to sensitivity to high carbon dioxide levels, or55
the aphid per capita growth rate will increase due to increased plant photosynthesis and aphid56
preference until it reaches a point whereby the CO2 concentration becomes too high for the57
aphids to thrive.58
Methods59
Experimental Design60
There were four different environments of CO2 for the plant-animal communities in plastic plant61
chambers. The chambers were labelled C, 1, 2, and 3, where C was the control chamber, 1 had62
the lowest added amount of CO2 , 2 had a medium amount of added CO2 , and 3 had the greatest63
added amount of CO2 . Six pots of A. thaliana were added to each of the four chambers in a64
staggered manner, as seen in Figure 1. This was done to ensure that aphids were isolated on a65
singular plant and were not able to interact with other plants in the habitat. These plants were66

previously seeded and grown for six weeks prior to the start of the experiment.67
Each of the 24 plants were inoculated on Day 1 of the experiment with two aphids at random68
locations of the plant. We took initial measurements of the stem length of each plant. If a plant69
had more than one deﬁnite stem, the largest one was taken. Each plant was labelled according to70
its chamber (C, 1, 2, 3) and a letter from A to F, based on the plant’s position in the chamber71
with A at the bottom left corner and F at the top right.72
The four CO2 monitors, combined with thermometers, measured CO2 concentration (ppm)73
and temperature (°C) in the chambers. A wire thermometer was taped to the interior of the74
chamber lid so that the tip hung from the center of the chamber until halfway to the bottom75
and a CO2 monitor was taped to the top of the lid. The monitors were attached to PASCO76
Spark devices (PASCO SPARK Science Learning System, PS-2008A) and were calibrated at77
the beginning of the experiment and whenever the lids were opened, taking measurements at 1578
minute intervals.79
Each chamber was subject to a speciﬁc amount of CO2 through the sublimation of dry ice80
(solid CO2 ), as depicted in Table 1. After the dry ice was added, the chambers were sealed with81
tape to create a closed system. On the days outlined in Table 1, we added CO2 to the chambers,82
recorded the number of aphids, and measured the stem height of the plant. To do so, we stopped83
the recording from the Spark devices and unsealed the chambers. We counted the number of84
aphids on each plant, with the use of dissection needles and magnifying glasses, and measured85
the plants with the same procedure as the initial measurements. The plants were watered once,86
on Day 8 of the experiment, with 25 mL of water.87
Statistical Analysis88
Statistical analysis was done using R 3.2.3 (written by Simon Urbanek, Hans-Jorg Bibiko, and89
Stefano Iacus, The R Foundation). After counting the number of aphids on each plant each90

data collection day, a linear model was created that compared the number of aphids to their91
respective chamber on the day that the measurement was taken. Since each plant in the study92
started with two aphids, this data was removed from the model. This model was normalized93
using a logarithmic transformation and an Analysis of Variance (ANOVA) was conducted. This94
model is denoted as Model 1. The same procedure was done for the measured stem height for95
each plant on each day and denoted as Model 2.96
It was noticed that there were some plants that sprouted throughout the experiment and some97
that did not. Since the error in our measurements was 0.05cm, a growth rate of ≤ 0.071cm per98
day was attributed to plants that did not ’boom’ (sprout) and these plants were given a binary99
value of 0. An x-y plot of their binary value can be seen in Figure 3. Those with a growth rate100
of > 0.071cm per day was attributed to plants that did “boom” and these plants were given a101
binary value of 1. A linear model was created using these “boom factors” and an Analysis of102
Variance was conducted. This model is denoted as Model 3. To caculate the boom factor, the103
growth rate Equation (1) was used.104
Stem length (last day)−Stem length (f ir st day)
Number o f days
(1)
Results105
The results for the CO2 levels in each chamber can be seen in Figure 6. The overall average106
CO2 concentrations were 287ppm, 2190ppm, 5843ppm, and 6640ppm for the Control, Low,107
Medium, and High chambers respectively.108
For Model 1, the ANOVA stated that the number of aphids is signiﬁcantly correlated with109
the chamber that the aphids were in (p = 0.0495). Further analysis showed that this was only110
true for the Medium chamber (p = 0.0246) and the number of aphids were not signiﬁcantly111
correlated with the Low or High chambers (p = 0.242, p = 0.810, respectively).112

