Defense

Question 1

Front

Answer 1

Hi, my name’s Olivia, I would like to take you through my bachelor thesis on glycogen allocation within the fungus cultivar Leucoagaricus gongylophorus of the leaf-cutter ants Acromyrmex echinatior and Atta colombica

Question 2

Focus of the study

Answer 2

The focus of my study was to understand the distribution of glycogen within the fungus cultivar, specifically looking at the three distinct layers and two tissue types, mycelium and staphylae

Question 3

Ant/fungus symbiosis

Answer 3

Leaf-cutter ants are known to practice leaf-cutter agriculture, where they cultivate or farm a fungus for food.
They live in an obligate, mutualistic symbiosis with the fungus, where the ants provide the fungus with plant substrate to grow on and protection, and the fungus cultivar in return provide the ants with nutrients, in the form of gongylidia, and shelter for the queen and brood.

Question 4

Why glycogen?

Answer 4

Glycogen is the stored form of glucose and can be metabolized by glycogen phosphorylase when energy is needed, or a lack of nutrients is experienced. This study chose to focus on glycogen because despite its role as a main storage molecule in both insects and fungi, little is known about its distribution within the fungus cultivar and its role in the ant-fungus symbiosis.

Question 5

Key assumptions

Answer 5

These hypotheses were based on key assumptions. The first two hypotheses were based on the same set of key assumptions, which overall state that
the ants are dependent on the fungus to provide them with glycogen, which means that the fungus has a way of actively allocating glycogen towards the ants.
And that optimized glycogen allocation and an increase in glycogen in the staphylae is correlated with an increase in the fitness of the ants.

The third and last hypothesis, was based on the key assumption that there is a passive transport of glycogen and a one way depletion as the fungus ages.

Question 6

Hypotheses

Answer 6

So the three hypotheses formulated on the basis of the aformentioned assumptions are…

Excpet to see higher concentrations in the staphylae compared to mycelium in middle layer

Expects to see highest concentrations in the middle, followed by top and lastly bottom layer

Expects to see highest concentrations at the top, followed by middle and lastly bottom layer.

Question 7

Methods

Answer 7

Due to a time limit, I will only give a quick review of the methods used during the experimental process. If you would like me to go through them in more detail, I would be happy to go back after the presentation.

Question 8

Step 1-4

Answer 8

We started by isolating the colony three days before sampling. On the sampling day we collected to samples from each layer and collected approximately 0.2mg mycelium from each layer and 25 staphylae from the middle layer.

During tissue extraction, we took the samples through multiple steps to break up the tissue and thereby release the glycogen, separating the glycogen from debris and deactivating glycogen degrading enzymes. Lastly we were left with a supernatant that we made a 4-fold dilution of and performed the glycogen analysis on.

Before the actual analysis we also had to prepare the reagents, such as taking reagents out ahead of time to thaw and making the enzyme mixes and glycogen standard.

Question 9

Glycogen analysis

Answer 9

The assay protocol included creating standard readings at the top row of the microplate with known glycogen concentrations.

The samples were added to four wells each, creating two replicates and two blanks.

Hydrolysis buffer was added to all the wells along with a master reaction mix, while hydrolysis enzyme mix was added to all the wells except the blanks.

Question 10

Visualization

Answer 10

For the visualization, we used the Ensight multimode plate reader and the kaleido data acquisition and analysis software t get our RFU values.

Question 11

Calculations

Answer 11

During the calculations, we found the corrected RFU values for the standard solution and created a calibration curve and a linear equation. We then plotted the corrected Rfus for our samples into the equation and got our glycogen concentrations. These were then divided by 25 and multiplied by 4 and 200. lastly, they were divided by the tissue mass.

Question 12

Statistical analysis

Answer 12

During our statistical analysis we used Rstudios, and found the mean glycogen concentrations for each layer and tissue type. We visualized these using box plots. We performed a Shapiro-Wilks test on the data to see if there was a normal distribution. We then performed 4 mixed-effects models on our data with different dependent and independent variables.

Question 13

ADH and VGH not supported

Answer 13

Our results showed that there were highest glycogen concentrations in the bottom layer, followed by the middle and lastly the top with the lowest concentrations.

We can see the mean concentrations here.

These results supported neither the ant distribution hypothesis or the vertical gradient hypothesis.

Looking at each genera, we can see that the atta colombica colonies actually have higher concentrations in the middle layer, followed by the bottom and then top. However, in this thesis we discussed the overall results.

We could also see that there was a larger variation in concentrations among the atta colomibca colonies compared to the acromyrmex echinatior colonies.

