With (human) wedding season fast approaching, we felt that it made sense to shed some light on the wedding traditions of other species. This week, we're reading another paper from the dynamic chemical ecology duo, Meinwald and Eisner, entitled "Pheromonal advertisement of a nuptial gift by a male moth (Utetheisa ornatrix)." The paper was originally published in the Proceedings of the National Acadamy of Sciences in 1991 and is freely available here.
Carolyn: So, I will admit that I chose this paper because of its intriguing title. Also, I knew that a classic Meinwald and Eisner paper promised to be both strange and highly informative.
This paper deals with the study of the nuptial gift of a male moth to the female moth during mating. First, let's just call the nuptial gift what it really is: it's a spermatophore. A spermatophore is a package of sperm plus nutrients and, in this case, defense compounds, transferred to the female upon mating. It had been found previously that male moths have circulating pyrolizidine alkaloids that they transfer in the spermatophore, and these alkaloids are potent defense compounds capable of warding off predators. The pyrolizidine alkaloids are derived from a plant (specifically the seeds of Crotalaria) that the moths eat.
To signal to females that he's carrying a load of alkaloid, the male moth converts a pool of the pyrolizidine alkaloids to a volatile pheromone called hydroxydanaidal. Hydroxydanaidal is emitted from a pair of brushes, also called "hair-pencils" or "coremata.” The male uses the hair-pencils to waft the pheromone toward the female in order to seduce her. According to this paper, "males endowed with hydroxydanaidal have a higher mating success than those devoid of the pheromone," so the strategy works.
The question at the center of this paper was whether the amount of pheromone on the hair-brushes (and advertised to potential mates) was correlated with the amount of alkaloid in the moth (i.e. the systemic alkaloid) or the amount transferred to the female via the spermatophore.
So my question for the ladies of Chemical Intuition is the following: given that the hydroxydanaidal is derived directly from systemic alkaloids, is there any reason to hypothesize that the pheromone content would not be correlated with the amount of alkaloid systemically or in the spermatophore, ahem! nuptial gift?
Alexandra: So given the assumption that these systemic alkaloids are precursors of the pheromone, then, at least in my extremely humble opinion, it seems highly likely that the quantities of the two would be correlated positively. In other words, their results did not make me fall out of my chair or anything.
Something that might be vaguely related though, and could be interesting to consider, is the potential evolutionary advantage of utilizing all precursor alkaloids for pheromone production. This is related to behaviors that evolutionary biologists call "cheating.”* Basically, if the male moths produced a ton of hydroxydanaidal, leaving no protective alkaloids for the marital gift, the females would basically be tricked into mating with them and would receive none of the advantages implied by their impressive pheromone endowment or whatever. Horrid! Do we think this could happen? And if we think deeply about how this would affect relative quantities, could this be related to the original question posed by the authors?
*This paper defines cheating as “engaging in behavior that exploits the cooperative behavior of conspecifics by imposing fitness costs on them, while providing fitness benefits to the cheater.”
Kristen: Do the precursor alkaloids serve any other purpose for the male beyond protection from predation? Is there huge energetic cost to converting these alkaloids to the pheromone? And how is the pathway to pheromone from alkaloid regulated? For instance, could an organism, as Alexandra suggested, increase pheromone production perhaps upon receiving some signal (perhaps a chemical signal that a single lady moth is flying about), leaving no protective alkaloids and effectively "cheat" just in time?
Carolyn: Wow! So many questions and so few answers :)
I think that many of our questions are centered trying to understand the possible reasons why the pheromone titre is positively and linearly correlated with the titre of systemic alkaloid. In other words, we're generally wondering what are the energetic costs and regulatory systems that lead to this result and why isn't cheating (i.e. over representation of systemic alkaloid content) prevalent?
