Glow-worm Larvae Bioluminescence Operates as an Aposematic Signal Upon Toads

While adult fireflies are famous for their courtship flashes, the function of the glowing behavior of the larvae is less well understood. Recent studies concluded that birds and wood ants find the larvae of Lampyris noctiluca, a European firefly, to be unpalatable, and that the larvae protect themselves by emitting a repellent odor when disturbed. This study proposes to demonstrate that the glow of the larvae serves to signal their unpalatability to nocturnal, visually guided predators, rendering them less susceptible to attack.

Toads are potentially important predators of firefly larvae because they share the same habitat, are nocturnal, and will readily attack most small moving items in sight. The toads and firefly larvae used in this experiment were obtained from the same location. It was assumed that, prior to being caught, the toads had experienced firefly larvae in the wild and had an opportunity to learn to avoid prey that glow.
The experiment consisted of three parts:

1. In the pre-learning experiment, wild-caught toads were exposed to a rotating drum with black cardboard stripes representing moving prey (the test dummies). LED lights embedded in these stripes were lit to represent glowing larvae, or left unlit, and the frequency with which the toads attacked lit versus unlit test dummies was compared. This test was designed to indicate if the toads had already learned in the wild to avoid glowing prey.
2. In the learning experiment, the toads in each trial were presented with live prey – two palatable (mealworms) and one unpalatable (a glowing firefly larva). Toads that avoided the firefly larva in five successive trials while still accepting the mealworms were defined as "learners." Twelve out of twenty-five toads were deemed learners.
   "Non-learner" toads were those who needed more than one larva in each trial to become learners. These were exposed to six additional trials with up to five larvae in each trial.
3. In the recognition experiment, the procedure was the same as in the pre-learning experiment – exposing the toads to the rotating drum and recording frequency of attacks on lit versus unlit test dummies – to discover if the toads' avoidance was a learned response from their bad experience eating larvae, and had resulted in a greater aversion to attacking the artificial glowing prey.

Results

Do wild-caught toads discriminate glowing from non-glowing prey?
Two of the 27 toads tested did not respond to the artificial prey at all, and were withdrawn from further experiments. Seven toads invariably snapped at both lit and unlit test dummies indiscriminately. The remainder of toads showed greater reluctance in attacking the glowing dummy prey than the non-lit ones – usually orienting toward the lit test dummies as if to attack, but not doing so.
Are glow-worm larvae disagreeable to the toads, and, if so, do the toads exhibit avoidance learning?
In the learning experiment, toads were offered live mealworms and firefly larvae. Mealworms were always eaten. Of the firefly larvae, 30% were left untouched; 66% were attacked and eaten, but of those eaten, 5% were spat out. The toads reacted adversely to the firefly larvae they swallowed (puffing, using their foreleg to attempt wiping the prey off their tongue, urinating, fleeing from the prey) but never toward the mealworms. The toads' willingness to attack firefly larvae decreased significantly during the course of the experiment from 80% to 40% – indicating they were learning to avoid this prey.
Do toads show stronger aversion to luminescent prey after avoidance learning?
Although learner toads could already discriminate between glowing and non-glowing prey, after the learning experiment their reluctance to attack luminescent prey increased significantly.
Do non-learner toads need more frequent encounters to learn avoidance?
Non-learner toads continued to attack a few larvae throughout the extra learning trials, but the number of larvae attacked decreased considerably over the course of these trials, demonstrating that the non-learners do learn avoidance with greater exposure to the larvae.
Do non-learner toads discriminate glowing from non-glowing prey after avoidance learning?
Non-learner toads did not discriminate between lit and unlit test dummies, even after the additional learning experiment. However, if exposed to live larvae just before the recognition experiment, they then discriminated between the glowing and non-glowing artificial prey as well.

Discussion

If bioluminescence plays a role in warning predators of unpalatability, one would expect wild-caught toads to display a reluctance to attack firefly larvae. The pre-learning experiment results indicate that toads collected from firefly habitats do discriminate between glowing and non-glowing prey.
The study demonstrates that toads vary in their ability to learn avoidance of glowing prey. Learner toads, although already hesitant to attack glowing prey, exhibited greater reluctance after the learning experiment. Non-learner toads were also disinclined to attack glowing prey, but only after being exposed to live larvae immediately prior to the recognition experiment. This suggests that bioluminescence did deter the non-learners, but that they did not retain the memory of disliking the larvae as well as had the learner toads.
While these experiments show that larval glow alone can serve as warning of unpalatability to at least one important predator, the researchers do not dismiss the possibility that other cues, such as color pattern and odors, are also involved in deterring predators.