Editor’s Choice @Biotropica 48(5): Volatile emission by fireflies to defend against predators

I am pleased to announce the Editor’s Choice Article for Biotropica 48(5): Fredric V. Vencl, Kristina Ottens, Marjorie M. Dixon, Sarah Candler, Ximena E. Bernal, Catalina Estrada, Rachel A. Page (2016) Pyrazine emission by a tropical firefly: An example of chemical aposematism? Biotropica 48(5): 645-655

Fireflies (Coleoptera: Lampyridae) are known for the flashing lights of their courtship displays, but they are also notable for emitting strong odors when bothered. Vencl and colleagues identified these volatile emissions from fireflies and did a series of feeding experiments with toads, ants, and bats to test the role of these volatiles, along with flash signals, as warnings of unpalatability. Their creative study and exciting results suggest that these multiple enemies exert conflicting selection on firefly defenses; you can read more about their results and motivations below.

Congratulations to Fredric and his collaborators.

EB

 


Fig. 1. The field site and camp where fireflies were observed and collected in Soberanía National Park, Republic of Panamá. The convergence of several ravines served to channel roving fireflies together at this spot to make observations and their collection easier (Photo: F. V. Vencl).

Fig. 1. The field site and camp where fireflies were observed and collected in Soberanía National Park, Republic of Panamá. The convergence of several ravines served to channel roving fireflies together at this spot to make observations and their collection easier (Photo: F. V. Vencl).

In response to Darwin’s query about why caterpillars, though not attracting mates, should be brightly colored like courting birds, Alfred Russell Wallace (1867) proposed that insects evolved conspicuous color patterns to advertise their unpalatability to potential predators. Wallace (1889) went on to elucidate the coincidence of unprofitability with conspicuous warning signals in what has become the well-known defense strategy of aposematism (Poulton 1890). The functional value of the aposematic defense is to forewarn a potential predator that it might be wasting its time and energy on a prey item that will prove to be inedible or poisonous.

In my field work ‘prospecting’ for unusual insect chemistry, I have made a habit of using my portable instruments – eyes, fingers, nose, mouth – to get a first impression of whether a new find has something new to offer. For example, if you pop a bug into your mouth and it tastes bitter, it may have some interesting alkaloids. If some creature sports yellow, black or red in contrasting patterns, it might be displaying a warning about its noxious chemicals (Fig. 1).

Fireflies taste bad. Temperate fireflies in the genus Photinus fireflies are known to synthesize steroidal pyrones, known as lucibufagens (LBGs), toxins that disrupt the Na+/K+ pump, an ion transporter whose function is absolutely essential for animal survival (Eisner et al. 1978; Agrawal et al. 2012). LBG-like compounds are also found in many anurans and in milkweeds, which are well-known to provide the dashing Monarch butterfly (Danaus plexippus) with its repugnant and emetic toxins that serve to thwart its vertebrate predators (Fig. 2). Evidence has accumulated supporting the hypothesis that fireflies also employ the aposematic defense strategy, wherein predators, such as bats and jumping spiders, associate their charismatic bioluminescent sexual displays with unpalatability (Moosman et al. 2009; Long et al. 2012). Moreover, because they are unable to synthesize their own LBGs, Photuris fireflies, like the subject of our study, eavesdrop on Photinus courtships in order to prey on and to expropriate their LBGs (Fig. 3). Unfortunately, we know next to nothing about the chemical ecology of tropical fireflies, despite the fact that their diversity far exceeds that of their temperate counterparts.

 Fig. 2. When disturbed, a Bicellonycha amoena firefly exudes droplets of blood from strategic locations around its body. Nothing is known about what compounds are present in these exudates. We now know that the cuticle is full of hydrocarbons, which may play a role in the final stages of courtship (Photo: F. V. Vencl).


Fig. 2. When disturbed, a Bicellonycha amoena firefly exudes droplets of blood from strategic locations around its body. Nothing is known about what compounds are present in these exudates. We now know that the cuticle is full of hydrocarbons, which may play a role in the final stages of courtship (Photo: F. V. Vencl).

Fireflies also smell bad. When we handled and then sniffed the locally abundant firefly, Photuris trivittata, we were impressed by the strong, somewhat sweet odors emanating from these disturbed individuals (Fig. 4). While being sniffed, the distressed fireflies were also flashing. Could these volatiles serve as an early warning signal, perhaps in conjunction with flash signals, to form an aposematic defense?

In order to reveal the nature of these unknown odors, I asked Catalina Estrada to ‘piggy-back’ some samples on her GC-MS. This sort of chemical fishing trip is better known as ‘exploratory research’. It paid off. We discovered a methoxy-pyrazine in the atmosphere surrounding (i.e., head space) of the upset Photuris firefly. While she was at it, Catalina also washed some fireflies in solvent to see what dissolved into solution. Violà: species-specific hydrocarbon profiles for each species. These profiles may be more important in courtship than in defense: nobody in the world knows! Bioassays with ants showed that they avoided intact fireflies, but readily attacked solvent washed fireflies. Moreover, ants were extremely sensitive to pyrazine, being driven away from very desirable sugar resource by remarkably small amounts of it. These findings represented the first discoveries bearing on the chemical ecology of any Neotropical firefly. They also inspired us to inquire whether pyrazine might affect interactions with other important firefly enemies.