Question 14

Mixed-effects on glycogen concentrations in layers

Answer 14

The mixed-effects model had …… as independent variables and glycogen concentration as dependent variable. The results of the model showed that Layer and Layer*Genera had an effect on the glycogen concentrations

and the following post-hoc showed that the significant differences were found between the layers of the Atta colombicacultivar. Specifically the top and bottom layer and the middle and top layer. There were no significant differences between the concentrations in the layers of Acromyrmex echinatior cultivar.

Question 15

Larval allocation hypothesis not supported

Answer 15

The first hypothesis, the larval allocation hypothesis was not supported either as the results showed that the mean glycogen concentration was lower in the staphylae compared to the surrounding mycelium in the middle layer.

Question 16

Mixed-effects model on middle layer

Answer 16

The mixed-effects model performed on the middle layer tissue types had glycogen concentrations as dependent variable and ….. as independent variables. The reusltsshowed thatthe significant effects were Tissue and Tissue*Genera, but not Genera. And the post-hoc showed that there was a significant difference between the glycogen concentrations of staphylae and mycelium in the middle layer for both genera.

Question 17

Staphylae mass no significant differences

Answer 17

Lastly, we also looked at the staphylaemass and glycogen concentration between the two genera. We can see here that Acromyrmex echinatior have a slightly larger staphyale than Atta colomiba, however, the mixed-effects model showed no significant difference between the tissue masses. No significant difference was found between the glycogen concentrations in the staphylae either.

This could indicate that the glycogen requirements from the fungus are the same for both genera.

Question 18

Discussion and future research

Answer 18

In my thesis I discussed possible explanations for the found distribution of glycogen in the cultivar, which I will now share with you. Additionally, I will also propose a possible way of testing these which can lay the groundwork for future studies.

Question 19

Discussion 1/1

Answer 19

I’ll start by discussing the glycogen distribution within the three layers of the cultivar.

In the thesis, I discussed that the distribution of glycogen could be due to differences in activity between the layers, and that the fungus might degrade glycogen in some layers of the fungus faster than it can synthesize and allocate it.
I wrote that the fungus might distribute the glycogen equally among the layers, but I want to correct this to say that the fungus might not distinguish between the layers in regards to glycogen allocation and synthesis.
Instead, the low concentrations in the top are accounted for by the higher activity, where the glycogen is metabolized at a higher rate than it’s synthesized. Meanwhile the bottom and middle layer are less active and therefore don’t metabolize glycogen as fast, instead accumulating glycogen stores.

Question 20

Future test for 1/1

Answer 20

My suggestion for an experimental setup would be as such: I would pick colonies from the two genera and measure the glycogen synthase activity in each layer. I would look at whether the activity is constant each layer and if there is a difference in activity between the layers. This could give an indication as to whether the fungus has assigned glycogen synthesis to a specific layer. And if the synthesis activity is the same in each layer, this could also indicate that each layer is responsible for each own glycogen stores.

Simultaneously, I would also measure the glycogen phosphorylase activity, the enzyme used to metabolize glycogen, in each layerto see if the level of activity is equal or varying among the layers. If the activity is higher at the top, this could be further support. Especially if the activity of glycogen phosphorylase is higher at the top than the glycogen synthase activity.

Question 21

Discussion 1/2

Answer 21

A second explanation could be that glycogen is actively synthesized and stored in the lower layers of the fungus. This could account for the high concentrations in the lower layers compared to the top layer. From here the glycogen would either be allocated towards over layers or brought along with the tufts of mycelium the ants would usually collect and place on the plant substrate at the top. As the ants usually pick mycelium from other layers to place on the plant substrate, it wouldmake sense that the fungus would store the glycogen so that it could be brought along and be used as an energy source.

Question 22

future test 1/2

Answer 22

This could be tested by measuring the glycogen synthase activity in each layer as mentioned in the previous experiment. Here we would again see if the activity is the same or higher in some layers. We would simultaneously measure the glycogen concentrations in each layer over the set period. If the glycogen concentrations don’t match the synthase activity it could be an indication that glycogen is being allocated to a specific layer, especially if one layer has a glycogen concentration higher than its synthase activity.

This test would also be paired with an observational aspect, where we would observe where the ants collect the mycelium from that they place on the plant substrate at the top and compare with the glycogen concentrations. Here we would look at layer preference of the ants and if there is a correlation between mycelium picked and high glycogen concentrations. If they seem to have a preferred layer to collect from which is also associated with higher glycogen concentrations, this could further support the hypothesis.

