This is the second installation of a new feature here at Chem Intuition, called "Casual Fridays," in which we discuss seminal papers in chemical ecology. Our conversations are driven by our own chemical intuition and curiosities of the moment. We encourage you to check out the paper we're reading for yourself and join the conversation!
This week we're reading "Defense of Phalangid: Liquid Repellent Administered by Leg Dabbing" which was published in Science in 1971. This is a paper from the labs of the dynamic chemical ecology duo of Cornell University, Professors Eisner and Meinwald. The paper deals with the identification and biological study of the repellant used by Vonones sayi toward their ant aggressors. Read it here!
Kristen: Another great classical entomology and chemical ecology paper chock full of amazing descriptions of the experimental procedures used to obtain and characterize the unknown glandular product, including descriptions of the "milking" of the Vonones (a daddy longlegs-like insect). I loved the simplicity of their initial experiments to assign the source and rough identity of the defensive fluid, such as the experiment where they fed dyed water to the insects and were able to conclude that the clear droplets formed upon agitation are in fact regurgitated fluid. Likewise, I loved the inclusion of the deductive reasoning that since the additive is brown, has a characteristic odor and tans human skin it is likely quinonoid. Genius.
So it appears these spider-like insects have evolved a rather unique method for distributing their defensive fluid:
1) regurgitate droplets of water
2) mix in a brown, repulsive quinonoid glandular substance
3) dip forearms into the mixture
and finally, 4) use forearms to rub- or "dab"- it against the offender.
The authors hypothesize that due to the general instability of quinones in water, the dilution of the repulsive substance immediately before use may be an evolutionary trick to retain the potency of quinones (as opposed to storing the quinones as an already dilute solution). But why dilute the quinones in the first place? Why not just "dab" the quinone solution itself? Any thoughts?
Carolyn: I considered the same questions while reading this. The authors state that the clear liquid serves only as a substance that dilutes the quinones, and has no activity on its own. Since the spider needs to move the substance from its point of excretion to its prey, I figured that the watery stuff aids in quinone delivery. In other words, I think that without dilution by water, the brown stuff, i.e. the mixture of the quinones, is either too viscous or too small a volume to be effectively transferred from the mouth area to the legs and finally, onto the victim.
I did however, enjoy the note that described the fact that as purified substances the quinones are crystalline at room temperature, but when they are mixed together the melting point of the mixture is depressed such that the brown stuff is liquid at room temperature. Go Nature, finding some good solutions to problems of states of matter.
My favorite part of this paper was the description of the experiments in which the Vonones were placed in an enclosure with a bunch of ants. The authors state, "During the several hours that Vonones were confined in the crowded arenas, only seven assaults took place, although casual encounters with ants occurred continuously and the ants were demonstrably aggressive (mealworms offered concurrently were promptly killed)."
First, why are ants so aggressive? Second, and maybe an easier question to consider, what is the mechanism by which the quinones are toxic to the ants (and other insects for that matter)?
Alexandra: A fun paper for sure. I have to admit, I got vaguely irritated at times with the excessive use of insect anatomy jargon, which I found unnecessary, and ultimately vaguely distracting from the main points of the paper. Once I got over that though, I thoroughly enjoyed the read. I especially liked imagining those feisty Vonones slappin' that regurgitated fluid on other insects.
Ditto Carolyn's assertion that the dilution is most likely to increase diffusion or to increase the volume so delivery is easier.
The question about toxicity and mechanism is interesting. Do the authors say in this paper that the quinones are toxic to the ants? Or just repellant? Toxic quinones are produced by many plants to deter insects and apparently these are usually secreted through the roots or leaves and are toxic upon ingestion. In the case of the Vonones the quinones are likely not being ingested, though I guess it's possible they are taken up through the exoskeleton. So I wonder if these quinones are similar to the insecticide quinones produced by plants? Additionally, is it possible that these quinones are basically mimicking the plant-produced quinones, in effect acting as a repellant without having to incur any sort of toxic effect?
Side note: quinones are known to have a large range of biological mechanisms: DNA interacalation, generation of free radicals, metal sequestration….but I haven't read up on which of mechanisms are the most common in insecticides.
Kristen: The mode of action of the quinones is definitely an interesting question, especially since the activity assay in this paper is more or less a qualification of "repelled ants or didn't repel ants” and the paper doesn’t provide much of any information about the actual mechanistic effects of the quinone mixture on the ants. Quinones are commonly used by other insects, such as millipedes, beetles and cockroaches, as defense compounds. Although I couldn't find too many studies on the mechanism of action of these compounds on their predators, I did come across a hypothesis that quinones, which are highly irritating and sometimes toxic to vertebrates, could act on predatory insects as irritants through “common chemical sense," which I like to think of as "chemical street smarts." This aligns with your hypothesis Alexandra, where the negative/toxic effects of quinones –whether plant or insect derived– could deter predators regardless of whether the specific quinone actually has toxicity.
The ubiquity of quinones as defensive chemicals in both plants and insects is interesting to contemplate. I also wonder if quinones are relatively easy to access from other essential metabolites and thus their use could have evolved independently in these different types of organisms?
Carolyn: Without doing a deep literature search to help answer the question of how exactly do Vonones biosynthesize or obtain their quinones used to repel insects my chemical intuition makes me want to say that the quinones could be biosynthesized either from shikimate pathway intermediates (the shikimate pathway is involved in the biosynthesis of aromatic amino aicds) or through some sort of polyketide synthase (PKS) pathway that uses acetate as a building block. What's interesting is that the shikimate pathway is found only in bacteria and plants, and insects are not generally known to utilize large, multimodular PKS type enzymes to biosynthesize secondary metabolites. So, from my perspective, it seems likely that there are bacterial symbionts of Vonones that may either completely biosynthesize the quinones or could provide late stage intermediates that the bug then, perhaps, oxidizes or hydrolyzes.
To address the question about the mechanism of action: what baffles me is that it seems as though the ants are repelled by the quinone excretion even when they are not in contact with it. So my question is, are the ants somehow getting volatile signals from this excretion (which seems unlikely, given the fact that the quinones are solids are room temperature) or, perhaps, is the ant that is put in contact with the gross brown goo sending out a secondary "alarm" signal to warn the other ants away?
We are perhaps unveiling all sorts of fun chemical ecology interactions beneath the surface of this paper....
Bottom line: Vonones are feisty! Who knew that insect secretions could lead us through such a winding discussion. While this "classic" paper pokes at the complex interactions between Vonones and their enemies, there is still much to be learned about how these chemical secretions work their repellant magic.