Drooling over plants (literally!)

It is really nice to be able to write about our latest paper, which just came out in Biology Letters to a fair bit of fanfare.  It got picked up by Science, New Scientist – including in their 23 July print issue – and even the Royal Society had a piece about it.  BBC Radio recently had me out to talk to them:

 

Why so much attention?  Well the paper is one of those quirky but interesting scientific discoveries.  And it is based on drool.  Yup, you read that right, the saliva of large mammals.

The paper goes back to about a decade ago when I was a postgraduate student in Canada.  At the time, I was based in the research group of Dawn Bazely.  Although Dawn has done a lot on plant population dynamics and plant invasions, her group in the early-2000s was working lots with plant endophytes.

Plant endophytes, as we would soon learn, are remarkably widespread in plants.  Your average leaf may be able to host up to 80 different species of fungus!  Many of these fungi are asymptomatic and probably have little impact on plant functioning.  But there are a few that do, and their discovery in pasture grasses only about 30 years ago has changed the face of modern livestock management.  That is because some of these endophytes were found to produce toxic alkaloids that can not only deter herbivory but poison animals.  Ryegrass staggers, one of the diseases caused by ingestion of toxic alkaloids produced by pasture grasses, dates back to Roman times.  It has been estimated that this disease caused $350 million in damages to the US cattle industry in the 1990s but its cause was only discovered, like I said, about 30 years prior.

So where does the saliva fit in?  Well, back in the Bazely group, we came across a paper that had recently been published in Oikos.  A Swede named Margareta Bergman had carried out a remarkable set of experiments on the regeneration of browsed willows.  Using jaws of moose from a natural history museum, she browsed plants, painted saliva on them, and found that they re-grew more quickly.  This was soon replicated by Tuulikki Rooke with goats – who incidentally is a past visitor to the Coomes group.

The importance of Margareta’s work is that it got me thinking about endophytes.  Endophytes typically reside in the basal portions of grasses and would be directly exposed when a large animal bites down on a plant.  Everyone in the endophyte field talks about plant-endophyte and plant-animal interactions, but what about the possibility for endophyte-animal interactions?  Surely if the animals had something that could combat the endophyte, it would be under positive selection?  This may be especially true for animals like moose, which feed on grasses – especially at certain times of the year – and they often defend territories, so can revisit the same plants.

With the help of the kind folks at the Toronto Zoo, and Zoo sauvage de St-Félicien, we set about trying to obtain saliva from these animals.  This proved a bit more difficult than I had expected.  Stories involving bananas are available in the pub.  But we were fortunate that the professional staff at the zoos were able to come through for us.  It turned out that when animals go under for medical procedures, their tongues need to be clipped so that they don’t swallow them.  That turns on a lovely saliva faucet that we were able to collect from.

Mark Vicari, who was one of the first to do his graduate work on endophytes and their interactions with wild mammals, kindly agreed to help me design and run all the experiments.  That’s him above collecting our main study species – red fescue (Festuca rubra) – from Presqu’ile Provincial Park in southern Ontario.  The first thing we did was isolate endophyte from grasses onto agar and applied moose and reindeer saliva.  What we found stunned us.  In about 36 hours, the growth of colonies exposed to saliva grew more slowly:

Endophyte colonies

Endophyte colonies in agar medium

Anti-fungal properties of saliva – in a way – are kind of unsurprising.  It makes good sense for all animals to have them for the sole reason that you don’t want to get a fungal infection in your mouth!  But whether these properties had an biological relevance for plant-herbivore interactions needed more work.  So we set out to do another experiment.

In this one, we used plants from the core of red fescue’s distribution in Sweden and from along its southern limit in southern Ontario.  Classic plant defense theory predicts that defenses should be consistently expressed in marginal habitats because plants can’t afford to lose biomass but growth takes precedence in more favorable environments, with defenses being switched on only when needed.  Therefore, if saliva works to inhibit fungal growth, we would expect declines in toxic alkaloid production across both populations.  However, if moose saliva has specific compounds that inhibit plant signalling pathways, we would expect a response in only the “favourable” populations with induced defenses.  A number of high profile papers, including in Nature and PNAS, have shown that these sorts of compounds may exist in the saliva of insects that often have to combat an array of nasty compounds.  So why wouldn’t they exist in large mammals?

Our experiment involved clipping plants from the different populations and then painting either saliva or water on them, as well as leaving some plants unclipped.  What we find here was even more remarkable!  The concentrations of one of the most potent alkaloids – ergovaline – increased by about 100% in the populations where we expected induced responses when we applied water with cutting.  Once we added the saliva, concentrations fell by about 40-70%.  There was no change in populations with constitutive defenses.  This strongly implicates inhibitors of signalling pathways as being present in moose saliva.  The lack of an effect in the constitutive populations – despite the growth effect observed in vitro – might be because we were dealing with established rather than growing hyphal networks.

Overall, I think this is a great example of one of those serendipitous scientific discoveries.  I’m still not sure it deals with the evolved aspect of the story, as we would probably need to test saliva from a non-herbivorous mammal in a way that got around the confounding issue of avoiding fungal infections.  Thankfully, there’s always more to do.

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