Question 23

Discussion 2/1

Answer 23

For our second discussion, we looked at the distribution of glycogen in the tissue of the middle layer.

Ifirst argued that the low glycogen concentrations in the staphylae could indicate that the cultivar does not actively provide the ants with glycogen or provide the ants with relatively small amounts of glycogen compared to the overall glycogen concentrations in the fungus. It was shown by another study that the staphylae contain high concentrations of glucose which is actively allocated towards the staphylae and colony. It could therefore be argued that the colony can synthesize their own glycogen from the glucose provided by the fungus and therefore don’t need much glycogen from the fungus.

Question 24

Future test 2/1

Answer 24

One possible way to test this could be to isolate a group of gardener ants and larvae to control their ingestion of staphylae.
After feeding them, they would be anesthetized at 4 degrees and divided into gaster and head-thorax samples.
These would then be measured for glucose and glycogen.
The glucose and glycogen enrichment measured in the head-thorax samples would be an indication of the concentrations present in the staphylae, while the difference in the concentrations measured in the gaster would indicate how much of the glucose ingested gets stored as glycogen.
These test would be performed over a certain period, as the ants need time to ingest the staphylae.

This would be an indication of whether the ants, and especially the larvae, can synthesize glycogen themselves and the first step to understand the ant’s glycogen requirements from the fungus.

Question 25

Discussion 2/2

Answer 25

Another possible explanation for the distribution was that the isolation before sampling caused the fungus to experience starvation, which ultimately affected the glycogen content in the staphylae.

We know that the fungus actively provide glucose to the ants as a part of their symbiosis (Shik et al., 2018). We also known that the fungus metabolizes glycogen when it experiences a lack of nutrients. So if the fungus experienced starvation during the isolation, the fungus might not have been able to produce the nutrients that the ants require, and in response metabolized its glycogen in the staphylae to meet the ants glucose requirements. Because we know that glucose plays a role in the ant-fungus symbiosis, it can be argued that glucose provisioning by the fungus gets prioritized over glycogen.

Question 26

future test 2/2

Answer 26

A way that this could be tested, could be to perform the same experimental procedure as I did in this study, but under two sets of conditions.
You would perform the glycogen analysis while not isolating the colony, which means including the colony in the feeding schedule, and isolating the colony as I did.
You would then sample on chosen days and compare the glycogen content under the two conditions. If the glycogen contents in the staphylae are higher in the period of no isolation, it could support the idea that the fungus metabolizes glycogen to provide glucose when nutrients are sparse.
Furthermore, you could measure the glucose concentrations of all layers and tissue types in both situations.
If the glucose concentrations vary in the mycelium depending on the situation, but the staphylae concentrations remain constant, it could be used as further support for glucose being prioritized in the ant-fungus symbiosis.

Question 27

Limitations

Answer 27

A major limitation of this study was that a wet-dry mass calibration curve wasn’t created and applied to the raw data.
We had taken caution to collect approximately 0.2mg of mycelium and 25 staphylae, but it was evident during collection that there was a difference in the water content of the tissue of each layer, with some being much more dry than others

Because dry tissue weighs less than wet tissue,the dry samples contain more tissue than the less dry samples, despite having a similar mass.this in turn would result in the final glycogen concentrations being incomparable, as the tissue mass is accounted for during our calculations.

We do expect that the application of the calibration curve will affect our final results, however, without actually creating and applying it to our data, there is no there is no way to know whether it will change enough to change the order of the layers. I would therefore suggest that further work be done on this study, by creating and applying the wet-dry mass calibration curve, to see if this could change the overall outcome of this study and possibly support any of the three hypotheses.

Question 28

Errors

Answer 28

During the experimental procedure, there were some changes made and some uncertainties that could have possibly affected the results. Once again I’ll just briefly go over them, but would love to come back to them if there are any questions.

One of these steps were removal of an extra step in the tissue extraction, which we did halfway through the study. This resulted in the removal of liquid loss, however, since this was done halfway through the study, it creates a discrepancy between the results before and after.

During tissue collection, we also had trouble placing the tissue in the caps, which resulted in some tissue landing outside the center of the caps, including the tissue in the total mass but not the tissue extraction.

Lastly, we had a glycogen analysis towards the end of the study, where something had gone wrong resulting in the readings for the standard solution being too high. We therefore couldn’t use the readings to create the calibration curve, so instead we took the mean of the previous standard solution RFUs and used them to create the calibration curve. Ofc this could have affected the final results, as the calibration curve wasn’t exactly suited for readings of this exact sample.