To get at the energetic costs, I think the best route to explore this topic is to investigate how the alkaloid, which is ingested from the favored food source of the moths, is converted to the hydroxydanaidal. Just looking at this structure (and using my chemical intuition!!) it looks as though there needs to be two hydrolysis reactions (to remove the linker bound by two ester groups) and two oxidation reactions (one to convert the alcohol to an aldehyde and another to oxidize the pyrolizidine ring to the pyrrole oxidation state). I think that this could conceivably occur nonenzymatically, but my bet would be that there are three, perhaps four enzymes that are involved in this biosynthetic pathway. Given that this reaction involves the oxidation of the ingested alkaloid, it could be possible that the production of the pheromone contributes to redox balance in the insect, i.e. the regeneration of reduced cofactors like FADH2 and NADH.
Regulatory systems: If we knew the genes involved in the biosynthetic pathway, we could look at the elements that regulate transcription of these genes. Are they constitutively expressed? Or only expressed when mating is imminent? In the paper we read, they compared the amount of hydroxydanaidal in both once-mated and virgin males. Based on these data, it looks as though the levels of pheromone are similar in these two groups. While I'm not an expert on moth physiology, this would imply to me, at least, that even without a female around to mate with, males produce significant quantities of pheromone that they display on their hair-pencils.
Evolutionary reasons to not be cheaters: I think the number one reason I can think of in this regard is that if the males convert all of their pyrozilidine alkaloids to pheromone, then they have no defensive compounds left to defend themselves. I think that the amount of pheromone produced is probably the result of an evolutionary trade-off between self-protection and successful advertisement to potential mates. Another reason to not be a cheater in this scenario is that the alkaloids transferred in the spermatophore go on to protect the eggs of the nascent moth baby; thus, the nuptial gift is, in a way, an insurance policy of the male to ensure that his progeny survives and goes on to propagate the genes of Papa Moth.
Alexandra: Great points Carolyn. You sent me on a whirlwind of thoughts that I hope are relevant.
Given the many advances in science technology since these experiments were performed, it's interesting to think about what experiments we would do now that were not feasible in 1991. I think Carolyn alluded to a few of them:
(1) DNA sequencing to determine the biosynthesis of the pheromone
(2) mRNA sequencing to determine when these pheromones are being produced.
(3) The scientific community’s new(ish) appreciation for the prevalence and importance of bacterial symbionts could lead us to investigate whether these molecules are actually being produced by a bacterial resident on the moth.
Finally, lets get to the role of the pyrolizidine alkaloids, about which I know very little. Apparently they are a pretty ubiquitous class of molecule made by a number of different plants. They are quite toxic (especially to hepatocytes, i.e. liver cells) because they can cross-link DNA with itself or with other proteins in the cell nucleus, causing intracellular mayhem. I think the assumption here is that these are defensive compounds to prevent herbivores from feeding on the plant; in fact grazing animals (like sheep or cows) can become fatally ill from feeding on pyrolizidine alkaloid-producing plants. Interestingly, the moth does not suffer from the cytotoxic effects of these compounds. Why? Are the alkaloids specifically toxic to hepatocytes or other cells found in, say, mammals, or do the moths have a self-defense mechanism?And what types of predators are moths warding off?
Kristen: Often organisms that sequester toxic chemicals from their diet for their own defense have methods for enzymatic detoxification and/or specialized organs for storage. Enzymatic detoxification, however, appears to not be at play in this system since the monocrotaline can be extracted directly from the males. And there’s no mention any specialized storage locations where the systemic compounds are localized…. Mystery unsolved.
Re: moth predators? it appears that these alkaloids are very important for warding off spiders and birds.
Carolyn: Well, here's to another Casual Friday, done come and gone. I think humans getting hitched this summer could learn a thing or two from the mating behaviors of moths. Mostly, that they should be honest about what they are each bringing to the relationship!
Further reading: Tom Eisner has some great narratives describing his observations in his entomology studies and how they led to his fruitful explorations into chemical ecology (and his decades long collaboration with Jerry Meinwald), including this nice little paper where he describes his initial observations and studies with these moths. For more stories, check out his book!