Day 27, Diary of a Research Assistant

by Leon Tran

I’ve been at the Rocky Mountain Biological Laboratory close to four weeks now. I’ve taken shelter in the cabin they call “Schofield,” with four other RAs, surviving on pasta and leftovers… During the day I leave my refuge to study whether or not female availability determines mate-locating strategy in the Gillette’s checkerspot butterfly, Euphydryas gillettii.

Leon gillettii
Euphydryas gillettii

My work entails patrolling the area known to harbor these creatures, observing the mate-locating behavior of the male butterflies. They can perch—a sit-and-wait strategy—or patrol—actively search for female butterflies—when pursuing Lepidopteran romance. However, these creatures don’t have the greatest sight among invertebrates. When they perch, I commonly see male butterflies pursuing other male butterflies and re-pursuing the same butterfly within seconds of realizing it is not the she-butterfly they seek. Sometimes they’ll even pursue the pinecones or flowers I occasionally throw into the treetops.

I also take temperature measurements in the treetops with a device called a HOBO—which in fact, is not homeless—to see if perch height temperatures affect the butterflies’ mate-locating strategy. One day in the field, I deployed my temperature HOBO into the treetops, only to discover that 30 minutes later, some mystery creature pilfered my temperature logger! I searched all over to no avail and while I was looking for said HOBO, the dead butterfly I was using to obtain body temperature measurements was also stolen! To my dismay, I looked up to find a bird perched in the tree to my right, singing what seemed to be a song of victory. I have no evidence this bird stole my butterfly, but if he did, he would soon learn that eating Gillette’s checkerspot butterfly will only bring him discomfort due to its distasteful nature. I can only hope my next butterfly survives the terror birds of Gothic—or else I’ll put a cage on it.

Breaking Diapause…

editor’s note: following an anything but quiescent academic year, the lab has emerged for field work.  With this post, we start up our blog again.

by Nicole Kish

Happy #PollinatorWeek, a celebration of the many insects that help to pollinate plants, thus providing us with fruits, seeds, and other crops! Unfortunately, pollinators also face a variety of threats ranging from habitat loss to insecticide misuse to disease. Populations of many pollinator species across the country have declined in recent years; for example, widespread honeybee losses due to colony collapse disorder were first reported in 2006, and the migratory monarch butterfly is facing threats due to habitat loss on both the overwintering and summer breeding grounds.

This summer, our lab has a contract with the South Carolina Army National Guard to conduct pollination network surveys at the McCrady Army National Guard Training Center. The South Carolina Army National Guard is committed to environmental stewardship, and because much of the land at the McCrady is undeveloped, the site hosts a wide variety of wildlife and acts as a refuge for several threatened and endangered species.

Twice a week, two undergraduate assistants and I make our way out to the fort, drive an ATV across the back roads to our study sites, and survey pollinator numbers and visits to flowers.  Because of the diversity of pollinating insects, this project is an opportunity for us as Boggs lab members to expand our knowledge of species identification: in addition to butterflies, we’re surveying beetles, bees, wasps, and flies. (And distinguishing between two species of bumblebee is tricky; they all look similar, move quickly, and females are armed with a stinger).

Even distinguishing between butterfly species while they are on the wing can be challenging.  For example, the larval diet of pipevine swallowtail, Battus philenor, makes the larvae and adults unpalatable to predators, and several other species, including the spicebrush swallowtail, red-spotted purple, and dark form eastern tiger swallowtail have evolved to mimic the pipevine.  All of these species can be found at McCrady, which prompted a lighthearted but frustrated “Not fair!” from one of my field assistants.  

We also have one more butterfly-centric goal: determining if, where, and how many monarch butterflies visit the McCrady Training Center. Monarchs typically pass through the region during early autumn on their southern migration, and to prepare for their arrival, we’ve located patches of their larval host plant (milkweed, various species of the genus Asclepias), which we examine periodically for eggs and larvae. We’re also setting up time-lapse trail cameras to catch any visits that might take place outside of our surveys.

