Mimicry in viceroy butterflies is dependent on abundance of the model queen butterfly
Geographic locations and survey techniques
We surveyed and collected specimens three times (June, July, and September) for 2 years (2003 and 2004) at eight locations in Florida, USA18. From south to north, the locations were Corkscrew, Collier County (26.361, −81.519); Lehigh Acres, Lee County (26.560, −81.678); Lake Istokpoga, Highlands County (27.296, −81.296); Cornwell, Highlands Country (27.396, −81.120); Leesburg, Lake County (28.788, −81.895); Cedar Key, Levy County (29.214, −83.021); Gainesville, Alachua County (29.637, −82.200); and Jena, Dixie County (29.667, −83.185) (Fig. 1). Relative abundance of each species was measured by calculating the rate of capture per species per hour per person. Two field surveyors sampled each site and the same two individuals surveyed all the sites. One surveyor sampled queens and the other sampled viceroys, switching their target species after 1 h. Twenty individuals were kept for chemical defense and palatability studies; any additional individuals were released once the survey was complete. Each field site was sampled for 2 h continuously along a 400 m × 10 m transect on sunny days between 900 and 1600 h. The number of larval hosts was also recorded at each site along the transect: the Carolina willow (Salix caroliniana) for the viceroy and the white twinevine (Funastrum clausum) for the queen butterfly. In separate analyses, we tested for an effect of queen abundance on viceroy abundance, an effect of F. clausum on queen abundance, and an effect of F. clausum on viceroy abundance (see Statistical Analyses, below).
Chemical analyses of viceroys and host plants
We investigated geographic variation in the defensive phenolic glycosides in the viceroy butterfly and its larval host plant, the Carolina willow18. Four samples of butterflies and four samples of willow were collected from each of the eight population locations in July 2003 and July 2004. A butterfly sample consisted of 5 adults (either all male or all female) (~0.5 g dry weight), while a willow sample consisted of 16 young leaves, two leaves from eight different plants (~10 g dry weight). All specimens were weighed, then air-dried at room temperature for 1 week and reweighed. Whole butterflies including their wings were used in the extraction. The extraction and identification protocol is described in through detail in previous research18. Analyses of each sample were conducted using an Agilent 1100 HPLC system tandem with Agilent MSD-Trap-SL ion trap mass spectrometer with the samples identity blinded from the technician. Calibration curves were constructed for the three phenolic compounds using the liquid chromatography–mass spectrometry (MS) protocol described above. For each compound, a characteristic product ion was chosen from its MS/MS as its quantification ion. Peak integration and quantification were performed automatically using Agilent Chemstation software (version A.10.01). The same samples were run twice for quantification to ensure consistency within a sample. The concentrations were considered consistent if runs 1 and 2 were within 10% of each other. If not, then the sample was re-injected until the two runs reached the consistency criteria. However, only the concentration of the first run was used for reporting and statistical analyses (N = 64 willow samples; N = 64 viceroy samples). In separate analyses, we tested for an effect of queen abundance on defensive phenolic glycosides in viceroys and an effect of queen abundance on defensive phenolic glycosides in willows. Each of these tests involved four different analyses that differed only in their response variable: total phenolic glycosides, salicin, salicortin, and tremulacin. We also tested for an effect of willow defensive phenolic glycosides on viceroy defensive phenolic glycosides. Note that we restricted these tests between willow and viceroy phenolic glycosides to a single compound or category. That is, we tested for an effect of total phenolic glycosides in willow on total phenolic glycosides in viceroys, an effect of willow salicin on viceroy salicin, an effect of willow salicortin on viceroy salicortin, and an effect of willow tremulacin on viceroy tremulacin. In testing the relationship between willow and viceroy defensive chemistry, we used the mean value of the concentration for each site/date combination as the predictor of the concentration of the corresponding compound in viceroys. For example, the total phenolic concentration in the two willows sampled at Corkscrew on 1 July 2013 was 62.8 and 38.1 mg g−1. We used the average, 50.45 mg g−1, as the predictor for the total phenolic concentration observed in viceroys.
