External Chemical Defenses; a natural history view

After a long hiatus (due to qualifying exams, a dissertation proposal, hundreds of Trichostema in the greenhouse and a couple manuscripts in progress), here is the long-awaited (by likely my parents, grandparents, and maybe 3 other people) blog post that outlines THE TOPIC OF MY DISSERTATION (in a rather digressive way). I took a long and circuitous route to find this topic, but its very exciting to me and when I’ve presented it publicly (at Ecological Society of America meeting last August and informally to many colleagues), it has been greeted with interest. This post will mostly deal with how I got to this topic and why it is a great thing for insect-plant biology, a later one will detail the implications (a paper on which was recently accepted!).

The stem of Trichostema laxum (Lamiaceae). The little bubbles on the tips of the glandular trichomes are full of an oily, wonderfully vinegary-smelling fluid, of unknown utility.

Coming from the northeast and botanizing and entomologizing in Massachusetts and Rhode Island, I knew my fair share of plants – herbs and trees, shrubs and aquatic plants – and many of their associated insects. Coming to California, I was exposed to more. A whole lot more! I met a great deal of new taxa – from aromatic plains of sagebrush, to strange long-horned moths (Adela) to smooth barked manzanitas and madrones to the most spectacular diversity of wildflowers I’ve ever seen to albatrosses wheeling offshore. As a natural historian, I had a lot to learn and I continue to learn new plants, insects and even occasionally birds, each time I venture out to a new spot, and often when I revisit old spots.

A Mormon cricket, Anabrus simplex. White Mountain, CA, Sept. 2012. A mind-blowingly large katydid.

In the first year, I bounced from project to project, trying to raise parasitoids from caterpillars, looking at galls and leaf miners for inspiration and tractable systems to look at the effects of plants and their chemistry on predators and parasitoids of the herbivores feeding on those plants. None of which really panned out individually, but I spent a lot of time wandering around with a net, hand lens and notebook. And I learned a lot of natural history of California, the coast, the coast range, the valley, and the west side of the Sierras, in the process.Then I happened upon the chenopod Blitum californicum and ended up spending a summer playing with various chenopod species and getting a paper out of it. This, coupled with work done with Billy Krimmel and Ian Pearse (see their research here), got me thinking about plants really hard. And I realized a major difference between plants back home and here: plants back home are generally rather glabrous (smooth-surfaced) and here they have a myriad of glandular trichomes, general stickiness, oiliness, resins, etc. and they often have strong smells (the latter point is an oft-mentioned feature of plants of Mediterranean climates and why many of our kitchen spices – e.g. sage, rosemary, oregano – hail from these areas).

The glandular trichomes on Aquilegia eximia (serpentine columbine: Ranunculaceae)
are extremely sticky and entrap enormous quantities of small flies and wasps.

So I spent more time seeking out these plants and examining them closely. I also spent time reading the literature on secretory tissues in plants. And I began to think, abstractly, what might the differences between compounds put onto plant surfaces and those inside a plant be? I made lists, I woke up in the middle of the night with ideas and eventually, I distilled these many ideas into five broad differences between the potentially-defensive chemical secretions and those sequested inside plant tissues. These form the meat of the accepted paper, to be detailed later. Instead I’ll briefly touch on how I experiment with external chemicals and why this is important and exciting.

The first approach I took to playing with (e.g. experimenting) external chemicals was testing their efficacy at preventing damage to plants. I did, and continue to do, this in two ways (in the chenopod paper as well as many small unpublished tests). The first way is to remove or reduce the defense, gently, using a paintbrush or a sponge, leaving the leaf surface intact. I then run choice or no choice (palatability) assays on these plants, usually using a the wonderfully generalist spotted cucumber beetle, which rarely fails to eat at least a little bit of a plant (but also will eat a LOT of a plant it likes). The second is to take the external chemical (either from the plant or the known chemical) and place it onto another plant, either in natural concentrations, or varying the concentrations, looking for changes in herbivory.

Lab-grown, highly glandular Antirrhinum californicum.