Focus on a lab member: An interview with Alison

Alison Ravenscraft is interviewed on the CEHG blog this week.  CEHG stands for “Computational, Evolutionary and Human Genetics at Stanford”.  Alison is a CEHG Fellow, as she finishes up her doctoral dissertation work.

The blog focuses on Alison’s research, how she got into science (and it’s a great “only Alison” story!), important mentors, and advice for grad students.

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https://stanfordcehg.wordpress.com/2015/12/01/fellows-feature-alison-ravenscraft/

 

The Mothia

by Carol Boggs

mothia poster

What would Halloween be without a departmental costume contest? The School of the Earth, Ocean & Environment’s second annual contest was this past Friday, Oct 30. Prizes were awarded for the best group costume and best individual costume. The best group costume award included a snazzy trophy, to be held by the winner until next Halloween, when it goes to the new winner – sort of like the Stanford/ UC Berkeley Axe trophy for football, and including the same competitive spirit!

This summer, our in-house punster, Rachel, nicknamed our lab group at RMBL “The Mothia”. So there was no question as to what we would do for the group costume. The presentation included a small skit on stage. We were missing a few undergrads, along with lab members who are at other universities. But the Mothia was still formidable, subdued only by the Field Marshal herself.

the mothia 2015 halloweenThe Mothia.
(Austin Clarridge, Nicole Kish, Rachel Steward, Angie Korabik, Miranda Hannah)

the mothia and field marshal backside 2015 halloweenThe Mothia, with the Field Marshal

Unfortunately, we were narrowly defeated based on the “clap meter” by a group of zombie legos (aka, the Z-lab; aka, Lori Ziolkowski’s lab). We’re plotting the comeback for next year!

Puddling and Problems

by Angie Korabik

As a Biology major in a Business Fraternity (long story), most of my friends are business majors, so when I explain to them that I am studying the nutrient preferences of native Lepidoptera, I am met with a blank look. To avoid vacant stares, I’ve started summarizing my research with the pithy phrase, “I force feed butterflies.”

In reality, my research in the Boggs lab is not nearly that violent. I am conducting a variation of Alison’s study that she describes in her post from August, “Why do some butterflies eat fruit instead of nectar?” In Alison’s study, she hypothesized that adult tropical butterflies seek nitrogen and salts when they feed on fruits, but as the density of fruit trees around Columbia, South Carolina is rather low, I wanted to investigate how else they might obtain these nutrients. Maybe, say, through puddling? For those who are not familiar with butterfly behavior, puddling occurs when butterflies feed on, well, mud puddles. Hopefully, this project will allow me to answer questions such as what nutrients butterflies are looking for when they puddle, whether there are any interspecific differences in nutrient preferences, and whether there is a specific gender-based preference for each of these nutrients.

To simulate puddling activity (and beware, this is where the force feeding comes in), I collected butteflies from various places around Columbia, SC and brought them back to the lab, where I kept them for a maximum of forty-eight hours. Four solutions were used to test feeding preferences: 0.01 M NaCl, 0.1 M NaCl, 10-4 M albumin, and 0.01 M casein. In the lab, each butterfly was held with their wings pinned by forceps and their abdomen resting on the surface, and with a pin, I unrolled their proboscis to place it into the solution. Each butterfly was offered each solution one at a time in random order. If the butterfly drank the offered solution, it was termed accept, if they did not, it was termed reject, After offering the butterfly each of the four solutions, I offered them water, which they were allowed to drink to satiation. If they accepted the water, I discarded the results of the previous trial and redid the experiment. After they were done being picky about what they ate, I placed them back into their breathable envelopes and released them back into the wild where they could frolic in peace. (Well, peace from me at least. I can’t make any guarantees about other predators or avid lepidopterists.)

The set up
The set up

I first started this experiment last September as a sophomore. Being my first time working with butterflies, I spent most of last fall learning how to catch a butterfly, how exactly I was going to set up my experiment, and how to get over my fear of breaking a butterfly while handling it. I managed to catch about 40 butterflies last fall with the help of my darling friends, Ash and Miranda, who were/are also part of the Boggs lab.