We evaluated whether or not the chemical profile of the viceroy defensive secretion varied by geographic location. Butterflies were caught at the eight field sites in July 2003 and July 2004, stored live at 8 °C, and analyzed within the next 3 days before being fed. The defensive secretion was sampled directly from the abdomen of the butterfly using a glass capillary18. Three males and three females were sampled from each site, and each individual butterfly secretion was analyzed separately (N = 64). Sample identity was blinded from the technician. Volatile compounds were quantified by the external standard method using a six-point standard curve with standards ranging from 0.005 to 5.0 mL mL−1. Calibration curves from triplicate injections of 2.0 ml were obtained using the gas chromatography (GC)–MS protocol above. Peak integration and quantification were performed automatically using Saturn 2100 Workstation software. Two millilitre of the secretion were collected from a disturbed butterfly and dissolved in 2.0 mL of ethyl acetate with 1.0 mL of 0.25 M p-chlorotoluene as the internal standard. Then 2.0 mL of this solution were injected directly into the GC column. Each butterfly sample was run on the GC twice for consistency. The concentrations were considered consistent if runs 1 and 2 were within 5% of each other. If not, then the individual butterfly was resampled until the two runs reached the consistency criterion. However, only the concentration of the first of those two runs was used for reporting and statistical analyses (N = 64). We tested for an effect of queen abundance on volatile defensive phenolics in viceroys; this involved a total of three separate analyses that differed only in the response variable: total phenolic volatiles, salicylaldehyde, and benzaldehyde.
Predator behavioral experiments
Laboratory-reared adult Chinese praying mantids, Tenodera sinensis (Mantodea: Mantidae) served as the experimental predator18,26. Chinese mantids are a known predator of butterflies and perform well in laboratory experiments. They are naturalized in the U.S. and have been observed preying on viceroys and queens at study locations in Florida and respond behaviorally to unpalatable prey similar to avian predators14,26. Fifteen egg cases were purchased from Carolina Biological Supply Company and reared to adults in 2003 and 2004. Mantids were reared in individual cages on a diet of fruit flies, houseflies, true bugs, and crickets. Mantids did not have access to butterfly species or distasteful prey before the experimental feeding trials. Each mantid was fed two adult crickets every night throughout the aversion learning and memory retention experiments.
All behavioral experiments were conducted in a laboratory arena consisting of three components: a rectangular perch for the predator, a square floor, and a cylindrical wall. The entire arena was painted a dark uniform gray26. The arena was illuminated by three full-spectrum halogen lamps (Solux-Eiko, 50 W, 4700 oK, 36o field of illumination). Each lamp was positioned 23 cm above the highest point of the perch and 20 cm from the other lamps. In all experiments, a trial began by placing a single mantid at the top of the perch inside the arena wall, such that the mantid’s longitudinal axis was perpendicular to the long axis of the perch. The mantid was allowed to acclimate for 5 min before trials began. All mantids remained at the top of the perch for all experiments and trials. Viceroys collected in July 2003 and July 2004 were used in this experiment and their identity was blinded from the human observer. Abdomens, rather than the entire insect, were used for consistency with previous experiments involving viceroys and avian predators14,28 and to remove potentially confounding effects of wing pattern and size variation40,41. Viceroy abdomens are black with white stripes, while the other butterfly abdomens used (Pieris rapae and Vanessa cardui) in the experiment were uniformly either white or light brown. A single butterfly abdomen was introduced to the arena by attaching one end of a string to a dowel then slowly dropping the attached abdomen from above within the field of view of the mantid. In separate analyses, we tested the effect of queen abundance on each of the two measures of palatability: predator learning aversion rate and predator memory retention.
The predator learning assay compared rates at which mantids learned an aversion for viceroys originating from different sites18,26. A single originally naïve mantid (N = 64) was fed only viceroys from a single locality. A trial ended either 5 min after a mantid attacked and ate the abdomen, or, if the mantid did not attack the abdomen, 5 min after the abdomen was presented to the mantid. After the trial ended, the butterfly abdomen or its remains were removed. If the mantid attacked the abdomen, it was returned to its holding cage after 2.5 min. If the mantid did not attack the abdomen, it was presented with a known palatable butterfly abdomen of similar size (Vanessa cardui or Pieris rapae) after 2.5 min to evaluate its hunger status. If the mantid attacked the palatable abdomen, it was evaluated as hungry. The mantid was not allowed to feed on the palatable abdomen because the abdomen might serve as a positive reward negatively affecting the aversion learning trials. This protocol prevented the mantid from associating its response to the viceroy abdomen with a palatable reward. A mantid was considered to show an aversion to the viceroy abdomen when it oriented to but failed to attack the abdomen, and subsequently attacked a palatable abdomen, in three consecutive trials. To test for a geographic difference between prey palatability and predator aversion learning rate, the number of trials until mantids reached aversion criterion was compared between geographic locations. All mantids were fed two crickets every evening in their cages to control hunger levels across treatments.