For Model 2, the ANOVA stated that the stem height was not significantly correlated with113
the chamber that the aphids were in (p = 0.0511). None of the individual chambers showed114
significant results either (p = 0.488, p = 0.118, p=0.340, for the Low, Medium, and High115
chambers respectively).116
For Model 3, the ANOVA stated that the relationship between chamber and whether or not117
plants “boomed” was insignificant (p = 0.206). However, further analysis showed that a plant118
being in the High chamber was significantly correlated with whether or not plants boomed119
(p = 0.0417). The Low and Medium chambers for this model were statistically insignificant120
(p = 0.477, p = 0.477, respectively).121
Discussion122
The null hypothesis stated high CO2 concentrations would have no effect on aphid population or123
plant growth rate. Meanwhile, our alternate theories expressed that CO2 concentrations would124
affect the growth rate either positively or negatively. Experimental results showed that elevated125
CO2 concentrations only had significant effects on aphid per-capita population growth and plant126
growth-rate under discrete values.127
Overall CO2 levels128
The average CO2 levels previously discussed are much higher than the predicted levels for the129
Earth’s atmosphere by the year 2100. This was done purposely to decrease the margin of error130
for the data. As shown by Figure 6, the CO2 levels were not steady over time. They were131
punctuated with spikes whenever dry ice was added to each chamber. This is because dry ice132
sublimates almost immediately. From this, we were only able to retrieve an average CO2 level133
for each chamber and associate a discrete value to it (control, low, medium, or high). While the134

low and medium chambers resulted in CO2 level averages that were proportional to the amount135
of dry ice added, the high chamber experienced a more rapid decrease in its CO2 levels, and136
thus, a lower than expected average concentration.137
Looking at Figure 6, it can be seen that the medium and high chambers only have four spikes138
coincident with when we added the dry ice, while the Low Chamber has five. While five doses of139
dry ice were given, the CO2 monitors in the medium and high chambers malfunctioned and did140
not measure an increase in the carbon dioxide levels. The result is that the calculated averages141
are lower than the actual chamber average.142
A final note about Figure 6 is that the CO2 levels for the Control Chamber sharply drop143
halfway through the study. While the cause of this is unknown, this decrease could have had an144
effect on the photosynthetic ability of plants as the diffusion of CO2 is a crucial part of plant145
photosynthesis.146
Direct effects of CO2 and aphid presence on plants147
What is immediately noticeable about the data is that the aphids in the Low, Medium, and High148
chambers thrived and reproduced under the high CO2 environments. As shown by Figure 4, the149
number of aphids grew almost exponentially in all the environments (excluding day 12 in the150
High chamber). This could be due to the absence of natural predators causing the creation of a151
niche space for the aphids to proliferate and overcome any negative effects of the CO2 . Another152
idea proposed by Absigold et al. (1994) was that pea aphids can compensate for changes induced153
by elevated CO2 levels by changing where they feed on the plant, their phloem-uptake rate, or154
their metabolism in general. It was observed by Absigold et al. qualitatively that while aphids155
tend to stay on the stem of the plant in low CO2 environments, many chose to move on top or156
underneath the leaves.157
After some statistical analysis, it was shown the aphids in the Medium chamber fared better158