I also do this in the field – something that is really hard to mimic with internal chemicals. Removal of exudates from plants can be difficult (e.g. Yerba Santa, with really tough leaf resins), but can also be really easy, as in the case of Antirrhinum californicum, the California snapdragon. In the field, removing the exudates of this snapdragon caused a really interesting response: insects – mostly a heliothine noctuid, caused more damage to the exudate-removed plants,as expected; but mammals (deer and/or black-tailed jackrabbits) ate preferentially the plants with exudates intact (highly significant result). This suggests that the exudates may be both a defense and a liability in nature, an interesting result that wouldn’t have been possible in a lab – and wouldn’t have been easy to find with an internal defense, as manipulation would have been more difficult.

Deer or jackrabbit eaten A. californicum from the experiment. 

Why is this exciting? Well, a manipulation like this wouldn’t really be possible with internal defenses. There are two general ways to look at the effect of internal chemical(s) on other organisms: (1) find or create lines that differ in concentrations of chemicals, or look comparatively across species that differ, or (2) create, via genetic techniques, knockout lines that lack a compound. The problem with both approaches is that there is pleitropy (one gene doing multiple things). If you have lines that differ in a compound, they also differ in other aspects. Similarly, if you have a gene knockout (or duplication), that will almost certainly have effects on other aspects of the plant. Of course, there are positives to both of those approaches: the comparative approach allows investigations on evolution of a trait and a genetic manipulation allows a level of integration and detail that no field study on a natural population will ever approach. In my systems, it seemed like a good way to get at defense mechanisms. But was I the first to do it? Absolutely not, in the marine world (Mark Hay’s lab) have been doing these sorts of exudate removals on seaweeds for decades. Yet terrestrial folks don’t cite these papers or think in quite the same way (the marine folks cite terrestrial chemical ecology all the time!). And even they weren’t the first! Thomas Hartmann pointed out in a 2007 paper on the history of plant-insect science, Ernst Stahl, in ~1900, removed the acid droplets secreted by evening primroses (Oenothera spp.) and found that the plants became far more palatable to herbivorous snails and slugs. However, despite the ease and history of these experiments, they are rare. And I’m not quite sure why.

The most satisfying part of these investigations so far (more to be detailed soon) has been combining a variety of approaches to think about a problem. I’ve made lots of natural history observations (examined lots of plants with secretions), thought about problems in creative ways (can I think of fundamental differences between internal and external chemicals, ecologically?), read the literature (from Darwin and earlier, to the present), planned and ran experiments, interpreted data (with such strange results as in the snapdragons) and am working on integrating it all into a dissertation, which I hope – in a few years – will be a cohesive body of work that other people will add to, build upon and apply to new systems and problems.

Acknowledgements

My exit seminar is this Tuesday, and (assuming all goes well) I’ll be Dr. Weinersmith by this Friday. There are a lot of folks I want to thank for helping me with my training to become a scientist, and I’m going to thank them here in addition to in my dissertation. I’ve been extremely lucky to work with so many amazing people. <3

ACKNOWLEDGEMENTS

            A lot of people and a handful of different labs were critical for making this dissertation a reality. While I’m inclined towards making jokes about “parasitizing many host labs”, Armand Kuris and Kevin Lafferty would point out that it would be more accurate to call me a micropredator, since I extracted resources from many labs without destroying them entirely. It is my hope that by the time I publish these manuscripts, they will think of me as something more akin to a mutualist. I suppose it’s time I actually get down to the acknowledgements.

First, I want to thank my awesome advisor, Dr. Andy Sih. It’s impossible to ask for a better role model. Andy is an endless spring of fascinating ideas, and a genuinely nice guy. He let me go off and try things on my own, yet was always available when I needed help or advice. I believe this combination of independence with a reliable safety net prepared me well for life after grad school, and certainly made grad school an awesome adventure. Working with the happiest man alive was a great reminder to pay attention to work-life balance as well. Thanks to Andy, also, for being so enthusiastic about Ada, and for making my near-simultaneous transition to motherhood and doctorhood as painless as possible. I’ll consider my career successful if I manage to be half as productive and happy as Andy. Andy, you’re AWESOME!