We evaluated predator memory retention by measuring the number of days until the mantid re-attacked a viceroy abdomen after reaching aversion criterion18,26. We compared predator memory retention among sites to evaluate the relative palatability of viceroy butterflies to predators. The same mantids (N = 64) used in the learning experiment were tested 2 days after the day they met the aversion criteria above and retested every second day thereafter. A trial ended either when the mantid attacked and consumed a viceroy, or when the mantid oriented to a viceroy, failed to attack, but subsequently attacked a palatable butterfly abdomen. A mantid was considered to have lost its aversive response when it attacked and partially or completely consumed a viceroy abdomen. All mantids were fed two crickets every evening in their cages to control hunger levels across treatments.
Statistical analyses
All analyses were performed in the R software package42, with the lme443 and lmerTest44 packages. For all analyses, we used generalized linear mixed-effect models, including collection year and site as random intercept effects. In analyses where the response variable was continuous, we used the lmer function of lmerTest and used the Satterthwaite method to approximate degrees of freedom. In analyses where the response variable was ordinal (i.e., abundance), we used the glmer function of the lme4 for Poisson regression. Degrees of freedom are not reported for the Poisson regression models, as they are not available for the glmer function43. For purposes of display and reporting means, we classified sites using K-means clustering based on the number of adult queens observed in abundance assays. Applying the elbow method to determine the number of clusters based on the total within sum of squares, K-means clustering identified two clusters: one cluster consisted of the four northern sites, with low queen abundance, and the other cluster consisted of the four southern sites, with high-queen abundance. Maps were created in R with inverse distance weighting using the sp45,46, gstat47, rgdal48, and raster49 packages.
Code availability
R scripts for all analyses can be found at https://github.com/jcoliver/viceroy-mimicry-geography and are archived on Zenodo50.
Reporting summary
Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.
How to Quit Ignoring the Abundance You Have
Mangos.
That’s what I noticed today on my morning walk, after we got past the tiny shih tzu that terrifies my much larger dog and slowed back to a reasonable pace.
I’ve been seeing them in the stores and at the produce stands, of course. All the varieties. And on our way home, I see them in bunches, dangling from the trees.
They’re on the road, in the ditches. Some smashed and rotten, some unripe, some perfect.
Today there were around fifty mangos on each side of the narrow road I walked. A giant tree loomed over the road, dropping mangos for anyone who walked by.
These were good mangos; medium sized, red-tinted, whole.
The kind they used to sell in the grocery stores in Missouri for $5 each.
Here they are, laying on the ground. Waiting for someone to notice them. Free for the taking. Abundant. Available.
A few days ago I overheard a few people discussing lack: lack of resources, in particular, lack of food resources. How difficult it is to find what you want. How you have to grocery shop in several stores to get everything. How inconvenient it is.
And here are the mangos, waiting.
Pick one up. It’s yours. Eat it for breakfast. Chop it up and put it on your salad for lunch. Dice it with red onions, peppers, cilantro and put it on anything: tacos, stir-fry, curry, grilled chicken, fish, rice and beans. Or spoon it straight into your mouth.
Abundance is everywhere, but we don’t see it.
We do the same thing with other resources. We focus on what’s lacking and overlook what is abundant, prolific, pouring itself into our waiting hands.
The result?
We operate in a victimized mindset, struggle to find inspiration, rage against the limitations, see our situations through a stilted viewpoint that present obstacles instead of opportunities.
Why we have resource blindness
We fixate on what we’re missing — rather than seeing what we have, in abundance — for these reasons:
We don’t ignore abundant resources on purpose, necessarily; it’s more that our mindset doesn’t allow us to see what is there.
The results of resource blindness
It prevents a sense of gratitude and supply.