than those in the Control chamber in a statistically significant fashion. This leads towards159
the idea that aphids fared better under a CO2 concentration of approximately 6000ppm than160
approximately 250ppm. However, the trend was not significant for the Low or High chambers,161
and it can only be concluded that aphids fare better under a 6000ppm CO2 level than increased162
CO2 levels in general. The reasons for this may be due to adaptation as proposed by Absigold et163
al. or preference.164
Interactive effect of CO2 and aphid presence on plants165
There are a few points to make before the statistics are analyzed, first focusing on the plants. The166
measurement technique used, stem height, is not the best measurement of plant performance;167
leaf area and plant biomass are generally more accepted as plant performance indicators (Wood168
& Roper, 2000; Wuyts, Dondht, & Inze, 2015). Due to the drawbacks of these data collection169
methods, mainly complexity and unsuitability. It’s no surprise that throughout the study, a170
majority of the aphids were present on the stems as opposed to on leaves. With this, plant171
performance was studied in the context of the aphids. The second reason is that it was the most172
feasible measure to study plant growth. While biomass would be the most accurate, it was173
infeasible due to the drawbacks of the study, mainly being complexity and unsuitability based174
on the limited research time frame.175
In future elevated CO2 environments, will growth rate and plant response to herbivores be176
affected? There are two antagonistic effects to consider: CO2 levels, and aphid presence, which177
is often detrimental to plant performance (Pollard, 2009). Despite the predicted increased178
plant growth due to the CO2 levels, the aphids provided a stressed environment which stunted179
plant growth. This interaction is consistent with several studies which also concluded plant180
growth is not highly affected by elevated CO2 environments combined with aphid presence181
(Salt et al. 1995; Hughes & Bazaaz, 1997; Hughes & Bazzaz, 2001). We conclude that in182

elevated CO2 environments, plant response to phloem-sucking insects such as aphids did not183
drastically change. Newman et al. (2003) constructed a mathematical model which concluded184
that aphid-population dynamics are largely dependent on the nitrogen soil concentration. This is185
also suggested in other studies (e.g. Hughes & Bazzaz, 2001; Risebrow & Dixon, 1987). Aphids186
have very specific selection of amino acid requirements; since CO2 affects phloem composition187
(Wang & Nobel, 1995), the aphid colonies will be affected since they are phloem-sucking insects.188
To consolidate this further, studies such as Docherty et al.’s (1997) have found that amino acids189
in phloem sap have declined at elevated CO2 , showing consistent results with the data collected190
in this study. It is important to note that the average CO2 concentration in the High chamber191
was not much different than that in the Medium chamber, yet the Medium chamber provided a192
statistically insignificant number of plants that did not “sprout”. Another potential reason for193
this result is not the average, but the maximum level of CO2 reached by each chamber. While194
the Low and Medium chambers reached a CO2 level of approximately 17 000 and 42 000 ppm,195
the High reached approximately 60 000 ppm. There could be a short-term threshold whereby a196
plant senses very high CO2 levels and, as a result, does not invest energy in stem growth.197
The data analysis showed that CO2 levels had a significant effect on aphid growth in one198
of the four chambers, as observed in Figure 4. Aphids moved more quickly through their life199
stages as was observed in forms of molting and fecundity. On top of that, it was noted that as200
the experiment progressed, there were more alates forming in the sealed chambers. This points201
to sexual reproduction which is specific to stressful aphid environments. The results show a202
statistically significant relationship between the aphid growth rate in the Medium CO2 chamber,203
which is the only chamber where winged alates were found (Leather, 1989). This observation204
points to medium-level CO2 concentrations being a big stressor of aphid colonies on plants.205
The Medium and High chambers had very similar CO2 concentrations, thus although the data206
was not significant for the high CO2 chamber, some extrapolations can be made that the same207
pattern is present in the high CO2 chamber. Similar studies have also found variance in aphid208