Because the Smithsonian Tropical Research Institute is a tight-knit community where scientific net-working is easy, Catalina and I approached Rachel Page and Ximena Bernal, respectively our resident bat and toad experts (Fig. 5). Along with their students, May Dixon, Sara Chandler, and Kristina Ottens, we were able to measure how pyrazine affected interactions with the two of the most important firefly enemies: bats and toads. We used mealworms coated with pyrazine to assess whether this chemical would repel these predators. Whereas toads found them acceptable, bats rejected intact fireflies. However, these predators had mixed responses to pyrazine. One bat species showed some reluctance to accept pyrazine-coated mealworms. We think small samples sizes limited our ability to detect stronger reactions to pyrazine.

Fig. 3. A Photuris trivittata female has captured and is devouring an Aspisoma physonotum male (Photo: F. V. Vencl). Her temperate counterparts sequester toxins from such prey, but we know nothing about what is going on chemically with this or any other tropical species.

Fig. 3. A Photuris trivittata female has captured and is devouring an Aspisoma physonotum male (Photo: F. V. Vencl). Her temperate counterparts sequester toxins from such prey, but we know nothing about what is going on chemically with this or any other tropical species.

Across diverse taxa, would-be prey warn potential predators of their unprofitability by displaying conspicuous visual, acoustic, or olfactory signals (Ratcliffe & Nydam 2008). Chemical compounds can also serve as early warnings to alert predators of prey unpalatability. Pyrazines are produced by a wide diversity of organisms (Moore et al. 1990; Dickschat et al. 2005). Due to their high volatility, ease of synthesis, and structural diversity, pyrazines are likely candidates for short distance signaling. In combination with bright coloration, other pyrazines, similar to our methoxy-alkyl version, also protect some insects from predation by functioning as aposematic signals (Ruxton et al. 2004). Pyrazines also function as pheromones in insects. For example, alkyl-dimethyl-pyrazines are pheromones in a number of insect species in the Order Hymenoptera (ants, bees, and wasps). Specifically, pyrazines serve as trail and alarm pheromones by some ant species and are also found in ant glandular secretions.  Pyrazines perform differently for humans. We detect them in association with the characteristic aromas and flavors of some of our favorite nutriments, such as wine, roasted nuts, maple sugar, coffee, BBQ beef, beer, and many fragrant fruits. They also add fresh, green, woody, ambery, musky, and minty scents. Thus, pyrazines have a very high odor impact relative to their molecular weight.

 

Fig. 4. Using a green LED, we were able to collect the male Photuris fireflies used in the study by imitating the female part of the courtship flash dialogue, which served to entice males to fly down from the forest canopy (Photo: F. V. Vencl).

Fig. 4. Using a green LED, we were able to collect the male Photuris fireflies used in the study by imitating the female part of the courtship flash dialogue, which served to entice males to fly down from the forest canopy (Photo: F. V. Vencl).

Our study is the first to establish a function for pyrazine in firefly ecology. We demonstrate that this compound functions differentially against a suite of ecologically relevant predators. As the next step, we wonder if perhaps flash cues, in conjunction with the pyrazine smell, might reduce attacks. The capacity to associate smells with visual cues has rarely been assessed directly by experiments and never in any tropical system. Our findings support the idea that tropical fireflies have evolved narrowly targeted chemical defenses due to selection imposed by a diverse enemy community. Overall, this study represents a fruitful collaboration that integrates bioassays, chemistry, and ecology that is the basis for further research to gain a deeper understanding of how different predators can impact the evolution of multiple resistance traits that form defense arsenals.

Fredric V. Vencl
Ecology and Evolution, Stony Brook University, Stony Brook, NY, U.S.A.
Smithsonian Tropical Research Institute, Balboa, Ancón, Republic of Panamá

Fig. 5. F. Vencl, C. Estrada, Rachel Page and Ximena Bernal.

Fig. 5. F. Vencl, C. Estrada, Rachel Page and Ximena Bernal.

 

 

References

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  2. Dickschat, J. S., Wagner-Döbler, I., & Schulz, S. 2005. The chafer   pheromone buibuilactone and ant pyrazines are also produced by        marine bacteria. J. Chem. Ecol. (4): 925-47.
  3. Eisner, T., Wiemer, D.F., Haynes, & L.W., Meinwald, J. 1978. Lucibufagins: defensive steroids from the fireflies Photinus ignitus and P. marginellus.  Proc. Nat. Acad. Sci. USA 75: 905.
  4. Long, S.M., Lewis, S., Jean-Louis, L., Ramos, G., Richmond, J., Jakob, E.M. 2012. Firefly flashing and jumping spider predation. Anim. Behav. 83: 81-86.
  5. Moosman, P.R., Cratsley, C.K., Lehto, S., Thomas, H. 2009. Do courtship   flashes of fireflies serve as aposematic signals to insectivorous bats? Anim. Behav. 78: 1019-1025.
  6. Moore, B.P., Brown, W.V., Rothschild, M. 1990. Methoxyalkylpyrazines in aposematic insects, their host plants and mimics. Chemoecology 1: 43-51.
  7. Poulton, E.B. 1890. The colours of animals: their meaning and use especially considered in the case of insects. Kegan Paul, Trench, Trubner & Co. Ltd, London, UK.
  8. Ratcliffe, J.M., Nydam, M.L. 2008. Multimodal warning signals for a multiple        predator world. Nature 455: 96-100.
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