Microbiota of Attine Ants' Gardens: Visualizing a Microbial Landscape by Scanning Electron Microscopy

We investigate the microbiota that live in fungus gardens on fungus-growing ants. We wanna know who they are and how they're organizing this microbial ecosystem. In particular, we wanna know how the microbiota interacts physically with the fungus the ants cultivate.

And these interactions are mostly mediated by biofilm. The microbial gardens cultivated by the ants is a complex and heterogeneous sample composed by the cultivated fungus, substrate added for cultivation and the microbiota. Besides being very delicate, it is challenging to find methods that preserve all garden components for imaging.

Our findings suggest that the microbiota integrates the fungus garden structures, also supporting that they may participate in the physiological responses of this environment. And we also provide the set of evidence that biofilms are important for these interactions. Our protocol preserves fungus structures in biofilms while simplifying other processes.

Yet it preserves the delicate garden structure, further detailing the microbiota's physical interaction and unveiling biofilm's spatial structure. Biofilm's are cellular systems in which microorganisms embedded in an EPS matrix form social networks. Scanning electro microscopy enable us to observe in unprecedented detail how the microbiota spatially organize in the fungus garden, surrounding hyphae and all over the substrate.

We believe that the preparation steps we suggest here will be widely applied to refine studies in other microbial ecosystems. To begin, locate and mark the attine ant species colony. Excavate a trench surrounding the nest area until the garden chamber is exposed.

Open the garden chamber laterally to prevent the soil from collapsing over the garden surface. Using entomological forceps, carefully collect garden samples. Transfer the garden samples to a clean plastic container containing a layer of plaster to balance the humidity.

After transferring the garden and ant workers, hermetically seal the container to prevent sample drying. Close the trench using the previously removed soil. Store the garden samples at 23 to 25 degrees Celsius, until further processing.

For sample fixation, use entomological forceps to remove workers, eggs, pupae, and larvae from the garden samples. Set aside garden fragments no larger than five cubic millimeters and add them to a two milliliter tube. Then with a pasteur glass pipette, add approximately one milliliter of Karnofsky fixative solution to the tubes to completely submerge the samples.

Agitate the tube gently to let the samples soak and incubate the samples at four degrees Celsius for at least 24 hours. To begin, obtain attine ant species colony fragments in Karnofsky fixative solution. After fixation, completely remove Karnofsky's fixative solution using a glass pipette without disrupting the sample.

Serially incubate the sample with one milliliter of increasing concentrations of ethanol, ensuring the sample remains undisturbed. After removing the ethanol completely, using forceps and spatula, carefully transfer the sample to a specimen container for the critical point dryer. Place the lid on the container and immerse it in a graduated glass beaker containing enough 100%ethanol to submerge the container.

Then transfer the specimen container to another graduated glass beaker containing enough 100%ethanol. Incubate for 10 minutes at room temperature. Then transfer the container to the critical point dryer.

To begin, obtain attine ant species colony fragments subjected to critical point drying. To prepare the sample holders, wrap the stubs with a piece of aluminum foil, covering only the top. Write the sample code or number on the bottom of each stub to identify them, before covering the upper part of the stubs with double-sided carbon tape.

Then open the lid of the specimen container and carefully transfer the dried sample to a glass Petri dish using forceps and a spatula. Carefully place fragments of the garden sample on the sticky surface of the tape-covered stub. After sputter coating with gold, place the stubs in the sample holder for scanning electron microscopy.

Use either the onscreen options or manual control to adjust the magnification and image position. Move the stage to obtain a comprehensive view of the sample. Employ the RDC function to focus on specific areas.

When observing an interesting structure, adjust the magnification, focus, brightness, contrast, and stigmation accordingly. For correct stigmation, using the manual user interface, move the stage in the X and Y directions. Adjust the raster rate to check the image resolution.

Adjust the magnification according to the part being imaged. To save the image, use the freeze function and click on the photo icon. Set up the file path for saving the image.

Finally, analyze at least three garden fragments, capturing images at all magnification ranges mentioned. Fungal hyphae in attine gardens were observed as intact tube-shaped structures covering the substrate surface, indicating effective sample preparation. Spread biofilms consisted of microbial cells forming a thin one to three-cell layer covering the substrate surface.