There is comfort and freedom in trusting that you are supported by a benevolent, generous universe; that there are options; that new resources are available; that having enough is the default.
It prevents efficiency.
You can waste an enormous amount of time and energy procuring what is difficult to get. When you have a plethora of something, freely and easily available, it’s much more efficient to find a way to use it. Adjust the means, and achieve the same end.
It limits your creativity.
When the resources you need are in limited supply, you become miserly with them. It’s a normal response to scarcity, but it’s not helpful for creative flow.
Creativity requires a kind of recklessness, a trust that there is more, always more; even within boundaries, you need to work with a sense of ongoing supply. Anxiety over provision will shut your creativity down.
How to see the abundance
You can shift your mindset to see what’s abundant and available rather than to focus on what might be scarce for you.
Step 1: Make a list of what seems to be in short supply.
Remember that there are many types of resources, tangible and intangible:
I’m sure you can think of more.
But what we’re concerned with are the resources you’re missing.
For me, as a mother of four who primarily works from home, solitude and privacy and focused time for work have long been a scarce resource. I wasted so much energy trying to create a routine that would allow me hours of uninterrupted work time.
I felt that’s what I needed; after all, isn’t that what writers and creatives do? Get up and write until lunch time? Or spend long unbroken blocks of time on creative work every afternoon? Or stay focused on one project until so many words or pages are completed?
I was comparing my life with someone else’s, thinking I needed to imitate their methodology in order to achieve similar goals.
The means are not the ends.
And the process — putting words down, on a page — may be the same, but the way you incorporate that process into your life can vary.
I discovered that I had a few things in abundance:
In order to use that time, I had to see it and recognize its potential. I had to see it as a welcome and suitable resource, not as a reject or impossibility.
I didn’t see it for a long time because it didn’t match my expectation. It didn’t line up, detail for detail, with the way I thought it should look.
Desperation will help you see resources you’ve been ignoring.
So if you’re feeling desperate about something, take heart. You’ll find your resource soon.
Try this
Make a list of needs, those resources you feel like you can never get enough of. Beside each one, list what you want it to do for you, provide for you. When you see what you get from a resource — the benefit or ability you’re missing — you can expand out and see other ways to receive that benefit or ability.
Step 2: Make a list of what you have in abundance.
You’ll need to start noticing for this.
Notice what comes to you without any effort at all. Notice what appears.
Don’t ignore the things that seem negative, like interruptions.
A negative is the single-perspective vision of a bigger thing. It’s one side of the coin. If you have something ‘negative’ in abundance, like interruptions, it means you have the inverse, or positive, of that thing in abundance, too.
You just have to see it.
For me, interruptions to work are very frustrating. Once I get my head in the research or the draft, it’s jarring to have to leave it to explain the snack options to a small child, for the fiftieth time that day.
But the interruptions mean that I have affection, support, and love. Seeing both sides of what you have in abundance can help you recognize the riches offered in it.
No, that doesn’t mean you have to tolerate things as they are. It’s good to ask people to respect your work, to find ways to minimize interruptions, etc.
Set boundaries, but see the abundance first. Then you can set boundaries with a sense of gratitude, rather than rage or defensiveness.
Asking for change from a perspective of gratitude is better:
(Because what I’m not exactly saying but kind of implying is that you are generally the source of all the conflict you experience. Change your self, adjust your perspective, reduce the inner conflict, and a lot of external conflict will disappear. But that’s another post for another day.)
Try this
Keep a log of what comes to you, unbidden, effortlessly. Write all of it down. The negative, the positive, the enjoyable, the frustrating. Try not to label it or reject it, simply become aware of it and note it. Once you have a list (give yourself a couple of days to do this, or even a week, so you have time to get a complete picture), continue to the next step.
Step 3: Look for the points of connection.
This is the thinking part of the process. Before this, you’ve just made two lists: what you need, and what you have.
Now it’s time to compare these two lists. It’s time to find the ways they connect.
What you lack — your scarce supply — isn’t important. Look instead at what you want to get from each thing you lack.
If you lack time alone, for example, what is it you want from that time alone? Is it a chance to work with focus? Is it time to get inspired? Is it to feel more yourself? Solitude? What do you want, what benefit or ability would that time alone provide?
