A Visual Guide for Studying Behavioral Defenses to Pathogen Attacks in Leaf-Cutting Ants

A visualized protocol provides a frame of reference for wider research on defenses and fungus-growing ants. The main advantage of this approach is that it will help researchers recognize key defensive behaviors to ensure a common understanding for future work. A reference for research in fungus-growing ants is likely to be particularly useful for young researchers who are not familiar with these behaviors.

A number of these behaviors are infrequent, so experienced researchers may never have observed them. This guide will facilitate comparisons across studies and ant species by illustrating key defensive behaviors along with consistent definitions. Furthermore, understanding behaviors in controlled conditions will help studies in natural habitats, where conditions are hard to control.

To begin, isolate Escovopsis strains from Acromyrmex echinatior laboratory colonies, by placing fungus garden fragments, with ants removed, on a Petri dish with moist cotton wool for several days, until Escovopsis germinates and sporulates. Transfer spores to potato dextrose agar plates, and incubate at approximately 23 degrees Celsius for two weeks. Using a sterile needle, remove mature spores from the plates.

Then, inoculate the spores on new plates under sterile conditions, and incubate them at approximately 23 degrees Celsius for two weeks. When the hyphae have grown to cover the entire plate and into mature brown spores, inoculate the spores onto new plates under sterile conditions. Using three original A.Echinatior colonies, obtain a total of 36 sub-colonies with 12 sub-colonies from each parent colony.

Four hours before ant introduction, add a teaspoon-sized piece of the original colony's fungus garden, some bramble leaves, and a piece of water-soaked cotton wool to the box. For the sub-colonies that are to be infected, fill an inoculation loop with Escovopsis spores. Then, inoculate the spores by gently tapping a small part of the fungus garden 10 to 20 times.

For the control sub-colonies, mimic the application of Escovopsis spores by tapping the fungus garden with a sterile inoculation loop 10 to 20 times. For the 72 hour post-infection observations, leave half of the sub-colonies undisturbed for 72 hours after the introduction of Escovopsis. For the zero hour post-infection observations, immediately add two brood, four minor workers, and four major workers from the parent colony to each box 30 minutes prior to recording.

Then, add the same mixture of workers to the 72 hour post-infection observations 30 minutes prior to recording. Perform video recording for four hours for each sub-colony. After recording all of the 36 sub-colonies, review the footage and score the behaviors of interest for each individual in each sub-colony.

The following clips provide a description of the key distinct behaviors to be observed during the observations. While reviewing the video, notice if an ant stops leg movement to initiate self-grooming. Check to ensure that the antennae pass through the antenna cleaners on the front legs.

Then, the ant should clean its legs and antenna cleaners with its mouth parts. Also, notice if an ant stops any leg movements at a fixed point in the fungus garden. Observe if the antennae are stationary and parallel, pointed towards the fungus and almost touching each other close to the tip of the mandibles.

Then, the glossa should emerge to lick the fungus. One or more ants may also groom each other. During this behavior, the ants stop all movement and stand closely together, maintaining physical contact.

Then, the grooming ant should point its antennae towards the receiving ant and lightly tap the receiver. Note that the upper and lower mouth parts of the grooming individuals are open with the glossa emerging to lick the receiver ant. Observe when the ant stops movement to initiate metapleural gland grooming.

The ant should lean to one side and reach one of its front legs back to rub the opening of the gland, while licking the other front leg with the glossa. Then, the ant will repeat the behavior with the opposite legs. When an ant stops leg movements at a fixed point on the fungus garden, note that the antennae are motionless and parallel, pointing towards a fixed point of the fungus garden.

Then, the ant will grab visible Escovopsis spores and carry them out of the nest. An ant may also stop leg movements at a fixed point on the fungus, point its antennae loosely towards a piece of fungus and tap the fungus piece with its antennae. After this, the ant will detach the piece of fungus from the rest of the fungal crop.

Another potential behavior may be observed when an ant stops leg movements at a fixed point on the fungus garden. The ant will bend its gaster and head towards each other to apply a drop of fecal fluid to its mouth parts. One at a time, the ant will pull its front legs through the mandibles, and the antenna through the antenna cleaners.

The main objective of this study was to create a catalog of short clips illustrating disease-defense behaviors in leaf-cutting ants. The behaviors described in the protocol were scored in an experimental infection scenario. In the control colonies, minor workers groomed the garden crop more than major workers.

In the infected colonies, there was a non-significant tendency for increased fungus grooming relative to the control colonies. A non-significant increase in fecal grooming was associated with infection, but no difference between minor and major workers was detected. Spore weeding and fungus weeding were both low in frequency, with no significant difference observed between infected colonies and controls.

A tendency for fungus weeding to increase with time since infection is observed. Few of the infected colonies with early-stage infections had visible spores remaining after the four hour observation period. Interestingly, in later-stage infection conditions, the ants were not capable of completely removing spores in any of the colonies.

The results of this experiment are thus an illustrative example of how the compilation of clips can be used for behavioral studies, to calculate average frequencies for minor and major worker behaviors. We documented behaviors that contribute to defenses in fungus-growing ants, and have systematically identified, described, and captured these behaviors on film. Our results reinforce research in the field suggesting why it's hard for a pathogen to infect colonies when faced an extensive set of behaviors and associated anti-microbial compounds.

We provide a new tool for future work in the field, and hope that the behavioral catalog will prove valuable in securing consensus and streamlined definitions, observation, and interpretation of behaviors.