This year, however, I’ve been encountering problem after problem as far as collecting is concerned. Last year a meadow-y spot off Gervais on the Columbia side of the river hosted an enormous colony of Agraulis vanillae, and on any given day I could go there and not only have few issues finding butterflies, but have to hurry to capture one butterfly before the four others that it was flitting about with got away. However, when I went to their colony this year, I discovered the site surrounded by orange construction mesh. Goodbye butterflies, hello parking lot. 😥

The release of the Agraulis vanillae!
The release of the Agraulis vanillae!

Not only did the loss of my wild Agraulis colony set me back as I had to search for new collection areas, but a few weeks ago, as I’m sure most of the country is aware, Columbia flooded due to massive storms from Hurricane Joaquin. I had been planning on trying to collect at least three days that week, but due to the fact that USC cancelled classes for the entire week and I ended up stranded in Chicago for a good portion of that time, it was simply not meant to be. Alas!

Fortunately, the butterflies survived the floods and I finally managed to go out collecting with Dr. Boggs and Nicole the following week. While most of the waters had receded by then, the fields we ended up visiting still had standing water remaining, and you could clearly see the level to which the flood waters had risen on the surround trees. It was quite astonishing. We ended up having a productive day though, catching nearly 20 butterflies including multiple Eurema, Pheobus, and other species.

Remaining water and the flood line.
Remaining water and the flood line.

I’ve been out a few more times since then, but the collecting continues on. So far I have about 74 butterflies in my data set, and hopefully by the end of this season, I’ll have enough data to start running some analyses. Until then, the local Lepidoptera will continue to fear ending up in my captivity. Muahaha!

Sustainable Carolina…and Rocky Mountain butterflies?

by Justin DuRant

This week, at Sustainable Carolina’s annual “Sustainability Showcase,” I presented a poster for a project that I did with Dr. Carol Boggs over the summer at Rocky Mountain Biological Lab in Gothic, CO. By analyzing old monitoring data from the past decade, we found that Euphydryas gillettii egg clusters have much higher survival to diapause if they are alone on a leaf compared to egg clusters that are layer right next to each other, merging into a single larval web.

Sustainable Carolina is the student branch of the University of South Carolina’s Office of Sustainability, and I have worked with them for the past two years. This fall, I was chosen to lead the Zero Waste Team. My group of interns manages the tailgate recycling program at home football games and works on other waste-related projects across campus. Other students in our organization plan and run events such as the Green Networking Breakfast, Reclaimed Runway (a fashion show using discarded materials), and the Green Career Fair.

Justin showing off at the Sustainable Carolina poster session.
Justin presenting his project at the Sustainable Carolina poster session.

Overall, the poster session was a satisfying experience. I spent some time talking with other folks I knew who were presenting, and I learned about some research on new battery technologies, aquaponics projects, permeable concrete, and more. I think I saw one person read through the entire poster (while I was not standing right next to it), so in this type of setting, images and captions might be the most important elements of a poster. By the end of the evening, I had a fairly effective elevator speech describing my project and another one for the question “So how does this relate to sustainability?” Standing for three hours at the end of a full day was pretty exhausting, but the coffee and bagels kept me on my feet.

The version of my poster that ended up being printed was missing a few important additions, like Dr. Boggs’ name as an author and some formatting adjustments. Thankfully, no one seemed to notice.

Field haikus and other poems…

By Rachel Steward

Anyone who has worked with me in the field knows I occasionally compose haikus and other short poems on the fly. I’ll contend it is endearing, though my field assistants say otherwise. I tend to be a bit lenient with the syllable count, as one might if one had spent the last several hours hunting for butterflies or surveying plants for signs of herbivory, so withhold your judgment. The poems below are only a small selection. Enjoy!

Here, beneath Gothic,
A scattering of cabins,
science hideaway.

IMAG00166
Gothic, the first week of June

How to spot science:
Giant forceps, ninety-one
Centimeter stick.

We go forth with nets.
With clipboxes and forceps,
To meet our fortune

Hunting butterflies.
But dammit, I have missed one
Distracted by another

Butterfly mothia:
The Speyeria await,
We will catch them all!

To which god does one pray
To keep larvae from turning
Into tubes of mush?