With the specific benefit of each resource in mind, look at your list of abundance. Look at the positive hiding behind each negative, and ask yourself to find the matches.
You can find them.
This is like those worksheets my 7-year-old does, a picture on one side of the sheet and the printed word on the other. You have to match them up correctly: cute kitten photo with the word “C A T.”
This is the need-and-resource version. Match up your lack and your abundance. There will be a match.
If there isn’t, dive deeper into your need.
How to dive deeper
Are you sure you know what you’re looking for?
Are you sure you know what you need, what the ultimate craving is beneath each stated desire, each seeming lack?
Focus on the present
I operate from a belief that has made my life much simpler and richer: what I have, right now, is exactly what I need.
What I am receiving — in any form, painful or pleasant — is a gift.
My job is not to judge the gift, but to unwrap it. To open it, to receive it. To accept it. To see its value, and — if I can’t see the value — to trust that it’s in there, somewhere.
That’s what you’re doing, right now: training yourself to see what you already have rather than to focus on what you think you’re missing.
What you have, right now, is precisely what you need. If there seems to be a lack, it’s because of your limited understanding, not due to an actual limitation of resource.
You may think that’s crazy. I get it.
But how much do you truly need right now, for this moment?
Not that much.
What throw us off is our refusal to keep our attention on the present. We get panicky. We’re afraid of contingencies. We want to have our hot little hands on everything we might need for the present and the foreseeable future.
But the future isn’t real. It doesn’t exist. It never will. All you will ever have is this present moment. If you want to put your precious energy into solving an invented scarcity for a pretend lack in your imaginary future existence, go for it.
Me, I have other things to do.
Rethink your goals
We think we know what we need and want, but often we don’t.
We compare with others, take on their achievements or appearances as our desires, and strive for something that doesn’t come from the heart.
If you can’t find what you need to accomplish some goal, stop and ask the bigger question: why can’t I do this thing?
Maybe the answer is simpler than you think.
Maybe you don’t really want to do the thing. Maybe it’s not your goal. Maybe the universe is trying to stop you from a colossal waste of time. Maybe you’re stopping yourself because you know your heart’s not in it.
Rethink your supply
We think we know what we have. But truly, we miss what is in front of us all the time.
Nothing in this universe is one-sided.
If you think you’ve getting only negative — angry boss, boring projects, endless interruptions, mundane coworkers — it’s up to you to look at it long enough to find the other side.
Find the inverse of each negative. Look for the positive. It’s there.
Find the gift. Find the abundant resource hidden, the one that’s been there all this time.
That angry boss might give the best, most valuable feedback you’ve ever had if you ask. Those boring projects may provide you with the day-dreaming time you need to plot your novel while you tick checkmarks off the spreadsheet at work. The mundane coworkers may b the dependable support system you’ve never had from your hip, interesting, flaky friends.
And if everything in your life is negative, if the thread that flows from the beginning to the end of your day is a painful one, here is an important thing to think about: it’s coming from you.
Suffering, ultimately, has nothing to do with your situation (external reality) and everything to do with your internal reality: your mindset, your choices, your way of viewing the world.
If that’s where you are, lost in pain and feeding on negativity, what the universe is giving you is precious. Do not reject it. Do not ignore it. It is the gift of desperation, of loss, of hopelessness, of frustration.
The gift of pain.
The gift of dissatisfaction, a gift which will cause you to look at your life hard and long and objectively enough to realize you are the common denominator.
You are the cause, you are the catalyst, you are the responsible party.
What a gift.
Now you know you have the power to change it. Now you have the resources you needed all along, which were nothing more than the understanding that you create your own reality with each choice you make.
Enjoy your abundant world.
Solving colibactin’s code
For more than a decade, scientists have worked to understand the connection between colibactin, a compound produced by certain strains of E. coli, and colorectal cancer, but have been hampered by their inability to isolate the compound.
So Emily Balskus decided to focus instead on the mess it leaves behind.
Balskus, a professor of chemistry and chemical biology, and her colleagues are the authors of a new study that seeks to understand how colibactin causes cancer by precisely identifying how the chemical reacts with DNA to create DNA adducts. The study is described in a Feb. 15 paper published in Science.
