We're starting a new format here at Chem Intuition, 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 "Purification of the Fire Ant Trail Substance" which was published in Nature in 1965. This is a paper from Harvard Professor Chris Walsh during his undergraduate days at Harvard University where he worked in the lab of renowned ecologist and ant researcher E. O. Wilson. The paper deals with the isolation of the active substance that ants use to mark their trail to food. Check it out here.
Alexandra: I really enjoyed reading this article. The whole description of how the ants are isolated and extracted is fantastic. The explicit use of a tea strainer to separate the ants from the debris: Love it. What really delights me about these classic papers are the in-depth descriptions of the methods. When I read recent papers I feel like I’m frantically looking for how the experiments were done and it's always hidden in some outlandishly long supplementary file that takes 20 minutes to download. Then you read it to find that they’ve actually just directed you to another paper. Repeat process. This is why PhDs take so long.
Kristen: Agreed- it’s a great paper. I loved that ~200,000 ants were used for the purification. That's a terrifyingly large number! It always amazes me in these classic chemical ecology and entomology papers how much biomass is required to isolate a minute amount of active compound. You can't help but admire the heroic efforts put forth by these authors to obtain the material of interest.
Carolyn: Hello! Jumping on the ant bandwagon, or perhaps I should say trail…
The first thing I loved about this paper was the wonderfully detailed description of how the authors gathered hundreds of thousands of ants and proceeded to obtain about 250 micrograms of "apparently homogeneous trail substance." Having come of (science) age in an era in which papers have six figures in the main text and twenty-plus figures relegated to the supplementary information, I felt that reading this brief report was a blast of fresh air.
Alexandra: So this paper ends with the isolation of an "active trail substance" but do we know if this substance has since been identified? Is the assumption that this substance is actually a mix of molecules? Or do they think that the activity is attributable to just one single molecule?
Kristen: An (admittedly) quick search did not turn up any papers further characterizing the "active trail substance" of this particular species (S. saevissima), but it appears some further work has been pursued with a related species, S. invicta. A series of farnesene compounds were isolated from the trail hormone of S. invicta, none of which were very active alone. However, when a subset of these compounds was combined at physiologically relevant concentrations, recruitment activity was observed. Perhaps such chemical synergy is also at work in the "active trail substance" of this species?
On another note, I love the hypothesis put forth at the end of the paper that the instability of this "active trail substance" may be evolutionarily advantageous for the ant: after the trail has served its purpose, it will disappear. As a chemist, I associate the instability of a chemical of interest with lab frustration and challenges, but it’s interesting to contemplate how this instability may be useful and selected for in Nature.
Alexandra: The stability hypothesis is definitely intriguing. It also makes me wonder if other species of ant – or even other insects – can recognize this same substance. I imagine that upon locating some amazing food source they probably want to keep it as exclusive as possible.
Kristen: And exclusive or not, one could imagine they probably don't want old trails leading to depleted food sources lying around-- no need for workers to waste energy pursuing an obsolete trail.
Carolyn: I don't think that the authors assume that the substance is a mix of molecules or one single molecule. Rather, they leave this as an open question by stating "While it is clear that the active material is contained within the peak observed by gas chromatography, the sample may still be contaminated with a large amount of an inactive compound with similar chromatographic properties." The authors had a limited amount of information from which to draw conclusions and they don't over interpret or stretch the data to support theories to questions like these.
The stability question is, to me, the most fascinating part of this paper. It seems as though the ants have evolved to make a disappearing ink trail substance: something that sticks around while they need it and degrades quickly when it's no longer benefiting the ants.
It made me think that this chemical instability is just one solution to a more general problem in chemical ecology. Specifically, chemicals are made as signals to indicate something to members of the same, or in some cases, different, species. Generally, the organisms need the signal to dissipate as soon as the indicated event is over. What are other solutions to this problem in Nature that you all know of? Signal instability and quick degradation is one solution. What are others?
Alexandra: Great point about the hesitancy of the authors to over-state their findings. Unfortunately, it seems as though, these days, the norm is to do the opposite. Researchers often over-hype the implications of their research in order to maximize the impact of the publication or to help obtain funding. To some extent this over-hyping is understandable. On the other hand, it can be very misleading to readers who may not have the expertise to judge the data for themselves.
Moving on. So you’re asking, what are other solutions to this problem in Nature? That's a tough question. Instability definitely seems like the best way to erase a chemical cue, but I suppose there could be more "deliberate" strategies - like enzymatically converting the molecule to an unrecognized derivative once it is no longer needed. This seems way more energetically unfavorable.
Kristen: In the same vein as signal instability, how about chemical volatility? Organisms can rely on diffusion for signal transport and only respond at a critically "higher" concentration i.e. when the signal is still freshly produced or when the organism receiving the cue is close to the signal’s source.
Methyl jasmonate is a volatile signaling molecule used by many species of plants. Its production is induced by herbaceous damage and can signal to neighboring plants to expend energy in order to raise their defenses. One can imagine these sorts are mechanisms are particularly abundant in/important for immobile organisms (such as plants) that can't disperse (or move away from) the chemical signal they produce.
Carolyn: Acylhomoserine lactones come to mind when thinking about the need for a certain concentration threshold for signaling to happen. AHL’s are used by a variety of bacteria in quorum sensing processes, most notably by the light-making symbionts of the bobtail squid
So, to recap, right now we have listed the following as ways that organisms control turning chemical signaling on and off:
· chemical instability, in which the molecule is unstable and breaks down (either enzymatically or spontaneously) into an inactive compound after some period
· volatility, in which the chemical, presumably a gas, is lost through the process of evaporation or diffusion
· concentration effects, in which signaling is triggered on or off when the concentration of the signal surpasses or does not reach threshold concentration
Alexandra: Another good example is cyclic AMP, which is a small molecule produced by amoeba upon starvation. This molecule signals to neighboring amoeba to chemotax (move) and differentiate (commencing their “social lifestyle” - a way for the organism to disperse and locate a new food source). However, rather than the amoebal response increasing linearly with the concentration of cAMP, the amoeba become desensitized to the signal once it reaches a certain concentration. So the response curve of amoeba to cAMP is like an upside-down U, like this “n”. This type of receptor desensitization is another adaptation that can allow organisms to respond differently to a signal based on concentration rather than any kind of change to or loss of the signal itself.
Carolyn: So did they purify the amoeba pheromone by getting the amoeba to sniff the HPLC fractions?
Alexandra: They evaluated their response by having an amoeba focus group fill out surveys after being exposed to different concentrations of cAMP.
Carolyn: "How did it make you feel?"
Understanding how ants use chemicals to communicate with their nest mates remains a very active area of research today. If you want to know more about these critters, E.O. Wilson and Burt Holldobler, pioneers in this field, have written some fascinating and comprehensive books about the social lives of ants and the evolutionary implications of their behaviors (as well as numerous other books on equally engaging topics).
The Ants - Burt Holldobler & Edward O. Wilson
The Superorganism: The Beauty, Elegance, and Strangeness of Insect Societies - Burt Holldobler & Edward O. Wilson
Journey to the Ants: A Story of Scientific Exploration - Burt Holldobler & Edward O. Wilson