performance in elevated CO2 environments (e.g. Docherty et al. 1997).209
Another potential factor that was not accounted for was the nitrogen concentration in the soil.210
Nitrogen concentrations have a direct impact on the composition of the phloem on the plants211
and therefore, a potential indirect effect, either positively or negatively, on the aphids (Newman212
et al., 2003). Because this was not accounted for, it could have affected the fitness of aphids on213
individual plants, causing potential variation in the chambers. This implies the combined net214
effect on plant performance of all of the unkown variables balanced out. Other studies suggest215
that increased plant performance and growth compensate for the increased insect proliferation216
and consumption, further emphasizing underlying, unmeasured reasons (Caulfield & Bunce,217
1994; Salt et al., 1995).218
Here we experience ambiguity with several studies coming to different conclusions. It is219
possible that aphids experience increased proliferation under high CO2 environments; however,220
the response from the plants could have stunted this growth. This interaction could be completely221
insignificant and unrelated, supporting the null hypothesis, or there could be an underlying222
balancing effect of the many combined factors of CO2 , nitrogen, and energy spent on defences223
as opposed to growth.224
Overall, this study shows limited evidence that aphids significantly affected plant response in225
elevated CO2 environments. Our results are consistent with those of many other similar studies,226
showing that positive effects of increased plant performance are balanced by negative effects of227
increased aphid proliferation (e.g. Hughes & Bazzaz, 2001; Newman et al., 2003, etc).228
Implications229
Our data has important implications in our understanding of the interactive effect of atmospheric230
carbon dioxide levels on aphids and Arabidopsis. Although, it is possible that higher CO2231
concentrations potentially cause environmental stress on the aphids, high CO2 levels do not232

immediately and significantly decrease aphid growth rate on Arabidopsis. In fact, very high233
concentrations of approximately 6000ppm cause aphid growth to increase. This has major234
applications in predicting the future health of ecosystems as CO2 levels rise on Earth and shows235
that such a rise will not significantly decrease green peach aphid populations in the short term236
and this result could potentially stand for other organisms as well.237
Another implication has to do with the “sprout” rates. In the future, it could be found that238
plants experiencing high CO2 levels may not invest energy in stem growth. This decreased239
height when competing for sunlight may negatively affect certain species that experience this240
result.241
Finally, due to the production of alates, yet no significant decrease in the number of aphids242
present on plants, it can be inferred that aphids, under elevated CO2 , might disperse to other243
plants more quickly than before. This could have serious implications on the health of ecosystems244
and could result in a changing ecosystem dynamic in the future as CO2 levels continue to rise.245
Future Directions246
Our experiment, while focused on aphid growth rate, did not account for aphid death rate or247
molting. We only counted the aphids we saw alive on the plant. We recorded that there were248
many molts, but we cannot know if the aphids went through their life stages more quickly or249
died more quickly as well. This could be amended by counting the amount of dead aphids,250
perhaps with the use of microscopes, to differentiate between dead aphids and cuticles. The251
amount of molting would also play an important role in understanding the effect increased CO2252
and plant interaction have on the aphid life cycle. This way, we could find correlations with not253
only growth rate, but death rate and aphid life cycle.254
A second future direction would be to replace dry ice with a more consistent avenue for255
allowing CO2 into the containers. When modeling the effects of greenhouse gases, it would be256

beneficial to have a method that disperses CO2 evenly over time instead of dry ice that sublimates257
immediately and provides a distinct, short term spike in CO2 levels. This would involve the258
setup of a more complex system, with direct CO2 uniformly vented into the containers. This259
would provide us with an environment that would better emulate the rising CO2 conditions on260
Earth.261
Further, we could assess the effects of increased temperature as well as CO2 on the plant-262
animal interaction. This would also better model the Earth’s environment in the coming decades263
and would provide a more comprehensive view on the effects of the greenhouse effect on264
ecosystem interactions.265
Conclusion266
The data collected helps answer the posed questions about how aphid-plant interactions are267
modified in elevated CO2 environments. We observed that as seperate treatments aphids thrived268
better in Medium CO2 concentrations, which are still very high compared to ambient, and plants269
fared better in a high CO2 environment. We conclude that this is likely due to the expected270
negative effect of aphid presence and the positive effect of elevated CO2 balancing each other out.271
Simultaneously, the aphid presence and elevated CO2 environments did not have a substantial272
effect on plant or aphid response, leading to the conclusion that projected atmospheric CO2273
levels alone will not severely affect aphid-plant interactions.274
Acnowledgements275
We would like to thank Dr. Susan Dudley and Sebastian Irazuzta for their ongoing support in276
this project, specifically for guiding us through how to plan and run such an experiment and277
giving valuable feedback at our defense. Additionally, we would like to thank Dr. Russ Ellis for278

