Defense Flashcards
(28 cards)
Front
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
Focus of the study
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
Ant/fungus symbiosis
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.
Why glycogen?
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.
Key assumptions
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.
Hypotheses
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.
Methods
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.
Step 1-4
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.
Glycogen analysis
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.
Visualization
For the visualization, we used the Ensight multimode plate reader and the kaleido data acquisition and analysis software t get our RFU values.
Calculations
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.
Statistical analysis
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.
ADH and VGH not supported
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.
Mixed-effects on glycogen concentrations in layers
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.
Larval allocation hypothesis not supported
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.
Mixed-effects model on middle layer
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.
Staphylae mass no significant differences
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.
Discussion and future research
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.
Discussion 1/1
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.
Future test for 1/1
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.
Discussion 1/2
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.
future test 1/2
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.
Discussion 2/1
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.
Future test 2/1
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.
