Casual Fridays: We Discuss Gardening Ants and Their Symbionts!

This week we bring you another “Casual Friday” post! While we aren’t actually posting this on a Friday, we are still bringing you a another casual discussion of a chemical ecology paper. This week’s paper is from the lab of Cameron Currie and is entitled “Black yeast symbionts compromise the efficiency of antibiotic defenses in fungus-growing ants.”

This paper is focused on a particularly fun ecological community: a three-part symbiosis between ants, fungi, and bacteria. Specifically, these are ants in the tribe Attini that cultivate fungus gardens. These farmed fungus-gardens are the ants’ primary source of food. Integral to health of the gardens are bacteria in the genus Pseudonocardia that live on the ants' appendages. The symbiotic bacteria are thought to make antibiotics that protect the gardens from invasion by parasitic fungi in the genus Escovopsis

Over the years Currie and co-workers discovered that in addition to the bacteria, the fungus AND the pathogen invaders, black yeasts are also very common colonizers of Attini ants. This paper was aimed at trying to figure out where in the ant-fungus garden-bacterial symbiont circle the yeasts fit in.

Specifically, they asked, what are the effects of a weak interaction from the yeasts on the ant/fungus/bacteria community? While chemical ecologists tend to focus on strong and long-lasting interactions between pairs of organisms, transient and non-specific interactions can have large ecological effects as well. Just ponder a moment the whole-body effects of a bacteria-induced case of food sickness: You are stuck in bed drinking fluids and your gut bacteria have to recover from having their neighbors flushed away!  
 

AMC:

Dearest ladies,

I am really struggling with the experiment shown in Figure 1.

This is basically the piece de la resistance of this paper. It shows that the fungal cultivar fares worse when the black yeast is around. This result—that the black yeast are hurting the ecological success of the ants by making the fungal gardens more susceptible to Escovopsis attack—is the impetus for all of the other work in the paper. Yet, to my eyes, it seems like the effect is very small: the two lines’ final values seem so similar. Their end points only differ by 0.2 and the standard error is reported to be <0.01.  How many times was this experiment done? Given the complicated nature of the experimental set up, it seems amazing to me that the error is so small.

 

CAB:  Well, Alexandra, how should we feel about the data that supports the question at the heart of this paper? I think for the moment we need to just assume that this is a repeatable, significant difference in the ant garden health when black yeasts are present. If we can trust their experimental set-up, then we might conclude that though this interaction isn't intense, it is real and reproducible.

I have a few questions about the first experiment investigating a hypothesis as to how the black yeasts were negatively impacting garden health.

This experiment compared growth of the black yeasts on two different solid medias (solid media meaning agar-based, so sort of like a dry jello): agar containing ground-up bacteria collected from the ants and agar with no additives. They saw significantly more black yeast growth on the agar containing bacterial biomass. From these data, they conclude the black yeast can use bacterial-derived nutrients for growth.

To me, if you want to make a conclusion about the specificity of the black yeasts consuming the symbiotic bacteria you would need to compare growth on agar with biomass from different bacteria, and include isolates that don’t come from the ants but from the surrounding environment.

 

KLS:

Hi ladies!

Interesting read - I always admire these types of experimental investigations. The natural world is incredibly complex!  Designing experiments to study these interactions is challenging and come with a caveat: the conclusions may not reflect what is going on in the environment as the interactions in Nature could be influenced by additional direct or indirect inputs, biotic or abiotic, that are not captured in the laboratory.  I suppose makes this an interesting paper to discuss! 

So onto your question Carolyn. I had the same thoughts when reading the experimental techniques. I guess I don't know too much about the feeding habits of fungi. Do they prefer very specific species/genera of bacteria? Or do they indiscriminately eat bacteria? If it is the former, then yes, the experiment you suggested seems very necessary.

So we are left asking if the Pseudonocardia bacteria are necessary for growth of the black yeasts on the ant or can the black yeasts obtain other food sources from the environment?

What happens to the fungal gardens when the Pseudonocardia bacteria are completely depleted from this network of organisms?

 

AMC: Regarding whether or not the black yeast like to feast upon Pseudonocardia: Are they really asking if the black yeast are eating the Pseudonocardia?  Because if they are, then why couldn't they do the experiment with live bacteria?  By adding dead Pseudonocardia bacteria to the media aren't you just enriching the media by adding more nutrients? Most laboratory media for growing bacteria contains ground-up yeast. As you two ladies have pointed out, it would be much more compelling if the black yeast opted for a Pseudonocardia bacteria meal over another bacterial option.

 

CAB:The questions keep coming while I consider another experiment. They wanted to figure out how the presence of the black yeasts impacted the general success of ant's bacterial symbionts. They grew the black yeasts together with Pseudonocardia on Petri dishes containing "nutrient medium" which allows for "good growth" of both the black yeast and bacteria. The two organisms were grown together on the plates, and the sizes of their colonies measured as an indicator of success. Their results lead to the conclusion black yeasts grow better with bacteria than alone, while bacteria grow worse with the black yeasts than they do alone.

How does the set-up of this experiment compare to Nature's set-up? I'm especially interested in the structure of the two communities on (around?) the ant. Are the bacterial colonizers well established on the ant before the black yeasts arrive? How do the inoculum sizes (i.e. the number of individuals cells of each organism present) differ? For instance, are there always 100-fold more bacterial cells than yeast cells (this would be my guess)? 

Second, we must take a lot for granted here in terms of the nutritional standards of the medium and their method for measuring growth. For instance, the yeast might obtain a limiting nutrient from the ants that isn't present in the nutrient medium; the ants might grow smaller colonies when the black yeast is present for a variety of reasons, such as the production of a diffusible factor by the yeast that inhibits large colony growth.

Alternatively, the ants might just take longer to get to the same colony size, and while their growth rate is slowed, perhaps that's never the issue in Nature as the ants are always well established by the time the yeasts show up.

 

KLS: I would imagine the ideal set up for this experiment would mimic the environment on the ant cuticle as best as possible. The authors mention that these cuticles are attached to ant glands that provide nutrients for the bacteria. What are these nutrients and how do they compare to the conditions used in this paper for growth?

I am fairly certain bacterial colonies are established early in an ants lifetime but the black yeasts may be established fairly early in the life of an ant as well. It seems we need answers to some central questions about the relative size and establishment of these microbial communities before we can think about a more “realistic” experimental set-up...

 

AMC: Since we are all about chemical ecology, maybe we can discuss the role of natural products in interpreting the results of this paper.  Of course there are the antifungal molecules produced by the Pseudonocardia - but what else? Are there other interactions that we think could be mediated by secondary metabolism?  Or do we think this interaction is more about competition for resources?  In other words, the black yeast is usurping nutrients used by the Pseudonocardia rather than actively trying to kill it.

 

CAB: OK, here’s me thinking more outside the box when it comes to the chemical ecology of this story. Maybe there are inhibitory compounds coming from the black fungus that stunts the growth of Pseudonocardia? Maybe the black yeasts physically block the ants from delivering their dosage of drugs to the fungal gardens to protect them from invaders. Not chemical, but it could be the culprit?