“It’s been known since 2006 that there’s a set of genes in certain gut-commensal bacteria — mostly in strains of E. coli — that gives them the ability to make molecules that can lead to DNA damage,” Balskus said. “Over the years, there have been a number of studies that have shown a correlation between the abundance of bacteria carrying this pathway and cancer in humans, and multiple mouse models of colitis-associated colorectal cancer have demonstrated that this specific set of genes … can effect tumor progression and invasiveness.”
But researchers have been in the dark about how it works.
“My lab started studying this because we were interested in this problem of how you can understand a molecule you can’t isolate,” Balskus said. “And the summary of our earlier work to understand colibactin was that, unexpectedly, we and other groups who worked on this pathway found that this natural product has what’s called a cyclopropane ring in it.”
It’s that chemical structure that Balskus and her colleagues believe forms the colibactin “warhead” — in part because similar structures are found in other, unrelated molecules capable of causing direct DNA damage by reacting with it.
“When we realized that, we hypothesized that a direct interaction with DNA may be important for colibactin’s genotoxic activity,” Balskus said. “That illuminated a new strategy for getting information about colibactin’s structure: Instead of trying to isolate the molecule itself, we could isolate and characterize the DNA adducts, or the products of the reaction with DNA.”
Isolating those DNA adducts, however, is no easy feat.
To do it, Balskus and her team turned to Silvia Balbo, a professor at the University of Minnesota School of Public Health, who developed a novel technique to identify DNA adducts based on how they fragment in a high-resolution mass spectrometer.
“What we did, which I thought was a very exciting experiment, was to take a strain of E. coli that could produce colibactin and a mutant strain with the same genotype, except it didn’t have the gene cluster that makes colibactin,” Balskus said. “We incubated those strains with human cell lines … and isolated the DNA from both sets of cells, put it in the mass spectrometer and compared the abundance of different DNA adducts in the samples. So we were able to find DNA adducts that were only generated in the cells that were treated with the genotoxin-producing bacteria.”
Armed with that information, Balskus said, her team’s next challenge was to understand the chemical structure of those adducts.
“It looked like they came from colibactin based on the fragmentation in the mass spectrometer,” Balskus said, “but that’s not enough to solve a chemical structure. What researchers in my lab did — and it was a heroic effort — was to chemically synthesize a standard. Then we compared it to the adducts produced in the cells, and they were the same.”
To demonstrate that the process was also at work in living animals, the team collaborated with Wendy Garrett at the Harvard T.H. Chan School of Public Health, to conduct an experiment in which germ-free mice were colonized with strains of E. coli that could and couldn’t produce colibactin.
“We showed that we were able to detect these same DNA adducts in the colonic epithelial tissue of the mice with the colibactin-producing strains,” Balskus said. “That tells us that all the chemistry that we and others have been doing ex vivo really might be relevant for what’s going on in vivo.”
Going forward, Balskus hopes to investigate whether those same adducts can be detected in samples from patients, and to understand the specific types of DNA damage caused by colibactin and whether they influence cancer development.
And now that the researchers have a good understanding of the chemical structure of the DNA adducts created by colibactin, Balskus said, they may be able to work backward toward the molecule itself.
“The adducts we identified are most likely coming from decomposition of a larger species,” she said. “So we’re still trying to solve this chemical mystery and working toward figuring out what the full structure might be.”
The findings also suggest that DNA adducts could be used as a key biomarker for the activity of compounds like colibactin and other potential carcinogens derived from the activity of gut microbes.
“Up until this point, when people were looking for organisms with the ability to make these DNA-damaging compounds, they were looking for the biosynthetic genes. That tells you about the genetic potential, but it doesn’t tell you that DNA damage has actually occurred,” Balskus said. “And we know from other areas of toxicology that if you have good biomarkers for predicting carcinogenesis, that can be powerful when thinking about assessing cancer risks.
“It’s still very early,” she continued, “but that is one area where our work could potentially lead. It’s still too early to know if colibactin plays a causal role in tumor development in humans, but we would like to have better ways of monitoring colon cancer susceptibility.”
This research was supported with funding from the Packard Fellowship for Science and Engineering, the Damon Runyon-Rachleff Innovation Award, the National Institutes of Health, the National Institute of Environmental Health Sciences, the Center for Environmental Health Sciences, and the U.S. National Institutes of Health and National Cancer Institute.
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