First instar (molt stage) Pieris macdunnoughii caterpillars.
First instar (molt stage) Pieris macdunnoughii caterpillars.

Mate, you dummies, mate!
You have nothing else to do.
Why do you spite me?

And a limerick to close things out:

There once were some Thlaspi ecologists
Who thought they could count all of it,
In one day they got
Through just 2.5 plots
And that evening felt far less confident.

But they continued their scientific endeavor,
Even as it promised to take them forever.
The density they lamented.
Ceci, in Mexico, they resented.
Still they went out no matter the weather.

Never did they succumb to negativity,
Chanting ‘1 basal, stage 5, no activity’
Now looking back on those days
Spent on those surveys
they question their standards of productivity…

IMAG00010 (2)
Ceci returned from Mexico and worked diligently on the Thlaspi surveys, so was forgiven.

Why do some butterflies eat fruit instead of nectar?

by Alison Ravenscraft

Ask anyone what an adult butterfly eats, and they’ll tell you flower nectar. But if you watch carefully, you’ll notice that butterflies will also eat stranger things. If you’re out hiking on a hot day, butterflies may land on you and drink your sweat. You might observe others sipping from mud puddles, dung, or carrion.

Travel to a tropical forest (or a butterfly house), and you’ll also find butterflies drinking the juices of fruits rotting on the forest floor. Fruit falls are only sporadically available in temperate climates, so North American butterflies haven’t specifically evolved to feed on them, but in the tropics fruit is available year-round and many butterflies have specialized on this resource. These butterflies often have modified probosces (the butterfly’s straw-like equivalent of a tongue). Some species have strong, thick, pointed probosces for piercing the skin of fruits, and others have probosces with brush-like tips for lapping up juice that has already oozed out into a fermented puddle. Frugivorous butterflies also evolved an intense attraction to the volatile chemical compounds in fermenting juice—particularly alcohol—and can expertly triangulate rotten fruit by smell even in the twilight of the forest floor.

So what do these strange foods do for a butterfly? Flower nectar is sugary, easy to obtain, and relatively clean compared to sweat, mud, dung, carrion and fermenting juices. Why go to all the trouble and mess?

As you might expect, when butterflies drink sweat, mud, or carrion, they’re looking for salt. Just like humans, butterflies crave salt because it is scarce in their natural diet. It’s even harder for herbivores (like butterflies) to acquire sodium than it is for us omnivores, because plant tissue is much lower in sodium content than animal tissue. Animal cells, but not plant cells, use sodium to control their internal water balance, so relatively high levels of sodium are critical for animals but useless—and potentially toxic—to plants. Research has shown that the sodium a butterfly eats gets incorporated into its eggs and improves the performance of its larvae. So when a butterfly risks getting swatted to drink an animals’ sweat, or braves pathogens to feed on dung, it’s doing it for the benefit of its offspring.

But what about that fermented fruit?

The general wisdom has been that some species evolved to feed on fruit because it provides another resource that is scarce in an herbivorous diet: biologically available nitrogen. Since the structural component of plant tissue is cellulose (a carbohydrate), plant tissue contains less nitrogen than animal tissue, whose structural component is protein (which is rich in nitrogen).

A collected fruit of Dipteryx panamensis. Butterflies love these fruits, which are similar to small, green mangoes. The peel is too thick for butterflies to pierce, but when the fruit falls to the ground, wild pigs chew off much of the pulp, leaving the fibrous, juicy pit behind for the butterflies
A collected fruit of Dipteryx panamensis. Butterflies love these fruits, which are similar to small, green mangoes. The peel is too thick for butterflies to pierce, but when the fruit falls to the ground, wild pigs chew off much of the pulp, leaving the fibrous, juicy pit behind for the butterflies
Both humans and butterflies have to acquire nitrogen from our food in the form of amino acids, proteins, or nucleic acids like DNA. Nectar is mostly just sugar water, and it is known to be particularly low in nitrogen (though it does contain some amino acids). Fruit, being a plant tissue, is also low in nitrogen, but even plant tissues have DNA and some proteins in them, so we would expect fruit to be richer in nitrogen than nectar. Frugivorous butterflies must therefore have evolved to feed on fermenting fruit because it is a richer resource than nectar and provides them with more nitrogen. …So the logic went.