allowing us to use his laboratory and providing us with all necessary equipment.279
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Tables and Figures340
Table and Figure Legends341
Figure 1: The plants were staggered in the chamber to maximize the distance between them. To342
stay consistent, the plants in each chamber were lettered, with A as the plant on the bottom left343
and F as the top right.344
Figure 2: The sealed system, complete with the chamber lids. The CO2 monitors, the blue345
devices going through the lid, were connected to the spark devices as seen on the platform at the346
top of the image. The white wire thermometers were taped to the interior of the lid.347
Table 1: This table summarizes the amount of dry ice that was inserted in the chambers at each348
collection date. The amount was changed each time to account for days of absence from the349
laboratory, but was kept consisent in terms of alternating between each iteration.350
Figure 3: A visual representation of the plants that "sprouted" and those that did not "sprout",351
where a plant that "sprouted" has a stem height growth rate of > 0.071cm per day. Those that352
"sprouted" were given a binary value of 1 while those that did not "sprout" were given a binary353
value of 0.354
Figure 4: A box plot representing the number of aphids present on each of the six plants in355
each chamber. Data was not available for day 7 for the control chamber. Overall, the plants356
experienced aphid growth over the 12 day period, and the increased number of aphids was357
statistically signiﬁcant for the medium chamber, leading to the conclusion that elevated CO2358
levels of approximately 6000ppm cause an increase in the number of aphids over time when359
compared to ambient CO2 levels.360
Figure 5: A box plot representing the stem height of each of the six plants in each chamber.361
Data was not available for day 7 for the control or low chambers. Overall, plants experienced362

stem growth over the 12 day period, however, no elevated CO2 chamber had a statistically363
signiﬁcant difference when compared to the control chamber. It is important to note that in the364
high chamber contained three plants that did not "sprout" and those make up the bottom portion365
of the high chamber box plot.366
Figure 6: A plot of the CO2 concentration in each chamber over time. The control shows what367
is expected - a ﬂuctuation in CO2 levels due to plant respiration. Meanwhile, the other plots368
show the varying CO2 levels over the 12-day period. Note that because of the much lower value369
in the control, the y-axis is not scaled to all of the other graphs for visual purposes.370
Figure 7: The residual graphs for the logarithmic transformation of the linear model that371
compares the stem height of plants to the chamber they were in and the day the measurements372
were taken. This model was run through an ANOVA and the p values for the low, medium, and373
high chambers were p = 0.488, p = 0.118, and p = 0.340 respectively.374
Figure 8: The residual graphs for the logarithmic transformation of the linear model that com-375
pares the number of aphids on each plant to the chamber they were in and day the measurements376
were taken. This model was run through an ANOVA and the p values for the low, medium, and377
high chambers were p = 0.242, p = 0.0246, and p = 0.810 respectively.378
Figure 9: The residual graphs for the linear model that compares the "sprout" factor for each379
plant to the chamber they were in. This model was run through an ANOVA and the p values for380
the low, medium, and high chambers were p = 0.477, p = 0.477, and p = 0.0417 respectively.381

Table 1
{Day of Experiment} Amount of CO2 added to the Chambers (±0.005g)
Control (C) 1 2 3
1 0 0.5 1.5 3
5 0 1.08 3 6.7
7 0 0.48 1.52 3
8 0 1.06 3 6.02
12 0 0.55 1.49 3.1

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Appendix382
Stem Height Linear Model383
Figure 7 shows the various plots from R 3.2.3 of the stem height linear model run for statistical384
analysis.385
Aphid Population Linear Model386
Figure 8 shows the various plots from R 3.2.3 of the aphid population linear model run for387
statistical analysis.388
"Boom" Linear Model389
Figure 9 shows the various plots from R 3.2.3 of the boom rate linear model run for statistical390
analysis.391

Effects of CO2 on aphid-plant interaction

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