I’m also indebted to members of the Sih Lab, who were the best kinds of labmates: those who set the bar extremely high and then help you get strong enough to reach it yourself. John Hammond showed me the ropes when I arrived at UCDavis, and made me feel right at home. Sean Fogenburg, Julia Saltz, and David Harris were majorly intimidating, and every conversation I have with them makes me think that I may never be able to work hard enough to catch up with how smart they are. Somehow I’ve tricked them all in to collaborating with me, and I love that we’re still working together beyond the Sih Lab. The Sih Lab postdocs were absolute powerhouses, and Julien Cote, Tomas Brodin, Maud Ferrari, Jonathan Pruitt, PO Montiglio, Orr Spiegel, Tina Wey, Adrienne Mora, and Louise Conrad helped me grow as a scientist and were amazing role models. I’m particularly indebted to Maud and Louise, who I got to know well through the POD project. I’m extremely happy that they had children before I did, since this made having a family while being an awesome scientist seem achievable. I’m very glad for their friendship, as well as the support and advice they gave me when Ada came along. Eric Payne, Matthew Young, Denise DeCarion, and Andrew Bibian were amazing collaborators; fun to work with, and super smart and hard working. I couldn’t have asked for a better group of colleagues.

Drs. Ryan Hechinger, Kevin Lafferty, and Armand Kuris at the Ecological Parasitology Lab at the University of California Santa Barbara generously took me into their lab and showed me the world of parasites. If I spend the rest of my days researching parasites I will consider my life well-lived, and I have these men to thank for introducing me to a topic that brings me so much joy. Armand’s parasitology course was a once-in-a-lifetime experience, and hardly a day goes by that I don’t draw on information I learned from that course. Kevin Lafferty provided fascinating conversations and lots of advice for being successful in academia. I’m particularly indebted to Ryan Hechinger. Ryan spent many hours chatting with me about my research, his research, how to do science, how to write about science, grant writing, reviewing manuscripts, and the list goes on (and on, and on, and on). After almost every conversation I engaged in serious literature searches. You kept the bar high (including the pull-up bar in the lab), which seriously motivated me to push myself to reach it (the metaphorical bar; definitely not the real bar). The time Ryan spent with me absolutely made me a better scientist (and was a lot of fun!).

My labmates at the UCSB Ecological Parasitology lab were fantastic as well. There were some super hectic times (qualifying exams, etc) while working at UCSB, and my office mates kept me sane. Many, many thanks to Sanna Sokolow, Chelsea Wood, and Maya Wolf for being great listeners and even better friends. The lab was filled with brilliant people who taught me a lot about parasitology, including Jennifer Shaw, Alejandra Jaramillo, John McLaughlin, Alice Nguyen, Sara Weinstein, Julio Lorda, Tara Stewart, Loren Merill, Erin Mordecai, Ana Elisa Garcia, and Gabi Navas. I also had the pleasure of working with Zöe Zilz, Chloe Warinner, Virginia Tan, Jessie Lei, and Deirdre Fuller. These students were wonderful to work with, and I hope I lived up to the task of mentor. I’m sure they realized I didn’t know what I was doing, but hopefully they knew I was trying! Finally, a million thank you’s to Alan Wood, who may be the most patient man on the planet. Thanks to Alan for being patient with all the times I screwed stuff up in the lab and all the stuff I needed. Alan is a real superhero.

Dr. Ryan Earley generously opened up his lab to me, and taught me a ton about mechanisms of behavior. Some of the most intellectually stimulating conversations that I’ve had in my entire life are conversations I had with him at his dining room table. I hope we have a lot more of these conversations in the future. He was also amazingly supportive when I announced my pregnancy. Ryan went above and beyond to make sure I completed my dissertation work while having a safe pregnancy.