When I went looking for evidence that fruit was higher in nitrogen than nectar, though, I realized that it didn’t exist! No one had ever actually bothered to test the assumption. So that’s what I set out to do, with the eventual goal of asking how butterflies respond to nutrients that are scarce in their primary food (fruit or nectar).

I collected samples of wild fruit and nectars from my field site, La Selva Biological station in Costa Rica. With help from collaborators, I used a technique called “Ultra Performance Liquid Chromatography” (UPLC) to quantify the sugars and nitrogenous compounds in my samples. I found that fruit does indeed contain more nitrogen than nectar: in fact, per unit of sugar, fermenting fruit juice contains 33 times more essential amino acids. I was also curious about sodium content, since salt is also often a scarce nutrient. I couldn’t collect enough material to measure the sodium content of my own samples, but I found enough data in the literature to conclude that on average the sodium concentration of wild fruits is over ten orders of magnitude (!!) higher than that of flower nectars.

Insects normally remove nectar from flowers so fast that there is none left to collect!  I enclosed flowers in mesh bags overnight so that I could collect the nectar in the morning. In the lower left corner, a nectivorous Urbanus teleus feeds on an adjacent flower (Stachytarpheta frantzii)
Insects normally remove nectar from flowers so fast that there is none left to collect! I enclosed flowers in mesh bags overnight so that I could collect the nectar in the morning. In the lower left corner, a nectivorous Urbanus teleus feeds on an adjacent flower (Stachytarpheta frantzii)
Finally, I wanted to know whether nectivorous and frugivorous butterflies respond differently to sodium and nitrogen. Since fruit is higher in both nutrients, I predicted that nectar-feeders would have stronger cravings for both, because animals often seek out nutrients that are scarce in their diets. (Think of your craving for salty snacks after you’ve been out sweating on a hot day.)

Baited fruit traps hanging in the forest. Attracted by the smell, butterflies fly into the open bottom, feed on the bait, then fly upwards towards the "light" (the white top of the mesh cylinder), where they get confused and wait to be collected.
Baited fruit traps hanging in the forest. Attracted by the smell, butterflies fly into the open bottom, feed on the bait, then fly upwards towards the “light” (the white top of the mesh cylinder), where they get confused and wait to be collected.
Testing these predictions required lots of wild butterflies of different species. To catch the fruit-feeding species, Michelle, an intrepid fellow lab member, and I decorated the Costa Rican rainforest with mesh traps baited with a fermented mix of mango, molasses and rum. To catch nectar feeders, we did a lot of good old-fashioned butterfly net flailing. (Median age of people who do this line of work: 7.) We caught over 400 wild butterflies belonging to 33 frugivorous and 38 nectivorous species. I offered each of these butterflies tiny droplets of water mixed with either amino acids or salt, and recorded whether they accepted (drank) or rejected each one.

The result: as predicted, nectar-feeding butterflies did have a much stronger craving for salt. On average, nectivores were almost three times more likely to drink the salt water droplet than frugivores. However, despite the fact that fruit has more amino acids than nectar, frugivorous butterflies were still four times more likely to accept the amino acid droplet than nectivores!

Live butterflies wait for the feeding trials in breathable envelopes. Each petri dish has droplets of a nutrient, which will be offered to the butterflies, and a soaked piece of filter paper for the butterflies to sit on, since they have taste receptors in their feet at well as their probosces.
Live butterflies wait for the feeding trials in breathable envelopes. Each petri dish has droplets of a nutrient, which will be offered to the butterflies, and a soaked piece of filter paper for the butterflies to sit on, since they have taste receptors in their feet at well as their probosces.
So what happened? Well, it’s possible that frugivores were more attracted to amino acids simply because they tasted more like their natural diet. But an additional, non-exclusive possibility is that the two feeding guilds’ nutrient requirements differ. My predictions rested on the assumption that all butterfly species have similar basic requirements for sodium and nitrogen, but my results suggest that this might not be the case for nitrogen. Fruit feeders are known to be longer-lived than nectar feeders—on average they live 2.7 times longer—and longer lifespans can lead to an increased requirement for nitrogen (which is used, among other things, for the maintenance of body tissues). Did an amino-acid rich, fruit-based diet allow butterflies to evolve longer lifespans, at the cost of an increased requirement for amino acids?

