Group 9 - PAIx Manuscript

THE EFFECTS OF ELEVATED CO2 LEVELS ON THEINTERACTION 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 l eng th (l ast d ay)−Stem l eng th ( f i r st d ay)

Number o f d ay s(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 35 0 1.08 3 6.77 0 0.48 1.52 38 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

Documents

Group 9 - PAIx Manuscript