The Earley Lab grad students are amazing, and became friends as well as colleagues. So many thank you and big hugs go to Amanda Hanninen, Mark Garcia, Elizabeth Lee, Adam Fuller, Kristy Marson, Stephanie Robinson, and Caleb Anderson. I also had the pleasure of working with amazing undergraduate students, including Tyler King, Jelani Grace, Madeleine Haddock, Cameron Calhoun, Lee House, Morgan Lancaster, and Walter Smith. My dissertation would not have been possible without these awesome people, and I can’t wait to see what amazing things they do with their lives. Finally, many thank yous to Jimmy Ramage, who was incredibly supportive and helpful throughout my time at UA. He was a real life saver.

I’m extremely grateful that Andy introduced me to my science twin, Violet Compton Renick. We not only share the same birthday, but we also both stumbled upon studying behavioral syndromes in California killifish (me with an emphasis on parasites, her with an emphasis on pesticides). She made my two big dissertation experiments possible, and I look forward to working with her for years to come!

Øyvind Øverli is an amazing collaborator. How many collaborators provide comments on your manuscripts, teach you about gene expression and metabolomics, and teach you how to shoot a rifle, drive a tractor, and use a chainsaw? Epic, right? Thanks for teaching me loads of cool stuff, and for the amazing Scandinavian adventure. Thanks also to the amazing folks in the Øverli lab: Patrícia Silva, Uniza Khan, Mario Gallardo, and Marco Vindas.

Zen Faulkes spearheaded Team Clone-Zombie, which took us on a fantastic adventure. Organizing the Zombie symposium for the Society of Integrative and Comparative Biology conference was a ton of work, but I’m so glad we did it. We met loads of cool people, heard fascinating talks, gathered together a bunch of awesome manuscripts for a fantastic special edition of Integrative and Comparative Biology, and got to meet in real life after collaborating on the internet for years. Looking forward to the next Team Clone-Zombie collaboration!

Andy Sih, Ryan Earley, Richard McElreath, and Judy Stamps made up my dissertation committee, and were amazing. Thanks for making me a better writer, a better critical thinker, and thanks for being so cool with the frantic way I go about getting things accomplished.

Reaching back even farther, I want to thank my Masters’ advisors at Bowling Green State University. Dr. Jeffrey Miner’s Population and Community Ecology course was so engaging that I decided to shift my career trajectory from med school to grad school after only a handful of lectures. I can’t imagine a career that would make me happier, so I’ll forever be indebted to Jeff for bringing his enthusiasm to the classroom, giving an engaging lecture, and encouraging me to go to grad school. Dr. Daniel Weigmann took a chance on me after a particularly awkward interview, and bent over backwards to put me in a position to be successful. He showed me the value of hard work, and that science could be loads and loads of fun. Dan opened a ton of doors for me, and I believe that most of the happiness in my life that I’ve achieved was available to me because Dan believed in me and invested in me. Thank you, Dan.

Thanks also to all the amazing friends who kept me sane during the crazier parts of this dissertation: Tiffany Holmes, Kristin Zarrello, Linda Novitski, Ann Chang, Marit Wilkerson, Laura Young, Matthew and Emily Zefferman, Lauren Camp, Cara Harwood, Rachel Schwartz, Sarah Fogenburg, Sarah Strand, Mara Evans, Nick DiRienzo, and Paul Smaldino.

I also could not have done this without the unwavering support of my mom, dad, and brother. You fostered my love of nature and animals from an early age. I’ll always remember catching snakes with dad, going to the lake with mom, and fishing with George. You always believed in me, and I knew you were behind me 100%. Thank you.

Finally, thank you to Zach Weinersmith. I really, really could not have done this dissertation without you. Few other partners would suffer being moved long distances five times in five years. Zach edited my e-mails, edited my manuscripts, helped me fund my research, helped me set-up experiments, and literally moved two tons of wet sediment (amongst many other sacrifices). On top of all that, he made me laugh during the roughest moments. And now we have a baby! Hi, Ada Bean! You, my little sweetie-pie, put my career in perspective, and have taught me a new kind of love. I’m having a ton of fun now, but I can’t wait until you get older and we can go on adventures together. Here’s to many more years of Weinersmith shenanigans!