This is all speculation, of course. Although we’re a little bit closer to solving the mystery of the frugivorous butterflies, I’ve managed to ask at least as many questions as I’ve answered. There’s always more work to do for the professional butterfly hunter!

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If you’d like to read more about Alison’s study on butterfly diets and responses to nutrients, you can find her paper here: http://link.springer.com/article/10.1007%2Fs00442-015-3403-6 (If you don’t have access, feel free to send her an email request.)

Changes coming to the blog

Summer is winding down (although no one told Columbia, SC’s heat index), and the Boggs lab field season is officially coming to a close. The lab’s undergraduate field assistants have returned from Colorado, and classes at South Carolina began on Thursday. Only Rachel remains at Gothic, and she will return to SC in a few weeks, since snow will likely begin falling at RMBL in late September.

As the fast-paced nature of the field season gives way to the steadier (but sometimes hectic) pace of the academic year, the format of this blog will also change. We’ll post regularly but less frequently–most likely one “science heavy” post every two weeks, with shorter posts here and there when the opportunity arises.

Thank you for following the lab’s adventures over the summer, and we look forward to sharing our work with you throughout the coming year!

The Friday Flutter– August 14, 2015

From Gothic

Suddenly Gothic is a ghost town. The undergrad program finished this week and a number of research groups, including our own, wrapped up their field seasons.

IMG_0847
For the past 13 years, Boggs lab members at RMBL have placed bets on the size of the E. gillettii population, with the winner getting a free ice cream from the town store. This year Carol won with a guess of 400– only 4 away from the MRR-based model prediction– and treated the entire lab to ice cream. The best possible outcome. [ from left to right: Rachel, Ceci, Claire, Carol, Justin, Minjia, Leon, Daniel]

For the Boggs and Watt labs, the gradual exodus that began with Hannah on the 3rd continued with Daniel flying out Wednesday.

Justin and Leon followed on Thursday, leaving their rain boots and several other souvenirs to be transported back by car. The University of South Carolina fall semester starts in just under a week.

Ceci departed on the bus to Denver early early this morning, and will split the next three weeks between her home in Connecticut and as many backpacking trips as possible before heading back to Brown.

Minjia will be winging her way back to Florida tomorrow with more than a month until classes start up again at Stanford. 

Carol and Ward also began the drive back to South Carolina and hope to be back in Columbia by Sunday night.

Rachel remains at RMBL with her 200 f1 butterflies. 

Pictures
(Above) The radish growth rack converted to hold butterfly mating cages. (Below) A mating pair.

From Stanford

Alison continues to work on data analysis and postdoc applications.

Microbial symbiosis fact of the week:
For every human cell in your body, there are 10 microbial cells.  This means that if we could somehow make all of your human cells invisible, a ghostly imprint of yourself would remain.  Your skin flora would show the outline of your body, and your nose and mouth flora would define the internal surfaces of these areas. But we would see the largest aggregation of microbes in your gut, where you host about 3 pounds of bacterial cells.  These gut flora have a metabolic function equivalent to your liver; in effect, you could say that our bodies include an entire organ composed of non-human cells.
If you’d like to learn more about the human gut flora, check out Michael Pollen’s article here: http://www.nytimes.com/2013/05/19/magazine/say-hello-to-the-100-trillion-bacteria-that-make-up-your-microbiome.html?_r=4&pagewanted=all&

From Elsewhere

Nicole is on vacation with her family at Hilton Head this week, and thanks to an impressive number of vacation homes with their beachfront lights off, I got a great view of the Perseids meteor shower. Other highlights included watching an osprey catch a fish and counting loggerhead sea turtle nests.

ground squirrel pretending to be a tree squirrel
Weird wildlife: A golden-mantled ‘ground’ squirrel 20 ft up in the tree across from Carol and Ward’s second-story window (photo by C. Boggs)